Limitations to yield in saline-sodic soils:

Quantification of the osmotic and ionic regulations

that affect the growth of crops under salinity stress

Ehsan Tavakkoli

BSc. Hon (Agricultural Engineering)

MSc (Agricultural Sciences)

School of Agriculture Food and Wine Faculty of Science

The University of Adelaide, Australia

Thesis by publication submitted to the

University of Adelaide for the degree of Doctor of Philosophy

January 2011

i

DEDICATION

I would like to dedicate this thesis to a number of people without whom I could not stand where I am today.

This thesis is dedicated to my late grandfather, who passed away at the age of 99 in the course of my Masters degree at UNE. His soul will live on within me for his encouragement and support enabling me to continue my studies in Australia.

Also this thesis is dedicated to my parents who were my first teachers and taught me the best kind of knowledge and for their love, endless support and encouragement. Finally, this thesis is dedicated to Professor Acram Taji who supported me all the way since the beginning of my studies in Australia, and who has been a great source of motivation and inspiration.

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Table of Contents

ABSTRACT ............................................................................................................................. v

DECLARATION ................................................................................................................... vii

Acknowledgments ................................................................................................................. viii

1. INTRODUCTION ................................................................................................................... 1

1.1Thesis outline ...................................................................................................................... 3

2. LITERATURE REVIEW ...................................................................................................... 5

2.1 Saline, sodic and saline-sodic soils: definitions .............................................................. 5

2.1.1 Salinity .................................................................................................................... 5

2.1.2 Sodicity .................................................................................................................. 7

2.1.3 Saline-sodic soils .................................................................................................... 9

2.2 The physical and chemical properties of saline-sodic soils ......................................... 10

2.2.1 Clay dispersion in sodic soils ................................................................................ 10

2.2.2 The chemical properties of saline-sodic soils........................................................ 15

2.3 Causes of salinity ............................................................................................................ 17

2.4 Growth responses of crop plants in saline-sodic soil ................................................... 19

2.4.1 Two-phase process of growth inhibition by salinity ............................................ 19

2.4.2 Effects of salinity on plant available water: Osmotic stress ................................. 23

2.4.3 Effects of specific ion toxicity in crops ................................................................ 27

2.4.3.1 Mechanisms of Na+ toxicity in plants ................................................... 28

Selectivity of potassium uptake at the plasma membrane ................. 31

Binding of calcium to the plasma membrane ................................... 32

2.4.4 Plant photosynthesis ............................................................................................. 34

2.5 Mechanisms of salt tolerance in crop plants ................................................................ 37

2.5.1 Osmotic adjustment .............................................................................................. 39

2.5.2 Reduced uptake and translocation ........................................................................ 40

2.5.3 Transport and compartmentation .......................................................................... 42

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2.6 Chloride dynamics in crop plants in relation to salinity responses ........................... 44

2.7 Salinity tolerance in barley and faba bean ................................................................... 50

2.7.1 Barley (Hordeum vulagare) .................................................................................. 50

2.7.2 Faba bean (Vicia faba) .......................................................................................... 51

2.8 Breeding for improving salt tolerance in crop plants ................................................. 52

2.8.1 Screening Methods ............................................................................................... 54

2.8.1.1 Germination ......................................................................................... 55

2.8.1.2 Photosynthesis and other physiological indicators .............................. 56

2.8.1.3 Field vs. controlled conditions ............................................................. 57

2.9 Salinity research ............................................................................................................. 59

2.9.1 Soil and nutrient culture systems ......................................................................... 59

2.9.1.1 Soil culture systems .............................................................................. 59

2.9.1.2 Nutrient solution systems ..................................................................... 60

2.9.1.3 Field studies ......................................................................................... 61

2.9.1.4 Growth conditions and salinity treatments .......................................... 62

2.9.2 Separating the osmotic stress from ionic toxicity ................................................ 63

2.10 Conclusions and further research ............................................................................... 65

List of articles presented for this thesis .............................................................................. 68

List of peer-reviewed conference papers presented for this thesis .................................. 69

3. Chapter 3 The response of barley to salinity stress differs between hydroponics and soil systems

4. Chapter 4 Growth of faba bean in saline-sodic soils: Monitoring of leaf development and water use dynamics enables the quantification of osmotic and ionic regulation at whole-plant level

5. Chapter 5 Additive effects of Na+ and Cl- ions on barley growth under salinity stress

6. Chapter 6 High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress

7. Chapter 6 Effective screening methods for salinity tolerance: pot experiments but not hydroponics are plausible models of salt tolerance in barley

8. Genotypic variations of faba bean in response to transient salinity at whole-plant level

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9. GENERAL DISCUSSION AND CONCLUSIONS ........................................................... 70

9.1 Introduction ..................................................................................................................... 70

9.2 Relative importance of ion (Na+ and/or Cl-) toxicity and osmotic effect to growth and yield reduction under different levels of salinity ............................................................... 74

9.3 Relative importance of Na+ and Cl- toxicity in growth reduction of barley and faba bean ............................................................................................................................... 76

9.3.1 Barley ................................................................................................................... 77

9.3.2 Faba bean ............................................................................................................. 78

9.4 Evaluation of crop salt tolerance in solution and soil cultures under controlled environmental conditions: Are they good surrogates for evaluating whole-plant response to salinity under field conditions? ...................................................................... 80

9.4.1 Barley ................................................................................................................... 81

9.4.2 Faba bean ............................................................................................................. 83

9.5 Conclusions ...................................................................................................................... 84

9.6 Recommended future research ....................................................................................... 86

References for literature review and general discussion ....................................................... 89

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ABSTRACT

Salinity reduces yields of agricultural crops in many arid and semi-arid areas of the

world where rainfall is insufficient to leach salts from the root zone. Salinity reduces

plant growth and yield by two mechanisms, osmotic stress and ion cytotoxicity. Munns

et al. (1995) proposed a two-phase model of salt injury where growth is initially

reduced by osmotic stress and then by Na+ toxicity. However, some uncertainty exists

regarding the relative importance of the two mechanisms. This is due to the difficulty in

separating the osmotic effect from specific ion effects because of the overlap in the

development of the two type stresses during the development of salinity stress. There

has also been some recent debate about the importance of soil Cl-, and by implication

plant Cl- uptake, as predictors of damage and yield loss, rather than electrical

conductivity. Where NaCl is high, increased uptake of Na+ ions will be associated with

high uptake of Cl- ions. Reliable and effective salt tolerance screening techniques to

predict field performances are important for breeding programmes. Thus, in

comparisons between results from laboratory and/or glasshouse soil and solution culture

screening techniques and field evaluations of salt tolerance, it is important to verify

whether or not the laboratory conditions can predict responses to field stresses. The

main objectives of this research were to:

determine which of the two ions most frequently implicated in salinity, Na+ and

Cl-, is most toxic to barley and faba bean

quantify the relative importance of ion (Na+ and/or Cl-) toxicity and osmotic

stress on growth and yield reduction under different levels of salinity

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investigate whether hydroponics and pot experiments under controlled

environmental conditions are useful surrogates for evaluating whole-plant

response to salinity under field conditions.

High concentration of Na+, Cl- and NaCl separately reduced growth, however the

reductions in growth and photosynthesis were greatest under NaCl stress and were

mainly additive of the effects of Na+ and Cl- stress (Chapter 5 and 6). The results

demonstrated that Na+ and Cl- exclusion among genotypes are independent mechanisms

and different genotypes expressed different combinations of the two mechanisms. The

results also suggested the two-phase model of salt stress may not be appropriate at all

levels of salt stress. Osmotic stress was the predominant cause of reduced growth at

high levels of salinity, while specific-ion toxicity was more important under mild

salinity stress (Chapters 3 and 4). In barley, the effects of salinity differed between the

hydroponic and soil systems. Differences between barley cultivars in growth, tissue

moisture content and ionic composition were not apparent in hydroponics, whereas

significant differences occurred in soil. Reductions in growth were greater under

hydroponics than in soil at similar EC values and the uptake of Na+ and Cl- was also

greater (Chapters 3 and 7). Early assessment of salinity tolerance at seedling stage was

found to be unsuitable. This work has also established sound screening procedures that

significantly correlated with field evaluation of grain yield in genotypes of barley and

faba bean (Chapters 7 and 8). Salt exclusion coupled with a synthesis of organic solutes

were shown to be an important component of salt tolerance in the tolerant genotypes

and further field tests of these plants under stress conditions will help to verify their

potential utility in crop improvement programs.

vii

Declaration

This work contains no material which has been accepted for the award of any other

degree or diploma in any university or other tertiary institution to Ehsan Tavakkoli and,

to the best of my knowledge and belief, contains no material previously published or

written by another person, except where due reference has been made in the text. I give

consent to this copy of my thesis when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act

1968. The author acknowledges that copyright of published works contained within this

thesis (as listed below) resides with the copyright holder(s) of those works. I also give

permission for the digital version of my thesis to be made available on the web, via the

University’s digital research repository, the Library catalogue, the Australasian Digital

Theses Program (ADTP) and also through web search engines, unless permission has

been granted by the University to restrict access for a period of time.

Tavakkoli E., Rengasamy P., McDonald, GK (2010) The response of barley to salinity stress differs between hydroponics and soil systems. Functional Plant Biology 37, 621-633. Tavakkoli E., Rengasamy P., McDonald GK (2010) High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany 61, 4449–4459. Tavakkoli E., Fatehi F., Coventry S., Rengasamy P., McDonald GK (2011) Additive effects of Na+ and Cl- ions on barley growth under salinity stress. Journal of Experimental Botany. 62, 2189-2203.

Date: 24/1/2011

Ehsan Tavakkoli

viii

Acknowledgements

Completing this thesis has been a long road, and without the help of many people along the way it would not have been possible. I would like to gratefully acknowledge the input and support of my principal supervisor, Associate Professor Glenn McDonald and my co-supervisor Dr Pichu Rengasamy, who have guided my research throughout this project and for their patience and the many discussions during all stages of the research. As I prepared this thesis they have spent many hours revising written material, and their availability, insights and assistance have been much appreciated but words alone cannot express the thanks I owe to them. Several other individuals have provided me with significant support over the course of this project. In particular, I am very grateful to my best friend, Dr Graham Lyons, for his invaluable advice, continuous support and friendship. His gentle reassurances and encouragement at various times in the course of my study are very much appreciated. I am very grateful to Waite Analytical Services (Teresa Fowles, Lyndon Palmer, Matthew Wheal and Deidre Cox) for accurate and timely analysis of soil and plant samples. I am also very grateful to my colleagues and friends in the Plant Nutrition Group, in particular Dr G. Lyons, Prof. R. Graham, Dr Y. Genc, Dr W. Bovill and Mr D. Keetch and in SARDI, Dr G. Sweeney, Dr. J. Emms and Mr. H. Drum for their support, frequent advice, technical assistance and friendship. My thanks go to Mr S. Coventry (National Barley Breeding Program, UA) and Dr J. Paull (National Faba Bean Breeding Program, UA) for their expert management of field trials and providing the seeds of barley and faba bean genotypes for this study. I am thankful also to Prof M. Tester for making the Lemna Tec imaging system accessible during this research and the Australian Centre for Plant Functional Genomics for enormous help in letting me to use the lab equipments for these studies. I am very grateful to Prof S. Tyerman, Dr R. Munns, Dr C. Grant, Dr A. McNeil, Dr D. Chittleborough, and Prof. D. Suarez for stimulating discussion on different aspects of this research project. Many people at the School of Agriculture, Food and Wine (UA) and South Australian Research and Development Institute have also assisted me in this research. I would like to thank Mrs. A. Marchuk, Mr. C. Rivers, Mrs. W. Sullivan and Dr. Y. Shavrukov for their assistance in the laboratory. Many thanks also to Mr. P. Ingram for his help with glasshouse and growth chambers arrangement. I wish to sincerely thank the Grains Research and Development Corporation for funding and the University of Adelaide, School of Agriculture, Food and Wine for funding and hosting this PhD study. Finally, and most importantly, I would like to thank my family: mum and dad, my sister Dr M. Tavakkoli and my brother in law Dr H. Fadavi for their positive source of encouragement, love and support throughout this study.

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Chapter 1

1. Introduction

With the world's population expected to grow from 6.8 billion now to 9.1 billion by

2050 (Nature 2010), a there is concern about our ability to produce sufficient food to

meet the burgeoning population. The world's population more than doubled from 3

billion between 1961 and 2007. According to the World Bank and the United Nations,

between 1 and 2 billion humans are now malnourished, due to a combination of

insufficient food, low incomes, and inadequate distribution of food. As the world

population increases, the food problem will become increasingly severe, conceivably

with the numbers of malnourished reaching 3 billion. For example, the per capita

availability of world grains, which make up 80 % of the world's food, has been

declining for the past 15 years (Kendall and Pimentel 1994). Certainly with a quarter

million people being added to the world population each day, the need for grains and all

other food will reach unprecedented levels. More than 99 % of the world's food supply

comes from the land, while less than 1 % is from oceans and other aquatic habitats

(Pimentel et al. 1973; Pimentel et al. 1994) and so maintaining soil fertility and

increasing crop production is of worldwide significance. Hunger and poverty must be

overcome by expanded food production by applying sustainable and viable soil fertility

and plant nutrition management.

Salinity is an important environmental factor that reduces crop productivity in many

agricultural areas, mainly in arid and semi-arid regions (Rengasamy 2010b). In

Australia, of the 7.6×106 km2 of agricultural land about 33% (2.5×106 km2) has sodic

soils that have a potential to develop transient salinity. Of the area of land devoted to

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cropping approximately 80% can be affected by some form of salinity - 16% from water

table-induced salinity and 67% from transient salinity - costing the farming economy

about $A1.330 billion per annum, in lost opportunity (Rengasamy 2006).

Salt in the soil solution inhibits plant growth for two reasons. First, the presence of salt

in the soil solution reduces the ability of the plant to take up water, and this leads to

slower growth. This is the osmotic or water-deficit effect of salinity (Munns 1993).

Second, excessive amounts of salt enter the transpiring leaves and this may further

reduce growth. This is the salt-specific or ion-excess effect of salinity (Munns 1993).

The physiology of plant responses to salinity and their relations to salinity resistance

have been much researched and frequently reviewed in recent years (Cheeseman 1988;

Yeo 1998; Flowers 2004; Chinnusamy et al. 2005; Munns and Tester 2008). However,

it has been difficult to assess with any confidence the relative importance of ion toxicity

and water deficit to reduction in growth (Rajendran et al. 2009). Despite the fact that the

osmotic effect on growth of more salt tolerant species such as wheat and barley is often

much greater than the salt-specific effect (Ueda et al. 2004; Munns and Tester 2008),

the relative importance of the mechanisms that regulate the growth rate are not well-

understood. Additionally, this may be further be complicated in soil-grown plants where

the effect of soil physical properties may interact with the soil solution to determine soil

water potential and water uptake by plants. Therefore many interpretations have been

proposed regarding the physiology and causes of growth and yield reduction in the

field, the question whether the cause of the reduced growth is water deficit or ion excess

is still not resolved.

In annual crop and pasture plants, research on salt tolerance has focussed on the effects

of Na+. However, there has been some recent debate about the importance of soil Cl,

3

and by implication plant Cl uptake, as predictors of damage and yield loss, rather than

electrical conductivity (Dang et al. 2006b; Dang et al. 2008). Chloride toxicity is known

to be important in some species, especially perennial plants, but there is little

information on its impact on annual broadacre crops. Little is known concerning the

primary acquisition mechanisms of Cl by plants, and knowledge about its subcellular

distribution and flux dynamics is scarce (Britto et al. 2004).

1.1 Thesis outline

Chapter 2 develops a conceptual framework of constraints caused by soil salinity and

sodicity in cropping systems, and the current literatures on the impacts of salinity on

soil, water and plant relationships in soil systems are discussed.

Chapter 3, is based on a paper published in the journal of Functional Plant Biology. In

this chapter the relative importance of different mechanisms of salinity tolerance in

hydroponics and in soil in two varieties of barley that are known to differ in their salt

tolerance and ability to exclude Na+ are compared. We also tested the hypothesis that

the responses to salinity in soil-based systems are different to those observed in

hydroponics.

Chapter 4 aims to quantify the relative importance of ion toxicity and osmotic stress to

growth reduction at different levels of salinity among two faba bean genotypes differing

in their salt tolerance. To describe the different phases of salt stress, we provide an

example of a non-destructive, real-time method to assess the growth of faba bean plants

during a greenhouse experiment using commercially available image capture and

analysis equipment (LemnaTec ‘Scanalyser 3D’). Daily measurements of plant growth

4

in parallel to plant water use allowed the continuous monitoring of responses to salinity

stress.

Chapters 5 and 6 are based on papers that have been published independently in the

Journal of Experimental Botany. In Chapter 5 the extent to which the Na+ and Cl

contribute to ion toxicity in barley is critically assessed on selected barley varieties with

different mechanisms of salt tolerance in both hydroponic and soil systems. In Chapter

6, the relative importance of toxicity of Na+ versus Cl in faba bean was assessed using

two genotypes of faba bean differing in their ion exclusion mechanisms and salt

tolerance in a soil-based experiment.

Chapter 7, is based on a paper that has been submitted to the Journal of Experimental

Botany. The aims of work in this study were to evaluate the genotypic variation for

salinity tolerance among 60 varieties of barley in a supported hydroponic system and to

investigate possible physiological traits that could be used as screening criteria in

selected genotypes in a soil-based experiment and in the field.

Chapter 8, is based on a paper submitted to the journal of Field Crops Research. This

study reports the results of two experiments conducted to compare the responses to

salinity in hydroponics and in the field in a diverse range of faba bean cultivars and to

assess the value of tissue Na+, Cl- and K+ concentrations as a criterion for salt tolerance

and assess the importance of different mechanism of salinity tolerance in two systems.

In chapter 9 the conclusions are drawn and a perspective into future research is given.

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Chapter 2

2.0 Literature review

This literature review develops a conceptual framework of constraints caused by soil

salinity and sodicity in a soil-based cropping system. The impacts of salinity on soil,

water and plant relationships in soil systems are discussed. The review attempts to

address the constraints to plant growth and nutrition in saline-sodic soils. Then the

mechanisms of osmotic stress and Na+ toxicity to plants are explained along with Cl-

dynamics in crop plants in relation to salinity responses. In addition, the mechanisms of

salinity tolerance and the relative importance of these various processes are discussed.

The different experimental techniques in salinity research and the advantages and

limitations of these experimental methods as well as some aspects of growing plants for

salinity experiments, comparisons between experiments and the relevance of

experiments to field situations are summarized. The review concludes with a summary

of the research hypotheses.

2.1 Saline, sodic and saline-sodic soils: definitions 2.1.1 Salinity

Salinity is the concentration of dissolved mineral salts in soil solution as a unit of

volume or weight basis (Ghassemi et al. 1995). The major ions present in a soil solution

are the anions chloride (Cl-), sulphate (SO4 2-), bicarbonate (HCO3-), carbonate (CO3 2-)

and nitrate (NO3 -), and the cations sodium (Na+), calcium (Ca2+), magnesium (Mg2+),

and potassium (K+). In hypersaline soils (originated from the evaporation of sea water),

other constituents can be present, such as barium (Ba), strontium (Sr), lithium (Li),

6

silicon dioxide (SiO2), rubidium (Rb), iron (Fe), molybdenum (Mo), manganese (Mn),

and aluminium (Al3+) (Tanji 1990), but such soils are agriculturally non-productive and

are not considered further. The formation of CO3 2- and HCO-3 is affected by pH and

these ions are only present in soils of pH 9.5 or greater (Rengasamy 2010b). Saline soils

are classified as those with ECe > 4 dS m-1 and an exchangeable Na+ percentage (ESP)

< 15, which equates to an approximate NaCl concentration of 40 mM (Rengasamy

2010b). The ratios of the ionic constituents in soil-water depend on the chemical

reactions that take place in soil-water-plant systems under different conditions.

Chemical analyses provide full details of salinity (pure water or soil-water extract) and

specific ion concentration. However, as a general predictor, salinity usually is described

as total salts irrespective of its constituents.

Electrical conductivity (EC) is used as a fast method to evaluate soil salinity and is

based on the fact that the electrical current transmitted between two electrodes under

standardised conditions changes with a change in soluble ionic salts. The basic SI unit

of EC is Siemens per metre (S m-1). In agriculture, EC is often low; thus deciSiemens

per metre (dS m-1) is widely used. The unit (mmhos cm-1) used in the past is

numerically equal to dS m-1. Electrical conductivity can be related to electrolyte

concentration for different solution conditions.

log Co = à +ϖ log EC

where Co is the salt concentration expressed in mmol L-1, ϖ and à are empirical

parameters which vary with different mixed solutions, and have values of about one.

Soil-water salinity depends on the water content at which the salinity needs to be

determined. Separating the soil solution from the soil sample is difficult (Dyer et al.

2008) and the quantity of extracted water within the normal plant available range is

7

usually insufficient to conduct chemical analyses. Therefore, an extra known volume of

water can be added to the soil sample before extracting the soil solution. The extraction

process can be performed after mixing a given weight of soil with a certain volume of

water. Different soil:water extract ratios have been used to predict soil salinity such as

1:5, 1: 2.5 and 1:1 extract. A good approximation of soil-water salinity is that measured

in a saturated soil paste extract (ECe). Saturated soil paste extract can be prepared in

which a given weight of soil is saturated and then the soil solution extracted. Since the

water content at saturation is nearly twice that of field capacity, the EC of the saturated

extract is approximately half that of the soil at field capacity (Rhoades et al. 1989). Soil

salinity in the field can be monitored using various instruments. Examples of these

instruments are a salinity sensor based on electronic conductivity, time domain

reflectometery (TDR), and inductive electromagnetic meter (Dudley 1994).

2.1.2 Sodicity

Sodic soils are generally defined by the parameter of exchangeable sodium percentage

(ESP) which can be calculated as follows (concentrations in cmolc kg

-1);

ESP = (100 × Exchangeable Na+) / Cation Exchange Capacity

The sodicity of irrigation water and soil solutions is defined using the parameter of Na+

adsorption ratio (SAR) and can be calculated as follows (where the concentrations Na+,

Ca2+ and Mg2+ are measured in mmol L–1.concentrations in mM);

SAR = [Na+] / [Ca2+ + Mg2+]1/2

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No universally accepted critical ESP for sodic soils exists. The current definitions for

sodic soils are based upon the critical ESP at which soil dispersion occurs, with the

critical value ranging between 5% (McIntyre 1979) and 15% (USSL 1954). In

Australia, three classes of sodicity, non-sodic (ESP < 6%), sodic (ESP 6%-15%) and

strongly sodic (ESP >15%) are assigned (Rengasamy 2006). These conflicting sodicity

classifications demonstrate that the behaviour of soils in the presence of exchangeable

Na+ differs according to numerous factors. These factors include the electrical

conductivity (EC) of the soil solution and leaching water, soil texture, clay mineralogy,

organic matter content, position within the profile, pH and the suite of accompanying

cations (Rengasamy 2010b). Thus, the sodicity classification of a particular soil needs

to be determined according to its behaviour in the environment and corresponding

limitations to productivity, rather than using a critical ESP value.

Using the measured ESP as an indicator of the level of Na+ on soil exchange surfaces

may have errors due to the difficulties in determining the CEC. Qadir and Schubert

(2002) concluded the reasons for incorrect estimation of ESP was because: (a) the

extraction of exchangeable Ca2+ and Mg2+ during the chemical analysis process might

cause some CaCO3 and MgCO3 to dissolve, erroneously leading to an increase of CEC,

especially in calcareous soils; (b) the CEC in variable charge soils depends on pH,

solute concentration and buffering capacity of soil-water extract; (c) the removal of Na+

by extraction from a source that does not contain a true form of exchangeable Na+, such

as Na+ zeolites. Furthermore, determining the CEC is time consuming. In contrast, SAR

is thermodynamically more appropriate because it approximates the activities of various

cations in solution. In addition, SAR requires fewer parameters (the concentration of

Na+, Ca2+ and Mg2+), and can be determined from the same soil water extract used to

9

evaluate the EC in soil solution. SAR, however, does not take into account the change

of Ca2+ concentration in soil solution as a result of change of solubility of the Ca2+

(Qadir and Schubert 2002). Sodium remains soluble and in equilibrium with

exchangeable soil Na+ all the time. Conversely, Ca2+ does not remain completely

soluble and might be raised in soil solution because of dissolution of soil minerals and

usually precipitates in the presence of carbonates, bicarbonates and/or sulphates in

solution.

Moreover, in Australia many saline-sodic soils, particularly subsoils, have higher

exchangeable Mg2+ than Ca2+. Rengasamy et al. (1986) concluded that the enhanced

clay dispersion in high magnesic saline-sodic soils is due to the lower flocculating effect

of Mg2+ compared to Ca2+. Therefore, there is a need to derive and define a new ratio of

these cations in place of SAR, which will indicate the effects of Na+, K+, Mg2+ and Ca2+

on soil structural stability. This may be achieved by using a formula analogous to the

SAR but which selectively incorporates the dispersive effects of Na+ and K+ on the one

hand with the flocculating effects of Ca2+ and Mg2+ on the other. The concept cation

ratio of soil structural stability (CROSS) proposed by Rengasamy and Marchuk (2010)

is of such instances.

2.1.3 Saline-sodic soils

A saline soil dominant in Na+ ions is saline–sodic and becomes sodic when salts are

leached. Similarly a sodic soil becomes saline–sodic when Na+ salts accumulate in soil

layers (Rengasamy 2010b). Generally saline–sodic soils have a spectrum of disorders

and the soil solutions have a range of values of SAR and EC. Saline sodic soils are

10

those with an ECe > 4 dS m-1 and an ESP > 15. Further, as the pH of the soil increases

above 8, it becomes alkaline and carbonates dominate the anions. Thus, salts affect

plants through adverse soil properties of alkalinity and sodicity, properties imposed on

the soil by mobile salts. Different categories of salt-affected soils that are generally

found in different parts of the world, with criteria mainly based on SAR and EC of the

saturation extracts of the soil and pH measured in 1:5 soil–water suspensions are shown

in Figure 1 (Rengasamy 2010b).

The increase of pH could cause a significant increase of the ESP. It has been shown that

there is a linear relationship between the ESP and the pH of the soil saturated paste. A

small increase in the pH could result in a large increase in ESP values. This suggests the

increase in the pH enhances the preference of Na+ to be adsorbed on clay colloids (Ezlit

et al. 2010). It also indicates the increase of ESP with pH is the main factor determining

the clay deflocculation at given sodicity and salinity levels. Thus, the negative effect of

pH on soil deflocculation may be due to the increase of the ESP.

2.2 The physical and chemical properties of saline-sodic soils

2.2.1. Clay dispersion in sodic soils

Saline-sodic soils are subject to severe structural degradation and restrict plant

performance through poor soil-water and soil-air relations (Rengasamy and Olsson

1991). Large proportions (86 - 2%) of saline-sodic soils in Australia have dense subsoils

with an alkaline pH (8-9-5) trend, and their subsoil clay is highly dispersible due to

adsorbed Na+.

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Figure 1. Categories of salt affected soils based on Na+ adsorption ratio (SARe) and

electrical conductivity (ECe) measured in soil saturation extract and pH1:5 measured in

soil water suspension and possible mechanisms of impact on plants. Toxicity,

deficiency or ion-imbalance due to other elements (e.g. B, K, N, P) will depend on the

ionic composition of the soil solution. The diagram also shows the cyclic changes of the

categories as influenced by the climatic factors and land management. (Note: In

Australia, 1:5 soil : water suspension is commonly used for measurements of EC (and

also for SAR) because of the easiness of measurements. Preparation of soil saturation

extract is laborious and costly. Saturation extract is prevalently used in USA and other

parts of the world and therefore to compare the research data, particularly salt tolerance

thresholds for crops based on ECe, conversion of EC1:5 to ECe (Kelly and Rengasamy

2006) has become a necessity (Rengasamy 2010b).

12

Clays are those mineral particles in the soil with a diameter of less than 0.002 mm. As

such, they make up a large proportion of the internal surface area of the soil and

contribute significantly to soil physical and chemical properties. The stability of the

aggregates in a soil depends upon the relative strength of the forces that exist between

the clay fraction of the soil and the soil solution (Sumner 1993). The clay particles in

dry aggregates are linked together by strong attractive forces and the distance between

adjacent clay particles is generally less than 1 nm (Rengasamy and Olsson 1991). When

a dry soil aggregate is hydrated however, interactive forces lower the potential energy of

the water molecules, with the resultant release of energy being used partially for the

structural transformation of the clay aggregate and partially in the release of heat

(Rengasamy and Olsson 1991). The structural transformation of the aggregates that

occurs upon their hydration may include swelling, slaking and dispersion. Dispersion

involves the breakdown of a soil into particles of < 2 μm, which then diffuse through

the dispersing solution (Churchman et al. 1993). The dominant soil factor contributing

to dispersion is exchangeable Na+ but non-soil factors, such as the application of

external stresses, also contribute (So and Cook 1993).

The diffuse double layer (DDL) is the interface between the surface of a clay mineral

and the soil solution and consists of the negative charge of the clay surface and the

cations in the soil solution. The thickness of the DDL is smaller when dominated by

divalent (e.g. Ca2+

) or trivalent (e.g. Al3+

) ions, but larger where monovalent (e.g. Na+)

ions predominate. The thickness of the DDL is also reduced by solutions with high

electrolyte concentrations (Rengasamy and Sumner 1998). When a soil has a high ESP

and the electrolyte concentration of the soil is sufficiently low, the distance between

clay particles upon hydration increases to such an extent that the particles begin to

13

separate, resulting in accentuated swelling. When sodic soils disperse and then dry, the

result is the formation of a massive structure, without any hierarchical arrangement of

clay particles into micro and macro-aggregates. Dispersion has numerous adverse

effects on the physical properties of saline-sodic soils including reduced hydraulic

conductivity, increased susceptibility to surface crusting and hard-setting, reduced water

infiltration, increased runoff and soil erosion, reduced soil aeration and poor soil

drainage (Quirk and Schofield 1955).

The hydraulic conductivity (HC) of a soil is a measure of its ability to transmit water

when exposed to a hydraulic gradient. The maintenance of stable soil aggregates is

important in sustaining soil HC, as HC is largely dependant on the structure of the soil

matrix. Macropores are primarily responsible for the transmission of water through a

profile and the return of aerobic conditions after a watering event, while micropores are

largely responsible for the storage of water within the soil profile (Quirk 1978).

Shainberg and Caiserman (1971) established that there is a significant negative

correlation between soil ESP and HC, even at low sodicity levels and that a constant

reduction in HC is achieved at higher sodicity levels. The nature of this relationship is

also highly dependent on the EC of the percolating solution and is influenced by all of

the aforementioned factors; hence soil ESP alone does not predict the HC of soils

(Quirk and Schofield 1955). The primary mechanism responsible for the decreased

permeability of saline-sodic soils at low EC values is the swelling of clay domains

(Quirk and Schofield 1955). It remains unclear to what extent the blocking of soil pores

upon clay dispersion contributes to reductions in the HC of sodic soils (Quirk 2001).

14

Infiltration is the movement of water into the soil. The primary soil constraint to

infiltration in many soils is the formation of a thin surface layer with higher strength,

smaller pores and lowers HC than the underlying soil (Bradford et al. 1987). The poor

aggregate stability exhibited by saline-sodic soils upon wetting contributes to this seal

or crust formation and consequent reductions in infiltration rate (Kazman et al. 1983).

Crops produced on saline-sodic soils frequently suffer aeration stress after irrigation or

rainfall (Jayawardane and Chan 1994) as restricted infiltration of water results in

waterlogging in the surface soil layers and restricted internal drainage results in

waterlogging in sodic subsoils (McIntyre 1979). When a soil becomes waterlogged, the

pore space in the soil structure that usually allows the exchange of gas between the soil

and atmosphere is filled with water and diffusion of oxygen is severely reduced. The

consequences of waterlogging for the plant may include reduced or ceased growth, the

death of root apices and changes to the patterns of nutrient accumulation (Barrett-

Lennard 2003). Given that Na+ uptake can be increased under hypoxia (Wetson and

Flowers 2010) and anoxia (Drew and Lauchli 1985), this can exacerbate salinity stress

in saline-sodic soils.

Plant Available Water Capacity (PAWC) is a phrase used to describe the amount of

water present in a soil between field capacity and permanent wilting point. A negative

correlation has been established between soil EC, ESP and PAWC (Dang et al. 2006b;

Hochman et al. 2007). This has been attributed to the loss of porosity in the PAWC-

range of saline-sodic soils due to the processes of swelling and dispersion (McCown et

al. 1976). The resultant reduction in water storage can lead to the crop suffering

premature water stress (Rengasamy 2002; 2010b). Similarly, restricted root growth in

15

saline-sodic soils may result in lower plant rooting depth and a lower effective profile of

available water (Nuttall et al. 2003; Dang et al. 2006a; Dang et al. 2006b).

Productivity of crops grown under dryland conditions depends on efficient use of

rainfall and available soil moisture accumulated in the period preceding sowing.

However, single or multiple factors of physical subsoil constraints (e.g. reduced

hydraulic conductivity, reduced water infiltration, reduced soil aeration and poor soil

drainage) are present in many saline-sodic cropping soils that restrict ability of crop

roots to access this stored water and nutrients. Planning for sustainable cropping

systems requires identification of the most limiting constraint and understanding its

interaction with other biophysical factors.

2.2.2 The chemical properties of saline-sodic soils

Saline-sodic soils (ECe > 4 dS m-1 and an ESP > 6) are often associated with a number

of chemical properties that affect the availability of nutrients to plants. These include

elevated pH, high soil solution Na+ and Cl- concentrations, altered exchange equilibrium

and changes in redox potential. While NaCl and other highly soluble salts can be found

in high concentrations in the soil solution, soils may contain sparingly soluble salts such

as gypsum in amounts much greater than can be held in the soil solution. These salts

precipitate out of the soil solution and may form layers of visible salt crystals within the

soil profile (Bernstein 1975).

The calcareous saline-sodic soils commonly used for cereal production in Australia tend

to have alkaline pH values because they are often found in weakly leached

16

environments (i.e. dry climates) (Isbell 1996). Soil alkalinity occurs as a result of the

cumulative effects of long-term inputs of bases and outputs of acids. Alkaline anions

such as bicarbonate, carbonate or hydroxide occur in the soils mainly as a result of

removal of hydrogen ions from the soil, through the weathering of silicate minerals. If

there is sufficient flushing of the profile with water containing dissolved CO2, then

alkaline bicarbonate salt weathering products are leached out of the soil profile and it

will not become alkaline. The contribution of the carbonate precipitation to the rise in

pH of saline-sodic soils can be determined using data collected by Cruz-Romero and

Coleman (1975). Their data indicated that at ESP values between 0-100, the pH of

montmorillonite clay without CaCO3 present increased from 7.2 to 7.7 with increased

ESP, and the pH of montmorillonite clay with CaCO3 present increased from 8.2 to 10.0

as ESP increased. Hence the presence of CaCO3 is necessary for large rises in pH, which

in turn explains the presence of some neutral and acid sodic soils.

High soil solution Na+ affects the availability of nutrients to plants due to changes in the

ion exchange equilibria and solubility of some compounds. The exchangeable cations

found in irrigated Vertosols consist largely of Ca2+ and Mg2+, with a small proportion of

K+ and variable proportion of Na+. Irrigation water may also contain appreciable

quantities of Na salts (USSL 1954). The equilibrium that exists between the cations in

the soil solution and the cations on the exchange sites is constantly changing according

to the moisture content of the soil.

The chemical properties of saline-sodic soils that can limit crop growth and water

extraction include high concentrations of Na+, Cl-, and high levels of extractable boron.

These tend to be co-correlated, and spatially variable, in the alkaline soils that dominate

17

large sections of the regions of the grains industry in Australia. Despite the apparent

significance of subsoil properties to crop production, few quantitative data are available

to define the relationship. There is a need to quantify the relative importance of a range

of subsoil constraints in explaining variation in crop yield to give a better understanding

of the specific contribution of each constraint.

2.3 Causes of salinity

There are three major types of salinity based on soil and groundwater processes:

groundwater associated salinity, transient salinity and irrigation salinity (Rengasamy

2006). Groundwater associated salinity, commonly known as dryland salinity, occurs in

discharge areas of the landscape where water exits from groundwater to the soil surface

bringing salts dissolved with it. In landscapes where the watertable is deep and drainage

is poor, salts, which are introduced by rain, weathering and aeolian deposits are stored

within the soil profile. The concentration of salts often fluctuates with season and

rainfall and salt accumulation in soil layers is a common feature in sodic soils regions.

This type of salinity is termed ‘transient salinity’ (Rengasamy 2002) and is also known

as dry saline land or magnesia patches in South Australia.

The term transient salinity was first used by Hutson (1990) to model salt accumulation

in the root zone under irrigation practices. Transient salinity is a term to denote the

temporal and spatial variation of salt accumulation in the root zone that is not

influenced by groundwater processes and a rising saline watertable. Sodic soils can be

particularly susceptible to the development of transient salinity. Transient root zone

salinity is caused by two major factors: water and solute flux and hydraulic conductivity

of the root zone layers (Hutson 1990), which are affected by sodicity. Water infiltration

18

is very slow if the subsoils are sodic and water does not move down below that layer

(Rengasamy 2002). This can cause temporary waterlogging in the subsoil and a

saturated zone. Salts, derived from rainfall and soil weathering reactions, accumulate in

the saturated zones in the soil profile. After the wet season, when the water evaporates

quickly, salt accumulation in the sodic subsoil layers is exacerbated. The development

of transient salinity can be strong in low rainfall environments because of the low rates

of leaching in sodic clays, low rainfall in dryland areas and high transpiration by

vegetation and high evaporation during summer (Rengasamy 2002). The amount of salt

accumulating is not large, but can be detrimental to crops (Rengasamy 2002). This zone

of high salinity fluctuates with depth and also changes with season and rainfall.

Secondary salinity is salinisation associated with human activity, mainly as a

consequence of improper methods of irrigation. Poor quality water is often used for

irrigation, so that eventually salt builds up in the soil unless the management of the

irrigation systems is such that salts are leached from the soil profile. According to

Flowers and Yeo (1995) too few attempts have been made recently to assess the degree

of human-induced secondary salinization and this makes it difficult to evaluate the

importance of salinity to future agricultural productivity. Anthropic salinization occurs

in arid and semi-arid areas due to waterlogging brought by improper irrigation (Bresler

et al. 1982). Secondary salt-affected soils can be caused by human activities other than

irrigation and include deforestation, accumulation of air-borne or water-borne salts in

soils, salinization caused by contamination with chemicals and overgrazing (Pessarakli

1991; Fitzpatrick et al. 1994; Szabolcs 1994; Bond 1998).

19

2.4 Growth responses of crop plants in saline-sodic soil

The deleterious effects of salinity on plant growth are associated with (1) low water

potential of the root medium which causes a water deficit within the plant; (2) toxic

effects of ions mainly Na+ and Cl−; and (3) nutritional imbalance caused by reduced

nutrient uptake and/or transport to the shoot (Munns and Termaat 1986; Hasegawa et al.

2000; Ashraf 2004). This section will discuss these responses and the consequences for

crop growth under salt stress.

2.4.1 Two-phase process of growth inhibition by salinity

The general response of plants to salinity is reduction in growth (Ghoulam et al. 2002)

that occurs in two phases (Figure 2), a model which was proposed by Munns and

Termatt (1986) and developed in a number of further papers (Munns et al. 1995; Munns

and Tester 2008). Phase I is the reduction in growth caused by osmotic stress, and phase

II is the growth reduction caused by ion toxicity. The two phases occur consecutively,

and the transition between phases I and II may occur after days or weeks, depending on

a range of factors including salt concentration, environmental conditions and plant

physiology (Figure 2) (Munns et al. 1995).

Phase I affects the rate of expansion of new leaves. The rate of cell division is reduced,

as is the size, but not depth of the cells, resulting in smaller, more succulent leaves

(Munns and Tester 2008). The duration of phase I will therefore be affected by a range

of variables. Increasing external salt concentration or temperature would reduce the

duration of phase I, as would a faster initial growth rate of the plant (Munns et al.

1995).

20

Figure 2. The growth response to salinity stress occurs in two phases: a rapid response to the increase in external osmotic pressure (the osmotic phase), and a slower response due to the accumulation of Na+ in leaves (the ionic phase). The solid line represents the change in the growth rate after the addition of NaCl. (a) The broken line represents the hypothetical response of a plant with an increased tolerance to the osmotic component of salinity stress. (b) The broken line represents the response of a plant with an increased tolerance to the ionic component of salinity stress based on (Munns et al. 1995). (c) The dashed line represents the response of a plant with increased tolerance to both the osmotic and ionic components of salinity stress (Munns and Tester 2008). Phase I would also be shorter for plants that accumulate salts faster, as the growth

reductions caused by osmotic stress would be overtaken by the toxic effects of salt

accumulation in the plant (Munns and Tester 2008). Phase II is the toxic phase of Na+

and/or Cl- accumulation in the shoot tissue. This phase is characterized by an increase in

senescence of older leaf tissue (Munns and Tester 2008). The effect of salt on growth

during this stage is determined by the rate of salt accumulation in the tissues, and the

degree to which the plant can tolerate high salt concentrations. Once salt concentrations

build up to toxic levels, leaf tissues die. Senescence occurs in older leaves due to the

longer time for salt accumulation, although plants may preferentially divert salt into

older tissues in order to protect new leaves (Munns et al. 1995). Phase II is salt specific,

and growth reductions depend on sensitivity to particular ions present. This point would

also coincide with the onset of visible symptoms of salt damage in terms of necrosis and

NOTE: These figures are included on page 20 of the print copy of the thesis held in the University of Adelaide Library.

21

senescence of salt affected tissue. Growth would then be reduced by both decreased rate

of new leaf production, and increased death of older leaves (Munns and Tester 2008).

While a two phase model is a useful description of the way in which salinity acts to

reduce plant growth, it must also be considered that it is a simplistic representation of a

complex process. The effect of salt on plants may initially be only osmotic, at the

moment in which the plant comes into contact with the salt, but when plant starts to take

up salt and accumulate to high concentrations, the toxic phase of salinity stress

commences. After the initial osmotic adjustment, ion uptake may appear to have limited

impact in terms of visible symptoms of salt stress, but the metabolic demands of

maintaining osmotic adjustment and water uptake, preventing excessive ion uptake,

compartmentalizing ions, and protecting cellular processes, will result in a lower growth

for the plant. This effect is compounded when the accumulation of salts in plant tissue

reaches concentrations that result in death of that tissue, so that the plant loses

photosynthetically active leaf area to supply growing tissues with assimilates, further

reducing the overall growth rate of the plant.

The transition between phases observed by Munns et al. (1995) is the point at which the

toxic effects of salinity increase from interrupting cellular processes, to causing tissue

death, accentuating the difference in growth rate. However, not all responses to salinity

are consistent with this model. Using Krichauff (a variety with a relatively good Na+

exclusion) wheat plants and several electrolyte solutions to impose different levels of

salinity, Rengasamy (2010a) clearly indicated the continuous operation of an osmotic

effect as the EC of the soil solution increases (Figure 3).

22

Figure 3. Dry matter production of wheat in relation to EC of the pot soil solution comparing NaCl, CaCl2, Na2SO4, and Hoagland nutrient solution treatments (Rengasamy 2010a).

The osmotic effect was predominant and severely restricting of plant growth above a

certain value of soil solution EC, which in that study was 25 dS m-1, corresponding to

an osmotic pressure of 900 kPa. It was shown that below this EC value, ionic effects

due to Na+, Ca2+, SO4 2–, and Cl– were significant at low EC values. Further

investigations are necessary to find out whether the individual ionic effects are due to

toxic effects or ion imbalance effects. The clear understanding of the mechanisms will

help plant scientists to develop strategies in selection and breeding of salt-tolerant plants

(Rengasamy 2010a). In summary, while the two-phase model is useful in explaining

how a plant responds to salt stress, its ability to explain differences among genotypes

that exhibit differences in salt tolerance have been less successful. Some uncertainty

exists regarding the relative importance of the two mechanisms. This is due to the

difficulty in separating the osmotic effect from specific ion effects because of the

NOTE: This figure is included on page 22 of the print copy of the thesis held in the University of Adelaide Library.

23

overlap in the development of the two type stresses during the development of salinity

stress which was not covered by two-phase model.

2.4.2 Effects of salinity on plant available water: Osmotic stress

One of the important effects of soil salinity on plant growth is to induce plant water

deficits. This occurs because of the decline in the osmotic potential of the soil solution

as salt concentrations increase and a consequent reduction in water uptake by roots.

Water availability is also affected by soil texture and the physical structure of the soil.

This is in contrast to the hydroponic systems that are used commonly in studies on

salinity, where water availability and uptake are determined only by the osmotic

potential of the nutrient solution. Understanding the differences between soil and

hydroponic systems is important to the development of experimental systems that can

replicate field responses and produce more reliable screening methods.

Water potential (Ψ) is a thermodynamic concept that helps to explain the movement of

water through the soil-plant system. It is defined as the potential energy per unit mass,

volume, or weight of water. Water uptake by plants, and their growth rate, is affected by

the water potential of the growth medium. Soil water is subject to a number of force

fields, which causes its potential to differ from that of pure, free water. Such force fields

resulted from the attraction of the solid matrix for water, as well as from the presence of

solutes and the action of external gas pressure and gravitation (Iwata et al. 1994). The

total soil water potential (Ψt) is the sum of several component potentials:

Ψt = Ψm + Ψo + Ψp + Ψg

24

where Ψm, Ψo, Ψp, and Ψg are the matric, osmotic, pressure, and gravitational potential

components (Campbell 1988). The total potential of water in soil, referenced to the

chemical potential of pure liquid, is equivalent to the chemical potential of the soil

water at a chosen temperature and pressure. Matric potential is due to the

adhesion/cohesion and surface tension forces between water and soil particles. It is the

main component of total soil water potential in non-saline soils. The osmotic potential

of the soil is due to the concentration of soluble salts in the soil solution, and makes a

significant contribution to total soil water potential in saline soils (Groenevelt et al.

2004). The effects of gravity (gravitational potential) are not generally considered in

calculations of total soil water potential (Cresswell et al. 2008). Under saline conditions,

the low osmotic potential associated with the high concentration of salts in the soil

solution will decrease total soil water potential and affect both the rate of water use, and

the final soil water content to which the plant can extract water. The importance of this

will change with soil water content due to the changes in the concentrations of salt.

The degree to which plant growth is reduced during stress largely depends on the

severity of the stress. Mild osmotic stress leads rapidly to growth inhibition of leaves

and stems, whereas roots may continue to elongate (Westgate and Boyer 1985; Nonami

and Boyer 1990). The degree of growth inhibition due to osmotic stress depends on the

time scale of the response, the particular tissue and species in question, and how the

stress treatment was given. Arrested growth can be considered as a way to preserve

carbohydrates for sustained metabolism, prolonged energy supply, and for better

recovery after stress relief (Bartels and Sunkar 2005). Continued root growth under salt

stress may provide additional surfaces for sequestration of toxic ions, leading to lower

salt concentration. For example, salt tolerance of barley was correlated with the better

25

root growth rates coupled with fast development and early flowering (Munns et al.

2000b).

Increase of salt in the root medium can lead to a decrease in leaf water potential and,

hence, may affect many plant processes (Sohan et al. 1999). At very low soil water

potentials, this condition interferes with the plants’ ability to extract water from the soil

and maintain turgor (Sohan et al. 1999). Thus, in some aspects salt stress may resemble

drought stress. However, at low or moderate salt concentrations (higher soil water

potential), plants can adjust osmotically (accumulate solutes) and maintain a potential

gradient to continue the influx of water. Under such conditions growth may be

moderated, but unlike drought stress, the plant is not water deficient. Several authors

have found that water potential and osmotic potential of plants declined with an increase

in salinity, whereas turgor pressure increased.

The effect of salinity on permanent wilting point (PWP) was modelled by Groenevelt et

al. (2004), using the soil water retention curve as a base. From this the two extremes of

soil water extraction were shown, from matric potential only, to the full effect of both

matric and osmotic potentials. Depending on a plant’s ability to osmotically adjust, with

no adjustment, the plant will experience either the full osmotic stress, or if the plant is

able to fully osmotically adjust it will overcome the osmotic potential component and

experience only the matric potential. Most plants fall in the range between these two

extremes. As the soil dries, the relationship between matric and osmotic potential, in

terms of their contribution to total soil water potential, changes (Groenevelt et al. 2004).

While osmotic potential decreases in a linear fashion, due to a simple

concentration/dilution effect with soil water content, matric potential decreases in a

26

manner proportional to the logarithm of soil water content, the exact relationship

determined by an individual soil’s water retention curve (Groenevelt et al. 2004).

A plant in drying soil is exposed to increasing levels of both water stress and osmotic

stress, because the matric potential and the osmotic potential decrease simultaneously

with decreasing soil moisture (Shalhevet 1993; Glen and Brown 1998). This is common

in arid soils, in which salts often concentrate near the surface as the soil dries between

rains, and in irrigated soils which can accumulate damaging levels of salts between

irrigations (McCree and Richardson 1987; Shalhevet 1993). Both low soil osmotic

potentials and low soil matric potentials (associated with reduced water content) cause

low water potentials in plants resulting in reduced leaf expansion rates, lower

photosynthetic rates per unit leaf area and reduced growth (Rawson and Munns 1984).

Studies in which plants were grown in drying soils at different salinities show a more

complicated response, in which soil salts actually mitigate some of the negative effects

of water stress. For example, plants in drying soils usually survive longer in saline than

in non-saline soils, because salt-stressed plants grow less and, therefore, deplete soil

moisture more slowly than non-stressed plants (McCree and Richardson 1987;

Shalhevet 1993). Studies of the combined effects of salt and water stresses on growth of

maize (Stark and Jarrell, 1980) and sorghum (Richardson and McCree, 1985) showed

that although salinity reduced the rates of leaf expansion under well-irrigated

conditions, it also allowed leaf expansion to continue down to lower leaf water

potentials under water stress. Furthermore, salt stress can increase instantaneous leaf

water use efficiency by reducing stomatal conductance to a greater extent than

photosynthesis, thereby allowing plants under salt stress to produce more dry matter

than plants in nonsaline soil on the same quantity of water (Richards, 1992). Finally,

27

salt stress can precondition plants to low soil water potential by allowing them to

osmotically adjust, enhancing their ability to survive as the soil dries (Shalhevet, 1993).

Thus the combined effects of salinity and water stress may be less detrimental to plant

growth than the sum of the separate effects.

In several plants, salt tolerance and drought tolerance are linked through a common

mechanism of salt uptake for osmotic adjustment (Flowers and Yeo 1995).

Physiological studies have often dealt separately with salt and water stresses, but in the

field, salt stress is usually accompanied by water stress. Despite their importance,

relatively few studies have considered the combined effects of water and salt stress on

plants.

2.4.3 Effects of specific ion toxicity in crops

The ion-specific phase of the response to salinity starts when salt accumulates to toxic

concentrations. If the rate at which they die is greater than the rate at which new leaves

are produced, the photosynthetic capacity of the plant will no longer be able to supply

the carbohydrate requirement of the young leaves, which further reduces their growth

rate. While the osmotic stress has an immediate effect on growth, the ionic stress

impacts on growth much later, and with less effect than the osmotic stress, especially at

low to moderate salinity levels. Only at high salinity levels, or in sensitive species that

lack the ability to control Na+ and/or Cl transport, does the ionic effect dominate the

osmotic effect. In the following sections the mechanisms of ion toxicity are discussed.

28

2.4.3.1 Mechanisms of Na+ toxicity in plants

With basic soil and solution culture experiments highlighting that Na+ (and Cl–) has an

effect on growth and nutrient accumulation in a variety of plant species, experiments

have also been conducted in order to determine the mechanisms of these effects. This

issue has largely been addressed in the context of the effects of NaCl salinity on nutrient

uptake. The effects of NaCl on plant growth are apparent at a number of different levels.

At the molecular level, NaCl stress is manifested in reduced binding of Ca2+ to the plant

plasma membranes (Yermiyahu et al. 1994). At the whole plant level NaCl stress is

manifested in reduced root (Kent and Lauchli 1985; Kurth et al. 1986) and shoot growth

(Cramer et al. 1989; Yeo et al. 1991; Cramer 1992) and changes in ionic composition of

the plant (Ben-Hayyim et al. 1987; Reid and Smith 2000).

In shoots, high concentrations of Na+ cause a range of osmotic and metabolic problems

for plants. Leaves are more susceptible to salt stress than roots because Na+ (and Cl–)

accumulates to higher concentrations in shoots than in roots (Munns and Tester 2008).

Roots tend to maintain fairly constant levels of NaCl over time, and can regulate NaCl

levels by export to the soil or to the shoot. Na+ is transported to shoots in the rapidly

moving transpiration stream in the xylem, but can only be returned to roots via the

phloem. There is limited evidence of extensive recirculation of shoot Na+ to roots,

suggesting that Na+ transport is largely unidirectional and results in progressive

accumulation of Na+ as leaves age (Apse and Blumwald 2007).

Sodium has the ability to compete with K+ for binding sites essential for cellular

function. More than 50 enzymes are activated by K+, and Na+ cannot substitute in this

29

role (Bhandal and Malik 1988). Thus, high levels of Na+, or high Na+/K+ ratios can

disrupt various enzymatic processes in the cytoplasm. Moreover, protein synthesis

requires high concentrations of K+, owing to the K+ requirement for the binding of

tRNA to ribosomes (Blaha et al. 2000) and probably other aspects of ribosome function.

The disruption of protein synthesis by elevated concentrations of Na+ appears to be an

important cause of damage by Na+.

Osmotic damage could occur as a result of the build up of high concentrations (possibly

several hundred mM) of Na+ in the leaf apoplast, since Na+ enters leaves in the xylem

stream and is left behind as water evaporates. This mechanism of Na+ toxicity was first

proposed by Oertli (1968), and direct supporting evidence has been provided by X-ray

microanalysis measurements of Na+ concentrations in the apoplast of rice leaves

(Flowers et al. 1991). These authors calculated that there was about 600 mM Na+ in the

apoplast of leaves of rice plants that were moderately salt-stressed.

The cellular toxicity of Na+ causes another type of osmotic problem. Plants need to

maintain internal water potential below that of the soil to maintain turgor and water

uptake for growth. This requires an increase in osmotica, either by uptake of soil solutes

or by synthesis of metabolically compatible solutes. This drought component of salinity

poses a dilemma for plants: the major, cheap solutes in saline soils are Na+ and Cl–, but

these are toxic in the cytosol. Compatible solutes are non-toxic, but are energetically

much more expensive. With high concentrations of Na+ in the leaf apoplast and/or

vacuole, plant cells have difficulty maintaining low cytosolic Na+ and, perhaps as

importantly, low Na+/K+ ratios (Gorham et al. 1990; Dubcovsky et al. 1996).

30

Sodium chloride salinity also affects the nature of root development, by altering the

Na+/Ca2+ ratio of the growth medium. Kurth et al.(1986) and Huang and Redman

(1995) found that roots grown in high Na+ and low Ca2+ mediums are shorter and

thicker than those grown in standard media. The Na+/Ca2+ ratio of the growth medium

also has an effect on the development of root hairs, reducing their length and density

(Shabala et al. 2003). A consistent feature of this body of experimentation is the

ameliorative effect of increasing the concentration of Ca2+ in the growth medium. The

addition of Ca2+ to a solution containing NaCl improves root elongation (Kent and

Lauchli 1985; Kurth et al. 1986) and shoot growth (Cramer et al. 1989; Yeo et al. 1991;

Cramer 1992), prevents symptoms of Ca2+ deficiency (Maas and Grieve 1987), reduces

root thickening (Kurth et al. 1986) and restores the growth and development of root

hairs (Shabala et al. 2003). At high salinities however, much of the growth inhibition by

NaCl can be attributed to osmotic effects and these occur independently of the Ca2+

concentration of the growth medium (Cramer et al. 1989). Kinraide (1999) found that

the negative effect of NaCl salinity on wheat roots could be restored by the addition of

Ca2+ if the concentration of Na+ was <130 mM. Additions of NaCl beyond this level

reduced root elongation regardless of the concentration of Ca2+, indicating the effects of

osmotic stress beyond this point which also infer that ionic stress may have an earlier

effect followed by osmotic stress. This is in contrast to the two-phase model of crop

growth under salinity stress. The ionic imbalance component of NaCl stress is related to

decreasing levels of Ca2+ activity at the plasma membrane of the plant roots. As the

concentration of Na+ in the growth medium increases, there is a corresponding decrease

in the activity of Ca2+ in the growth medium (Cramer and Lauchli 1986; Shabala et al.

2003). Thus, concentrations of Ca2+ that are adequate for growth in low Na+ mediums

may not be adequate in high Na+ mediums (Cramer and Lauchli 1986; Cramer et al.

31

1986). The amelioration of Na+ toxicity by Ca2+ involves increasing the activity of Ca2+

in the solution (Reid and Smith 2000).

Selectivity of potassium uptake at the plasma membrane

The existence of a relationship between the Na+ content of the growth medium and the

K+ content of plant shoots and roots was determined initially in bean and barley plants

by Epstein (1966). Early work suggested that K+ and Na+ uptake occurred via dual

mechanisms in the plasma membrane, one of high K+ affinity and one of low K+

affinity. Na+ was considered to compete effectively with K+ for uptake at the low K+

affinity mechanism but the high K+ affinity mechanism was considered to be highly

selective for K+ (Epstein 1966). The positive affect of Ca+2 on plant growth and ionic

composition under conditions of high Na+ was attributed to the maintenance of

selectivity of the high K+ affinity mechanism (Epstein 1961).

The ability to differentiate between K+ influx and K+ efflux led Cramer et al. (1985) to

determine that the use of supplemental Ca2+ on cotton grown under high NaCl

conditions has no direct effect on K influx. The beneficial effect of Ca2+ under these

conditions was instead attributed to the maintenance of the integrity of the membrane

and thus the prevention of K+ leakage from the root cells. The factor most significantly

correlated with the increasing influx of K+ into the cotton plants grown in various NaCl

solution concentrations, is increasing root weight (Cramer et al. 1987).

32

Binding of calcium to the plasma membrane

Calcium has a role the promotion of cell wall development and the maintenance of

structure in the plasma membrane (Pooviah and Reddy 1987). Removal of Ca2+ from

the plasma membrane of a cell by EDTA results in leakage of K+ from the cell

(Weimberg 1983). Similar results have also been obtained under conditions of high Na+

in the growth medium (Cramer et al. 1985; Ben-Hayyim et al. 1987; Yermiyahu et al.

1994).

Yermiyahu et al. (1997) measured the effect of various concentrations of Na+ and Ca2+

on the binding of Ca2+ to the plasma membrane of melons and the consequent level of

root elongation. A correlation was observed between the salt-tolerance of melons and

the extent of Ca2+ binding to the plasma membrane, with the more salt tolerant melon

variety having a lower critical Ca2+ binding percentage at which optimal root growth

occurred. The role of Na+ displacement of Ca2+ from the plasma membrane in NaCl

toxicity has also been established through the analysis of the growth of wheat seedlings

in solutions of varying Na+, Ca2+ and K+ concentrations (Kinraide 1998).

Sodium-induced depletion of K+ from plant tissue has been cited as a mechanism for

NaCl toxicity by a range of authors (Cramer et al. 1985; Ben-Hayyim et al. 1987;

Yermiyahu et al. 1997). The difficulty in determining the role of Na+-induced K+

deficiency in NaCl toxicity occurs in trying to establish a causal relationship from a

variable that may be confounded. Kinraide (1999) used 30 solutions of varying

concentrations of Ca2+, Na+ and K+ in order to try and determine the existence of such a

relationship in wheat seedlings. Not only was Na+-induced reduction in root or shoot K+

33

concentration not observed, contradicting the K+ depletion hypothesis but also, root and

shoot elongation was found to occur at optimal levels at low K+ concentrations. These

results contradict those observed in a variety of other experiments. For example,

Shabala et al. (2003) found that critical levels for K+ deficiency were reached in barley

seedlings cultivated under conditions of NaCl salinity over a period of several days, due

to K+ efflux from roots. Chen et. al. (2007), in a large-scale glasshouse trial including

nearly 70 barley cultivars, showed that a plant’s ability to maintain high K+/Na+ ratio

(either retention of K+ or preventing Na+ from accumulating in leaves) is a key feature

for salt tolerance in barley. It is important to consider that the experiment undertaken by

Kinraide (1999) was short-term, with 2 day old wheat seedlings being grown for only 2

days. In larger plants grown over a longer period, such as those cultivated under a field

situation, the loss of K+ from plant roots could result in deficiency.

Excessive Na+ influx into plant tissues has also been cited as a mechanism for NaCl

toxicity by a number of authors (Cramer et al. 1987; Yermiyahu et al. 1997; Reid and

Smith 2000). Increased tissue concentrations of Na+ have been found in plant roots and

shoots under increasing levels of NaCl (Kinraide 1999; Reid and Smith 2000) and

increasing Na+ influxes measured under conditions of NaCl salinity. Again, the

difficulty in determining the role of Na+ influx in NaCl toxicity occurs in trying to

establish a causal relationship between Na+ and plant growth reductions when Cl- also

has the potential to reduce plant growth. A reduction in Na+ influx has been observed on

numerous occasions with the addition of Ca2+ to a high NaCl medium (Cramer et al.

1987; Yermiyahu et al. 1997; Kinraide 1999; Reid and Smith 2000). This would lend

support to the hypothesis that Na+ influx is related to NaCl toxicity under conditions of

low Ca2+. Kinraide (1999) however found that high Na+ concentrations in plant shoots

34

reduce plant growth due to osmotic effects, but that there was no direct effect of high

root Na+ concentrations on plant growth. Again, this experiment was only conducted

using 2-day-old wheat seedlings for 2 days. According to the two-phase growth

response model to salinity (Munns et al. 1995) comparison between genotypes with

contrasting rates of Na+ uptake and long term differences in salt tolerance showed that

both genotypes had the same growth reduction for 4 weeks in 150 mM NaCl, and it was

not until afterwards that a growth difference between the genotypes was clearly seen

(Munns et al. 1995; Munns et al. 2006) so most likely over a longer period of time, high

root or shoot Na concentrations might reduce plant growth.

2.4.4 Plant photosynthesis

It is well documented that photosynthetic capacity of plants grown under saline

conditions is depressed (Downton 1977; Longstreth and Nobel 1979; Berry and

Downton 1982; Bongi and Loreto 1989; Ziska et al. 1990; Melesse and Caesar 1992;

Ashraf 2001; Flexas et al. 2004; Netondo et al. 2004; Chaves et al. 2009) depending on

type of salinity, duration of treatment, species and plant age. The effects can be direct,

as the decreased CO2 availability caused by diffusion limitations through the stomata

and the mesophyll (Flexas et al. 2004; Chaves et al. 2009) or the alterations of

photosynthetic metabolism (Lawlor and Cornic 2002). Photosynthetic response to

salinity stress is highly complex. However, there has been a long-standing controversy

whether salinity stress limits photosynthesis primarily through stomatal closure and by

greater diffusive resistances or by metabolic impairment (Boyer 1976; Sharkey 1990).

Devitt et al. (1984) reported that the primary effect of salt on photosynthesis in wheat

and sorghum is due to decreased stomatal conductance to CO2 diffusion. On the other

35

hand, James et al. (2002) reported that the reductions in assimilation rate were initially

due to stomatal effects and, with time, were due to a combination of stomatal and non-

stomatal limitations. The non-stomatal limitations were associated with a build up of

salt above 250 mM.

Acclimation responses under osmotic stress, which indirectly affect photosynthesis,

include those related to growth inhibition or leaf shedding that, by restricting water

expenditure by source tissues, will help to maintain plant water status and therefore

plant carbon assimilation. Osmotic compounds that build up in response to a slowly

imposed dehydration also have a function in sustaining tissue metabolic activity.

Acclimation responses to salinity also include synthesis of compatible solutes as well as

adjustments in ion transport (such as uptake, extrusion and sequestration of ions). These

responses will eventually lead to restoration of cellular homeostasis, detoxification and

therefore survival under stress.

Stomata close in response to leaf turgor decline, to high vapour pressure deficit in the

atmosphere or to root generated chemical signals. Under mild salt and/or water stress, a

small decline in stomatal conductance may have a protective effect against stress, by

allowing the plant to save water and improving plant water-use efficiency (Flexas et al.

2002; Flexas et al. 2004; Chaves et al. 2009). Salinity stress also results in an apparent

reduction in mesophyll conductance to CO2 (Flexas et al. 2007). It was suggested that

leaf internal diffusion conductance was depressed under water-stress conditions (Jones

1976). However, this model assumed that CO2 concentration in the chloroplast was

close to zero or to the compensation point, which was later shown to be untrue

(Farquhar and Sharkey 1982).

36

Changes in leaf biochemistry that result in down-regulation of the photosynthetic

metabolism may occur in response to lowered internal CO2 under prolonged stresses

(Chaves et al. 2009). For example, a de-activation of Rubisco by low intercellular CO2

(Ci) has been observed (Meyer and Genty, 1998). Following stomatal closure and the

fall in CO2 concentration in the intercellular airspaces of leaves, other enzymes have

been shown to decrease their activity; this change was quickly reversed when CO2 in

the surrounding atmosphere was increased. Under salt stress, metabolic limitations of

photosynthesis resulting from increased concentrations of Na+ and Cl- in the leaf tissue

do occur (Munns et al. 2006). The fast changes in gene expression during salt stress that

have been observed (Chaves et al. 2009) suggest that alterations in metabolism start

very early. When, in addition to salinity and drought, plants are subjected to other

environmental stresses such as high light and temperature, photoinhibition is likely to

occur. These photoprotective mechanisms compete with photochemistry for the

absorbed energy, leading to a decrease in quantum yield of PSII (Genty et al. 1989).

In conclusion, an association between increases in plant ion concentration under saline

conditions and decreases in CO2 assimilation was observed, and it was due partially to

non-stomatal effects, suggesting of ion toxicities. However, these finding were only

correlative (James et al. 2002). Further experiments using genotypes with greater

contrasts in Na+ and Cl- accumulation and/or osmotic tolerance will be necessary to

assess the stomatal and non-stomatal limitations on plant photosynthesis upon salinity

stress.

37

2.5 Mechanisms of salt tolerance in crop plants

Crop salt tolerance is defined as the ability of the plants to survive and produce

economic yields under the adverse conditions caused by salinity (Bresler et al. 1982).

Salt tolerance is usually assessed as the biomass produced under salt stress compared to

that produced under controlled (non-stressed) conditions (Munns et al. 2002). A large

degree of genetic variation in salt tolerance exists within the plant kingdom (Figure 4).

Of the cereals, rice (Oryza sativa) is the most sensitive and barley (Hordeum vulgare) is

the most tolerant (Figure 4). Bread wheat (Triticum aestivum) is moderately tolerant and

durum wheat (Triticum turgidum ssp. durum) is less so. Tall wheatgrass (Thinopyrum

ponticum, syn. Agropyron elongatum) is a halophytic relative of wheat and is one of the

most tolerant of the monocotyledonous species; its growth proceeds at concentrations of

salt as high as in seawater. The variation in salinity tolerance in dicotyledonous species

is even greater than in monocotyledonous species. Some legumes are very sensitive,

even more sensitive than rice (Munns and Tester 2008), although alfalfa (or lucerne)

(Medicago sativa) is very tolerant, and halophytes such as saltbush (Atriplex spp.)

continue to grow well at salinities greater than that of seawater.

Salt tolerance in crops might be improved by exploiting variation present in our existing

crops, including landraces and progenitors; using inter-specific, or where possible, inter-

generic variation to enhance tolerance in some crops; or through transgenic approaches

(Colmer et al. 2005). Domestication of halophytic plants to develop new crops that are

highly salt tolerant is also an option.

38

Figure 4. Diversity in the salt tolerance of various species, shown as increases in shoot dry matter after growth in solution or sand culture containing NaCl for at least 3 weeks, relative to plant growth in the absence of NaCl. Data are for rice (Oryza sativa) (Aslam et al. 1993), durum wheat (Triticum turgidum ssp durum) (Colmer et al. 2005), bread wheat (Triticum aestivum) (Colmer et al. 2005), barley (Hordeum vulgare) (Colmer et al. 2005), tall wheatgrass (Thinopyrum ponticum, syn. Agropyron elongatum) (Colmer et al. 2005), Arabidopsis (Arabidopsis thaliana) (Cramer 2002) alfalfa (Medicago sativa) (Kapulnik et al. 1989), and saltbush (Atriplex amnicola) (Aslam et al. 1986) (Munns and Tester 2008).

These approaches to improve salt tolerance have been reviewed extensively (Flowers

and Yeo 1995; Flowers et al. 1997; Yeo 1998; Munns et al. 2002; Munns and James

2003; Flowers 2004; Munns et al. 2006). Improvement in salt tolerance may be based

on one or more of the following strategies: (i) minimising accumulation of salt in shoots

by reducing uptake and translocation of salt to the shoots which effectively excludes salt

from the leaves; (ii) osmotic tolerance to salt-induced water deficits and (iii)

compartmentation of Na+ and Cl- within the vacuoles. The important mechanisms of

tolerance to soil salinity are discussed below in more details.

NOTE: This figure is included on page 38 of the print copy of the thesis held in the University of Adelaide Library.

39

2.5.1 Osmotic adjustment

Plant water uptake is largely driven by an osmotic gradient between the soil water and

plant tissues (Yeo 1998). As the concentration of salt in soil increases, the osmotic

potential of the plant tissues must decrease in order for the plant to maintain water

uptake. This may occur through increased uptake of ions or synthesis of osmotic

solutes. Both glycophytes and halophytes use a combination of strategies, with tolerant

species storing salts in the vacuole and apoplasm, and synthesized organic solutes in the

cytoplasm (Munns and Tester 2008).

The organic osmotic solutes that are produced in response to salt, drought or cold stress

allow osmotic adjustment, and protect proteins and membranes when high salt levels are

present (Ashraf and Harris 2004; Diego et al. 2008). Betaines, polyols, sugars and

amino acids are the major types of solutes produced (Ashraf and Harris 2004). The use

of organic molecules for osmotic adjustment is particularly important in the cytoplasm

where high concentrations of Na+ and Cl- may interfere with cellular processes. These

compounds may also provide protection against oxidative stress that can increase under

saline conditions. Osmotic solutes synthesized in the leaves can be translocated to other

tissues (Cram 1976; Shomer-Ilan et al. 1991).

Achieving osmotic adjustment through uptake of ions is simpler for the plant than

synthesizing organic osmotic solutes because the ions needed are available in high

concentrations in the soil and are readily supplied through the transpiration stream.

However, problems occur for the plant in balancing uptake of sufficient ions for

osmotic adjustment, while avoiding accumulation of toxic concentrations in the plant.

40

The plant also needs to maintain uptake of essential nutrients. Under saline conditions,

beyond the tolerance mechanisms of the plant, maintenance of low tissue salt

concentrations may not protect against reduced photosynthetic activity if the plant’s

osmotic adjustment is not sufficient to balance the reduction in water potential. Osmotic

adjustment however, may also be insufficient to protect photosynthetic function, if plant

tissues have accumulated excessively high salt concentrations. To achieve osmotic

adjustment without disrupting cell processes, Na+ and Cl- must be kept out of the cell

cytoplasm, while K+ and Ca2+ concentrations in the cytoplasm must be maintained. At

high concentrations, Na+ and Cl- enter the cytoplasm via diffusion along an

electrochemical gradient. Calcium and K+ concentrations in the cytoplasm of 0.2 mM

and 100 to 200 mM respectively are maintained via cation carrier proteins of varying

specificity. Under saline conditions where apoplastic Na+ is very high, cytoplasmic Na+

needs to be regulated via H+/Na+ antiports which pump Na+ back to the apoplast. The

effectiveness of the H+/Na+ antiports, and the ability of the various cation channels to

discriminate between Na+, Ca2+ and K+, and maintain a low cytoplasmic Na+/K+ ratio

are important traits in regard to salinity tolerance.

2.5.2 Reduced uptake and translocation

Transport of Na+ and Cl- from the roots to the rest of the plant tissue occurs via the

xylem in the transpiration stream. The plant may control Na+ and Cl- transport through

xylem loading, via selective removal of ions from the xylem flow, and by control over

the destination tissues. The concentration of salt in the xylem affects the rate of

accumulation in plant tissues. Hence, control over root uptake and xylem loading of Na

and Cl is an important tolerance mechanism. Salt tolerant barley has a xylem Na+

41

concentration about 4% of the external solution (Wolf et al. 1990), while salt sensitive

lupin (Lupinus albus) has a xylem concentration of about 10% (Munns 1988). While the

concentration of Na+ in the xylem sap is small compared to the concentration in the soil

solution, the high volume of flow in the xylem due to transpiration means that a small

increase in the concentration in the xylem stream leads to rapid accumulation of salt in

the shoots (Munns 1985). The transpiration stream drives upward movement of water

and ions within the plant, but the concentration of ions in the xylem flow is governed by

a range of complex control mechanisms in the root cells (Cheeseman 1988). Uptake of

Na+ and Cl- increases with increasing external concentrations, but is relatively

independent of fluctuations in transpiration flow, so that the concentration of Na+ and

Cl- in the xylem reduces as flow volume increases (Munns 1985). External NaCl

concentration does however have a significant effect on the volume of xylem flow,

which in wheat decreased by 44% as external NaCl concentration increased to 100 mM

(Termaat et al. 1985).

The second aspect of salt tolerance in relation to xylem transport is the ability of the

plant to allocate salt to different tissues, especially if the plant does not effectively

exclude Na+ and Cl- at the roots. While Na+ and Cl- concentrations in the xylem sap

may be the same for the whole plant, the rate at which accumulation occurs would be

different because of the variation in transpiration rates between tissues.

Phloem transport is important in terms of the distribution of salt and other nutrients in

the plant (Jeschke and Pate 1991). While both Na+ and Cl- can be transported in the

phloem, Na+ movement occurs to a much larger extent. In lupin, the concentration of

Na+ in the phloem increased as the sap flowed towards the developing tissues, with a

42

maximum concentration of 70 mM. Under moderately saline conditions, the

concentration of Na+ in leaves increased until they were fully expanded, and was then

stable, indicating equal amounts of import and export through xylem and phloem

(Munns et al. 1988). A gradient in concentration along the leaf is observed in both

xylem and phloem sap as Na+ is deposited, and then moved within the leaf. Prior to

emergence, barley leaves are supplied with phloem sap which is high in K+ and very

low in Na+. Once the leaf emerges it begins to accumulate Na+, supplied via the xylem

sap, and K+ from the phloem. Then as the leaf ages and continues to accumulate Na+, K+

is exported to growing tissues in the phloem (Wolf et al. 1990). Other mechanisms of

using xylem or phloem sap to manage salt within the plant occur less commonly.

Sodium may also be removed from the shoots by the translocation to the roots. Rice

plants exposed to NaCl for a short period during vegetative growth showed a decrease

in total Na+ as the plant grew after salt stress had been removed. A decrease in shoot

Na+ concentration would be expected, due to dilution of accumulated Na+ through

increase in total biomass. The reduction in total Na+ in the plant does however indicate

that some Na+ is re-translocated back into the growth medium. This occurred to a

greater extent during the vegetative stage than the reproductive stage, possibly due to

the re-allocation of phloem sap towards developing organs at this stage (Castillo et al.

2007).

2.5.3 Transport and compartmentation in cells

Transport and compartmentation of Na+ and Cl- within the cells and tissues of the plant

is controlled by a range of active and passive transport mechanisms (White and

43

Broadley 2001; Tester and Davenport 2003; Apse and Blumwald 2007). While some

plants effectively exclude Na+ and Cl-, others are able to tolerate accumulation of high

concentrations of these ions in their shoot tissue. The key aspect to tolerating high

internal salt concentrations is how the salt is stored both within and around the cell.

Both halophytes and glycophytes are intolerant of salt accumulation in the cytoplasm,

so the apoplasm or vacuoles are used to store salts so that metabolic processes are not

disrupted. Many of the transport mechanisms present in cells in the shoot tissue are the

same as those found in root cells. In the shoot tissue however, efflux into the apoplastic

space does not remove excess ions from the plant, or conveniently relocate the problem

via xylem flow. Movement of Na+ into the cell occurs via diffusion or passive transport,

according to a concentration and charge gradient. Entry can occur through non-specific

cation channels, and high and low affinity cation transporters. The cytoplasm is

negatively charged, and apoplastic space positively charged due to the activity of H+

pumps. Na+ and Ca2+ diffuse across the plasma membrane into the cytoplasm, reaching

concentrations in the range of 1 to 10 and 0.2 mM respectively (Apse and Blumwald

2007).

By holding salts in the cell wall or apoplasm, there is no energy cost of transfer across

membranes into the vacuole. However, once high concentrations accumulate, the

cytoplasm may shrink due to water stress and dehydration of leaf tissues (Marschner

1995). Storage of salts in the apoplasm also requires the cell to maintain an ever-

increasing concentration gradient across the plasma membrane, which has the potential

for abrupt failure and cell death. Wheat cells in suspension had a sudden increase in Na+

influx when exposed to salt, resulting in high internal Na+ concentrations. After a short

period however, Na+ efflux increased so that the concentration inside the cell slowly

44

decreased (Babourina et al. 2000). This suggests that wheat could maintain low

cytoplasmic Na+ concentrations by returning salts to the apoplasm. Accumulation of

salts in the vacuole is another strategy which contributes to the osmotic adjustment of

the cell. This incurs an energy cost of moving salts across membranes into the vacuole,

and maintaining the concentration gradient between the vacuole and cytoplasm. In

addition, once the capacity of the vacuole to store salts has been reached, salts will then

accumulate in the cytoplasm where they will have a toxic effect on enzyme function.

Increase in endoplasmic reticulum and vesicles in cells in order to export and

compartmentalize Na+ ions may occur. The effectiveness of storing salts in the vacuole

as a mechanism for salt tolerance depends on the membrane ion transporters across the

tonoplast. Salinity tolerance in Arabidopsis is linked to the function of vacuolar Na+/K+

and Na+/H+ antiports to reduce cytoplasmic Na+ and Cl- concentration (Apse et al.

1999).

2.6 Chloride dynamics in crop plants in relation to salinity responses

Chlorine is a Group VII halogen element and is of interest in plant physiology and in

soil-plant relations both because of its role as an essential element and because of its

accumulation in plants growing in salt-affected soil. Chlorine occurs predominantly as

chloride (Cl-) in the soil with the concentration of 1–5 mM in soil solution (Eaton

1966), and in some cases Cl- can reach phytotoxic levels, particularly in coastal

environments, such as salt marshes, where it can exceed 800 mM (Britto et al. 2004).

The Cl- anions do not form complexes readily and, since exchange sites on layer

silicates in soil clays are negatively charged, Cl- tends to be repelled from mineral

45

surfaces contained in many soil particles. Thus the concentration of Cl- in the bulk

solution is greater than in the diffuse layer surrounding soil particles.

In plants, chlorine is present mainly as Cl-, although plants do contain compounds with

covalently bound Cl-. Chloride is a major osmotically-active solute in the vacuole and is

involved in both turgor and osmoregulation (Xu et al. 2000; White and Broadley 2001).

In cytoplasm it regulates the activities of enzymes. The growth of many plants is

reduced substantially in Cl--free media (Xu et al. 2000), and environmental factors that

enhance growth rate increase susceptibility to Cl- deficiency. Deficiency causes reduced

leaf growth and wilting, followed by chlorosis, bronzing and, finally, necrosis. Roots

become stunted and the development of laterals is suppressed.

Chloride in soil is highly mobile, and at neutral and basic pH values Cl- is not adsorbed

on the exchange complex, however at low pH, retention or anion exchange may occur in

variable charge soils. Because Cl- is mostly present in the soil solution, it is easily

leached and Cl- is held in soils as counterion for high concentrations of Na+. Over time

however, Cl- and free Na+ (not retained by the cation exchange capacity) can leach from

the soil, and the soil becomes sodic rather than saline (Sumner 1995).

A minimal Cl- requirement for crop growth of 1 g kg-1 dry weight has been suggested.

This quantity can generally be supplied by rainfall, and Cl--deficient plants are rarely

observed in agriculture or nature (Xu et al. 2000). The least sensitive plants are beans

(Phaseolus spp.), squash (Cucurbita maxima), barley (Hordeum vulgare), maize (Zea

mays) and buckwheat (Fagopyrum esculentum) (White and Broadley 2001). The tissue

Cl- concentration at which deficiency symptoms are observed varies between about 0.1

46

and 5.7 mg g-1 of dry weight (Xu et al. 2000). To some extent, Br- can replace Cl-, but

this is of no natural significance since the abundance of Br in the Earth’s crust is about

100-fold less than that of Cl-.

High tissue Cl- concentration can be toxic to crop plants, and may restrict agriculture in

saline environments. The growth response of plants to high Cl- concentration in the

external medium ([Cl-] ext) can be divided into four categories (Greenway and Munns

1980) (Figure 5). Species can be grouped into I) halophytes which can be subdivided

into IA) species whose growth is stimulated by increases in Cl-, such as Sueda

maritima, Atriplex nummularia, or IB) species whose growth is little affected by 200

mM [Cl-]ext like Spartina spp. and sugar beet (Beta vulgaris); II) halophytes and non-

halophytes whose growth is reduced substantially by 100 mM [Cl-]ext, which can be

subdivided into tolerant species like Festuca rubris, cotton (Gossypium sp) and barley

(Hordeum vulgare); intermediate species like tomatoes (Solanum lycopersicum) and

sensitive species like soybean (Glycine max) and III) very salt sensitive non-halophytes

like citrus and woody plants species. Many important cereals, vegetable and fruit crops

are susceptible to Cl- toxicity which is a major constraint to horticultural production.

The critical tissue Cl- concentration for toxicity is about 4-7 mg g-1 for Cl- sensitive

species and 15-50 mg g-1 for Cl- tolerant plant species, respectively (White and

Broadley 2001). Differences between cultivars in the ability to withstand Cl- toxicity are

frequently related to the ability to restrict Cl- transport to the shoot. This has been

observed in soybean, wheat, barley, stone fruit trees, grapevine and citrus (Greenway

and Munns 1980; Storey and Walker 1999). Eaton (1942) found a linear, or nearly

linear, relationship when growth depressions of many crops were plotted against the

concentration of Cl- in an external solution, but the slope of the curves of various plants

47

were different. This slope is one indication of the degree of plant sensitivity to C1-. In

general, Cl- tends to accumulate in plant tissues, particularly leaves, to toxic levels. At

the same time, Cl- accumulation in plants is closely related to Cl- concentration in the

external solution and genotype. Salt tolerance of avocado, grapefruit, orange, and grape

varieties is related to the C1- accumulation properties of rootstocks , that is, to the

retention of Cl- by the roots (Hajrasuliha 1980).

Figure 5. Growth responses of different species to salinity. Growth was determined after 1 to 6 months at high [Cl-]ext. Curve 1 represents extreme halophytes (Group IA). Curve 2 represents halophytes (Group IB). Curve 3 represents plants whose growth is reduced substantially by 100 mM [Cl-]ext (Group II). Curve 4 represents very salt-sensitive non-halophytes (Group III). Figure redrawn from Greenway and Munns(1980).

At low concentrations, entry of Cl- into the cell occurs via active transport through Cl-

transport proteins. Influx of Cl- across the plasma membrane occurs through ATP driven

Cl--/H+ symports, and outwardly-rectifying Cl- channels (Sanders 1980; Felle 1994).

NOTE: This figure is included on page 47 of the print copy of the thesis held in the University of Adelaide Library.

48

The plasma membrane also contains a range of Cl- channels for Cl- efflux, activated by

Ca2+ concentration, membrane polarization and other cellular signals (Skerrett and

Tyerman 1994). Under non-saline conditions, Cl- influx and efflux are used to control

cell turgor and osmoregulation through Cl- channels (Skerrett and Tyerman 1994; White

and Broadley 2001). Two channels have been identified, a Cl- channel, which also

transports malate, and a malate activated Cl- channel, which also transports a range of

organic and inorganic anions. The second channel has been identified in CAM plants,

some halophytes and Arabidopsis (White and Broadley 2001). Skerrett and Tyerman

(1994) showed passive uptake could also occur in saline conditions if the membrane

potential is depolarized and cytosolic Cl- is at low millimolar concentrations. Cytosolic

Cl- appears likely to be in the range of 10 to 20 mM, but it may be higher in saline

conditions. Felle (1994) showed that the cytoplasmic concentration doubled (from 15 to

33 mM) within minutes of increasing the external Cl- concentration from 0 to 20 mM.

Yamashita et al. (1994) found an increase in Cl- permeability of protoplasts isolated

from barley roots after plants were pre-treated with 200 mM NaCl, supporting such a

role for Cl- channels. However, there clearly needs to be more work comparing Cl-

transport in lines with different levels of Cl- accumulation in the shoot to test the

significance of different transport processes in whole plant accumulation. Radioactive

tracer studies have shown that net Cl- loading into the root xylem is lower in grapevine

genotypes that have lower shoot Cl- accumulation. This may be due to reduced loading

of Cl- via anion channels, but it may also be due to increased active retrieval of Cl- from

the xylem stream. Sites of accumulation indicating retrieval from the xylem are in

petioles, in woody stems and roots. Biochemical approaches (effects of different salts

on protein synthesis or enzyme activity in vitro) are equivocal as have been estimations

of concentrations in the cytoplasm or of organelles such as chloroplast and

49

mitochondria. Yet tissue concentrations as high as 400 mM are tolerated by most

species, and even the sensitive ones like citrus can tolerate tissue concentrations of 250

mM, so the Cl- must be compartmentalized in the vacuole. The thermodynamics and

mechanisms of Cl- transport at the tonoplast are largely unknown, and difference in

properties between tolerant and sensitive lines obscure.

An increase in Cl- uptake and accumulation has been observed to be accompanied by a

decrease in shoot NO3– concentration. Various authors have attributed this reduction to

Cl- antagonism of NO3 - (Bar et al., 1997) while others attributed the response to

salinity’s effect on reduced water uptake (Lea-Cox and Syvertsen 1993). The NO3–

influx rate or the interaction between NO3- and Cl- has been reported to be related to the

salt tolerance of different plants. Kafkafi et al. (1992) found that the more salt-tolerant

tomato and melon cultivars had higher NO-3 flux rates than the more sensitive cultivars.

Little is known concerning the mechanisms of Cl- uptake by broadacre crops, and

knowledge about its subcellular distribution and flux dynamics have not been well

documented and afflicted by controversy. For example, Drew and Saker (1984)

proposed that the flux to the shoot as a major regulator of influx across the plasma

membrane of roots, a relationship that is important given that Cl- accumulation in the

shoot appears to be a major determinant of Cl- sensitivity in plants. The elucidation of

mechanisms of Cl- toxicity also requires knowledge of the subcellular localization and

quantification of Cl- pools; for instance, one may ask whether the toxic effect is due

primarily to the osmotic effect of high Cl- in the cell walls (Öertli 1968), or to Cl-

accumulation in the cytosol, where it can affect protein synthesis (Gibson et al. 1984)

and enzyme activity (Flowers et al. 1977). In general, there appears to be no consensus

50

regarding either the regulation of Cl- uptake (Flowers 1988; Xu et al. 2000), or the

mechanisms of Cl- toxicity.

The question here is that why most studies on salt tolerance of plants have been focused

on Na+, rather than on both Na+ and Cl-. Genetic methodologies have shown promise in

assessing the relative toxicity of Na+ and Cl-. For wheat, the genetic variation in salinity

tolerance correlates with leaf Na+ but not Cl- accumulation (Gorham et al. 1990).

However for citrus it correlates with leaf Cl- accumulation (Storey and Walker 1999).

There has also been some recent debate about the importance of soil Cl, and by

implication plant Cl- uptake, as predictors of damage and yield loss, rather than

electrical conductivity. Where NaCl is high, increased uptake of Na+ ions will be

associated with high uptake of Cl- ions (Dang et al. 2006a; Dang et al. 2006b). Soil Cl-

has been suggested to be a better predictor of growth and grain yield of dryland cereal

crops under salt stress in southwest Queensland where there are high levels of gypsum

(CaSO4) in the soil (Dang et al. 2006a). Under such conditions, electrical conductivity

is high because of the high concentrations of CaSO4 in the soil but the level of Na+

uptake may be relatively low and the uptake of Cl- relative to Na+ may be high. Cl-

toxicity is known to be important in some species, especially perennial plants (Wieneke

and Läuchli 1979; Hajrasuliha 1980; White and Broadley 2001), but there is little

information on its impact on most of broadacre crops. Little is known about the primary

acquisition mechanisms of Cl- by plants, and knowledge about its subcellular

distribution and flux dynamics is scarce.

2.7 Salinity tolerance in barley and faba bean

2.7.1 Barley (Hordeum vulagare)

51

Barley is considered to be one of the most salt tolerant crops, along with cotton and

sugar beet (Maas and Hoffman 1977). There is also significant genetic variation in salt

tolerance within barley, but there has been little progress in developing more salt

tolerant varieties. Almost 5000 lines of barley were screened (for germination, seedling

vigour, the vigour of the adult plants and their ability to produce filled grain) in Syria

(Srivastava and Jana 1984) and while promising lines were identified, the results have

not led to the generation of new cultivars (Colmer et al. 2005). Varietal differences in

leaf Na+ and Cl- concentrations have been reported (Forster et al. 1997; Forster et al.

2000) although for a larger number of genotypes (124 lines) under field conditions

neither leaf Na+ nor Cl- were found to be useful predictors of salt tolerance in barley

(Royo and Aragüés 1999). The Kna1 locus (codominant gene controlling K+/Na+

discrimination), appears to be absent in barley, as judged by the high Na+ and low

K+/Na+ in leaves compared with bread wheat. This was the case for 37 barley and 3 H.

vulgare subs. Spontaneum accessions (Gorham et al. 1994).

2.7.2 Faba bean (Vicia faba)

Faba bean (Vicia faba) is a valuable protein-rich food that has sustained large human

populations and provides an alternative to soybean meal for animal feed in temperate

regions (Stoddard et al. 2006). Yields are often variable due in part to the crop’s

shallow root growth and poor drought tolerance (Morgan et al. 1991; Turner et al.

2001). Many of the soils on which faba bean is grown are saline (Jensen et al. 2010;

Rispail et al. 2010) and relative to other crops, faba bean is sensitive to salinity (Maas

and Hoffman, 1976). In the case of faba bean, little information is available in the

literature regarding genetic variability for salt tolerance but the information available

suggests the sources of salinity tolerance may be more limited than in many other crops

52

(Malhotra 1997; Del Pilar Cordovilla et al. 1999). High levels of salinity tolerance were

found in a small number of accessions from provinces in China with extensive alkali–

saline soils (Li-juan et al. 1993; Duc et al. 2010). Further screening for salinity

tolerance among 504 landraces and breeding lines from worldwide sources found only

16 tolerant genotypes from China, Greece, Egypt and Australia (Duc et al. 2010). Little

is known about the levels of salt tolerance in current Australian germplasm.

2.8 Breeding for improving salt tolerance in crop plants

Salinity control through reclamation of salinized land or improved irrigation techniques

is often prohibitively expensive and provides only a short-term solution (Ashraf 1994;

Shannon 1997; Singh and Singh 2000). If global food production is to be maintained, it

seems reasonable to predict that enhancement of the salt tolerance of crops will be an

increasingly important aspect within a widening number of plant breeding programs.

The goals of plant breeding in this endeavour are to develop cultivars that can grow and

produce economic yields under moderately saline conditions (Epstein et al. 1980;

Flowers and Yeo 1995). Plant species and cultivars within a crop species vary greatly in

their response to salinity. Exploiting this genetic diversity within a crop species,

therefore, provides a practical means for breeding for improved salt tolerant cultivars

(Epstein et al. 1980; Flowers and Yeo 1995). Several screening and selection schemes

have been proposed for salt tolerance improvement in wheat and other crop species

(Kingsbury et al. 1984; Munns et al. 2002; Munns and James 2003). Field screening

procedures in saline soils are confronted by spatial heterogeneity of soil chemical and

physical properties as well as seasonal fluctuations in rainfall (Richards 1983; Richards

et al. 1987). Hence, many screening experiments for salt tolerant genotypes were

53

conducted either under in vitro or under controlled environmental conditions

(Kingsbury et al. 1984; Rawson et al. 1988; Munns et al. 2000a). The complexity and

polygenic nature of salt-stress tolerance are important factors contributing to the

difficulties in breeding salt-tolerant crop varieties. Classical screening methods are

based on assessment of yield responses to salt. Although screening based on yield

represents the combined genetic and environmental effects on plant growth and includes

integration of the physiological mechanisms conferring salinity tolerance at the whole

plant level, it is more convenient and practical if indirect indicators of salt tolerance can

be employed at the whole plant, tissue, or cellular levels (Ashraf and Harris 2004). For

example, plant species and even cultivars differ in their capacity to regulate ions.

Selection can be made for high leaf K+/Na+ ratios or high K+ concentrations in the

presence of salinity, and high K+/Na+ discrimination has been described as a

physiological index for salinity tolerance in bread wheat (Dvořak et al. 1994) and

durum wheat (Munns et al. 2000a). Enhancement of resistance against both

hyperosmotic stress and ion toxicity may also be achieved via molecular breeding of

salt-tolerant plants using either molecular markers or genetic engineering (Forster et al.

1997; Forster et al. 2000; Munns 2007). Plant growth and development can be affected

by salinity stress at any time during the crop life cycle, but the extent and nature of

damage and the impact on yield will depend on the developmental stage at which a crop

encounters the stress. In general, cereal crops are most sensitive to salinity during the

vegetative and early reproductive stages and less sensitive during flowering and the

grain filling periods. However, plant genotypes may respond differently to salt stress at

different growth stages (Kingsbury et al. 1984).

54

In developing a breeding program to improve the salt tolerance of a crop, it is necessary

to gain knowledge concerning the genetics and physiology of tolerance mechanisms and

then to understand the stress and environmental interactions. Once the mechanisms and

the interactions have been identified, different genotypes must be studied in order to

determine genetic variability and the relationship with performance under natural saline

conditions. Screening procedures can then be developed to select for desired

characteristics in either parental or segregating materials. Indeed, the literature abounds

with results of empirical studies of salt tolerance, providing a foundation from which

screening procedures can be developed. Although all aspects of such studies cannot be

presented in this review of literature, this review is intentionally limited to potential

screening procedures for salt tolerance in crop plants.

2.8.1 Screening Methods

Breeding crop plants for salinity stress tolerance has been difficult and slow. The

quantitative nature of salt tolerance traits and the problems associated with developing

appropriate and replicable testing environments make it difficult to distinguish salt

tolerant lines from sensitive lines. The speed of the screening methods has a great

bearing on how the germplasm can be incorporated into breeding programs for salt

tolerance. Faster screening methods can be employed for identification of potential

parents in a breeding program. It was argued that rapid screening methods should avoid

the need to grow plants under controlled conditions (Munns and James 2003), that

future development of molecular markers, gene discovery using microarray approaches,

and pyramiding genes can reduce the work involved in phenotypic screens, yet such

promises have yet to be fulfilled. While a comprehensive review by Munns and James

(2003) discussed various screening methods, the focus of this section is to highlight

55

some novel research areas among the disciplines currently applied to crop improvement

for salt tolerance.

2.8.1.1 Germination

Seed germination is the first stage of crop development at which salt stress can be

encountered. Salinity stress reduces germination, seedling emergence, and

establishment. Tolerance of salinity is obviously necessary at the whole plant level

through the complete life cycle in grain-producing and forage species. Determination of

germination potential of seeds in saline conditions presents simple and useful

parameters, provided that tolerance at the seedling stage reflects enhanced salinity

tolerance at the adult plant level. However, most investigators have been unable to

demonstrate a relationship between germination under laboratory high salt conditions

and later growth stages across a range of species, including bread wheat (Ashraf and

McNeilly 1988; Francois et al. 1994; Francois and Maas 1999), durum wheat

(Almansouri et al. 2001) and barley (Norlyn and Epstein 1982). Possible reasons can be

that the processes which control cell expansion during germination and subsequent

growth are entirely different. Many species, such as wheat and barley, are capable of

germinating at very high salt concentrations (over 300 mM NaCl), but the emerging

radicle cannot grow further at this level of salinity (Munns and James 2003). Such

tolerance among species at germination could be explained by the physicochemical

nature of the enlarging process during this developmental stage. Germination

percentage is a convenient test for screening large numbers of genotypes in a rapid

manner but must first be correlated to tolerance during emergence, vegetative growth,

flowering, and maturity if it is to be of value (Ashraf 2004; Krishnamurthy et al. 2007).

56

2.8.1.2 Photosynthesis and other physiological indicators

Several studies have focused on the response of the photosynthetic apparatus to salinity

stress. Salinity decreases both net photosynthesis and growth in higher plants (James et

al. 2002). It was found that salinity changes photosynthetic parameters, including

osmotic and leaf water potential, pigment compositions, transpiration rate, leaf

temperature, and relative leaf water content (Munns et al. 2002). The study of osmotic

potential in crop plants has undergone renewed interest recently because of its

importance in osmotic adjustment (James et al. 2008). The application of chlorophyll

fluorescence measurements to screening barley genotypes for salinity tolerance has been

demonstrated by Belkhodja et al. (1994), and a significant correlation between

chlorophyll fluorescence and salinity tolerance was shown. It was suggested that

chlorophyll fluorescence could be used as a tool for screening cultivars for salinity

tolerance. In a study on the relationship between grain yield, carbon isotope

discrimination, canopy temperature, stomatal conductance, and grain ash content in a set

of barley cultivars grown under saline conditions, it was concluded that none of the

studied characteristics would be useful in screening for high yield under saline

environments (Isla et al. 1998). Leaf injury could be considered another screening

criterion for salinity tolerance and can be measured by membrane damage (leakage of

ions from leaf discs), premature loss of chlorophyll (using a handheld meter), or damage

to the photosynthetic apparatus (using chlorophyll fluorescence). These methods have

been reported to only discriminate between genotypes tolerating low or moderate

salinities within a range of 50–100 mM NaCl (Munns and James 2003).

Under salinity stress conditions, osmotic adjustment is usually achieved by the uptake

of inorganic ions from the growth media. This accumulation of ions is often

accompanied by mineral toxicity and nutritional imbalance. The first adaptation

57

mechanism to high salinity is exclusion of Na+ ion from Na+ sensitive sites, which has

been proposed to be a function of a Na+/H+ antiporter and Na+- ATPase (Serrano et al.

1999). Also, glasshouse experiments have shown that landraces with low Na+

accumulation yield better than high Na+ genotypes at moderate salinity (Husain et al.

2003) which indicates that Na+ exclusion is a robust trait that should help to confer

salinity tolerance in the field. The trait of Na+ exclusion has a high heritability, and

recently a QTL has been mapped and a molecular marker for this trait was identified

(Munns et al. 2002), which is being used in a breeding program. Chloride (Cl−) also has

been found to be excluded in some crop species. Therefore, Na+ exclusion (Garcia et al.

1995), K+/Na+ discrimination (Asch et al. 2000), and Cl− exclusion (Rogers and Noble

1992; Rogers et al. 1997) traits have proven valuable in screening germplasm for

salinity tolerance.

2.8.1.3 Field vs. controlled conditions

Most studies evaluating genetic variation in salt resistance in crop plants have been

performed in controlled or semi-controlled environments at a single level of salt stress

with no validation of the results under field conditions. Furthermore, studies under

controlled conditions generally involve imposing salinisation on seedlings over a

relatively short period (often 1-2 days) whereas the salinity stress in the field may show

a greater level of spatial and temporal variation. The variation in salt stress in the field

also means that plants can be exposed to a range in salt concentrations at different

growth stages, but it is not clear which is the most appropriate salinity level for

screening and what stage of development best relates to genetic differences expressed in

the field. This information is necessary to develop efficient breeding and selection

methods for salt tolerance in crops, and it needs to be compared with results of studies

58

carried out in naturally saline field environments (Richards 1983; Richards et al. 1987;

El-hendawy et al. 2005). Genotypic differences in flowering or maturity times can also

cause large variations in yield if the ambient conditions are variable during the

flowering or grain-filling periods. Hence, screening large numbers of genotypes for

salinity tolerance in the field is difficult. A field study using advanced durum wheat

lines indicated significant genetic variation for salt tolerance whilst the confounding

effects of drought stress made it difficult to discriminate the genotypes for salt tolerance

(Srivastava and Jana 1984).

While a number of mechanisms relating to improved salt stress adaptation in crops have

been suggested, the fact remains that their association with genetic gains for yield and

their relative importance in different salinity-prone environments are still only partially

defined. Therefore, a well-focused approach combining the molecular, physiological,

and metabolic aspects of abiotic stress tolerance is required for bridging the knowledge

gaps between short- and long-term effects of the genes and their products and between

the molecular or cellular expression of the genes and the whole plant phenotype under

stress. Because different abiotic stresses are most likely to occur simultaneously under

field conditions, a greater attempt must be made to mimic these conditions in laboratory

studies. The timing of the salinity stress event with respect to the developmental stage

of the plant should also be addressed. Although plants can differ in their sensitivity to

various abiotic stresses during different developmental stages including germination,

vegetative growth, reproductive cycle, and senescence, from a strictly agronomic point

of view there appears to be only one main consideration (Mittler and Blumwald 2010):

How would this interaction between stress and development affect overall yield?

59

2.9 Salinity research

The effects of salinity on the physical and chemical characteristics of soils have been

well characterised. The limiting effects of saline-sodic soils on the growth and nutrition

of crops, that occur both directly as a result of the low availability of water for the plant,

increasing concentration of Na+ and/or Cl- and indirectly as a results of the physical

limitations, are less clear and more difficult to quantify. There exists a vast body of

literature in which field, soil and nutrient based culture systems have been used to try to

quantify the impacts of elevated ions concentrations on plant nutrition. The results of

these studies vary considerably however, due to the variety of factors that contribute to

the physical and chemical conditions of a soil at a given salinity level, species and

varietal differences in plant response to salinity and the frequent confounding of

salinity, sodicity and/or poor soil physical condition with different effects of elevated

levels of Na+ on plant growth and nutrition. This section will compare studies that have

been done on soil or solution culture systems. The advantages and limitations of these

experimental methods are discussed. Some aspects of growing plants for salinity

experiments, comparisons between experiments and the relevance of experiments to

field situations are summarized below.

2.9.1 Soil and nutrient culture systems

2.9.1.1 Soil culture systems

Numerous authors explored the effects of Na+ on plant nutrition in the glasshouse

through the addition of Na+ salts (e.g. NaHCO3 or NaCl) to soils. The consistent

outcomes of this type of experimentations have been that increases in soil salinity result

in elevated plant tissue Na+ and Cl- concentration. Bernstien and Pearson (1956) and

Bains and Fireman (1964) also observed negative correlations between the growth of a

range of crop plants and soil ESP. As soil ESP increased, the Na+ concentration of the

60

crop plants generally increased and the crop K+ and Ca2+ concentration generally

decreased, although considerable differences existed between the various crop species.

Wright and Raiper (2000) also measured correlations between increasing soil Na+ and

increasing levels of Na+, decreasing K+/Na+ ratio and decreasing Ca2+ concentrations in

wheat.

The methodologies used in these soil culture systems have limitations however, as the

additions of Na+ salts increases soil sodicity, unless the excess salt is leached from the

soil, confounding salinity with sodicity. Wright and Raiper (2000) used NaHCO3 salt to

sodify a clay loam, raising the ESP from 1 to 38% but also increasing the ECe from 1.8

to 6.1. The precipitation of carbonates is also largely responsible for the high pH values

commonly found in calcareous saline-sodic soils. Thus, using carbonate salts to vary

soil salinity can increase soil pH to excessive levels and confound salinity with artificial

extremes of pH. For example in the above mentioned experiment conducted by Wright

and Raiper (2000) the increase in ESP from 1 to 38% was also associated with increase

in pH (1:5 H2O) from 6.3 to 8.7.

2.9.1.2 Nutrient solution systems

Numerous authors have explored the impacts of salinity on plant nutrition in the

glasshouse through the use of nutrient solution experiments (Munns et al. 2002; Munns

and James 2003; Genc et al. 2007). Solution culture experiments are useful in the study

of interactions between salinity, sodicity and plant growth and nutrition because they

effectively enable saline-sodic soil solution chemistry to be separated from poor soil

physical condition. Mass and Grieve (1987) grew corn in a nutrient solution, with the

addition of iso-osmotic concentrations of NaCl and CaCl2 and it was observed that Na+

61

had a negative effect on plant growth and uptake of Ca2+ at Ca2+/Na+ ratios less than

5.7. Aslam et al. (2003) used nutrient solution with 100 mM NaCl and varying

concentration of Ca2+ to grow safflowers and observed that increasing solution

concentrations of Ca2+ decreased plant uptake of Na+ and that solution Na+ decreased

the uptake of K+.

The solution culture methodology used in these studies also has limitations however,

which are largely related to difficulties of their design and application to practical

situations. Firstly, it is difficult to change the concentration of a nutrient in the solution

without also altering the osmotic potential or the concentration of another nutrient

confounding both. Secondly, it is difficult to conduct nutrient cultures experiment at

concentrations that mirror those in the soil, especially for nutrients that occur in low

concentrations in the soil solution, such as K and P. Thus a relationship, which may

arise in the high nutrient concentrations of a nutrient culture system, may not be

apparent under soil conditions and vice versa.

2.9.1.3 Field studies

Research on responses of crop plants to soil salinity including breeding and screening

germplasm for salt tolerance is further complicated by genotype × environment

interactions, and variability of the salt-affected field in its chemical and physical soil

composition. The recent discovery of widespread salinity in the commonly occurring

sodic subsoils in the Australian cropping areas has sparked great interest in breeding

crops, especially wheat and barley, which cope better with salinity. The presence of

subsoil salinity in the field amplifies some of the issues pertaining to pots and brings in

other issues. Subsoil salinity is often accompanied by sodicity and boron toxicity.

62

Sodicity can increase the danger of hypoxia because sodic soils are finely porous and

can become almost saturated with water in wet conditions, leaving little space for air;

hypoxia can worsen the exposure to salinity (Barrett-Lennard 1986; 2003; Wetson and

Flowers 2010). Furthermore, field screening procedures in saline soils are confronted by

spatial heterogeneity of soil chemical and physical properties as well as seasonal

fluctuations in rainfall. Richards (1983) argued that where such variation is large and

includes a substantial area not strongly affected by salt, it is better for breeders to select

for high yield in benign conditions rather than for salinity tolerance, as the yields of

crops in the benign areas of a highly variable field typically account for most of the total

yield from such a field. The primary salinity that is common in sodic subsoils in

Australia often varies widely within a paddock.

2.9.1.4 Growth conditions and salinity treatments

The majority of experiments focusing on the effects of salinity on plant response are

currently using solution culture method to screen for Na+ exclusion (Fortmeier and

Schubert 1995; Munns et al. 2003; James et al. 2006; Genc et al. 2007; Moller et al.

2009; Genc et al. 2010a; Shavrukov et al. 2010). Solution culture is the preferred

screening method as it is completely controlled; therefore the results should not be

affected by environmental variation. The ability of solution culture to identify

genotypes that have optimum yield under saline conditions in the field is still needs to

be researched. In solution culture water uptake is only reduced by the osmotic potential

generated by salts, and this osmotic potential is maintained at a constant level, however

in soil, matric potential will be added to the equation and so the solution culture

experiments are not suitable method for determining the importance of osmotic stress.

63

Addition of Na+ reduces Ca2+ activity in solutions. If this is not compensated for by

addition of Ca2+ with the Na+, uncertainty remains about whether the effects of Na+

addition are due to the increase in Na+ or the decrease in available Ca2+. Thus, salt

treatments need to include supplemental Ca2+ to maintain stable Na+/Ca2+ ratios, or

constant Ca2+ activity (Genc et al. 2010b). When plants are grown in solution or sand

culture, or using supported hydroponics, the ionic conditions can be tightly controlled,

facilitating comparisons of results between different experiments. When plants are

transpiring at a reasonable rate, processes affected by transpiration, such as the net

delivery of Na+ to the shoot, will be most likely to be occurring in ways that are most

similar to those found in the field.

The time of exposure to salinity, and the level of the salt treatment, determines the

physiological and molecular changes that are found. A high salt treatment for a sensitive

plant like Arabidopsis will induce changes predominately associated with senescence;

however a low salt treatment may not result in discernable changes in gene expression

and metabolite levels. Finding the right balance is critical. The duration of the plant

establishment (pre-treatment) and salinity (treatment) stages of the experiment is an

important consideration when looking at the outcome of an experiment. The length of

the initial phase of plant growth before treatments are applied, and the time taken to

increase the level of salinity treatments to final concentration will have a significant

impact on the results.

2.9.2 Separating the osmotic stress from ionic toxicity

Separating the osmotic and ionic effects experimentally is difficult, and requires using

media with equal osmotic potential to compare with the plant’s response to osmotic

64

stress independently of Na+ and/or Cl-. This has often been achieved by using a solution

culture system where high molecular weight polyethylene glycol (PEG), mannitol or

sorbitol is used to impose osmotic stress (Almansouri et al. 2001; Chen et al. 2003). A

major weakness of this technique is that PEG breaks down and is taken up by plants,

where it can have a toxic effect or reduces O2 movement within the nutrient solution by

increasing solution viscosity, resulting in a hypoxic root environment (Lawlor 1970).

Furthermore, experiments which use organic osmotica do not adequately address the

role of ions taken up by the plant in osmotic adjustment within the plant. Some species

adjust to osmotic stress by taking up ions, while others synthesis osmotic solutes. Thus,

the type of stress imposed on the plant by NaCl and PEG are different, and act through

different mechanisms (Murillo-Amador et al. 2002); if the plant is required to use

different methods of osmotic adjustment because of the type of treatment applied, then

comparing the results of the different treatments has little value.

Of the experiments assessing the adverse effects of salinity on plant growth, several

attribute toxicity to either Na+ or Cl- when NaCl, or NaCl + Control (with basal nutrient

application), are the only salt treatments used (Lauchli and Wieneke 1979 ; Chauhan

and Chauhan 1985; Munns 1985; Cramer et al. 1994; Nuttall et al. 2006). Results for

the other ions are sometimes not even presented. Another group of experiments replaces

either Na+ or Cl- with other ions to determine whether or not there is a toxic effect.

Chloride is commonly compared with SO42-, such as a comparison of NaCl and Na2SO4,

while Na+ is replaced with K+ or Ca2+ (Chavan and Karadge 1980; Manchanda and

Sharma 1989; Kinraide 1999). This is a better model than using only one salt, but

allows the potential for false negative results, for example, a comparison of NaCl and

Na2SO4 may not show Cl- toxicity if Na+ is also having a substantial toxic effect on the

65

plant; or false positives if the comparison ion has a beneficial effect on plant growth, or

precipitation occurs (e.g. CaSO4).

2.10 Conclusions and further research

The responses of plants to a particular level of soil salinity depend upon a variety of soil

and plant characteristics. Several plant factors contribute to Na+ and Cl- toxicity and the

importance of these will vary with plant species and the time-scale over which the

interaction is studied. Given the complexity of the plant-soil interactions that control the

growth, water uptake and nutrient accumulation of crops in saline-sodic soils, it is

difficult to specify a single mechanism for yield reduction under such conditions.

Studies using solution culture methods have failed to address the actual plant response

under field conditions, where several environmental factors are also involved. Thus,

there is a clear need to characterise the relative contributions of the various soil and

plant factors causing growth and yield reduction over the length of a growing season in

soil-based systems.

Much of the recent focus in salt tolerance studies has been on the transport and

accumulation of Na+, while the role of Cl- in yield reduction of grain crops has been

relatively ignored. While symptoms of Cl- toxicity are documented in plant species,

especially woody perennials (Xu et al. 2000), much less information is available on Cl-

toxicity in grain crops. There is a need to improve the understanding of the effects of Cl-

in saline-sodic soils on salt tolerance of grain crops.

Under dry-land conditions, concurrent changes in matric and osmotic potentials

determine plant water uptake (Rengasamy 2002). The influence of soil texture and type

of clay on plant-available water compounds the effect of matric and osmotic potentials

66

(Rengasamy 2006). However, the influence of salt on the water availability in saline-

sodic soils under dryland conditions has not been comprehensively tested.

This review has identified the soil chemical, soil physical and plant factors that

contribute to the patterns of growth and nutrient accumulation that occur in crops grown

in saline-sodic soils. The following chapters investigate the relative importance of these

factors in barley and faba bean growth in saline-sodic soils. The results of this study are

expected to contribute to understanding of the relative importance of different

mechanisms of plant tolerance to salinity which is an important step in improvement of

agricultural production in saline environments. A better understanding of these factors

is important to assess the limitations placed on farming system, and will facilitate

development of improved varieties while allowing to implement effective crop and soil

management strategies in saline-sodic fields.

The aim of the research conducted in this thesis focused on identifying and quantifying

the physiological responses of crop plants to osmotic stress and to Na+ and Cl- toxicity

under controlled conditions. Field trials in the project focused on identifying

correlations between potential subsoil salinity and sodicity constraints, and plant growth

and also to verify the results of studies on genotypic variation of plants response to

salinity stress in controlled environments. Barley (Hordeum vulgare L.) and faba bean

(Vicia faba) were selected as the test species for the experiments. Barley is one of the

most economically important and widely grown crops in Australia (second in only to

wheat) and occupies a large geographic area (almost 4 million hectares). Faba bean is an

important winter legume crop and is gaining increasing levels of interest from farmers

67

as a rotation crop for cereals. The total area of production across Australia has increased

steadily since the start of the 1990s and is currently at 113000 hectares.

The aims of the work in this thesis are to:

1) determine which of the two ions most frequently implicated in salinity, Na+ and

Cl-, is most toxic to barley and faba bean

2) quantify the relative importance of ion (Na+ and/or Cl-) toxicity and osmotic

stress on growth and yield reduction under different levels of salinity

3) investigate whether hydroponics and pot experiments under controlled

environmental conditions are useful surrogates for evaluating whole-plant

response to salinity under field conditions.

68

List of articles presented for this thesis

1. Tavakkoli E., Rengasamy P., McDonald, GK (2010) The response of barley to

salinity stress differs between hydroponics and soil systems. Functional Plant Biology

37, 621-633.

2. Tavakkoli E., Rengasamy P., McDonald GK (2010) Growth of faba bean in saline-

sodic soils: Monitoring of leaf development and water use dynamics enables the

quantification of osmotic and ionic regulation at whole-plant level. Journal of

Experimental Botany. (Submission May 2011).

3. Tavakkoli E., Fatehi F., Coventry S., Rengasamy P., McDonald GK (2011) Additive

effects of Na+ and Cl- ions on barley growth under salinity stress. Journal of

Experimental Botany. 62, 2189-2203.

4. Tavakkoli E., Rengasamy P., McDonald GK (2010) High concentrations of Na+ and

Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean

under salinity stress. Journal of Experimental Botany 61, 4449–4459.

5. Tavakkoli E., Fatehi F., Rengasamy P., McDonald GK (2010) Effective screening

methods for salinity tolerance: pot experiments but not hydroponics are plausible

models of salt tolerance in barley. Journal of Experimental Botany. Under review

(submitted Dec 2010).

6. Tavakkoli E., Paull J., Rengasamy P., McDonald GK (2010) Genotypic variations of

faba bean in response to transient salinity at whole-plant level. Field Crops Research.

Accepted. In Press.

69

List of Peer-reviewed conference papers for this thesis

1. Tavakkoli E., Coventry S., Paull J., Jones B., Rengasamy P., McDonald GK (2010)

On-farm assessment of sub-soil salinity and sodicity constraints to faba bean and

barley production in areas of southern Australia. Proceedings of the 15th ASA

Conference, 15-19 November 2010, Lincoln, New Zealand (Oral presentation).

2. Tavakkoli E., Rengasamy P., McDonald GK (2010) Insight into the mechanism of

salt tolerance in faba bean using destructive and non-destructive techniques. Gordon

Research Conference on Salt and Water Stress. Les Diablerets Conference Center,

Switzerland.

3. Tavakkoli E., Cozzolino D., Roumeliotis S., Coventry S., McDonald GK. (2010)

Application of near-infrared spectroscopy in analysis of chemical/physical

characteristics of soil and plant mineral nutrients. The 14th ANISG conference. NIR

in action: it makes economic sense. National Wine Centre of Australia, Adelaide,

Australia.

4. Tavakkoli E., Rengasamy P., McDonald GK (2009) Screening for salinity tolerance

of barley genotypes using destructive and non-destructive techniques. ACPFG

symposium on the Genomics of Salinity. Nov 2009. Adelaide, Australia.

5. Tavakkoli E., Rengasamy P., McDonald GK (2009) Phenomics-based screening for

salinity tolerance: A case study for the evaluation of the impact of salinity on growth

of barley and faba bean. 1st International Plant Phenomics Symposium: from Gene to

Form and Function. Discovery Centre, CSIRO, Canberra, Australia, 21 - 24 April

2009.

6. Tavakkoli E., Rengasamy P., McDonald GK (2008) A critical analysis of osmotic

and ionic effects of salinity in two barley cultivars, Global Issues Paddock Action.

Proceedings of the 14th Australian Agronomy Conference. September 2008, Adelaide

South Australia (Oral presentation).

7. Tavakkoli E., Rengasamy P., McDonald GK (2008) Soils Do It Differently: An

Assessment of Comparative Response of Barley Genotypes to Salinity Stress in

Hydroponic and Soil Systems. Proceedings of ANZ Soils Conference, Massey

University, Palmerston North, New Zealand from 1-5 December 2008 (Oral

presentation).

Chapter 3

Thesis: Limitations to yield in saline-sodic soiis: Quantification of osmotic and ionic

regulations that affect the growth and nutrition of crops under salinity stress. By Ehsan

Tavakkoli,201,L

CHAPTER 3

TITLE OF PAPERThe response of barley to salinity stress differs between hydroponics and soil systems.

AUTHORS OF PAPER:Ehsan Tavakkoii, Pichu Rengasamy and Glenn McDonald

School of Agriculture, Food and wine, Waite campus, The university of Adelaide, SA, 5064

Name of journal - year of publication, issue, page numbers

Functional Plant Biologlt 37, 621-633.

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

Tavakkoli, E., Rengasamy, P. and McDonald, G.K. (2010) The response of barley to salinity stress differs between hydroponic and soil systems. Functional Plant Biology, v.37 (7), pp.621-633, July 2010

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1071/FP09202

Chapter 4

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

1

Growth of faba bean in saline-sodic soils: Monitoring of leaf

development and water use dynamics enables the

quantification of osmotic and ionic regulation at whole-plant

level

Ehsan Tavakkoli*, Pichu Rengasamy and Glenn K. McDonald

School of Agriculture, Food and Wine,

The University of Adelaide,

*To whom correspondence should be addressed:

Ehsan Tavakkoli

School of Agriculture, Food and Wine,

Waite Campus

The University of Adelaide,

PMB 1 Glen Osmond, South Australia, 5064

Phone: +61-8-8303 6533

Fax: +61-8-8303 7109

Email: ehsan.tavakkoli@adelaide.edu.au

Word count: 6375

Number of figures: 8

Number of tables: 2

Number of supplementary tables: 2

2

Abstract

Soil salinity reduces the growth of crops by a combination of osmotic stress and ion-

specific toxicity mechanisms. The responses to salt are generally described in terms of

the two phase model in which growth is initially reduced by osmotic stress and then Na+

toxicity. Despite the large volume of work on salt stress, the relative importance of

these mechanisms to salt injury and the contributions of Na+ and Cl- to salt stress are

still not understood well. A high resolution image capture and analysis system was used

to monitor the growth of the plants non-destructively and gas exchange measurements

were used to examine the effects on photosynthesis. The results suggested that

responses of faba bean growth to salinity stress may dependent on the severity of salt

stress. Osmotic stress was the predominant cause of reduced growth at high levels of

salinity, while specific-ion toxicity was more important under mild salinity stress. Fiesta

maintained greater whole-plant tolerance to salinity by dual mechanisms of ion

exclusion and osmotic adjustment, compared to Cairo. A reduction in C assimilation

was partially due to non-stomatal effects, suggesting ion toxicity was an important

reason.

Keywords: osmotic stress, ionic toxicity, faba bean, sodium, chloride, image analysis

3

Introduction

Approximately 800 million ha throughout the world are affected by salinity (including

both saline and sodic soils), equating to more than 6% of the world’s total land area

(FAO 2008). Saline soils contain a variety of salts, such as Na2SO4, MgSO4, CaSO4,

MgCl2, KCl and Na2CO3, but are normally dominated by NaCl (Rengasamy 2006).

Faba bean (Vicia faba L.) is a valuable protein-rich crop which is often grown on saline

soils (Jensen et al. 2010; Rispail et al. 2010) and is sensitive to salinity compared to

many other crops (Maas and Hoffman 1977). Surveys of commercial crops of faba bean

in South Australia demonstrated that faba bean accumulates considerably more Na+ (up

to 3 times higher) in the shoot to the more salt tolerant field pea (Pisum sativum) (G.

McDonald unpublished data). However, few studies have been published on its

response to salinity and therefore very little information is available on its genotypic

variation and physiological mechanisms in response to salt tolerance (Del Pilar

Cordovilla et al. 1999; Malhotra 1997).

Salt stress is caused by a decrease of the substrate osmotic potential and by ion-specific

toxicity, which makes investigations of the mechanisms of salt-induced growth

inhibition difficult (Hasegawa et al. 2000; Munns and Tester 2008). Plants have evolved

various mechanisms to adapt to salt stress (Munns and Tester 2008), including control

of Na+ and/or Cl- uptake and xylem loading, intracellular compartmentation of Na+ and

Cl- into the vacuoles and salt excretion. However, reducing Na+ and/or Cl- accumulation

is considered to be the most efficient approach to improving salt tolerance, because by

reducing uptake, the range of other mechanisms including osmotic adjustment and

tissue tolerance for dealing with excess Na+ and/or Cl- do not need to be invoked.

Osmotic adjustment involves the plant’s ability to tolerate water deficit and cell osmotic

4

potential associated with the lowered water potential of the salt-affected soil (Boyer et

al. 2008).

Understanding how the separate effects of salinity combine to reduce growth of plants,

is an important step in developing appropriate selection criteria for improved salt

tolerance. One of the important steps in understanding the effects of salt stress was the

two-phase model of growth response to salt stress proposed by Munns et al. (1995).

According to this model exposure to high concentrations of NaCl leads to a gradual

accumulation of Na+ and Cl- by plants, but the reduction in growth is caused by two

different, but overlapping effects. Growth is initially reduced by salt-induced osmotic

stress and is manifested as an inhibition of leaf expansion. This initial inhibition of

growth is not ion-specific but is caused by a decrease of the osmotic potential in the

substrate. The second phase of growth reduction is associated with specific ion toxicity

when a accumulation of salt to toxic concentrations has occurred in the leaves (Munns

et al. 1995; Munns and Tester 2008).

The relative importance of osmotic and ion-specific toxicity effects in the response of a

salt-sensitive plant such as faba bean, with poor capacity to exclude Na+ and Cl– from

the transpiration stream, is not clearly established. In the present study, an attempt was

made to establish the relative importance of osmotic and ion-specific toxicity effects on

plant growth in salt-stressed faba beans using plants cultivated in soil treated either with

iso-osmotic solutions (Termaat and Munns 1986), NaCl or CaCl2. To describe the

different phases of salt stress it is necessary to make daily observations of plant growth

over time. Technologies that allow non-destructive measurements using machine vision

are now available and their application to research in plant stress physiology is in its

infancy (Rajendran et al. 2009; Tavakkoli et al. 2009). In this paper, we provide an

5

example of a non-destructive, real-time method to assess the growth of faba bean plants

during a greenhouse experiment using commercially available image capture and

analysis equipment (LemnaTec ‘Scanalyser 3D’). Daily measurements of plant growth

in parallel to plant water use allow the continuous monitoring of responses to salinity

stress (Rajendran et al. 2009; Tavakkoli et al. 2010a).

Grain crops growing in soil of dryland farming systems experience variable soil water

contents depending on rainfall, leaf area and evaporative demand. Salinity may reduce

the availability of this water because of its effect on soil water potential and it may also

reduce total water use because leaf area, transpiration and growth are all reduced by

salinity. Therefore, productivity of crops on salt-affected soils will depend on whether

they are able to use all of the available water, and on how efficiently the water is used.

Therefore, manipulating water use and water-use efficiency genetically or through

management are important criteria that should be considered to genetically improve salt

tolerance and productivity in saline soils.

The rate of photosynthetic CO2 fixation of glycophytes typically declines with salt

stress, and this decline is generally found to be at least partially a consequence of

stomatal closure, although the extent to which such closure limits photosynthesis during

salt stress (by reducing the intercellular CO2 concentration) has been less often

quantified. Effects of salt stress on the non-stomatal components of photosynthesis have

also been considered, but there are few quantitative assessments of the effects of salt on

physiological and biochemical processes independent of altered stomatal limitations on

photosynthesis. In this study, measurements of ambient photosynthesis and stomatal

conductance were used to assess the magnitude of stomatal limitations on

photosynthesis as a consequence of salt stress. We have also utilized the response of

6

CO2 fixation to varying intercellular CO2 concentration to determine to what degree

salinity affects the efficiency of certain biochemical reactions of photosynthesis.

Much of the recent focus in salt tolerance studies has been on Na+ transport and

accumulation, while the role of Cl- in yield reduction of grain crops has been somewhat

neglected despite the fact that Cl- is the major anion in saline soils. Recent field studies

into the effects of salinity on yields of wheat and chickpea suggested that the effects of

Cl- toxicity can be high and may be more important than that of Na+ (Dang et al. 2008).

There are also reports that legume species are particularly sensitive to high levels of Cl-

(Maas and Hoffman 1977; Marschner 1995). Severe leaf chlorosis and depression of

photosynthesis were found for common bean (Hajrasuliha 1980), and high

concentrations of Cl- led to a decrease in growth rate (Cachorro et al. 1993). However

Slabu et al. (2009) concluded that Na+ is the primary toxic ion while Cl- has a secondary

effect. They found that high Na+ uptake interferes with K+ and disturbs efficient

stomatal regulation resulting in unproductive water loss and necrosis while high Cl-

causes chlorophyll degradation in faba bean. Therefore the objective of this study was to

quantify the relative importance of ion (Na+ and/or Cl-) toxicity and osmotic stress to

growth reduction at different levels of salinity among two faba bean genotypes differing

in their salt tolerance. The effects of Na+ and Cl- were also compared, given the

importance of both ions in salinity.

Materials and Methods

The experiment examined the responses to Na+ and Cl- in two varieties of faba bean,

Cairo and Fiesta which differed in their salt tolerance. In a previous hydroponic

screening experiment, Fiesta showed a superior ability to exclude Na+ (1126 versus

1754 mmol kg-1 DW) and Cl– (858 versus 1888 mmol kg-1 DW) than Cairo when grown

7

at 75 mM NaCl, which was associated with greater salt tolerance (81% versus 41%

relative growth). These differences in the concentrations of Na+ and Cl– between Fiesta

and Cairo were confirmed in measurements from field trials at sites with moderately

high levels of salinity and the differences in ion exclusion were also related to grain

yield at the sites (E Tavakkoli et al., unpublished data).

The experiment was conducted in a glasshouse using salt-amended soil. The A horizon

(topsoil) of a sandy loam red Chromosol (Isbell 1996) was collected from Roseworthy

(34.51° S, 138.68° E), South Australia, air dried and ground to pass through a 5 mm

sieve. A soil water characteristic curve was determined using the pressure plate method

(Klute 1986) and the soil moisture content at field capacity (–10 kPa, equivalent to 37%

w/w) was estimated. Ten soil treatments were prepared which consisted of: control (no

amendment); mild salinity (control soil plus 25 mmol NaCl kg–1 or 12.5 mmol CaCl2 kg–

1); moderate salinity (control soil plus 50 mmol NaCl kg–1 or 25 mmol CaCl2 kg–1); high

salinity (control soil plus 100 mmol NaCl kg–1 or 50 mmol CaCl2 kg–1); low osmotic

potential (two times concentrated macronutrients, designated CNS1); moderate osmotic

potential (four times concentrated macronutrients; CNS2), and high osmotic potential

(eight times concentrated macronutrients; CNS3). The three concentrated macronutrient

solutions were designed from a modified Hoagland solution (Lisle et al. 2000) and had

ECFC (EC of soil solution at soil moisture content of field capacity) values equivalent to

the low, moderate and severe levels of salinity.

Pots, 15 cm in diameter and 20 cm deep, were lined with plastic bags and filled with

1700 g of air dry soil to a bulk density of 1.35 Mg m-3. Prior to transplanting, basal

fertilisers the composition of which (in mg pot-1) was: NH4NO3 (380), KH2PO4 (229),

CaCl2 (131), MgCl2 (332), CuCl2 (10.7), ZnCl2 (11), Na2MoO4 (6.84) and H3BO3 (15),

8

was applied to the soil and thoroughly mixed. The soils were then wet to field capacity

with deionised water and were allowed to equilibrate for 4 days at 25°C. The

experiment was conducted in a temperature-controlled glasshouse with day/night

temperatures of approximately 23/19C. The PAR (photosynthetically active radiation)

intensity was measured using a model LI-1000 quantum meter (Li-Cor, Lincoln, NE,

USA) and it varied from 450 to 600 mol m-2 s-1. Faba bean seeds were germinated in

potting mix and one seedling was transplanted to each pot. The salt treatments were

imposed 7 days later, when seedlings were at the 3 leaf stage. The salt or CNS

treatments were added in solution to each pot in a quantity sufficient to return the soil to

field capacity over 4 days. Because of possible P toxicity from the concentrated nutrient

solutions, the concentration of P remained at 2 mM in all solutions. The pots were

weighed and watered to field capacity regularly. The experimental design was a

factorial, completely randomised design comprised of 10 treatments two faba bean

genotypes (Cairo and Fiesta) with four replicates, giving a total of 80 pots.

Measurements

Non-destructive measurements of plant growth were taken periodically through the

experimental duration using a plant image capture and analysis system (Scanalyser 3D,

LemnaTec, Würselen, Germany). Three high resolution images were taken of every

plant, one from the top and two from the side at a 90° horizontal rotation. These pictures

were used to produce false colour images where the plant could be identified from the

background of the photograph for calculation of total plant area. The projected shoot

area was calculated by an image based leaf sum (IBLS) model, where the three areas

measured by the imaging system were summed and used as a parameter for non-

destructive plant growth analysis (Rajendran et al. 2009). To ensure the captured

images were an accurate measure of growth they were compared to destructive

9

measures of the total shoot fresh and dry weight and the leaf area of the same plants at

the time of harvest. There was a strong linear relationship between the area calculated

by the imaging system and shoot fresh weight (r=0.95), dry weight (r = 0.94) and leaf

area (r=0.96). A sigmoidal curve was fitted to the leaf area data relating the increase in

total leaf area to time. Final leaf area was calculated as the upper asymptote (a) of the

sigmoidal curve (Aguirrezabal et al. 2006). The function of fitted curves is given as:

The osmotic potential of leaf sap was measured at the time of harvest. A disc of

Whatman GF/B glass micro-fibre paper was placed in the barrel of a 2 mL plastic

syringe so that it covered the outlet hole. A fresh leaf was then put in the barrel, the

plunger was re-inserted, and the tip of the syringe was sealed with Blu-Tack. The

syringe was frozen in liquid nitrogen and, still sealed, was thawed to ambient

temperature. When temperature equilibration was complete, the plunger and Blu-Tack

were removed and the barrel of the syringe was placed in a 15 mL centrifuge tube, with

its tip resting inside a 1.5 mL ependorff tube. After centrifugation at 2500 g for 10 min

at 40C, the osmolality of a 10 µl sample was measured in a Vapro pressure osmometer.

Photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (T) were

determined on the mid-portion of the fourth leaflet of the last fully emerged leaf.

Measurements were made with a LI-COR 6400 (LI-COR, Lincoln, NE, USA) portable

gas exchange system. All measurements were taken 4-5 h into a 9-h photoperiod, and

settings were chosen to match glasshouse conditions. Leaf temperature was maintained

at 250C, light intensity was set at 800 μmol photons m-2 s-1 with a red/blue light source,

and the CO2 concentration was set at 400 μmol mol-1. Leaf to air VPD was maintained

at 1.1 kPa. The effect of salinity on non-stomatal control of photosynthetic capacity was

10

analysed by measuring CO2 assimilation-intercellular CO2 (A:Ci) response curves. A:Ci

response curves for individual leaves were obtained with a series of measurements,

where A was initially measured after 10–15 min at ambient CO2 (400 μmol mol−1). To

determine the initial slope of the A:Ci curve, the CO2 concentration was gradually

deceased to 0 μmol mol−1 (300, 200, 100, 30 and 0 μmol mol−1). The CO2 concentration

was then returned to 300 μmol mol−1 before progressively increasing to 800 μmol mol−1

(300, 500, 600, 800 μmol mol−1) to complete the curve.

Plants were harvested 49 days after transplanting. Fresh weight and dry weight of the

plants were recorded and shoot moisture content estimated. To determine the ionic

composition of whole shoots, the shoots were digested in 40 mL of 4% HNO3 at 95°C

for 6 hours in a 54-well HotBlock (Environmental Express, Mt Pleasant, South

Carolina, USA). The concentrations of Na+ and K+ in the digested samples were

determined using a flame photometer (Model 420, Sherwood, Cambridge, UK).

Chloride concentrations of the digested extracts were measured using a chloride

analyser (Model 926, Sherwood Scientific, Cambridge, UK). Recovery of Na+, Cl- and

K+ from plant standards (Australasian Soil and Plant Analysis Council) were 98, 99 and

98% respectively.

At the completion of the experiment, soil samples were taken from each pot and oven-

dried at 105°C. The samples were moistened to field capacity (water potential at -10

kPa) and centrifuged at 4500 rpm for 30 min to extract the soil solution and passed

through 0.25 µm filter paper. Electrical conductivity and osmotic potential of the

solutions were measured and these represent the EC (ECFC) and osmotic potential (FC)

of the soil solution at field capacity. Additional soil samples were weighed, oven dried

and re-weighed for determination of gravimetric water content.

11

Statistical analysis

Statistical analyses were performed in R 2.5.0 (R Development Core Team 2006). Data

for growth, ion content and moisture content were analysed using two-way ANOVA to

determine if significant differences were present among means. Variances were checked

by plotting residual vs. fitted values to confirm the homogeneity of the data. Differences

among the mean values were assessed by Least Significant Differences (LSD).

Relationships between individual variables were examined using simple linear

correlations and regressions which were performed using SigmaPlot (version 10).

Results

Non-destructive measurement of plant growth and water use dynamics under salt stress

Under control conditions, plants of Cairo and Fiesta showed exponential growth over

the experimental period (Fig. 1 and 2 and Table S1). Under salt stress however, there

was a significant reduction in plant growth between 11 and 14 days after transplanting;

the reduction occurred earlier under higher EC and in the Cairo. To quantify the relative

contribution of osmotic stress and specific ionic toxicity to plant biomass reduction at

the completion of the experiment, the growth of control plants at the final measurement

was compared to those under NaCl, or concentrated nutrient solution. This indicated the

relative importance of osmotic stress and ionic toxicity varied according to the severity

of salt stress as well as between varieties. In Fiesta grown at 4.2 dS m-1, the NaCl and

CNS treatments reduced the crop biomass production by 20% whereas the growth of

Cairo was reduced by 37% by NaCl and by 15% in the CNS, which suggests osmotic

stress dominated the response to NaCl in Fiesta, while the effect of Na+ and Cl- toxicity

exacerbated the osmotic stress in Cairo (Fig. 1 and 2). At ECFC 8.5 dS m-1 NaCl and

CNS treatments reduced the growth of Fiesta by 41 and 30% whereas the contribution

of these to the decline in growth of Cairo was 55 and 44%, suggesting that toxicity of

12

Na+ and Cl- was becoming more important in Fiesta, while osmotic stress was

increasing in Cairo. This trend continued at ECFC 15.5 dS m-1: NaCl treatment reduced

growth of Fiesta about 20% more than CNS; however, there was no difference between

NaCl and CNS treatments in Cairo.

(d)CairoEC 4.2 dS m-1

CNS 1

Dai

ly w

ater

use

(g p

ot-1

)

0

10

20

30

40

50

60(e)CairoEC 8.5 dS m-1

CNS 2

(f)CairoEC 15.5 dS m-1

CNS 3

(b)CairoEC 8.5 dS m-1

CNS 2

(c)CairoEC 15.5 dS m-1

CNS 3

(g)CairoEC 4.2 dS m-1

CNS 1

Time (days)

0 5 10 15 20 25 30 35

Cum

ulat

ive

wate

r use

(g p

ot-1

)

0

100

200

300

400

500

600

700(g)CairoEC 8.5 dS m-1

CNS 2

Time (days)

0 5 10 15 20 25 30 35

(j)CairoEC 15.5 dS m-1

CNS 3

Time (days)

0 5 10 15 20 25 30 35

LSD0.05 LSD0.05

LSD0.05 LSD0.05 LSD0.05

LSD0.05 LSD0.05LSD0.05

(a)CairoEC 4.2 dS m-1

CNS 1LSD0.05

Tota

l lea

f are

a (p

ixel

×103 )

0

100

200

300

400

500

600

700

800

Figure 1. Non-destructive characterizations of plant growth (a-c): the dynamics of total leaf expansion, (d-f) the daily changes of water use and (k-m) cumulative water use at different levels of soil ECFc generated from NaCl (■), CaCl2 (□) or concentrated macro-nutrient solution (○) compared with control (●) treatments in Cairo. The vertical arrow in (a) indicates the time of treatment application. In (a-c) a sigmoidal curve fitted to the data and the parameters are presented in Table S2. Values are means (n=4).

13

(a)FiestaEC 4.2 dS m-1

CNS 1To

tal l

eaf a

rea

(pix

el×1

03 )

0

100

200

300

400

500

600

700

800(b)FiestaEC 8.5 dS m-1

CNS 2

(c)FiestaEC 15.5 dS m-1

CNS 3

(d)FiestaEC 4.2 dS m-1

CNS 1

Dai

ly w

ater

use

(g p

ot-1

)

0

10

20

30

40

50

60(e)FiestaEC 8.5 dS m-1

CNS 2

(f)FiestaEC 15.5 dS m-1

CNS 3

(g)FiestaEC 4.2 dS m-1

CNS 1

Time (days)

0 5 10 15 20 25 30 35

Cum

ulat

ive

wate

r use

(g p

ot-1

)

0

100

200

300

400

500

600

700(h)FiestaEC 8.5 dS m-1

CNS 2

Time (days)

0 5 10 15 20 25 30 35

(j)FiestaEC 15.5 dS m-1

CNS 3

Time (days)

0 5 10 15 20 25 30 35

LSD0.05LSD0.05 LSD0.05

LSD0.05 LSD0.05 LSD0.05

LSD0.05LSD0.05 LSD0.05

Figure 2. The same as Figure 3, but for Fiesta.

Daily water use in the control treatment increased throughout the experiment but

salinity and the CNS treatments reduced water use and in many cases caused it to

plateau or decline (Fig. 1 and 2). There was little difference in daily water use among

the treatments initially but differences developed after 15 - 20 days at 4.2 dS m-1 and

after 5 days at 15.5 dS m-1. The effects of the EC treatment of daily water use tended to

appear sooner in Cairo (Fig 1d-f) than in Fiesta (Fig 2d-f). The two genotypes did not

differ in their water use under control conditions but Fiesta had a higher water use under

salt and CNS treatments (Fig. 2d-f). There was a strong correlation (r = 0.95, P <

14

0.0001) between total leaf area and cumulative water use at the completion of the

experiment.

Daily water use efficiency, estimated from the daily changes in the plant leaf area and

water use throughout the experiment, initially increased and reached a peak at about 20

days after sowing, then declined (Fig 3). There were few consistent differences in WUE

among the salt and CNS treatments until about 20 days, when the NaCl and CNS

treatments caused a reduction in WUE, especially at moderate to high ECFC. The

reduction due to NaCl was greater than that with CNS. The higher efficiency of water

use in Fiesta was indicated at all levels of salinity.

The gravimetric water content of soil at the end of the experiment was calculated for

different ECFC levels generated from NaCl treatments (Fig 4). This essentially

represents the drying of the soil by plants in each treatment as the decrease in soil

osmotic potential associated with each salt treatment will affect water uptake by the

plants (Groenevelt et al. 2004). As the plants in the control treatment grew, their water

use increased and they dried the soil to wilting point. However, when soil salinity

increased, rates of water used decreased rapidly (Fig 2, 3). The responses of the two

genotypes differed significantly (Genotype × Treatment interaction, P < 0.05) with

Cairo clearly being less able to utilise the soil moisture compared to Fiesta (Fig 4). As

well, the high salt concentration in the soil lowered the soil OP and effectively reduced

the availability of moisture, so that available soil moisture reserves were depleted at

higher soil moisture contents.

15

(a)CairoEC 4.2 dS m-1

CNS 1W

ater

use

effi

cien

cy (P

ixel

g-1

)

0

500

1000

1500

2000

2500

3000(b)CairoEC 8.5 dS m-1

CNS 2

(c)CairoEC 15.5 dS m-1

CNS 3

(d)Fiesta

EC 4.2 dS m-1

CNS 1

Time (days)

0 5 10 15 20 25 30 35

Wat

er u

se e

ffici

ency

(pix

el g

-1)

0

500

1000

1500

2000

2500

3000(e)Fiesta

EC 8.5 dS m-1

CNS 2

Time (days)

0 5 10 15 20 25 30 35

(f)Fiesta

EC 15.5 dS m-1

CNS 3

Time (days)

0 5 10 15 20 25 30 35

LSD0.05 LSD0.05LSD0.05

LSD0.05LSD0.05 LSD0.05

Figure 3. The daily changes of water use efficiency at different levels of soil ECFc generated from NaCl (■), or concentrated macro-nutrient solution (○) compared with control (●) treatments in Cairo (a-c) or Fiesta (d-f). Values are means (n = 4).

Shoot dry matter and plant moisture content in relation to salinity

Increases in ECFC by either increased salt concentration or osmotic potential of the soil

solution significantly (P < 0.001) reduced dry weight and moisture content of Cairo and

Fiesta, although there was a significant difference in their response (Fig. 5). The two

genotypes did not differ significantly in shoot dry matter when grown in non-saline soil

(mean shoot dry weight Cairo 9.5 g, Fiesta 10.1g plant-1). Fiesta showed a significantly

higher tolerance at all levels of ECFC than Cairo, except at CNS 1 where there was no

significant difference between the two genotypes (P > 0.05). In Cairo, at ECFC 4.2 dS m-

1, NaCl and CaCl2 and CNS treatments reduced the dry weight up to 23%, 26% and

15% respectively, whereas there was no significant difference between these treatments

in Fiesta.

16

Figure 4. The relationship between gravimetric water content of soil at harvest and ECFC of soil solution generated from NaCl in Cairo (a) and Fiesta (b). The upper dotted line indicates the soil water content at FC, the lower, short-dash line indicates PWP from matric potential alone (-900 kPa) and the solid line indicates the water content from a combined matric and osmotic potential of -1500 kPa where the mass of salt in solution remains constant (soil solution concentration increases as soil water content decreases). Values are means (n=4).

With increasing salinity, the difference between NaCl, CaCl2 and CNS treatments

became less in Cairo but relative shoot dry matter of Fiesta in CNS3 treatments was

30% higher than NaCl. In Fiesta, the reduction in growth due to increased CaCl2 was

less compared to NaCl but in Cairo this was only seen at low and moderate salinity (Fig.

5 a, b). The whole shoot moisture content significantly decreased as the ECFC increased,

with Cairo being about 20% lower than Fiesta (Fig. 5 c, d). In both varieties, the

moisture content when grown with CNS was greater than when pant were grown with

NaCl or CaCl2.

Shoot ionic concentration and leaf osmotic potential in relation to salinity

For plants in non-saline soil, tissue Na+ concentration concentrations were approx 90

mmol kg-1 DW and no difference was apparent between the two genotypes. Tissue Na+

(b) Fiesta

Soil solution ECFC (dS m-1)

0 2 4 6 8 10 12 14 160.15

0.20

0.25

0.30

0.35

0.40(a) Cairo

Soil solution ECFC (dS m-1)

0 2 4 6 8 10 12 14 16

Gra

vim

etri

c w

ater

con

tent

(g/g

)

0.15

0.20

0.25

0.30

0.35

0.40

17

concentration increased with salinity in both genotypes (P < 0.001), but the

concentrations in the shoots of Fiesta were significantly less than those of Cairo. The

Na+ concentration of plant tissue increased to 400 mmol kg-1 or by 4.5-fold for Fiesta

and to 700 mmol kg-1 or 7.5-fold for Cairo over the range of ECFC levels in the NaCl

treatment (Fig. 6a). Relative shoot dry matter declined with increasing Na+

concentration in both cultivars, but the decline was significantly less in Fiesta compared

to Cairo (Fig. 6a). The concentration of Cl- also increased with the concentration of

NaCl and the Cl- concentrations were consistently greater than the tissue Na+

concentrations in each NaCl treatment by up to 2.5-fold. The responses of the two

genotypes differed significantly (Genotype × Treatment interaction, P < 0.01) (Fig. 6b).

Chloride concentrations in Fiesta were up to 45% lower than those in Cairo (P < 0.01)

consistently over all salinity levels. The reduction in relative shoot dry matter was

highly correlated with increasing Cl- concentration in the whole shoot both in NaCl (r =

-0.99, p<0.001) and CaCl2 treatments (r = -0.95, P < 0.001).

Potassium concentrations were high in the control plants of both genotypes, but Cairo

had significantly lower concentrations (687 mmol kg-1 DW) compared with those in

Fiesta (768 mmol kg-1 DW). Potassium decreased significantly in the tissues of plants

exposed to salinity and the genotypes differed in their response (P < 0.001). At ECFC 15

dS m-1, tissue K+ concentration decreased by 70% and 25% in Cairo and Fiesta

respectively (Fig. 6c). Leaf osmotic potential also decreased with increasing salinity

level but the changes up to ECFC 4.2 dS m-1 were not significant (Fig 6d), but after this

there were large and significant declines in osmotic potential. Cairo had significantly

lower leaf osmotic potential compared to Fiesta.

18

(a)CairoLSD0.05

0 2 4 6 8 10 12 14 16

Shoo

t dry

mat

ter (

g)

2

3

4

5

6

7

8

9

10

11

NaCl r = -0.87CaCl2 r = -0.99CNS r = -0.99

(b)FiestaLSD0.05

0 2 4 6 8 10 12 14 162

3

4

5

6

7

8

9

10

11

NaCl r = -0.97CaCl2 r = -0.97CNS r = -0.92

(c)CairoLSD0.05

ECFC (dS m-1)

0 2 4 6 8 10 12 14 16

Plan

t moi

stur

e co

nten

t (g/

g)

5

6

7

8

9

10

NaCl r = -0.87CaCl2 r = -0.89CNS r = -0.96

(d)FiestaLSD0.05

ECFC (dS m-1)

0 2 4 6 8 10 12 14 165

6

7

8

9

10

NaCl r = -0.98CaCl2 r = -0.99CNS r = -0.98

Figure 5. Relationship between soil electrical conductivity (ECFC) and (a and b) shoot dry matter and (c and d) tissue moisture content of Cairo (a, c) and Fiesta (b,d) grown at different levels of soil salinity generated from NaCl (●), CaCl2 (○) or concentrated nutrient solution (▲) for 49 days. Fitted curves are derived from linear or exponential decay regressions. Values are means (n=4).

Contribution of organic and inorganic solutes to leaf osmotic potential under salt stress

Analysis of the sap of leaf tissue showed that the concentration of several compounds

altered markedly in response to both NaCl and CNS treatments. The soluble sugars,

sucrose, glucose and fructose, increased in shoots of both genotypes, with the increases

measured in Fiesta greater than those in Cairo (Table S2). A similar pattern was

observed for the betaine, proline and trigonelline concentrations, with Fiesta showing

19

higher capacity to accumulate these compounds. However, Cairo had a higher proline

concentrations under osmotic stress compared to Fiesta.

At different ECFC levels, inorganic solutes (Na+, K+ and Cl-) accounted for 38-70% of

the measured total solute potential (Ψs) in Cairo and 40-51% in Fiesta where the total

would include inorganic and organic osmolytes (Table 1). Organic ion contribution to

measured Ψs was 24-29% for the salt-sensitive Cairo, and 36-44% for Fiesta among the

different salt treatments. Fiesta which showed a high ability to exclude Na+ and Cl- also

exhibited a very strong capacity for accumulation of inorganic solute to contribute to

total Ψs as EC increased. However, in Cairo, the major contribution to Ψs was from the

high concentrations of Na+ and Cl-. Interestingly in Fiesta, the major contribution to

estimated inorganic osmolytes was by K+ and it did not change at different levels of soil

EC, whereas it fell by 45% in Cairo (Table 1).

Gas exchange characteristics under salt stress

The response in photosynthesis to salt treatment differed between the two varieties. The

photosynthetic rate (A) in Cairo declined by approximately 64% over the range of NaCl,

CaCl2 and CNS treatments. In Fiesta, A declined by 42% in NaCl and CaCl2 treatments

and 33% over CNS treatments. The decline in A was associated with a large reduction

in stomatal conductance (gs; Fig 7 c, d) as well as in intercellular CO2 concentration (Ci;

Fig 7e, f). While the decline in Ci:Ca value was only 2% in Fiesta at EC 4.2 dS m-1 ,

values of gs decreased by 16% which suggest that the reduction in photosynthetic

capacity at low salinity level of Fiesta is mainly associated with stomatal closure. This

was also found with Cairo in the CNS1 treatment where Ci value did not decrease

significantly but there was a 20% reduction in gs. Overall, the value of Ci:Ca declined

from approx 0.79µbar/µbar to 0.30 µbar/µbar in Cairo and to 0.55 µbar/µbar in Fiesta

20

over the range of salt treatments meaning that stomatal limitations on photosynthetic

CO2 fixation increased about 60 and 30% in these two genotypes respectively (Fig 7).

The salt treatments caused a greater reduction in Ci:Ca than the CNS treatment and this

effect tended to increase more in Fiesta as ECFC increased (Fig. 7e and f).

(a)LSD0.05

Na+ concentration (mmol kg-1 DW)

0 200 400 600 800

Shoo

t dry

mat

ter (

g)

2

3

4

5

6

7

8

9

10

11(b)LSD0.05

Cl- concentration (mmol kg-1 DW)

0 400 800 1200 1600 20002

3

4

5

6

7

8

9

10

11

(c)LSD0.05

K+ concentration (mmol kg-1 DW)

0 200 400 600 800 1000

Shoo

t dry

mat

ter (

g)

2

3

4

5

6

7

8

9

10

11(d)LSD0.05

Leaf osmotic potential (-MPa)

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.22

3

4

5

6

7

8

9

10

11

Figure 6. The relationship between plant relative shoot dry matter and (a) Na+

concentration (mmol kg-1 DW), (b) Cl- concentration (mmol kg-1 DW), (c) K+ (mmol kg-

1 DW) and (d) leaf osmotic potential (-MPa) of Cairo (●,○,▲) and Fiesta (■,□,) grown at different concentrations of NaCl (●,■), CaCl2 (○,□) or concentrated nutrient solution (▲,) for 49 days in soil. Fitted curves are derived from linear or exponential decay regression. Values are means (n=4).

21

Table 1. Estimated contribution of organic and inorganic ions to leaf osmotic potential. The contribution of individual solutes to measured osmotic potential was determined using the van’t Hoff equation, where the calculated Ψs, was based on solute concentration on a fresh weight basis. The percentage value is based on the measured value of leaf OP.

Van’t Hoff equation Ψs (MPa) = –csRT, where cs = Osmolarlity (mol L–1), R = 0.0083143 L MPa mol–1 K–1, and T = 293 K were considered. Contribution = (Ψs calculated/Ψs measured) × 100.

Estimated Contribution (MPa)

Sucrose Glucose Fructose Betaine Proline Trigonelline Na+ K+ Cl-

% Organic

contribution

% Inorganic ion

contribution

% Total

Contribution

Cairo

Control -0.03 -0.02 -0.02 -0.02 -0.01 -0.0002 -0.03 -0.19 -0.03 15 38 53

EC 4.2 -0.04 -0.04 -0.04 -0.02 -0.02 -0.0016 -0.13 -0.13 -0.24 24 71 95

EC 8.5 -0.08 -0.07 -0.07 -0.04 -0.13 -0.0021 -0.21 -0.13 -0.50 29 62 92

EC 15.5 -0.10 -0.09 -0.10 -0.08 -0.18 -0.0036 -0.32 -0.10 -0.82 28 61 89

Fiesta

Control -0.02 -0.02 -0.02 -0.01 -0.01 0.0002 -0.02 -0.21 -0.03 13 40 53

EC 4.2 -0.06 -0.05 -0.05 -0.04 -0.04 0.00038 -0.06 -0.22 -0.07 36 51 87

EC 8.5 -0.09 -0.07 -0.07 -0.07 -0.11 -0.0114 -0.10 -0.22 -0.16 44 51 95

EC 15.5 -0.14 -0.11 -0.10 -0.11 -0.17 -0.0211 -0.15 -0.22 -0.25 42 40 82

22

Assimilation rate as a function of intercellular CO2 concentration:

The initial linear slope (Table 1) and upper plateau of the A:Ci curve declined with

increasing salinity in Cairo, however, in Fiesta while there was a significant difference

between the control and salt-affected plants there was no differences between the

various salt treatments. The initial slope of this response estimates the RuBP-

carboxylase-limited CO2 fixation capacity of plants (Caemmerer and Farquhar 1981;

Seemann and Berry 1982). The maximum photosynthetic rate of Cairo was about 28%

lower under NaCl stress than CNS treatment at ECFC 4.2 dS m-1 whereas no difference

was observed between these two treatments in Fiesta plants (12% reduction under NaCl

and CNS treatments relative to control) (Fig 8a and c). A very different pattern was

observed when plants were grown at 15.5 dS m-1 (Fig. 8 b and d). All treatments

reduced the photosynthetic capacity by 50% in Cairo whereas there was a more negative

effect of NaCl compared to CNS and CaCl2 in Fiesta.

Discussion

Salinity stress is dynamic as osmotic and ionic stresses vary over time (Munns 2002).

Consequently the response of plants to salt stress and the importance of different

tolerance mechanisms changes as the relative effects of osmotic and ionic stress vary

(Tavakkoli et al. 2010b). In the current study, plant growth was strongly reduced by

salinity (Fig 1 and 2), but Cairo was more sensitive than Fiesta. Using high resolution

image analysis to measure growth repeatedly during the development of salt stress (Fig

1 and 2) indicated the relative importance of the ion toxicity and osmotic stress on final

biomass production varied with: (1) the degree of stress encountered (mild, moderate,

and severe); (2) the variety; and (3) the duration of the stress. Under mild salinity stress

(ECFC =4.2 dS m-1), growth of Fiesta was reduced mainly by osmotic stress due to its

efficient exclusion of Na+ and Cl-. However, the effectiveness of ion exclusion declined

23

at higher levels of salinity (ECFC=8-15 dS m-1) (Fig 2) and specific ion toxicity became

the main cause of reduced growth. Around day 30-32, there was an indication that Na+

and Cl- had accumulated to toxic concentrations as growth in the NaCl and CaCl2

treatments ceased while that in the concentrated nutrient solution continued. At the

higher level of salinity stress the exclusion of Na+ and Cl- in Fiesta seemed to contribute

less to salt tolerance, however, its ability to maintain relatively high plant moisture

content indicated it had a higher level of osmotic tolerance.

On the other hand, the reduction in growth of Cairo, a genotype with a poor Na+ and Cl-

exclusion compared to Fiesta, was a result of ion toxicity rather than pure osmotic

treatments at low salinity stress. However, the osmotic stress became the major cause of

growth reduction with increasing levels of soil salinity (Fig 3). An interpretation of

these results is that the difference between the two genotypes at low ECFC values is an

ion-specific effect, while the rapid decline in growth above EC 8.5 dS m-1 is a result of

both osmotic and ion-toxicity effects. Variation in growth between different salt

treatments and between Cairo and Fiesta came about primarily because of differences in

total water use which was very similar to variation in dynamic plant growth (Fig 1 and

2). Water use efficiency fell below the control at the intermediate and high salinity

levels. A determinant of productivity in saline soils is the total amount of water used by

plants. In this experiment more water was left behind in the soil as salinity increased

(Fig 5). This could be due to plants being unable to lower their water potential

sufficiently to match the lower potential of the drying saline soil thereby leaving water

behind. The differences observed between Cairo and Fiesta is consistent with the

differences in the final soil water content that the two genotypes were able to extract to,

and the overall salinity tolerance of these genotypes (Fig. 1 and 6).

24

(c)CairoLSD0.05

gs (

mol

m-2

s-1

)

0.2

0.4

0.6

0.8

NaClCaCl2CNS

(b)FiestaLSD0.05

NaClCaCl2CNS

(d)FiestaLSD0.05

NaClCaCl2CNS

(e)CairoLSD0.05

ECFC (dS m-1)

0 2 4 6 8 10 12 14 16

Ci :

Ca

0.2

0.4

0.6

0.8

1.0

NaClCaCl2CNS

(f)FiestaLSD0.05

ECFC (dS m-1)

0 2 4 6 8 10 12 14 16

NaClCaCl2CNS

(a)CairoLSD0.05

A (µ

mol

CO

2 m-2

s-1

)

4

6

8

10

12

14

16

18

20

NaClCaCl2CNS

Figure 7. Relationship between gas exchange parameters (a and b) net CO2 assimilation rate (A);( c and d) stomatal conductance (gs); (e and f) the ratio of partial pressures of CO2 inside of the leaf and in the air (Ci:Ca); and soil electrical conductivity (ECFC) in fourth leaflet of Cairo and Fiesta grown at different concentrations of NaCl (●), CaCl2 (○) or concentrated nutrient solution (▲) for 49 days. A general linear model was used to fit a regression line to data. The resulting F-test indicated significant difference between slopes (P < 0.05) for each graph. Values are means (n=4).

25

(a)CairoLSD0.05

0 100 200 300 400 500 600 700 800

A (µ

mol

CO

2 m

-2 s

-1)

0

5

10

15

20

25

30

ControlCNSNaClCaCl2

(b)CairoLSD0.05

0 100 200 300 400 500 600 700 8000

5

10

15

20

25

30

(c)FiestaLSD0.05

Intercellular CO2 concentration (µmol mol-1)

0 100 200 300 400 500 600 700 800

A (µ

mol

CO

2 m

-2 s

-1)

0

5

10

15

20

25

30(d)FiestaLSD0.05

Intercellular CO2 concentration (µmol mol-1)

0 100 200 300 400 500 600 700 8000

5

10

15

20

25

30

ECFC = 4.2 dS m-1 ECFC = 15.5 dS m-1

ECFC = 4.2 dS m-1 ECFC = 15.5 dS m-1

Figure 8. The response of net photosynthetic CO2 fixation to variation in the intercellular CO2 concentration (Ci) (A:Ci curve) of Cairo (a and c) and Fiesta (b and d), grown at two soil EC levels generated from NaCl (■), CaCl2 (□) or concentrated macro-nutrient solution (○) compared with control (●). Values are means of four individual measurements in leaves at a similar age.

In dryland farming systems saline-sodic subsoils have an ECe between 2 and 16 dS m-1

which can dramatically affect crop production through osmotic effects (Rengasamy

2010). Osmotic potential added to matric potential renders subsoil water unavailable to

crops, as indicated in Figure 6. While matric potential indicates water availability to

plants, plant uptake of water is governed by total water potential, which includes the

soil osmotic potential. Figure 6 shows that, in the absence of salt (ECFC~1.3 dSm–1), the

26

plants can fully use the available soil moisture but when the soil is saline (ECFC of 2.4

dS m–1 or higher), the plants cease to take up water even when the soil dries only to

30% water content because the total water potential (matric plus osmotic) at that point,

reaches –1500 kPa. This indicates that a significant proportion of the difference in

salinity tolerance between genotypes is due to difference in ability to access soil water

under saline conditions. The points at which plants are unable to maintain water

extraction to permanent wilting point as determined by sum of matric and osmotic

potential is an important indicator of salt tolerance (Rengasamy 2002; Rengasamy

2010).

A common mechanism in response to salt stress is the accumulation of compatible

solutes which may be interpreted as a symptom of injury caused by stress or some type

of adaptive response. This poses the question of whether salt-tolerant genotypes also

have a superior ability to accumulate higher concentrations of compatible solutes. In

this study, salt stress caused an increase in ions and organic solutes in both genotypes,

but the more salt tolerant variety, Fiesta, had significantly higher concentration of

soluble sugars (glucose, fructose and sucrose), glycine betaine, proline and trigonelline

(Table 1). Ion accumulation in plants can also play a major role in osmotic adjustment

to high salinities. It would seem, however, from the relationship between ion

accumulation and water status observed here for Cairo and Fiesta that the simple

accumulation of Na+ and Cl- alone can not account for the osmotic behaviour of this

species. In Cairo, despite Na+ and Cl- accumulating high enough to affect an osmotic

adjustment, there was a marked decrease in shoot water content (Fig. 1c). The inability

of Cairo to maintain its shoot water content at high salt concentration could be

explained by an osmotic imbalance which results from an inability to regulate the

accumulation of Na+ and Cl- ions in the shoot. While the ability to restrict Na+ and Cl-

27

accumulation could prevent the development of an osmotic imbalance in Fiesta, the

concentration of Na+ and Cl- accumulated when considered together with the reduction

in shoot K+ would seem to necessitate the synthesis of additional osmotically active

solute in order to prevent an osmotic imbalance with respect to the external salt in soil

solution. The increases observed in soluble sugars, glycine betaine and proline amount

to a total of 1570 µmol g-1 DW and could therefore represent an important component

of the shoot osmotic potential (Table 1). The present study suggests that salt exclusion

coupled with a synthesis of organic solutes are important components of salt tolerance

in the tolerate genotype, Fiesta.

The concentrations of Na+ and Cl- increased in faba bean on exposure to salt (Fig 2),

with tissue Cl- concentrations generally exceeding those of Na+ when grown in soil.

Increases in plant ion concentrations are not novel and occur with all plants exposed to

NaCl. What is generally not clear, however, is at what concentration ions reach toxic

concentrations and whether toxicity can be assigned to Na+ or Cl-. The most striking

differences were with respect to Cl-, the concentration of which increased dramatically

with increasing salinity and could not be accounted for by reduced biomass (Fig 1 and

2). In Cairo Cl- increased to an average of 3-times that of Na+ in salinised plants, while

in Fiesta, which excludes Cl- it increased by an average of 1.7-times in salinised plants.

So if any consistent relationship between yield and ion concentration exists between

genotypes, then Cl- would be the most promising candidate ion with which to screen for

salinity tolerance in faba bean. Mamo et al. (1996) reported a 5-fold increase in shoot

Cl- concentrations for chickpea growing under salinized conditions. The critical toxic

concentrations of Na+ and Cl- are considered to be in a range of 300-950 mmol kg-1 DW

and Cl- 250-900 mmol kg-1 DW respectively (Reuter and Robinson 1997).

28

In many studies on salt tolerance it has not been possible to determine whether the toxic

effects observed are due to Cl-, Na+ or to a contribution of the two since Na+ and Cl- are

combined with their counteranions/countercations. The above results indicate that, in

faba bean, many physiological disturbances by salinity are linked to both leaf Cl- and

Na+ build up and the reductions in dynamics of growth and final shoot dry matter due to

Cl- were similar to those of Na+ (Fig 1, 2 and 5). It was shown that a reduction in

growth due to CaCl2 was slightly less than NaCl and therefore clearly indicates that,

when leaf Cl- concentration is high, the presence of Na+ as the dominant cation

exacerbates the severity of stress which suggests that high concentrations of Na+ and Cl-

are limiting the growth through different mechanisms but simultaneously (Tavakkoli et

al. 2010a).

Stomatal closure is generally associated with salinization of salt-sensitive species

(Downton 1977; Longstreth and Nobel 1979). The results presented here for faba bean

indicate that stomatal conductance is reduced by salinity; however, the extent to which

stomatal closure affects photosynthetic capacity is indicated by the magnitude of the

reduction in Ci (Berry and Downton 1982; Farquhar and Sharkey 1982). The salt-

induced decline in Ci of up to 30% in faba bean (Fig. 7), would be responsible for a

nearly equal decline in the photosynthetic rate, as the A:Ci response is nearly linear over

such intercellular CO2 concentrations (Fig. 8). In mangroves, Ball and Farquhar

(1984a,b) found that long-term growth at high salinity caused a reduction in both the

initial slope and CO2-saturated portions of the A:Ci response, whereas short-term

exposure to high salinity primarily affected only the upper portion of the curve. These

responses are similar to those found following water stress of P. vulgaris, where short-

term stress reduced the CO2-saturated portion of the A:Ci response, while severe stress

29

produced a decline in both the initial slope and CO2-saturated region (Caemmerer and

Farquhar 1981; Farquhar et al. 2001).

Conclusion

The present approach enables the analysis of key determinants of a salt tolerant

phenotype as there is an increasing realisation that molecular and genomics data have to

be understood in the context of physiological responses the so-called phenotype gap

(Miflin 2000). Application of continuous, non-destructive leaf area determination of

both varieties could help to estimate plant reaction to salt stress. The results indicated

that the response of crop growth varies with severity of salt stress. Osmotic stress was

the predominant cause of reduced growth at high levels of salinity, while specific-ion

toxicity was more important under mild salinity stress. Fiesta maintained greater whole-

plant tolerance to salinity by dual mechanisms of ion exclusion and osmotic adjustment,

compared to Cairo. The reduction in plant growth was correlated with both Na+ and Cl-

concentration in plant tissues, which indicates that both Na+ and Cl- are contributing

significantly to growth reduction under saline conditions, but with evidence to suggest

that Cl- toxicity is more important. This study also showed an association between an

increase in ion concentration and decrease in A. This was partially due to non-stomatal

effects, suggesting ion toxicity was an important reason. It is likely that stress-tolerant

faba bean plants accumulate compatible solutes and field tests of these plants under

stress conditions will help to verify their potential utility in crop-improvement

programs.

Acknowledgments

We thank Waite Analytical Services (The University of Adelaide) for their help with the

elemental analyses, Mr. D. Keetch for his help with the experiments, Mr. S. Coventry

30

(The University of Adelaide) for excellent HPLC guidance and Dr G. Lyons for critical

comments on this manuscript. We particularly would like to express our gratitude to

Prof M. Tester (ACPFG) for making the Lemna Tec imaging system accessible during

this experiment and Dr B. Berger and Mr. J. Eddes for help with image analysis.

Funding provided by the Grains Research and Development Corporation to ET, and by

the University of Adelaide is gratefully acknowledged.

31

References Aguirrezabal L, Bouchier-Combaud S, Radziejwoski A, Dauzat M, Cookson SJ, Granier C (2006) Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes. Plant, Cell and Environment 29, 2216-2227. Berry JA, Downton WJS (1982) Environmental regulation of photosynthesis. In 'Photosynthesis: development, carbon metabolism, and plant productivity'. (Ed. Govindjee) pp. 263-343. (Academic Press: New York Londo) Boyer JS, James RA, Munns R, Condon TAG, Passioura JB (2008) Osmotic adjustment leads to anomalously low estimates of relative water content in wheat and barley. Functional Plant Biology 35, 1172-1182. Cachorro P, Ortiz A, Cerdá A (1993) Growth, water relations, and solute composition of Phaseolus vulgaris L. under saline conditions. Plant Science 95, 29-32. Caemmerer S, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 376-387. Dang YP, Dalal RC, et al. (2008) High subsoil chloride concentrations reduce soil water extraction and crop yield on Vertisols in north-eastern Australia. Australian Journal of Agricultural Research 59, 321-330. Del Pilar Cordovilla M, Ligero F, Lluch C (1999) Effect of salinity on growth, nodulation and nitrogen assimilation in nodules of faba bean (Vicia faba L.). Applied Soil Ecology 11, 1-7. Downton WJS (1977) Photosynthesis in salt-stressed grapevines. Aust. J. Plant Physiol. 4, 183-192. FAO (2008) FAO Land and Plant Nutrition Management Service. Available at http://www.fao.org/ag/agl/agll/spush [Verified 24 May 2010] FAOSTAT. FAO. Farquhar GD, Sharkey TD (1982) Stomatal conductance and photosynthesis. Annu. Rev. Plant Physiol. 33, 317-345. Farquhar GD, von Caemmerer S, Berry JA (2001) Models of Photosynthesis. Plant Physiol. 125, 42-45. Groenevelt PH, Grant CD, Murray RS (2004) On water availability in saline soils. Australian Journal of Soil Research 42, 833-840. Hajrasuliha S (1980) Accumulation and toxicity of chloride in bean plants. Plant and soil 55, 133-138. Hasegawa PM, Bressan RA, Zhu J-K, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 463-499. Isbell RF (1996) 'The Australian Soil Classification.' (CSIRO: Melbourne)

32

Jensen ES, Peoples MB, Hauggaard-Nielsen H (2010) Faba bean in cropping systems. Field Crops Research 115, 203-216. Klute A (1986) Water retention: Laboratory methods. In 'Methods of soil analysis. Part 1'. (Ed. A Klute) pp. 635-662. (Agron. Monogr. 9. ASA and SSSA: Madison, WI) Lisle ML, Lefroy RDB, Blair GJ (2000) Methods for rapid assessment of nutrient supply capacity of soils. Communications in Soil Science and Plant Analysis 31, 2627-2633. Longstreth DJ, Nobel PS (1979) Salinity effects on leaf anatomy. Consequences for photosynthesis. Plant Physiol 63, 700-703. Maas EV, Hoffman GJ (1977) Crop salt tolerance-current assessment. J. Irrig. Drain. Div. ASCE 103, 115-134. Malhotra RS (1997) Evaluation techniques for abiotic stresses in cool season food legumes. In 'Recent Advances in Pulses Research'. (Eds AN Asttranu and A Masood) pp. 459-473. ( Indian Society of Pulses Research and Development: Kanpur, India) Mamo T, Richter C, Heiligtag B (1996) Salinity effects on the growth and ion contents of some chickpea (Cicer arietinum L.) and lentil (Lens culinaris Medic) varieties. J Agron Crop Sci 176, 235-247. Marschner H (1995) 'Mineral nutrition of Higher Plants.' (Academic Press) Miflin BJ (2000) Crop Biotechnology. Where Now? Plant Physiology 123, 17-28. Munns R (2002) Comparative physiology of salt and water stress. Plant, Cell and Environment 25, 239-250. Munns R, Schachtman DP, Condon AG (1995) The significance of a two-phase growth response to salinity in wheat and barley. Australian Journal of Agricultural Research 22, 561-569. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651-681. R Development Core Team (2006) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Rajendran K, Tester M, Roy SJ (2009) Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment 32, 237. Rengasamy P (2002) Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. Australian Journal of Experimental Agriculture 42, 351-361. Rengasamy P (2006) World salinization with emphasis on Australia. Journal of Experimental Botany 57, 1017-1023.

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Rengasamy P (2010) Soil processes affecting crop production in salt-affected soils. Australian Journal of Soil Research 37, 613-620. Reuter DJ, Robinson JB (Eds) (1997) 'Plant Analysis. An Interpretation Manual (2nd edn).' (CSIRO publishing: Melbourne) Rispail N, Kaló P, et al. (2010) Model legumes contribute to faba bean breeding. Field Crops Research 15, 253-269. Seemann JR, Berry JA (1982) Interspecific differences in the kinetic properties of RuBP carboxylase protein. Carnegie Inst. Washington Year Book 81, 78-83. Slabu C, Zörb C, Steffens D, Schubert S (2009) Is salt stress of faba bean (Vicia faba) caused by Na+ or Cl- toxicity? J. Plant Nutr. Soil Sci. 172, 644-650. Tavakkoli E, Rengasamy P, Mcdonald GK (2009) Phenomics-based screening for salinity tolerance: A case study for the evaluation of the impact of salinity on growth of barley and faba bean. In '1st International Plant Phenomics Symposium: from Gene to Form and Function'. Discovery centre, CSIRO, Canberra, Australia. (Ed. RT Furbank) Tavakkoli E, Rengasamy P, Mcdonald GK (2010a) High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany 61, 4449–4459. Tavakkoli E, Rengasamy P, Mcdonald GK (2010b) The response of barley to salinity stress differs between hydroponics and soil systems. Functional Plant Biology 37, 621-633. Termaat A, Munns R (1986) Use of concentrated macronutrient solutions to separate osmotic from NaCl-specific effects on plant growth. Australian Journal of Plant Physiology 13, 509-522.

34

Table S1. The parameters of fitted sigmoidal curves to leaf area dynamics in figure 6. The equation of fitted curves is given in materials and methods. a b Xo r2 Cairo Control 1.108×103 7.414 2.758×101 0.9973 ECNaCl 4.2 5.319×102 6.005 1.988×101 0.9995 ECNaCl 8.5 4.889×102 5.315 2.190×101 0.9993 ECNaCl 15.8 3.763×102 6.292 1.894×101 0.9971 ECCaCl2 4.5 6.974×102 6.789 2.294×101 0.9978 ECCaCl2 8.1 5.056×102 5.989 2.029×101 0.9987 ECCaCl2 15.5 3.886×102 6.160 1.856×101 0.9986 CNS 1 7.724×102 6.935 2.377×101 0.9961 CNS 2 4.921×102 5.315 1.835×101 0.9992 CNS 3 3.781×102 5.848 1.748×101 0.9995 Fiesta Control 8.809×102 5.621 2.185×101 0.9996 ECNaCl 4.2 7.742×102 6.000 2.192×101 0.9992 ECNaCl 8.5 6.195×102 5.744 2.041×101 0.9996 ECNaCl 15.8 5.253×102 6.691 2.100×101 0.9995 ECCaCl2 4.5 7.732×102 5.993 2.187×101 0.9996 ECCaCl2 8.1 6.728×102 5.744 2.100×101 0.9995 ECCaCl2 15.5 6.275×102 6.484 2.211×101 0.9995 CNS 1 8.308×102 6.219 2.273×101 0.9996 CNS 2 7.321×102 5.744 2.175×101 0.9996 CNS 3 5.253×102 6.691 2.100×101 0.9989

35

Table S2. Changes in concentration of soluble sugars, glycine betaine, proline and trigonelline [µmol g-1 DW] in Cairo and Fiesta plants in response to increasing soil EC (dS m-1). Values are means (n=4).

Concentration (µmol g-1 DW) Sucrose Glucose Fructose Betaine Proline Trigonelline Cairo

Control 91 85 85 55 25 0.55 EC 4.2 125 102 114 61 66 4.5 EC 8.5 199 175 195 103 348 5.6 EC 15.5 215 205 218 175 401 7.8 CNS 1 105 114 101 56 85 3.3 CNS 2 131 143 126 101 324 3.5 CNS 3 155 185 155 117 341 4.1 Fiesta Control 75 80 81 42 38 0.86 EC 4.2 181 155 145 125 110 12.3 EC 8.5 255 195 215 211 322 33.5 EC 15.5 391 225 244 288 422 48.5 CNS 1 61 43 143 65 195 12.8 CNS 2 75 63 154 172 201 14.5 CNS 3 88 91 156 215 215 16.8 LSD0.05 (genotype×salt) 7.5 4.6 4.1 6.3 26.5 0.29

Chapter 5

Thesis: Limitations to yield in saline-sodic soils: Quantification of osmotic and ionicregulations that affect the growth and nutrition of crops under salinity stress. By EhsanTavakkoli,20ll.

CHAPTER 5

TITLE OF'PAPERAdditive effects of Na* and cl- ions on barley growth under salinity stress

AUTHORS OF'PAPER:Ehsan Tavakkoli, Foad Fatehi, Stewart Coventry, Pichu Rengasamy and Glenn McDonaldSchool of Agriculture, Food and wine, Waite campus, The university of Adelaide, SA, 5064Name of journal paper submitted:Exp er ime ntal B o t any, 2 0 I 0, Ac c ept e d, DOL. JXBERQ42}. In press.

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

Tavakkoli, E., Fatehi, F., Coventry, S., Rengasamy, P. and McDonald, G.K. (2011) Additive effects of Na+ and Cl- ions on barley growth under salinity stress. Journal of Experimental Botany, v.62 (6), pp.2189-2203, March 2011

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1093/jxb/erq422

Chapter 6

Thesis: Limitations to yield in saline-sodic soils: Quantification of osmotic and ionicregulations that affect the growth and nutrition of crops under salinity stress. By EhsanTavakkoli,20IL

CHAPTER 6

TITLE OF PAPERHigh concentrations of Na* and Cl- ions in soil solution have simultaneous detrimental effectson growth of faba bean under salinity stress

AUTHORS OF PAPER:Ehsan Tavakkoli, Pichu Rengasamy and Glenn McDonaldSchool of Agriculture, Food and wine, Waite campus, The university of Adelaide, SA, 5064

Name of journal - Accepted paperJournal of Experimental Botany, 2010 61,44494459

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

Tavakkoli, E., Rengasamy, P. and McDonald, G.K. (2010) High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany, v.61 (15), pp.4449-4459, October 2010

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1093/jxb/erq251

Chapter 7

Thesis: Limitations to yieid in saline-sodic soils:regulations that affect the growth and nutrition ofTavakkoli,201l.

CHAPTER 7

Quantification of osmotic and ioniccrops under salinity stress. By Ehsan

TITLE OF PAPEREffective screening methods for salinity tolerance: pot experiments but not hydroponics are

plausible models of salt tolerance in barley

AUTHORS OF PAPER:Ehsan Tavakkoli*, Foad Fatehi, Pichu Rengasamy, and Glenn K. McDonald

School of Agriculture, Food and wine, Waite campus, The universþ of Adelaide, SA, 5064

Name of journal paper submitted:Journal of Experimental Botany, 2010, submítted paper.

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

1

Effective screening methods for salinity tolerance: pot experiments but not hydroponics are plausible models of salt tolerance in barley

Ehsan Tavakkoli*, Foad Fatehi, Pichu Rengasamy, and Glenn K.

McDonald

School of Agriculture, Food and Wine,

The University of Adelaide,

*To whom correspondence should be addressed:

Ehsan Tavakkoli

School of Agriculture, Food and Wine,

Waite Campus

The University of Adelaide,

PMB 1 Glen Osmond, South Australia, 5064

Phone: +61-8-8303 6533

Fax: +61-8-8303 7109

Email: ehsan.tavakkoli@adelaide.edu.au

Word count: 6846

Number of figures: 9

Number of tables: 4

Number of supplementary tables: 2

Submitted article to Journal of Experimental Botany, December 2010

2

Abstract

Success in breeding crops for yield and other quantitative traits depends on the use of

methods to accurately evaluate genotypes under field conditions. Although many

screening criteria have been suggested to distinguish between genotypes for their salt

tolerance under controlled environmental conditions, there is a need to test these criteria

in the field. In this study, we investigated the salt tolerance, ion concentrations and

accumulation of compatible solutes of genotypes of barley with a range of putative salt

tolerance using three growing conditions (hydroponics, soil in pots and natural saline

field). It was found that solution culture was not a reliable indicator of the differences in

salt tolerance between barley plants that are evident in soil-based comparisons.

Significant correlations were observed in the rankings of genotypes on the basis of their

grain yield production in the field and relative shoot growth in pot experiment at ECe

7.2 (Spearman’s rank correlation (rs) = 0.79) and ECe 15.3 (rs = 0.82) and the crucial

parameter of leaf Na+ (rs = 0.72) and Cl- (rs = 0.82) concentrations at ECe 7.2 dS m-1.

Early assessment of salinity tolerance at the seedling stage was found to be unsuitable.

This work has established screening procedures that correlated well with grain yield at

sites with high levels of soil salinity. This study also showed that several mechanisms

are involved in salt tolerance and that the relative importance of these traits may differ

with the severity of the salt stress. Salt exclusion coupled with a synthesis of organic

solutes were shown to be an important components of salt tolerance in the tolerant

genotypes and further field tests of these plants under stress conditions will help to

verify their potential utility in crop-improvement programs.

Keywords: Salinity tolerance, osmotic stress, specific ion toxicity, barley, screening,

physiological traits, soil, hydroponics

3

Introduction

Broadacre cropping in Australia is based on rainfed systems in a semiarid environment,

where the efficient uptake and use of water is the main driver of productivity. However,

more than 60% of the 20 million ha of cropping soils in Australia are sodic, which

together with low rainfall and high rates of evapotranspiration have contributed to the

development of transient salinity (Rengasamy 2002). Saline subsoils adversely affect

the ability of crops to use subsoil water and this imposes a significant constraint on

productivity. In the last three decades, considerable effort has been directed towards

gaining a better understanding of how plants respond to salinity, and in particular the

physiological and molecular bases of salinity tolerance (Munns and Tester 2008).

A range of engineering and farm management solutions is available to control soil

salinity, but their costs and slow adoption mean that substantial soil salinisation is

inevitable. To maintain crop production in regions with saline soils and water, a genetic

approach, involving breeding cultivars with an enhanced ability to grow on salt-affected

land, has been proposed in conjunction with the normal reclamation and management

practices. The majority of the work on developing selection criteria for improved salt

tolerance has been done using solution culture, either in hydroponic or supported

hydroponic systems (Munns et al. 2002; Genc et al. 2007), or using sand-based systems

(Munns et al. 2002), with the implicit assumption that differences in salinity tolerance

expressed in hydroponic systems will result in improved performance in the field.

Strong evidence to support this is lacking and the ability of solution culture to identify

genotypes express its salt tolerance under stressed conditions in the field needs to be

critically evaluated (Gregory et al. 2009). Recently, Tavakkoli et al. (2010b)

4

demonstrated that solution culture may not be able to discern differences in salt

tolerance between genotypes of barley that are expressed when grown in soil.

Most studies evaluating genetic variation in salt resistance in crop plants have been

performed in controlled or semi-controlled environments at a single level of salt stress

with no validation of the results under field conditions. Furthermore, studies under

controlled conditions generally involve imposing salinisation on seedlings over a

relatively short period (often 1-2 days) whereas the salinity stress in the field may show

a greater level of spatial and temporal variation. The variation in salt stress in the field

also means that plants can be exposed to a range in salt concentrations at different

growth stages, but it is not clear which is the most appropriate salinity level for

screening and what stage of development best relates to genetic differences expressed in

the field. This information is necessary to develop efficient breeding and selection

methods for salt tolerance in crops, and it needs to be compared with results of studies

carried out in naturally saline field environments (Richards 1983; Richards et al. 1987;

El-hendawy et al. 2005).

Efforts to enhance crop yields under salinity stress have also had a limited success

because available knowledge of the mechanisms of salt tolerance has not been turned

into useful selection criteria to evaluate a wide range of genotypes within and across

species. Attempts have been made to evaluate salt tolerance at germination and

emergence stages in wheat and barley, and large genotypic differences were reported

(Munns et al. 2000; Chen et al. 2008; James et al. 2008), but this early evaluation

appears to have little relation with overall performance under saline conditions (Munns

et al. 2002). Though Na+ exclusion and K+/Na+ ratios have been suggested to be reliable

5

traits for selecting salt tolerant crops (Munns et al. 2002; Munns and James 2003;

Poustini and Siosemardeh 2004), the value of this trait has not been used routinely in

plant breeding programs. Therefore, there is a need to identify traits associated with

salinity tolerance, and develop simple, high throughput, repeatable screening methods to

evaluate large number of genotypes. Studies on salt tolerance among the major cereals

have concentrated on Na+ transport and accumulation, while the role of Cl- in growth

and yield reduction of grain crops has been neglected. It is generally considered that Cl-

toxicity is not a major cause of reductions in growth of grain crops (Kingsbury and

Epstein 1986; Kinraide 1999) but some recent work at both field and glasshouse has

questioned this assumption (Dang et al. 2008; Tavakkoli et al. 2010b; a).

The aims of this work were to first evaluate the genotypic variation for salinity tolerance

during the early vegetative stage among a variety of barley entries, and to investigate

possible physiological traits that could be used as screening criteria in selected

genotypes in a soil-based experiment and in the field. Plant Na+ and Cl- concentrations

were also compared given the importance of both ions in salinity.

Materials and Methods

Experiment 1: Hydroponic screening

Sixty genotypes of barley were screened for their tolerance to salinity (Table 1).The

genotypes were a selection of varieties and breeding lines that have been used in barley

breeding trials in South Australia and were representative of the range of genetic

material that has been grown in the region. The pedigrees of these genotypes are

diverse, coming from a range of genetic backgrounds. The experiment used a supported

hydroponic system (Genc et al. 2007). Plants were grown in cylindrical PVC tubes (4

6

cm diameter × 28 cm depth) filled with cylindrical black polycarbonate pellets

(approximately 2–4 mm long and 1–2 mm in diameter) in a series of 50 L tubs each of

which contained 42 PVC tubes. Two tubs were served by a single tank of 80 L nutrient

solution. Each tub was filled and drained with 25 L of nutrient solution every 30 min. A

modified Hoagland’s solution was used, the composition of which (in mM) was:

NH4NO3 (0.2); KNO3 (5); Ca(NO3)2 (2); MgSO4 (2); KH2PO4 (0.1); Na2SiO3 (0.5);

NaFe(III)–hydroxyethyl ethylenediamine triacetic acid (HEDTA) (0.05); H3BO3 (0.01);

MnCl2 (0.005); ZnSO4 (0.005); CuSO4 (0.0005); and Na2MoO3 (0.0001). Solutions

were changed every 7 days, at which time the pH was adjusted to 6.0. The experiment

was conducted in a temperature-controlled growth chamber with day/night temperatures

of approximately 23/19C. The intensity of photosynthetically active radiation was

measured using a LiCor quantum sensor meter (Model LI-1000, Li-Cor, Lincoln, NE,

USA) and varied from 380 to 410 mmol m-2 s-1.

Uniformly sized seeds of each genotype were surface sterilized in 70% ethanol for 1

minute, followed by soaking in 3% sodium hypochlorite for 5 minutes and three lots of

rinsing with deionized water. Seeds were germinated on filter paper in Petri dishes at

room temperature for 3 days. The seedlings were then transplanted into PVC tubes (one

seedling per tube) filled with cylindrical black polycarbonate pellets. A NaCl

concentration of 150 mM was used as the salinity stress treatment. This concentration

was selected on the basis of applied salt treatment in most of the current studies on

salinity tolerance of barley (Garthwaite et al. 2005; James et al. 2006; Huang et al.

2008; Britto et al. 2010; Munns et al. 2010; Shavrukov et al. 2010).

7

Table 1. The genotypes of barley used in the Experiment 1.

Var/line Origin Source/Reference Albecta - - Arivat USA (Aslam et al. 1984) Arta Syria (Muehlbauer et al. 2009) Arupo CIMMYT - Barque Australia (McDonald 2006) Barque 73 Australia - Baudin Australia - Beecher Australia (Rawson 1986) Briggs USA (Lynch and Lauchli 1985) Buloke Australia (Nuttall et al. 2010) California Mariout North Africa (Halperin et al. 1997) Capstan Australia - Chevron USA (Gorham et al. 1994) Cl-3576 North Africa - Clipper Australia (Tavakkoli et al. 2010b) Club Mariout North Africa (Richards et al. 1987) CM67 North Africa (Gorham et al. 1994) CM72 North Africa (Cramer et al. 1990) CPI 71284-48 Iran (Shavrukov et al. 2010) CPI 77146-32 Iran - Dhow Australia - Dobla Spain (Royo and Aragüés 1999) Egmont ICARDA (Flowers and Hajibagheri 2001) Er/Apm Syria (Othman et al. 2006) Flagship Australia - Fleet Australia (Ellis et al. 2002) Franklin Australia (James et al. 2006) Gairdner Australia (Tajbakhsh et al. 2006) Gerbel UK (Royo and Aragüés 1999) H. Spont 41.1 Syria - Halycon UK - Harmel Syria (Othman et al. 2006) Hindmarsh Australia - ICARDA 382 Syria - ICARDA 391 Syria - Kaputar CIMMYT - Keel Australia (Harris et al. 2010) Maritime CIMMYT (Browning et al. 2006) Mundah Australia (Harris et al. 2010) O2D/20 Australia - Parent 08 Syria - Parent 12 Syria - Parent 15 Syria - Parent 16 Syria - Parent19 Syria - Prato USA (Ramagopal 1987) Ratna USA (Nair and Khulbe 1990) Sahara North Africa (Tavakkoli et al. 2010b) Schooner Australia (James et al. 2006) Skiff Australia (Munns and James 2003) Sloop Australia (Jiang et al. 2006) Tadmor Syria (Muehlbauer et al. 2009) Vlamingh Australia - WI 2198 Australia - WI 3416 Australia - WI 3788 Australia - WI 4262 Australia - Yarra Australia - YU 6472 China (Tajbakhsh et al. 2006)

8

At 8–10 days after transplanting, when the third leaf was beginning to appear, the salt

treatment was imposed in increments of 25 mM NaCl per day, together with

supplemental calcium as CaCl2 until the final concentration of 150 mM NaCl was

achieved. Plants were harvested 42 days after transplanting. Fresh weight was measured

and plants dried at 80 °C for 72 hours and dry weights were recorded. The whole shoot

moisture content was calculated from the fresh and dry weights. The salt tolerance was

calculated as the percentage ratio of shoot dry matter production in salt treatment to

control.

The osmotic potential of leaf sap was measured. A disc of Whatman GF/B glass micro-

fibre paper was placed in the barrel of a 2 ml plastic syringe so that it covered the outlet

hole. A fresh leaf was then put in the barrel, the plunger was re-inserted, and the tip of

the syringe was sealed with Blu-Tack. The syringe was frozen in liquid nitrogen and,

still sealed, was thawed to ambient temperature. When temperature equilibration was

complete, the plunger and Blu-Tack were removed and the barrel of the syringe was

placed in a 15 ml centrifuge tube, with its tip resting inside a 1.5 ml Ependorff tube.

After centrifugation at 2500 g for 10 min at 40C, the osmolality of a 10 µl sample was

measured in a Vapro pressure osmometer.

The high performance liquid chromatography (HPLC) Dionex DX 500 system

consisting of an AS40 Autosampler, GP40 gradient pump, AD20 UV/Visible

absorbance detector, ED40 electrochemical detector and LC20 chromatography

enclosure was used to quantify levels of compatible solutes in plants. Immediately

following harvest, the leaf sap was extracted as described for osmotic potential

measurement. One mL of methanol:chloroform:water (60:25:15 by vol.) was added to

9

each sample and the samples were vortexed for 1 min before centrifugation for 10 min

at 10000 g at 40C. The supernatant was removed and the samples were freeze-dried. The

samples were resuspended in 200 µL of milliQ water prior to injection into the HPLC.

A mixture of standards (glycine betaine, sucrose, glucose, fructose, mannitol,

trigonelline and sorbitol), was prepared in methanol:water (50:50, v:v) at 0.5 µg µL-1 for

glycine betaine and 2.5 µg µL-1 for the remaining solutes. Ten µL of the standard

solution was injected into the HPLC while running each batch of samples. The

contribution of organic and inorganic ions to leaf osmotic potential was determined

using the van’t Hoff equation, where the calculated contribution of individual solutes to

measured Ψs, was based on solute concentration on a molar basis (Marigo and Peltier

1996).

The dried shoots were digested in 40mL of 4% nitric acid (HNO3) at 950 C for 4 hours

in a 54-well HotBlock (Environmental Express, Mt Pleasant, SC, USA). The

concentration of Na+ and K+ in the digested samples was determined using a flame

photometer (Model 420, Sherwood, Cambridge, UK). Chloride concentrations of the

digested extracts were determined using a chloride analyser (Model 926, Sherwood

Scientific, Cambridge, UK). Plant standards (Australasian Soil and Plant Analysis

Council) were included in every batch of analysis and the recovery of Na+, Cl– and K+

from these were 95%, 91% and 92%, respectively.

Experiment 2. Responses in growth and ion concentration of 15 genotypes of

barley to different soil salinity levels

Based on the results of Experiment 1 as well as previous screening work for salt

tolerance (E. Tavakkoli, unpublished data; S. Coventry, personal communication) a

10

subset of 15 barley genotypes showing different levels of ion exclusion and salt

tolerance was selected for further study in soil. The15 barley genotypes were Fleet,

Flagship, Buloke, Hindmarsh, WI4263, Schooner, Parent 19, Gairdner, ODZ/20, Yara,

Sloop, Maritime, Capstan, Keel and Baudin.

The soil of the A horizon (topsoil) of a sandy loam red Chromosol (Isbell 1996) was

collected from Roseworthy (34.51 °S, 138.68 °E), South Australia. Following

collection, the soil was air-dried and ground to pass through a 5-mm sieve. A soil–water

characteristic curve was determined using the pressure plate method (Klute 1986) and

the soil moisture content at field capacity (–10 kPa, equivalent to 37% w/w) was

estimated. Basal fertiliser was thoroughly mixed through the soil at the following

concentrations (in mg pot-1): NH4NO3 (380), KH2PO4 (229), CaCl2 (131), MgCl2 (332),

CuCl2 (10.7), ZnCl2 (11), Na2MoO4 (6.84) and H3BO3 (15). Two salt treatments:

moderately saline (ECe ~ 7.2 dS m-1) and highly saline (ECe ~ 15.3 dS m-1) and a

control treatment (ECe ~ 1.2 dS m-1) were compared in this experiment. The amounts of

NaCl required to achieve the nominal treatments were determined in an assay using 0 to

2000 mM NaCl and the actual soil. The saline soils were prepared by dissolving NaCl

salt in milliQ H2O and spraying the solution on a 2 cm layer of soil to reach field

capacity moisture content. Each soil was covered with plastic to control evaporation and

left for 3 days at 25°C to reach equilibrium, then mixed thoroughly and air-dried

(Tavakkoli et al. 2010a). Samples of the saline-synthesised soils were moistened to field

capacity (water potential at -10 kPa) and centrifuged at 4000 g for 30 minutes to extract

the soil solution which was passed through 0.25 µm filter paper. Electrical conductivity,

ΨO and ion concentrations of the solutions were measured.

11

The plants were grown in pots, 10.4 cm in diameter and 32 cm deep in which there were

two layers of soil; 2200 g of air dry soil (subsoil) which contains the salt treatment and

800 g of untreated soil above (topsoil). Each layer was packed to a bulk density of 1.35

Mg m-3. The subsoil and topsoil were separated by a 3 cm layer of plastic beads (120 g)

to prevent salt rising to the topsoil through capillarity action. The top 3 cm of the pot

was also covered by plastic beads to minimise the water evaporation from the soil

surface. A polypropylene tube (14 cm long, 2 cm internal diameter) was inserted into

the upper 10 cm zone of each pot for watering the subsoil and referred to here as a

subsoil watering tube. During the early vegetative phase (3 weeks), plants were watered

with reverse osmosis (RO) water from the top. As the roots established, watering was

done only through the subsoil tubing.

Uniformly sized seeds of each genotype were surface sterilized in 70% ethanol for 1

min, followed by soaking in 3% sodium hypochlorite for 5 min, then rinsed three times

with deionised water. Five barley seeds were sown in each pot and thinned to three per

pot after 5 days. The experiment was conducted under the same growth conditions as

described in experiment 1. The pots were weighed and watered to 90% (Week 1-4) and

65% (week 5-10) of field capacity regularly and daily water use calculated. Plants were

grown for 10 weeks after germination. Three harvests were taken at 30, 50 and 70 days

after germination, respectively. At each harvest, the whole shoot from seedlings was

sampled for measurements of biomass and ion concentration and the youngest fully

expanded leaf for osmotic potential and organic solutes as explained in experiment 1.

The experimental design was a factorial, completely randomised design comprised of 3

treatments 15 barley genotypes with three replicates, giving a total of 135 pots.

12

Experiment 3: Field study

A field trial was conducted to assess the genotypic variation among 13 barley genotypes

(selected from experiment 2) in response to salinity stress at Hart, South Australia

(latitude 33.75994oS and longitude 138.41326oE). The region has a Mediterranean-type

climate and received 404 mm of rainfall in 2009, compared to the long term average of

460 mm. The soil at Hart is a calcareous gradational clay loam, classified as Vertic,

Pedal, Hypercalcic Calcarosol (Isbell 1996) and is the most extensive soil of the region

(Hall et al. 2009). The topsoil is alkaline, non-saline and non-sodic but the subsoil is

strongly alkaline (pH ≥ 9) and the ECe and exchangeable Na+ percentage (ESP), soluble

Na+ and Cl- concentrations increased with depth (Fig 1).

A randomized, complete block design with four replications was used. The trial was

sown using a custom-built cone seeder at a depth of 30 mm. Basal fertiliser was applied

with the seed as 12 kg P/ha of triple superphosphate (N:P:K:S = 0:17:0:0). Granular

urea (46:0:0:0) was applied by hand immediately prior to sowing and as a post emergent

application. Sowing rate was adjusted based on individual seed weight and germination

percentage with the aim of establishing 180 plants m2. The plots were 6 rows x 20 m

with an inter-row spacing of 225 mm. Weeds and disease, when present, were

controlled by a range of herbicides and fungicides.

At Zadoks growth stages (ZGS) 45, 65 and 92, five randomly-selected plants from each

plot were sampled. The plants were washed and separated into the upper and lower

leaves of the main stem for dry weight measurements, ionic analysis, leaf osmotic

potential and organic solutes as explained in experiment 1. At ZGS65, ten soil cores

were randomly taken from a soil depth of 0–100 cm.

13

Figure 1. The selected physical and chemical characteristics of soil at Hart site. All the

analyses were made on soil solution extracted from saturated paste extract. Soil samples

were taken on July 2009. The bars are standard errors of the means (n=10).

Electrical conductivity (ECe), pH, soluble Na+, Ca2+ and Mg2+ were determined in a

saturated paste extract. ESP was calculated from the values of soluble Na+, Ca2+ and

Mg2+ according to Rowell (1994). Chloride concentration was determined using a

chloride analyser (Model 926, Sherwood Scientific, Cambridge, UK). The plots were

machine harvested using a Wintersteiger plot harvester to determine grain yield.

pH

7 8 9 10D

epth

(cm

)

0

20

40

60

80

100

120

Na+ (mg/kg)

0 300 600 900

Dep

th (c

m)

0

20

40

60

80

100

120

Cl- (mg/kg)

0 400 800 1200

ECe (dS/m)

1 2 3 4 5 6 7 8

ESP (%)

0 10 20 30 40 50

K+ (mg/kg)

0 200 400 600

14

Statistical analysis

Statistical analyses were performed in R 2.10.1 (R Development Core Team 2006).

Data for growth, ion content and moisture content were analysed using two-way

ANOVA to determine if significant differences were present among means. Variances

were checked by plotting residual vs. fitted values to confirm the homogeneity of the

data. Differences among the mean values were assessed by Least Significant

Differences (LSD). Relationships between individual variables were examined using

simple linear correlations and regressions which were performed using SigmaPlot

(version 10). Spearman’s rank correlation test (rs) was used to examine consistency in

the rankings of genotypes for salt tolerance and grain yield production between the three

experiments. The heretiability of salt-tolerant traits were estimated by using of the

residual maximum likelihood (REML) statistical method to obtain unbiased estimates of

the variance components 2g and 2c, and the best linear unbiased predictions (BLUPs)

of the performance of the 60 genotypes in the first experiment and 15 genotypes in the

second experiment. Broad sense heritability was estimated as h2 = 2 g / (2 g + 2 c).

The significance of genetic variability among genotypes was assessed from the standard

error of the estimate of genetic variance 2 g, assuming the ratio 2 g /SE (2 g) to be

normally distributed.

Results

Hydroponics

Large genotypic variation in salt tolerance was evident (Fig 2) and it ranged from 39%

in CPI77146-32 to 95% in Halycon and Cl-3576 (Fig 2). The Na+ concentration in the

whole shoot varied over 3.5-fold among the 60 genotypes (Fig 3a), ranging from 862

mmol kg-1 DW in Skiff to 2818 mmol kg-1 DW in CPI71284-48. There was also more

15

than a 2.8-fold variation in the concentrations of Cl-, ranging from 759 mmol kg-1 DW

in Chevron to 2162 mmol kg-1 DW in CPI71284-48 (Fig 3b). The salinity tolerance of

barley genotypes was not associated with their ability to exclude Na+ and/or Cl-. The

observed variations among the genotypes in K+ concentrations were unrelated to salinity

tolerance (Fig. 3c) and the Na+ concentration. The concentrations of Na+ and Cl- were

significantly related (P < 0.01), with Cl- concentrations being lower than Na+

concentrations except in two genotypes (Parent 19 and Tadmor) which had similar

concentrations of Na+ and Cl- (Fig 3d, Table 1S).

Pot experiment

A significant linear relationship was found between biomass production of 15 genotypes

under salinity and under non-saline conditions at all three harvests, however, the range

of variation and ranking of genotypes varied significantly (Fig 4). The largest variation

in salt tolerance was found in the third harvest and it varied from 56% in Flagship and

Schooner to 89% in WI4262 at ECe 7.2, and 43% in Schooner to 84% in WI4262 at

ECe 15.3 respectively (Table 2S).

There was up to 2-fold variation in shoot Na+ and a 1.7-fold variation in shoot Cl-

concentrations among the 15 genotypes. Shoot Na+ and Cl- concentrations under saline

conditions were significantly and negatively correlated with the salt tolerance only at

the third harvest at ECe 7.2 and at the second harvest at ECe 15.3 dS m-1 (Fig. 5 and 6,

P ≤ 0.001). WI4262, Hindmarsh and Capstan which were the most tolerant varieties

also had the lowest leaf Na+ and Cl- concentrations. Shoot Na+ and Cl- concentrations

were low in the control treatment and did not show any relationships either with the

shoot biomass ratio or actual shoot biomass under control (data not shown). Leaf

16

osmotic potential varied significantly among genotypes and it was significantly related

to salt tolerance only at the second harvest at ECe 7.2, albeit weakly, and more stongly

at the second and third harvest at ECe 15.3 (Fig. 7, P ≤ 0.001). Shoot K+ concentration

and K+:Na+ ratios at the third harvest under ECe 7.1 were significantly related to the salt

tolerance (r = 0.70, P<0.05) whereas it was not related to salt tolerance at ECe 15.1

(data not shown). The heritability of salt tolerance (relative shoot biomass) for the three

harvests ranged from 0.36 to 0.66 and there was a trend of increase in these values with

increasing age of the plants sampled.

Field study

Genotypic variation in ion concentration and leaf osmotic potential in relation to grain

yield

There was a wide range in plant grain yield and Na+ and Cl- concentrations among the

13 genotypes (Fig 8). Grain yield production ranged from 3320 kg ha-1 in Maritime to

5538 kg ha-1 in Capstan. Significant genotypic variation occurred in Na+ and Cl-

concentrations as well as leaf osmotic potential in the flag leaf blade (Fig 8). Sodium

concentrations varied widely, ranging from 345 to 556 mmol kg-1 DW. As well Cl-

concentration varied about 1.5-fold ranging from 415 to 670 mmol kg-1 DW. As in the

experiment 2, leaf osmotic potential varied significantly ranging from -1.2 to -1.65

MPa. Leaf Na+ and Cl- concentrations and osmotic potential were negatively related to

the grain yield (Fig. 8, Table 3S, P ≤ 0.001).

17

SEM

Salt tolerance (%)

0 10 20 30 40 50 60 70 80 90 100

Cl-3576 Halycon

Parent19Gerbel

KaputarSloop

Parent-08 California Mariout

BeecherParent12

EgmontCapstan

SkiffSchooner

WI2198 Barque

CPI71284-48ICARDA-382

CM67 CM72 Arupo

ClipperEr/Apm Mundah

ArtaSahara

ICARDA-391Club Mariout

Ratna Barque73

WI3416Maritime

YU6472BaudinHarmel

Proto Parent15

FranklineVlamingh

AlbectaHindmarsh

ArivatDhowDobla

WI4262FlagshipWI3788

Chevron Gairdner

KeelParent16

O 2D/20H.Spont 41.1

FleetBriggsYarra

BulokeTadmor

CPI77146-32

Figure 2. The variations in salinity tolerance of 60 genotypes of barley grown in

supported hydroponic system for 7 weeks. The salt tolerance was calculated as the ratio

of dry matter production in salt treatment (150 mM NaCl) to control condition. The

coefficient of variation of experiment was 4.15%. The horizontal bar indicates the

standard error of the means (n=4).

18

Figure 3. The relationship between whole plant salt tolerance and shoot concentration

of (a) Na+ (mmol kg-1 DW), (b) Cl- (mmol kg-1 DW), (c) K+ (mmol kg-1 DW) and (d)

relationship between shoot Na+ and Cl- concentration of 60 barley genotypes grown at

150 mM NaCl for 7 weeks in a supported hydroponic system. The open circle is a

genotype of the wild barley (Hordeum spontaneum) and the closed circles are

domesticated genotypes of barley (Hordeum vulgare). Values are means (n=4).

(a)

Na+ concentration (mmol kg-1 DW)

500 1000 1500 2000 2500 3000

Salt

tole

ranc

e (%

)

0

20

40

60

80

100(b)

Cl- concentration (mmol kg-1 DW)

500 1000 1500 2000 2500 3000

Salt

tole

ranc

e (%

)

0

20

40

60

80

100

(c)

K+ concentration (mmol kg-1 DW)

0 500 1000 1500 2000 2500 3000

Salt

tole

ranc

e (%

)

0

20

40

60

80

100(d)

Na+ concentration (mmol kg-1 DW)

500 1000 1500 2000 2500 3000

Cl- c

once

ntra

tion

(mm

ol k

g-1 D

W)

500

1000

1500

2000

2500

3000

r = 0.70

y=x

19

Figure 4. The relationship between shoot dry matter of 15 genotypes of barley under

non-saline stress and two levels of soil salinity a) 7.2 and b) 15.1 dS m-1 at three harvest

points. Values are means (n=3).

Contribution of organic and inorganic solutes to leaf osmotic potential under salt

stress

Analysis of the sap of leaf tissue showed that the concentrations of sucrose, glucose,

fructose, betaine and proline increased markedly in response to salinity treatments. The

contribution of organic and inorganic solutes to leaf osmotic potential (Ψs) was assessed

for all genotypes but just the results from three tolerant (WI4262, Capstan and Fleet)

and three sensitive (Flagship, Schooner and Maritime) varieties identified in experiment

2 are reported here (Table 2). In hydroponic experiment, inorganic solutes accounted for

88-95% of the measured total solute potential in all genotypes whereas organic ion

contribution to measured Ψs was only 3-8%. In contrast, the contribution of organic

solutes to leaf Ψs in both ECe levels of experiment 2 and in the field was significantly

(a)ECe 7.2

Shoot dry matter under control condition (g)

0 2 4 6 8 10 12 14 16 18

Shoo

t dry

mat

ter

unde

r sa

linity

stre

ss (g

)

0

2

4

6

8

10

12

14

16

18

1st harvest r = 0.932nd harvest r = 0.743rd harvest r = 0.74

(b)ECe 15.3

Shoot dry matter under control condition (g)

0 2 4 6 8 10 12 14 16 18

1st harvest r = 0.752nd harvest r = 0.653rd harvest r = 0.69

y=x y=x

20

higher and ranged from 4-40%. The ranges in the contribution from organic osmolytes

in Experiment 2 and the field experiment were similar. Three tolerant and high-yielding

varieties Capstan, WI4262 and Fleet which had a better ability to maintain lower leaf

Na+ and Cl- concentration also exhibited a very strong capacity for accumulation of

inorganic solute to contribute to total Ψs. However, in Flagship, Schooner and

Maritime, the major contribution to Ψs was from the high concentrations of Na+ and Cl-

(Table 2).

First harvest

Salt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.22 (n.s)

Second harvest r = -0.40 (n.s)

Third harvest

r = -0.75

(a)ECe 7.2

(b)ECe 7.2

(c)ECe 7.2LSD0.05

First harvest

Na+ concentration (mmol kg-1 DW)

150 300 450 600 750

Salt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.27 (n.s)Second harvest

Na+ concentration (mmol kg-1 DW)

150 300 450 600 750

r = -0.78Third harvest

Na+ concentration (mmol kg-1 DW)

150 300 450 600 750

r = -0.29 (n.s)

(d)ECe 15.3

(e)ECe 15.3LSD0.05

(f)ECe 15.3

Figure 5. The relationship between whole plant salt tolerance and shoot Na+

concentrations of 15 genotypes of barley at three different growth stages under two

levels (7.2 and 15.3 dS m-1) of soil salinity. Values are means (n=3).

21

First harvestSalt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.29 (n.s)Second harvest

r = -0.39 (n.s)Third harvest

r = -0.77

First harvest

Cl- concentration (mmol kg-1 DW)

150 300 450 600 750

Salt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.29 (n.s)Second harvest

Cl- concentration (mmol kg-1 DW)

150 300 450 600 750

r = -0.79Third harvest

Cl- concentration (mmol kg-1 DW)

150 300 450 600 750

r = -0.28 (n.s)

(d)ECe 15.3

(e)ECe 15.3LSD0.05

(f)ECe 15.3

(a)ECe 7.2

(b)ECe 7.2

(c)ECe 7.2LSD0.05

Figure 6. The relationship between whole plant salt tolerance and shoot Cl-

concentrations of 15 genotypes of barley at three different growth stages under two

levels (7.2 and 15.3 dS m-1) of soil salinity. Values are means (n=3).

Consistencies and discrepancies between the three systems

Despite the imposition of stress to approximately the same degree, based on the EC of

the respective solutions in hydroponics and soil culture, the genotypic variation in salt

tolerance was much greater in soil as evidenced by the much greater salt×genotype

interaction and increased genotypic variation in response to stress. Screening in the pot

experiment identified three salt-tolerant genotypes (Capstan, Fleet and WI4262) which

were also identified as high yielding genotypes at a saline site in the field. However,

there was also a large discrepancy between hydroponic-based ranking of seedlings and

soil-culture-based ranking of seedling when salt tolerance was expressed as relative

growth. For example the cultivars Fleet and WI4262 were two of the sensitive

22

genotypes in hydroponics, but were overall the most tolerant and high yielding varieties

in soil and in the field.

First harvestSalt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.09 (n.s)Second harvest

r = -0.55Third harvest

r = -0.40 (n.s)

First harvest

Leaf osmotic potential (-MPa)

0.3 0.6 0.9 1.2 1.5 1.8 2.1

Salt

tole

ranc

e (%

)

20

40

60

80

100

r = 0.11 (n.s)Second harvest

Leaf osmotic potential (-MPa)

0.3 0.6 0.9 1.2 1.5 1.8 2.1

r = -0.85Third harvest

Leaf osmotic potential (-MPa)

0.3 0.6 0.9 1.2 1.5 1.8 2.1

r = -0.90

(a)ECe 7.2

(b)ECe 7.2LSD0.05

(c)ECe 7.2

(d)ECe 15.3

(e)ECe 15.3LSD0.05

(f)ECe 15.3LSD0.05

Figure 7. The relationship between whole plant salt tolerance and leaf osmotic potential

of 15 genotypes of barley at three different growth stages under two levels (7.2 and 15.3

dS m-1) of soil salinity. Values are means (n=3).

To quantify further the relation between salt tolerance of seedlings in hydroponics and

plants in pot screening with grain yield production in the field the phenotypic

correlation of genotypic means among the three techniques were examined (Table 3).

Shoot Na+, Cl- concentrations and leaf osmotic potential of plants grown in the field and

soil-culture were significantly correlated to grain yield production and salt tolerance

(Table 3). In contrast there were no significant correlations between those traits from

plants grown in hydroponics and grown in soil-culture or in the field. There was no

23

significant correlation between shoot K+ concentration and grain yield of plants grown

in the field (Table 3). The ranking of genotypes for their salt tolerance was evaluated by

Spearman’s rank test (Table 4). The ranking of 13 genotypes on the basis of grain yield

production in the field was significantly correlated with their ranking on the basis of

their salt tolerance at two different ECe levels in pot experiment. In contrast, the ranking

of the 13 genotypes in hydroponic experiment differed completely from soil-culture and

field screening (Table 4). There was little difference between the pot and field

experiments and selection of the tolerance and sensitive genotypes gave also an

excellent agreement between the two experiments.

Discussion

A critical aspect of improving salt tolerance of crop plants is identifying intraspecific

differences in capacity for growth under salt stress. A number of different physiological

traits contribute to the variation in salt tolerance and some screening procedures will be

more effective than others in identifying phenotypic responses based on specific

mechanisms. Selecting the most effective procedure to examine genetic variation in salt

tolerance based on specific physiological factors, and which predicts the differences in

salt tolerance in the field, is often overlooked, yet arguably this is the crucial step in

developing robust screening methods for salt tolerance. Several authors (Epstein et al.

1980; Greenway and Munns 1980; Flowers and Yeo 1986) have reviewed the

knowledge of the physiology of salt tolerance. In all cases many mechanisms are

implicated. Further, the relative importance of different mechanisms can vary between

closely related species (Rush and Epstein 1981) and varieties (Yeo and Flowers 1983)

and also with severity of salinity stress (Tavakkoli et al. 2010b). The study reported

here addressed three important questions for use of physiological mechanisms as a

24

selection criterion for improving salt tolerance: 1) Is there genetic variation in the

efficiency of the mechanism? 2) Is the targeted mechanism important in affecting the

whole plant tolerance? and 3) Is the screening method used for selecting a variety with a

mechanism of salt tolerance under controlled conditions, useful in predicting grain yield

in the field?

Screening in hydroponics failed to predict differences in salt tolerance and ion uptake in

soil, whether under controlled conditions or in the field. Despite the emphasis that has

been placed on Na+ and/or Cl- exclusion as a selection criterion for salt tolerance

(Munns et al. 2006), no relationship was observed between the level of exclusion and

salt tolerance in the genotypes used in hydroponic study based on early growth (Fig 3).

The different rankings in salt tolerance and in the relationships between ion

concentration and salt tolerance between soil and hydroponics suggest there are

fundamental differences in the responses to salinity between plants grown in the two

systems (Tavakkoli et al. 2010b). This has important implications for the development

of salt tolerant germplasm and for elucidating the relative importance of the

mechanisms of salt tolerance in the field.

Genetic differences in Na+ and Cl- exclusion among barley genotypes were not

associated with salt tolerance in hydroponics (Fig. 3). A similar result has been found

for wheat (Genc et al. 2007), which brings into question the rationale for using

hydroponic screening, at least at the salt concentrations commonly used in the much of

the current work (100-150 mM NaCl). In the pot experiment and in the field, however,

genetic differences in Na+ and Cl- exclusion and its association with plant growth were

25

(a)

Na+ concentration (mmol kg-1 DW)

300 400 500 600 700

Gra

in y

ield

(kg

ha-1

)

3000

3500

4000

4500

5000

5500

6000

r = -0.82

(b)

Cl- concentration (mmol kg-1 DW)

300 400 500 600 700

r = -0.93

(c)

Leaf osmotic potential (-MPa)

1.2 1.4 1.6 1.8

r = -0.78

Figure 8. The relationship between grain yield and leaf concentration of (a) Na+ concentration (mmol kg-1 DW), (b) Cl- concentration

(mmol kg-1 DW), and (c) leaf s (-MPa) of 13 barley genotypes grown at Hart site in 2009. The results are from youngest emerged leaves

at ZGS 65. Fitted curves are derived from linear regression. The vertical and horizontal bars are LSD at 95%. Values are averages (n=4).

26

Table 2. Estimated contribution of organic and inorganic ions to leaf osmotic potential (s). The contribution of individual solutes to measured s was determined using the van’t Hoff equation, where the calculated Ψs, was based on solute concentration on a fresh weight basis. The percentage value is based on the measured value of leaf s.

Leaf OP Na+ Cl- K+ Sucrose Glucose Fructose Betaine Proline Inorganic contribution Organic contribution Total

Hydroponic experiment

WI4262 -2.88±0.23 -0.81 -0.88 -0.85 -0.02 -0.01 -0.008 -0.01 -0.05 88 3 92

Capstan -2.65±0.21 -0.78 -0.81 -0.75 -0.03 -0.05 -0.01 -0.02 -0.06 88 6 95

Fleet -2.85±0.20 -0.85 -0.7 -0.81 -0.05 -0.03 -0.04 -0.008 -0.11 83 8 91

Flagship -2.87±0.21 -0.86 -0.85 -0.71 -0.05 -0.06 -0.04 -0.02 -0.05 84 8 92

Schooner -2.57±0.25 -0.83 -0.85 -0.75 -0.02 -0.02 -0.01 -0.005 -0.04 95 4 98

Maritime -2.61±0.26 -0.85 -0.79 -0.75 -0.05 -0.02 -0.01 -0.008 -0.05 92 5 97

Pot experiment EC 7.2 Harvest 3

WI4263 -1.28±0.03 -0.18 -0.22 -0.38 -0.09 -0.06 -0.09 -0.11 -0.03 61 30 91

Capstan -1.38±0.05 -0.15 -0.19 -0.44 -0.11 -0.09 -0.08 -0.12 -0.05 57 33 89

Fleet -1.21±0.04 -0.16 -0.21 -0.40 -0.12 -0.10 -0.11 -0.05 -0.02 64 33 97

Flagship -1.59±0.06 -0.35 -0.42 -0.31 -0.08 -0.05 -0.04 -0.01 -0.06 68 15 83

Schooner -1.58±0.08 -0.48 -0.50 -0.31 -0.02 -0.05 -0.02 -0.02 -0.02 82 8 90

Maritime -1.66±0.09 -0.52 -0.65 -0.32 -0.05 -0.02 -0.01 -0.002 -0.01 90 6 95

Pot experiment EC 15.3 Harvest 3

WI4263 -1.32±0.03 -0.18 -0.31 -0.29 -0.07 -0.09 -0.11 -0.15 -0.05 59 36 95

Capstan -1.65±0.05 -0.21 -0.29 -0.35 -0.11 -0.09 -0.10 -0.18 -0.11 52 36 87

Fleet -1.29±0.06 -0.21 -0.28 -0.25 -0.15 -0.08 -0.09 -0.15 -0.05 57 40 98

Flagship -2.25±0.08 -0.49 -0.59 -0.35 -0.11 -0.13 -0.15 -0.01 -0.02 64 19 82

Schooner -1.88±0.09 -0.48 -0.51 -0.45 -0.05 -0.08 -0.05 -0.04 -0.05 77 14 91

Maritime -2.11±0.08 -0.60 -0.71 -0.35 -0.04 -0.05 -0.04 -0.05 -0.05 79 11 90

Field experiment

WI4263 -1.15±0.05 -0.25 -0.21 -0.28 -0.05 -0.08 -0.09 -0.05 -0.09 64 31 96

Capstan -1.21±0.09 -0.22 -0.25 -0.31 -0.08 -0.05 -0.05 -0.02 -0.11 64 26 90

Fleet -1.18±0.11 -0.15 -0.23 -0.25 -0.11 -0.07 -0.07 -0.05 -0.12 53 36 89

Flagship -1.45±0.10 -0.33 -0.4 -0.25 -0.05 -0.08 -0.05 -0.07 -0.08 68 23 90

Schooner -1.62±0.15 -0.45 -0.51 -0.25 -0.02 -0.05 -0.05 -0.09 -0.08 75 18 93

Maritime -1.65±0.13 -0.49 -0.55 -0.22 -0.05 -0.08 -0.05 -0.08 -0.05 76 19 95

27

Table 3. Correlation coefficients (r) between pairs of physiological attributes of salt stressed barley plants grown in different cultures. *, **, *** = significant at 0.05, 0.01, and 0.001 levels, respectively. (n = 13).

Field Field Field Field Field Field Pot ECe 7.2 Pot ECe 7.2 Pot ECe 7.2 Pot ECe 7.2 Pot ECe 7.2 Pot ECe 7.2

Grain yield Leaf OP Na+ Cl- K+ K+:Na+ ST (Harvest 3) Na+ Cl- K+ K+:Na+ Leaf OP

Field Gain yield

Field Leaf OP -0.78**

Field Na+ -0.82** 0.74**

Field Cl- -0.93*** 0.82** 0.84**

Field K+ -0.31 0.15 0.11 0.34

Field K+:Na+ 0.75** -0.73** -0.94*** -0.74** 0.20 Pot ECe 7.2 (Harvest 3) ST 0.78** -0.88** -0.71** -0.86** -0.02 0.74**

Pot ECe 7.2 Na+ -0.77** 0.88** 0.78** 0.85** 0.13 -0.77** -0.91***

Pot ECe 7.2 Cl- -0.87** 0.89** 0.80** 0.91*** 0.29 -0.74** -0.89** 0.94***

Pot ECe 7.2 K+ 0.55* -0.70** -0.57* -0.67** -0.07 0.62* 0.72** -0.78** -0.76**

Pot ECe 7.2 K+:Na+ 0.71** -0.87** -0.72** -0.80** -0.06 0.75** 0.90*** -0.94*** -0.91*** 0.93***

Pot ECe 7.2 Leaf OP -0.75** 0.80** 0.63* 0.79** 0.37 -0.54* -0.74** 0.80** 0.77** -0.73** -0.80**

Pot ECe 15.3 ST 0.76** -0.83** -0.66* -0.74** -0.06 0.66* 0.90*** -0.88** -0.85** 0.58* 0.82** -0.75**

Pot ECe 15.3 Na+ -0.37 0.46 0.69** 0.52* -0.05 -0.69** -0.43 0.55* 0.43 -0.66* -0.60 0.54

Pot ECe 15.3 Cl- -0.35 0.29 0.36 0.37 0.20 -0.32 -0.22 0.46 0.47 -0.71** -0.56 0.55

Pot ECe 15.3 K+ 0.18 -0.31 -0.16 -0.06 -0.37 0.07 -0.12 0.02 -0.17 0.01 0.00 -0.06

Pot ECe 15.3 K+:Na+ 0.46 -0.57 -0.72 -0.52* -0.20 0.65* 0.32 -0.50 -0.51 0.63* 0.56 -0.56

Pot ECe 15.3 Leaf OP -0.63* 0.83** 0.59* 0.69** 0.21 0.23 0.08 -0.14 -0.05 0.10 -0.79** 0.77**

Hydroponic ST -0.12 0.11 -0.08 -0.01 0.39 0.23 0.08 -0.14 -0.05 0.10 0.10 0.35

Hydroponic Na+ 0.30 -0.25 -0.23 -0.19 -0.29 0.09 0.08 -0.03 0.00 -0.22 -0.08 -0.41

Hydroponic Cl- -0.01 0.08 0.07 0.00 -0.35 -0.22 -0.06 0.28 0.14 -0.26 -0.25 0.08

Hydroponic K+ 0.10 0.27 -0.02 0.10 -0.18 -0.05 -0.20 0.25 0.21 -0.25 -0.25 -0.02

Hydroponic K+:Na+ -0.38 0.53* 0.33 0.36 0.20 -0.24 -0.32 0.25 0.22 0.02 -0.15 0.47

Hydroponic Leaf OP -0.10 0.20 0.11 0.03 0.32 -0.07 0.09 -0.09 0.08 -0.23 -0.09 0.11

28

Table 4. The Spearmans’s rank correlations between grain yield of field-grown plants and salt tolerance of plants grown in the hydroponic and pot (n=13).

Field Pot ECe 7.2 Pot ECe 15.3

Grain yield Salt tolerance Salt tolerance

Field Grain yield

POT EC 7.2 Salt tolerance 0.79**

POT EC 15.3 Salt tolerance 0.82** 0.81**

Hydroponic Salt tolerance -0.22 0.11 -0.12

expressed, although the value of this to salt tolerance appeared to depend on the severity

of the salt stress (Figs 5 and 6). However, just as important was the observation that the

rankings in Na+ and Cl- exclusion in hydroponics were unrelated to the rankings found

in soil and in the field. Genetic differences in Na+ exclusion have been previously

demonstrated in hydroponic studies (Schachtman et al. 1991; Houshmand et al. 2005;

Munns et al. 2006; Genc et al. 2007), but the different results for selected barley

genotypes suggest that the level of discrimination is much lower in hydroponics than in

soil (Tavakkoli et al. 2010b; Rivandi et al. 2011), although the cause of this difference

between the two systems is not yet understood. Drew and Lauchli (1985) showed an

oxygen-dependent exclusion of Na+ ion from shoots by roots of maize. Under fully

aerobic conditions, roots partially excluded Na+ from the shoots over a wide range of

NaCl concentration (0.2-200 mM). With root anoxia, the exclusion mechanism broke

down so that much greater amounts of Na+ reached the shoots, with simultaneous

inhibition of K+ transport. While the supported hydroponic system used in this study

and by many other researchers (Munns et al. 2002; Genc et al. 2007) was filled and

drained with 25 L of nutrient solution every 30 min to provide aeration, the quantity of

oxygen at the root surface may not be sufficient for an efficient Na+ exclusion.

Moreover, for soil-grown plants, the salt concentration in the soil solution may not only

29

change due to mass flow exceeding uptake, but also as a result of decreasing water

content in the vicinity of the roots due to high transpirational demand and low

unsaturated hydraulic conductivity. This does not occur in solution culture, where the

matric potential is zero as is resistance to water movement (Vetterlein et al. 2004)

Maintenance of high K+ concentrations in salt-tolerant genotypes was observed only

among plants grown in soil at ECe 7.2 dS m-1 in experiment 2, which may be one of the

mechanisms underlying their higher salt tolerance (Table 3). However, for plants grown

in the hydroponics, soil ECe ~ 15.3 dS m-1 and in the field there was no significant

relationship between salt tolerance and/or grain yield and the shoot concentrations of

K+. The ratio of K+:Na+ has been associated with salt tolerance (Gorham et al. 1990;

Dvořak et al. 1994; Chen et al. 2007), however, in the current study, the significant

positive correlation between grain yield and K+:Na+ and negative correlation with Na+

and no significant relationship with K+ indicates that it is the genotypic variation in

shoot Na+ concentration that is driving the correlation rather than maintenance of high

shoot K+ concentration.

In soil-grown plants measuring the biomass production at 70 days (Harvest 3) after

germination has provided an accurate screen for tolerance of the relative biomass

production under saline conditions, and has revealed substantial variation among

genotypes at both levels of salinity stress which was also predictive of grain yield

production in the field (Table 3). However, there was no significant correlation between

salt tolerance of 15 genotypes after 70 days (Harvest 3) and their salt tolerance at earlier

growth stages. This finding confirmed the unsuitability of using an early assessment of

salinity tolerance at the seedling stage (Krishnamurthy et al. 2007). Tolerance to salinity

30

is obviously necessary at the whole plant level through the complete life cycle in grain-

producing species. While determination of salt tolerance in saline conditions presents

simple and useful parameters, the differences in the levels of salt tolerance at the

seedling stage did not reflect enhanced salinity tolerance at the adult plant level.

Similarly, most investigators have been unable to demonstrate a relationship between

tolerance under laboratory high salt conditions and later growth stages across a range of

crops, particularly bread wheat (Kingsbury et al. 1984; Ashraf and McNeilly 1988),

durum wheat (Almansouri et al. 2001) and barley (Norlyn and Epstein 1982). It is

possible that the processes which control cell expansion during early stage and

subsequent growth are entirely different. Many crops, such as wheat and barley, are

capable of germinating at very high salt concentrations (over 300 mM NaCl), but the

emerging radicle cannot grow further at this level of salinity (Munns and James 2003).

Such tolerance among crops at early stage could be explained by the physicochemical

nature of the enlarging process during this developmental stage. Nevertheless while

using relative growth at early stage seems to be a convenient test for screening large

numbers of genotypes in a rapid manner, it must first be demonstrated that it is

correlated to tolerance during vegetative growth, flowering, and maturity if it is to be of

value (Maas 1986; Ashraf and Harris 2004). The heritability for salt tolerance, which

range from 0.36 to 0.66, show that genetic differences explain a major part of the

phenotypic differences. There may be scope to further improve the screening efficiency

for shoot biomass ratio and thereby the operational heritability values by sampling

larger numbers of plants at one time.

Salinized crop plants may be able to produce osmotically active organic substances,

which often accumulate in the cytoplasm to balance the vacuole solute potential.

31

Soluble sugars, proline, and betaines are some of the compatible organic solutes found

in glycophyte plants (Hasegawa et al. 2000; Ashraf and Harris 2004). In our study, salt

stress caused an increase in ions and organic solutes in all genotypes, but the more salt

tolerant varieties had significantly higher concentration of soluble sugars (glucose,

fructose and sucrose), glycine betaine and proline when grown in soil or in the field

(Table 2). Cram (1976), showed that of the various organic osmotica, sugars contribute

up to 50% of the total osmotic potential in glycophytes subject to saline conditions. Ion

accumulation in plants can also play a major role in osmotic adjustment to high

salinities. It would seem, however, from the relationships between ion accumulation and

measured leaf osmotic potential observed here for genotypes that the simple

accumulation of Na+ and Cl- alone can not account for the osmotic behaviour of these

varieties (Table 2). While the ability to restrict Na+ and Cl- accumulation could prevent

the development of an osmotic imbalance in some genotypes, the concentration of Na+

and Cl- accumulated when considered together with the reduction in shoot K+ would

seem to necessitate the synthesis of additional osmotically active solute in order to

prevent an osmotic imbalance with respect to the external salt in soil solution (Table 2).

However, the contribution of organic solutes to osmotic potential of hydroponic-grown

plants was not significant. The leaf osmotic potential of plants grown under hydroponic

system was significantly lower than those grown in soil which reflects the large

difference between the two cultures in terms of the rate of ion uptake by plants (Table

2). Much previous research has involved exposing plants suddenly to such high

concentrations of NaCl (>100 mM) that cause osmotic shock rather than osmotic stress,

which induces major trauma that rarely if ever occurs in nature. Although we tried to

overcome the trauma of osmotic shock by increasing the concentration of salt gradually

in several small steps over a few days rather than in one large step (see Materials and

32

Methods), even this cautious approach may have been too sudden to identify useful

genetic variation in salt tolerance. The important point is that it can take weeks for such

variation to become evident in soil and especially under field conditions, and soil-grown

plants will have more time to adapt to the salt concentration than plants in hydroponic

systems (Passioura 2010). This is of particular importance for an adaptation mechanism

such as osmotic adjustment, which requires uptake of ions and the formation of

compatible solutes which are absent in hydroponics (Vetterlein et al. 2004; Tavakkoli et

al. 2010b).

A lack of correlation between the hydroponics and other two methods (Table 3),

suggests that hydroponics is distinct and it cannot be substituted for methods using

soils. An important finding of this study was that the correlations with Na+ and Cl-

concentrations and salt tolerance in the pots (Fig 5 and 6 c, e) was strongest when the

plant Na+ and Cl- concentrations were close to those found in field study (Fig 8 a, b) and

this was related to the differences in the relationship between Na+, Cl- and salt tolerance

at the three different harvests. This also occurred at different harvests, depending on the

level of soil salinity at which plants were grown. Chloride toxicity has not been

considered to be a major factor in salt tolerance in cereal crops. However, to a large

extent, the different responses to elevated Na+ between hydroponics and soil were also

seen with Cl–. In both pot screening and field study, the concentrations of Cl– were

higher than those of Na+, but in hydroponics the Na+ concentration was generally higher

than Cl- (Fig 9). This difference between hydroponics and soil has been found

previously (Tavakkoli et al. 2010b; Tavakkoli et al. 2011).

33

There has been the hope that a better understanding of the physiological basis of salinity

tolerance will also result in the identification of critical genes for which breeders might

select, or new genetic resources that could be manipulated by the tools of molecular

biology, but decades of research by physiologists have not identified a single gene or

genetic resource that has been used by breeders to improve salinity tolerance. Research

has been very fruitful in identifying some characteristics that are important in

accounting for differences in salt tolerance between and within species (Rawson et al.

1988; Munns and Tester 2008). However, so far these have not been useful in breeding.

Within any given field, large fluctuations in salinity, drought and extremes of

temperature can occur. As a consequence, a large degree of heterogeneity between the

stress levels that impact different plants in the same field can be present. This

heterogeneity, in turn, can affect plant performance and yield. In addition to

heterogeneity in saline conditions in differing parts of a given field, the simultaneous

occurrence of different abiotic stresses should also be addressed. Abiotic stresses such

as salinity and drought, salinity and heat, and distinct combinations of drought and

temperature, or high light intensity are common to many agricultural areas and could

affect plant productivity. It was recently shown that the response of plants to a

combination of salinity and heat stress is unique and cannot be directly extrapolated

from the response of plants to salinity or heat stress applied individually (Keles and

Oncel 2002; Koussevitzky et al. 2008). Because different abiotic stresses are most

likely to occur simultaneously under field conditions, a greater attempt must be made to

mimic these conditions in laboratory studies.

34

Figure 9. The relationship between shoot Na+ and Cl- concentration of 13 genotypes of barley grown in a) hydroponic, b) pot and c) field. Values are averages (n = 3 or 4).

(c)Field

Na+ concentration (mmol kg-1 DW)

300 400 500 600 700

300

400

500

600

700

r = 0.84

(a)Hydroponic

Na+ concentration (mmol kg-1 DW)

900 1200 1500 1800 2100 2400

Cl- c

once

ntra

tion

(mm

ol k

g-1 D

W)

900

1200

1500

1800

2100

2400

r = 0.76

(b)Pot ECe 7.2

Na+ concentration (mmol kg-1 DW)

300 400 500 600 700

300

400

500

600

700

r = 0.94

y=xy=x y=x

35

The timing of the salinity stress event with respect to the developmental stage of the

plant should also be addressed. Although plants can differ in their sensitivity to various

abiotic stresses during different developmental stages including germination, vegetative

growth, reproductive cycle, and senescence, from a strictly agronomic point of view

there appears to be only one main consideration (Mittler and Blumwald 2010) : How

would this interaction between stress and development affect overall yield? Most crops

are highly sensitive to abiotic stresses during flowering, with devastating effects on

yield (Sanchez et al. 2002; Humphreys et al. 2006; Barnabas et al. 2008). Another key

difference between laboratory studies and field conditions is the intensity and duration

of the stress. In the field salinity conditions are generated gradually during a period of

several weeks and months and plants do not experience a sudden stress. Thus, artificial

soil mixtures containing a high content of peat moss, vermiculite, sand or high organic

matter and solution culture methods should be avoided because they cannot reproduce

natural soil drying conditions (Mittler and Blumwald 2010; Tavakkoli et al. 2010b).

Conditions of water deficiency similar to those occurring in the field can be mimicked

in the laboratory by growing plants under limited daily amounts of water rather than by

withholding water altogether (see materials and methods).

Conclusions

This study demonstrated that solution culture may not allow differences in salt tolerance

between genotypes to be discerned and the diverse genotypic variation found in

hydroponics did not correlate with pot and field experiments. The unsuitability of using

early assessment of salinity tolerance at seedling stage was demonstrated. The exclusion

of Na+ and Cl- significantly contributed to salt tolerance and grain yield production in

pot and field studies. This work has also established sound screening procedures

36

(Experiment 2) that correlated with field evaluation of grain yield of the barley varieties

at a moderately saline site. This study also shows that several processes are involved in

salt tolerance and that the relative importance of these traits may differ with the severity

of the salt stress. The osmotic stress was the predominant cause of reduced growth at

high levels of salinity, while specific-ion toxicity was more important under mild

salinity stress. The present study also suggests that salt exclusion coupled with a

synthesis of organic solutes are important components of salt tolerance in the tolerant

genotypes and further field tests of these plants under stress conditions will help to

verify their potential utility in crop-improvement programs.

Acknowledgements

This work was supported by a grant from the Grains Research and Development

Corporation to ET and by the University of Adelaide. We thank Mr. S. Coventry (The

University of Adelaide) for kindly supplying the seed for this study and excellent HPLC

guidance, Mr P. Hooper (manager of the Hart Field Site) for his support with the field

study and Dr G. Lyons, for useful comments on the manuscript.

37

References: Almansouri M, Kinet JM,Lutts S (2001). Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.). Plant and Soil 231(243-254), 547-550.

Ashraf M,Harris PJC (2004). Potential biochemical indicators of salinity tolerance in plants. Plant Science 166(1), 3-16.

Ashraf M,McNeilly T (1988). Variability in salt tolerance of nine spring wheat cultivars. J Agron Crop Sci 160, 14-21.

Aslam M, Huffaker RC,Rains WD (1984). Early effects of salinity on nitrate assimilation in barley seedlings. Plant Physiol 76, 321-325.

Barnabas B, Jager K,Feher A (2008). The effect of drought and heat stress on reproductive processes in cereals. Plant, Cell & Environment 31, 11-38.

Britto DT, Ebrahimi-Ardebili S, Hamam AM, Coskun D,Kronzucker HJ (2010). 42K analysis of sodium-induced potassium efflux in barley: mechanism and relevance to salt tolerance. New Phytologist 186(2), 373-384.

Browning LS, Bauder JW,Phelps SD (2006). Effect of Irrigation water salinity and sodicity and water table position on water table chemistry beneath Atriplex lentiformis and Hordeum marinum. Arid Land Research and Management 20(2), 101 - 115.

Chen Z, Pottosin I, Cuin TA, Fuglsang AT, Tester M, Jha D, Zepeda-Jazo I, Zhou M, Palmgren MG, Newman IA,Shabala S (2007). Root plasma membrane transporters controlling K+/Na+

homeostasis in salt-stressed barley. Plant Physiol. 145(4), 1714-1725.

Chen Z, Shabala S, Mendham N, Newman I, Zhang G,Zhou M (2008). Combining ability of salinity tolerance on the basis of NaCl-Induced K+ flux from roots of barley. Crop Sci 48(4), 1382-1388.

Cram WJ (1976). Negative feedback regulation of transport in cells. The maintenance of turgor, volume and nutrient supply. Encyclopaedia of Plant Physiology. U Luttge,MG Pitman. Berlin, Springer-Verlag. 2: 284-316.

Cramer GR, Epstein E,Läuchli A (1990). Effects of sodium, potassium and calcium on salt-stressed barley. I. Growth analysis. Physiol Plant 80, 83-88.

Dang YP, Dalal RC, Mayer DG, McDonald M, Routley R, Schwenke GD, Buck SR, Daniells IG, Singh DK, Manning W,Ferguson N (2008). High subsoil chloride concentrations reduce soil water extraction and crop yield on Vertisols in north-eastern Australia. Australian Journal of Agricultural Research 59, 321-330.

Drew MC,Lauchli A (1985). Oxygen-dependent exclusion of sodium ions from shoots by roots of Zea mays (cv Pioneer 3906) in relation to salinity damage. Plant Physiol. 79(1), 171-176.

Dvořak J, Noaman MM, Goyal S,Gorham J (1994). Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination. TAG Theoretical and Applied Genetics 87(7), 872-877.

El-hendawy SE, Hu Y,Schmidhalter U (2005). Growth, ion content, gas exchange, and water relations of wheat genotypes differing in salt tolerances. Australian Journal of Agricultural Research 56, 123-134.

Ellis RP, Forster BP, Gordon DC, Handley LL, Keith RP, Lawrence P, Meyer R, Powell W, Robinson D, Scrimgeour CM, Young G,Thomas WTB (2002). Phenotype/genotype associations

38

for yield and salt tolerance in a barley mapping population segregating for two dwarfing genes. Journal of Experimental Botany 53(371), 1163-1176.

Epstein E, Norlyn JD, Rush DW, Kingsbury RW, Kelley DB,Cunningham GA (1980). Saline culture of crops: a genetic approach. Science 210, 399-404.

Flowers TJ,Hajibagheri MA (2001). Salinity tolerance in Hordeum vulgare: ion concentrations in root cells of cultivars differing in salt tolerance**. Plant and soil 231(1), 1-9.

Flowers TJ,Yeo AR (1986). Ion relations of plants under drought and salinity. Aust. J. Plant Physiol. 13, 75-91.

Garthwaite AJ, von Bothmer R,Colmer TD (2005). Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl- into the shoots. J. Exp. Bot. 56(419), 2365-2378.

Genc Y, McDonald GK,Tester M (2007). Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant, Cell and Environment 30(11), 1486-1498.

Gorham J, Jones WRG,Bristol A (1990). Partial characterization of the trait for enhanced K+-Na+ discrimination in the D genome of wheat. Planta 180, 590-597.

Gorham J, Papa R,Aloy-Lleonart M (1994). Varietal differences in sodium uptake in barley cultivars exposed to soil salinity or salt spray. Journal of Experimental Botany 45(7), 895-901.

Greenway H,Munns R (1980). Mechanisms of salt tolerance in nonhalophytes. Ann. Rev. Plant Physiol. 31, 149-190.

Gregory PJ, Bengough AG, Grinev D, Schmidt S, Thomas WTB, Wojciechowski T,Young IM (2009). Root phenomics of crops: opportunities and challenges. Functional Plant Biology(36), 922-929.

Hall J, Maschmedt D,Billing B (2009). The soils of Southern South Australia Department of water, land and biodiversity conservation, Government of South Australia.

Halperin SJ, Kochian LV,Lynch JP (1997). Salinity stress inhibits calcium loading into the xylem of excised barley (Hordeum vulgare) roots. New Phytologist 135(3), 419-427.

Harris B, Sadras V,Tester M (2010). A water-centred framework to assess the effects of salinity on the growth and yield of wheat and barley. Plant and soil, 1-13.

Hasegawa PM, Bressan RA, Zhu J-K,Bohnert HJ (2000). Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51(1), 463-499.

Houshmand S, Arzani A, Maibody SAM,Feizi M (2005). Evaluation of salt-tolerant genotypes of durum wheat derived from in vitro and field experiments. Field Crops Research 91(2-3), 345-354.

Huang S, Spielmeyer W, Lagudah ES,Munns R (2008). Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance. Journal of Experimental Botany 59(4), 927-937.

Humphreys MRS, Cairns AJ, Turner LB,Humphreys J (2006). A changing climate for grassland research. New Phytologist 169, 9-26.

Isbell RF (1996). The Australian Soil Classification. Melbourne, CSIRO.

39

James RA, Caemmerer SV, Condon AGT, Zwart AB,Munns R (2008). Genetic variation in tolerance to the osmotic stress component of salinity stress in durum wheat. Functional Plant Biology 35(2), 111-123.

James RA, Munns R, Von Caemmerer S, Trejo C, Miller C,Condon AG (2006). Photosynthetic capacity is related to the cellular and subcellular partitioning of Na+, K+ and Cl- in salt-affected barley and durum wheat. Plant, Cell and Environment 29(12), 2185-2197.

Jiang Q, Roche D, Monaco TA,Durham S (2006). Gas exchange, chlorophyll fluorescence parameters and carbon isotope discrimination of 14 barley genetic lines in response to salinity. Field Crops Research 96(2-3), 269-278.

Keles Y,Oncel I (2002). Response of antioxidative defense system to temperature and water stress combinations in wheat seedlings. Plant Science 163, 783-790.

Kingsbury R,Epstein E (1986). Salt Sensitivity in Wheat. A case for specific ion toxicity. Plant Physiology 80, 651-654.

Kingsbury R, Epstein E,Pearcy RW (1984). Physiological responses to salinity in selected lines of wheat. Plant Physiology 74, 417-423.

Kinraide TB (1999). Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects. Journal of Experimental Botany 50(338), 1495-1505.

Klute A (1986). Water retention: Laboratory methods. Methods of soil analysis. Part 1. A Klute. Madison, WI, Agron. Monogr. 9. ASA and SSSA. 2nd: 635-662.

Koussevitzky S, Suzuki N, Huntington S, Armijo L,Sha W (2008). Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination. J. Biol. Chem 283, 34197-34203.

Krishnamurthy L, Serraj R, Hash C, Dakheel A,Reddy B (2007). Screening sorghum genotypes for salinity tolerant biomass production. Euphytica 156(1), 15-24.

Lynch J,Lauchli A (1985). Salt stress disturbs the calcium nutrition of Barley (Hordeum vulgare L.). New Phytologist 99(3), 345-354.

Maas EV (1986). Salt tolerance in plants. Appl. Agric. Res 1, 12-25.

Marigo G,Peltier JP (1996). Analysis of the diurnal change in osmotic potential in leaves of Fraxinus excelsior L. Journal of Experimental Botany 47, 763-769.

McDonald GK (2006). Effects of soil properties on variation in growth, grain yield and nutrient concentration of wheat and barley. Australian Journal of Experimental Agriculture 46, 93-105.

Mittler R,Blumwald E (2010). Genetic engineering for modern agriculture: challenges and perspectives. Annu. Rev. Plant Biol 61, 443-462.

Muehlbauer GJ, Feuillet C, Maccaferri M, Sanguineti MC, Giuliani S,Tuberosa R (2009). Genomics of tolerance to abiotic stress in the Triticeae. Genetics and Genomics of the Triticeae, Springer New York. 7: 481-558.

Munns R, Hare RA, James RA,Rebetzke GJ (2000). Genetic variation for improving the salt tolerance of durum wheat. Aust. J. Agric. Res. 51, 69-74.

40

Munns R, Husain S, Rivelli AR, James R, Condon AG, Lindsay M, Lagudah ES, Schachtman DP,Hare RA (2002). Avenues for increasing salt tolerance of crops, and the role of physiologically based selection traits. Plant and Soil 247, 93-105.

Munns R,James RA (2003). Screening methods for salinity tolerance: a case study with tetraploid wheat. Plant and soil 253, 201-218.

Munns R, James RA,Lauchli A (2006). Approaches to increasing the salt tolerance of wheat and other cereals. Journal of Experimental Botany 57(5), 1025-1043.

Munns R, James RA, Sirault XRR, Furbank RT,Jones HG (2010). New phenotyping methods for screening wheat and barley for beneficial responses to water deficit. Journal of Experimental Botany 61, 3499-3507.

Munns R,Tester M (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651-681.

Nair KPP,Khulbe NC (1990). Differential response of wheat and barley genotypes to substrate-induced salinity under North Indian conditions. Experimental Agriculture 26(02), 221-225.

Norlyn JD,Epstein E (1982). Barley production: irrigation with seawater on coastal soil. Biosaline research: a look to the future. A San Pietro. New York, Plenum: 525-529.

Nuttall JG, Hobson KB, Materne M, Moody DB, Munns R,Armstrong RD (2010). Use of genetic tolerance in grain crops to overcome subsoil constraints in alkaline cropping soils. Australian Journal of Soil Research 48, 188-199.

Othman Y, Al-Karaki G, Al-Tawaha AR,Al-Horani A (2006). Variation in germination and Ion uptake in barley genotypes under salinity conditions. World Journal of Agricultural Sciences 2, 11-15.

Passioura JB (2010). Scaling up: the essence of effective agricultural research Functional Plant Biology 37, 585-591.

Poustini K,Siosemardeh A (2004). Ion distribution in wheat cultivars in response to salinity stress. Field crops research 85, 125-133.

R Development Core Team (2006). A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.

Ramagopal S (1987). Salinity stress induced tissue-specific proteins in barley seedlings. Plant Physiol. 84(2), 324-331.

Rawson HM (1986). Gas exchange and growth in wheat and barley grown in salt. Australian Journal of Plant Physiology 13, 475-489.

Rawson HM, Richards RA,Munns R (1988). An examination of selection criteria for salt tolerance in wheat, barley and triticale genotypes. Australian Journal of Agricultural Research 39, 759-772.

Rengasamy P (2002). Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. Australian Journal of Experimental Agriculture 42, 351-361.

Richards RA (1983). Should selection for yield in saline regions be made on saline or non-saline soils? Euphytica 32(2), 431-438.

41

Richards RA, Dennett CW, Qualset CO, Epstein E, Norlyn JD,Winslow MD (1987). Variation in yield of grain and biomass in wheat, barley, and triticale in a salt-affected field. Field Crops Research 15(3-4), 277-287.

Rivandi J, Miyazaki J, Hrmova M, Pallotta M, Tester M,Collins NC (2011). A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait. Journal of Experimental Botany 62, 1201-1216.

Rowell D (1994). Soil science : methods and applications. New York, Wiley.

Royo A,Aragüés R (1999). Salinity-yield response functions of barley genotypes assessed with a triple line source sprinkler system. Plant and soil 209(1), 9-20.

Rush DW,Epstein E (1981). Comparative studies on the sodium, pottasium, and chloride relations of a wild halophytic and a domestic salt-sensitive tomato species. Plant Physiology 68, 1308-1313.

Sanchez AC, Subudhi PK, Rosenow DT,Nguyen HT (2002). Mapping QTLs associated with drought resistance in sorghum (Sorghum bicolor L. Moench). Plant Mol. Biol 48, 713-726.

Schachtman DP, Munns R,Whitecross MI (1991). Variation in sodium exclusion and salt tolerance in Triticum tauschii. Crop Sci 31(4), 992-997.

Shavrukov Y, Gupta N, Miyazaki J, Baho M, Chalmers K, Tester M, Langridge P,Collins N (2010). HvNax3—a locus controlling shoot sodium exclusion derived from wild barley (Hordeum vulgare ssp. spontaneum). Functional &amp; Integrative Genomics 10(2), 277-291.

Tajbakhsh M, Zhou MX, Chen ZH,Mendham NJ (2006). Physiological and cytological response of salt-tolerant and non-tolerant barley to salinity during germination and early growth. Australian Journal of Experimental Agriculture 46, 555-562.

Tavakkoli E, Fatehi F, Coventry S, Rengasamy P,Mcdonald GK (2011). Additive effects of Na+ and Cl- ions on barley growth under salinity stress. Journal of Experimental Botany 62, 2189-2203.

Tavakkoli E, Rengasamy P,Mcdonald GK (2010a). High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany 61(15), 4449–4459.

Tavakkoli E, Rengasamy P,Mcdonald GK (2010b). The response of barley to salinity stress differs between hydroponics and soil systems. Functional Plant Biology 37(6), 621-633.

Vetterlein D, Kuhn K, Schubert S,Jahn R (2004). Consequences of sodium exclusion on the osmotic potential in the rhizosphere- Comparing of two maize cultivars differing in Na+ uptake. Plant Nutrition and Soil Science 167, 337-344.

Yeo AR,Flowers TJ (1983). Varietal differences in the toxicity of sodium ions in rice leaves. Physiologia Plantarum 59(2), 189-195.

42

Table 1S. The whole shoot concentrations of Na+, K+ and Cl- in 60 genotypes of barley in experiment 1. Values are means (n = 4). Na+ Cl- K+ CPI77146-32 2174 1226 971 Tadmor 1613 1830 1600 Buloke 2215 1518 1183 Yarra 1855 1225 952 Briggs 1229 1028 781 Fleet 1747 1010 1299 H.Spont 41.1 2185 1221 1027 O2D/20 1736 1112 1534 Parent16 1478 886 1082 Keel 1477 897 1259 Gairdner 2243 1375 1285 Chevron 902 760 775 WI3788 2151 1261 1105 Flagship 1950 1057 1481 WI4262 1632 1083 968 Dobla 1879 1460 1020 Dhow 1418 912 997 Arivat 1361 1054 845 Hindmarsh 1816 1152 1129 Albecta 1813 1016 1296 Vlamingh 1788 1172 1376 Frankline 2425 1451 1517 Parent15 1330 891 880 Proto 1771 1069 1046 Harmel 1780 1006 1324 Baudin 1673 1388 1516 YU6472 1466 1061 1168 Maritime 1292 1011 1105 WI3416 1862 1001 1395 Barque73 1656 1006 1424 Ratna 1316 972 1137 Club Mariout 1148 1012 874 ICARDA-391 1283 957 868 Sahara 1479 998 1206 Arta 2181 1200 1144 Mundah 1717 1014 1446 Er/Apm 1453 993 1049 Clipper 1728 1153 1168 Arupo 1966 1161 1126 CM72 1838 1004 1020 CM67 1263 1099 878

43

ICARDA-382 1119 903 887 CPI71284-48 2819 2163 2023 Barque 983 858 852 WI2198 944 830 712 Schooner 873 1050 816 Skiff 863 840 672 Capstan 1613 963 1240 Egmont 1149 999 903 Parent12 1327 1037 806 Beecher 1841 1061 1427 California Mar 1470 1140 854 Parent-08 984 803 835 Sloop 1676 991 1081 Kaputar 2138 1440 680 Gerbel 1403 946 2209 Parent19 1864 1838 2550 Halycon 1647 1029 1176 Cl-3576 1093 841 1295

44

Table 2S. The dry matter production of 15 genotypes of barley (experiment 2) in control conditions and in two levels of soil salinity (ECe = 7.2 and 15.3 dS m-1) at three harvests. Values are means (n = 3). Control EC1 EC1 EC2 EC2 DW DW ST DW ST First harvest Fleet 3.73 2.96 79 2.55 68 Flagship 2.23 2.12 95 1.63 73 Buloke 2.44 2.34 96 2.32 95 Hindmarsh 2.32 1.90 82 1.58 68 WI4263 3.06 2.55 83 2.15 70 Schooner 3.01 2.85 95 2.78 92 P19 3.01 2.60 86 2.32 77 Gairdner 2.46 2.08 85 1.62 66 ODZ20 3.53 3.03 86 2.52 71 Yara 3.25 2.85 88 1.91 59 SloopSA 3.40 3.01 89 2.88 85 Maritime 3.21 3.05 95 2.55 79 Capstan 3.55 3.25 92 3.05 86 Keel 3.65 3.01 82 2.65 73 Baudin 3.15 2.75 87 2.71 86 Second harvest Fleet 8.44 7.06 84 6.85 81 Flagship 10.16 7.12 70 6.94 68 Buloke 8.79 6.66 76 6.64 76 Hindmarsh 5.67 5.03 89 4.91 87 WI4263 6.38 5.91 93 4.93 77 Schooner 8.21 6.72 82 3.95 48 P19 7.91 5.85 74 4.85 61 Gairdner 6.51 5.15 79 4.05 62 ODZ20 6.24 4.98 80 4.81 77 Yara 8.44 6.85 81 5.37 64 SloopSA 7.55 5.15 68 4.85 64 Maritime 8.15 5.05 62 4.85 60 Capstan 7.32 6.25 85 5.85 80 Keel 7.88 5.15 65 5.05 64 Baudin 6.33 4.05 64 3.15 50 Third harvest Fleet 14.9 10.5 71 9.1 61 Flagship 12.6 6.8 54 6.5 52 Buloke 11.6 7.5 65 6.8 59 Hindmarsh 12.5 10.1 81 8.9 71 WI4263 9.6 8.5 89 8.1 84 Schooner 11.6 6.8 59 5.8 50 P19 10.1 8.9 88 6.5 64

45

Gairdner 9.5 6.4 67 5.6 59 ODZ20 13.9 10.9 78 10.5 75 Yara 16.2 9.9 61 7.8 48 SloopSA 9.9 6.5 66 5.0 51 Maritime 12.2 6.8 56 5.2 43 Capstan 15.1 13.1 87 10.5 70 Keel 14.6 10.9 75 8.8 60 Baudin 10.6 6.1 58 5.1 48

46

Table 3S. The grain yield (kg ha-1), leaf osmotic potential (OP) and leaf Na, Cl and K concentration (mmol kg-1 DW) of barley varieties grown at Hart site in 2009. The results for leaf OP and ion concentrations are from youngest emerged leaves at ZGS 65. Values are averages (n=4).

grain yield OP Na Cl K

Maritime 2988 1.65 556 689 665 Schooner 3132 1.61 455 615 685 Flagship 3355 1.63 415 675 695 Baudin 3792 1.68 450 580 655 Gairdner 3813 1.53 465 577 678 SloopSA 3820 1.58 395 550 665 Yarra 3990 1.45 420 568 670 Buloke 4001 1.38 425 560 615 Keel 4105 1.62 455 585 645 Hindmarsh 4355 1.18 365 480 633 WI4262 4545 1.15 370 485 690 Fleet 4755 1.39 361 465 625 Capstan 4985 1.21 345 470 677

Chapter 8

Thesis: Limitations to yield in saline-sodic soils: Quantification of osmotic and ionic

regulations that affecf ,6" g;ofih and nutrition of crops under salinity stress' By Ehsan

Tavakkoli,20ll.

CHAPTER 8

TITLE OF PAPERGenotypic variations of faba bean in response to transient salinity at whole-plant level

AUTHORS OF PAPER:Ehsan Tavakkoli*, Jãfã.y Paull, Pichu Rengasamy, and Glenn K' McDonald

School of Agriculture, Food and wine, Waite campus, The university of Adelaide' SA' 5064

Name of journal Paper submitted:io*rot i¡fiuta cript Research, 2010, submitted paper'

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

1

Genotypic variations of faba bean in response to transient

salinity at whole-plant level

Ehsan Tavakkoli*, Jeffrey Paull, Pichu Rengasamy and Glenn K.

McDonald School of Agriculture, Food and Wine,

The University of Adelaide,

Email:

ehsan.tavakkoli@adelaide.edu.au,

jeffrey.paull@adelaide.edu.au,

pichu.rengasamy@adelaide.edu.au,

glenn.mcdonald@adelaide.edu.au

*To whom correspondence should be addressed:

Ehsan Tavakkoli

School of Agriculture, Food and Wine,

Waite Campus

The University of Adelaide,

PMB 1 Glen Osmond, South Australia, 5064

Phone: +61-8-8303 6533

Fax: +61-8-8303 7109

Email: ehsan.tavakkoli@adelaide.edu.au

Word count: 4740

Number of figures: 5

Number of tables: 4

Number of supplementary tables: 1

Submitted article to the journal of Field Crops Research, December 2010

2

Abstract

Faba bean is an important pulse crop in the Mediterranean region, west Asia, China and

Australia where it is adapted to neutral-alkaline soils. In many cases faba beans are

grown as a rainfed crop on saline-sodic soils where growth and yield are limited by

salinity. Developing more salt tolerant varieties of faba bean would help improve

productivity on these soils. This study aimed to assess the genotypic variation of faba

bean plants to salinity stress and the suitability of various physiological traits to screen

11 faba bean genotypes for salt tolerance. The work validated the value of these traits by

comparing genotypes with different levels of salinity stress, identified under controlled

conditions and in the field. Significant genotypic variation for salinity tolerance in faba

bean was measured among the varieties screened in hydroponics and in the field. The

whole-plant sodium (Na+) and chloride (Cl-) concentrations at 75 mM NaCl were

significantly correlated with biomass production under controlled conditions (r = -0.97

and -0.95) and ranked the genotypes with their grain yield production in the field.

However, the importance of Na+ and Cl- exclusion to salt tolerance was diminished by

osmotic tolerance at higher levels of salinity. The present study also suggests that salt

exclusion coupled with a synthesis of organic solutes are important components of salt

tolerance in the tolerant genotypes. While Na+ and Cl- exclusion is an important primary

mechanism of salt tolerance, the development of more salt-tolerant germplasm is likely

to be accelerated if screening based on ion exclusion also takes into account genotypic

differences in osmotic tolerance.

Keywords: Salinity tolerance, osmotic stress, specific ion toxicity, faba bean,

screening, physiological traits

3

1. Introduction

Production systems in agriculture have been predominantly cereals-based but recently,

economic and environmental developments have revived interest in grain legumes as

rotational crops that break cereal disease cycles, fix nitrogen and produce a valuable

grain for feed and food uses (Robson et al. 2002). Faba bean (Vicia faba L.) is a

valuable protein-rich legume crop that is predominantly grown on neutral and alkaline

soils in west Asia, China and Australia. Yields are often variable due in part to the

crop’s shallow root growth and poor drought tolerance (Morgan et al. 1991; Turner et

al. 2001). Many of the soils on which faba bean is grown are saline (Jensen et al. 2010;

Rispail et al. 2010) and relative to other crops, faba bean is sensitive to salinity (Maas

and Hoffman, 1976). Surveys of commercial crops of faba bean in South Australia

revealed that compared to field pea (Pisum sativum) faba bean accumulates

considerably more Na+ in the shoot (G. McDonald unpublished data). Faba bean’s

intolerance of salinity may exacerbate its sensitivity to dry conditions because the crop

can not make full use of available soil moisture.

Genetic variation in tolerance to salinity has been reported in all the major food crops

such as wheat (Munns et al. 2000; Munns and James 2003), barley (Epstein et al. 1980),

rice (Yeo and Flowers 1983), triticale (Norlyn and Epstein 1984), oats (Verma and

Yadava 1986), sorghum (Krishnamurthy et al. 2007), alfalfa (Noble et al. 1984) and

millet (Kebebew and McNeilly 1996). However in the case of faba bean, little

information is available in the literature regarding genetic variability for salt tolerance

but the information available suggests the sources of salinity tolerance may be more

limited than in many other crops (Del Pilar Cordovilla et al. 1999; Malhotra 1997).

High levels of salinity tolerance were found in a small number of accessions from

provinces in China with extensive alkali–saline soils (Duc et al. 2010; Li-juan et al.

4

1993). Further screening for salinity tolerance among 504 landraces and breeding lines

from worldwide sources found only 16 tolerant genotypes from China, Greece, Egypt

and Australia (Duc et al. 2010).

Developing appropriate screening methods to select for improved salt tolerance relies

on understanding the causes of yield loss from salinity. Reductions in growth and yield

are due to three mechanisms. First, osmotically-driven uptake of water, which is

necessary for cell enlargement, may be inhibited by low water potentials (Ψ) in the root

space caused by the high salt concentrations of the soil solutions (osmotic stress).

Second, specific solutes such as K+, Ca2+ and NO-3 needed for normal cell functions

may not be available at sufficient quantities because of competition by Na+ or Cl- for

uptake (nutrient imbalance). Third, plants may not be able to regulate the net uptake of

Na+ and Cl- to maintain ion concentrations within the physiologically-active range and

concentrations may build up to eventually disrupt cell metabolism due to toxic effects

(ion toxicity). Plants have evolved various mechanisms to adapt to salt stress (Munns

and Tester 2008), including control of Na+ and/or Cl- uptake and Na+ and/or Cl- xylem

loading, Na+ and/or Cl- retrieval from the xylem, Na+ and/or Cl- extrusion from the root,

intracellular compartmentation of Na+ and Cl- into the vacuoles and salt excretion.

Osmotic adjustment involves the plant’s ability to tolerate water deficit and cell osmotic

potential associated with the lowered water potential of the salt-affected soil (Boyer et

al. 2008). This action maintained a favourable osmotic force for water uptake and a high

turgor pressure in the cells (Bernstein 1961). However, reducing Na+ and/or Cl- uptake

should be the most efficient approach to control Na+ and/or Cl- accumulation in most

crop plants, as a way to improve their salt resistance, since if uptake is reduced, the

range of other mechanisms for dealing with excess Na+ and/or Cl- do not need to be

5

invoked. Therefore, much of the past research on improving salt tolerance in crop plants

has focussed on selecting for ion exclusion and particularly Na+ exclusion.

Screening for salt tolerance should be ideally carried out in the field at sites where

salinity is a problem. However, screening in the field can be problematic because of the

inherent variability of salinity within fields (Richards 1983) and potential interactions

with other environmental factors (Flowers 2004). The difficulties encountered in field

screening can be overcome by the use of soil (Poustini and Siosemardeh 2004) and

solution-based screening methods (Munns and James 2003), together with trait-based

selection criteria (Noble and Rogers 1992). Screening methods based on hydroponics or

supported hydroponics have become the preferred method for most researchers because

it gives a high degree of control and reproducibility. Despite the large volume of

literature on hydroponic screening for salt tolerance, few studies have reported salt

tolerance rankings of genotypes and the relationship between vegetative and grain yield

responses under controlled and field conditions.

To improve salt tolerance in faba bean, there is a need to develop reliable screening

protocols. Therefore two experiments were conducted with the following aims: (1) to

compare the responses to salinity in hydroponics and in field in a diverse range of faba

bean cultivars and (2) assess the value of tissue Na+, Cl- and K+ concentration as a

criterion for salt tolerance and assess the importance of different mechanisms of salinity

tolerance in the two systems.

2. Materials and Methods 2.1. Plant material

Eleven genotypes of faba bean were screened for their tolerance to salinity: Cairo,

Farah, Fiesta, Fiord, Manafest, Nura, Icarus, Acc 1608/2, Acc 1477/4, Acc 1512/2 and

6

Acc 1487/7. The genotypes were obtained from the National Faba Bean Breeding

Program, University of Adelaide, and were a selection of varieties and breeding lines

that were representative of the range of genetic material that has been grown in the

region and are selected from natural parents of different geographical origin, growth

habitats, seed size and earliness (Table 1).

Table 1. Genotypes used in the present study

Variety/Line Pedigree ICARDA Accession

Origin of source population

Cairo Selection from an open pollinated population of Acc 972, pollinator unknown

ILB2282 Greece

Farah Ascochyta blight resistant selection of Fiesta VF

- Spain

Fiesta VF Selected from ILB 955 BPL1196 B8817 Spain

Fiord Acc 59, Selection from landrace - Greece

Manafest Selection from landrace ILB 3026 Ecuador

Nura Icarus/Ascot (Ascot = Ascochyta resistant selection of Fiord)

Icarus = BPL 710 Ecuador & Greece

Icarus Selected from LAT81-24857-1 BPL 710 Ecuador

Acc 1477/4 Selection from landrace Crete

Acc1487/7 Selection from landrace ILB 917 Algeria

Acc 1512/2 Selection from landrace ILB 759 Syria

Acc 1608/2 Selection from landrace BPL 5250 Spain

Acc = Accession in University of Adelaide faba bean collection

ILB and BPL = Accessions in ICARDA faba bean collection

7

2.2. Experiment 1: Hydroponics screening

Growth condition

A hydroponics experiment was conducted to examine the responses to salinity (Na+ and

Cl-). A factorial experiment consisting of a control (0 mM NaCl) and two

concentrations of NaCl (75 and 150 mM) was arranged in a completely randomised

design with four replicates. The experiment used a supported hydroponic system (Genc

et al. 2007). Plants were grown in cylindrical PVC tubes (4 cm diameter × 28 cm depth)

with mesh bottom and filled with cylindrical black polycarbonate pellets (approximately

2–4 mm long and 1–2 mm in diameter) in a series of 15 L tubs. Each of these tubs

contained 24 PVC tubes and they were placed on and connected to another tub which

contained 24 L of nutrient solution. Every 30 minutes, nutrient solution was pumped

from the bottom tub into the top tub where it remained for 30 min after which the pump

was switched off and nutrient solution in the top tub drained back into the bottom tub.

Roots remained moist between the on and off cycles and there was no evidence of water

stress on growing seedlings. A modified Hoagland’s solution was used, the composition

of which (in mM) was: NH4NO3 (0.2); KNO3 (5); Ca(NO3)2 (2); MgSO4 (2); KH2PO4

(0.1); NaFe (III) (HEDTA) (0.05); H3BO3 (0.01); MnCl2 (0.005); ZnSO4 (0.005);

CuSO4 (0.0005); and Na2MoO3 (0.0001). Solutions were changed every 7 days, at

which time the pH was adjusted to 6.0. The experiment was conducted in a temperature-

controlled glasshouse (23/19C). The photosynthetically active radiation was measured

(model LI-1000, Li-Cor, Lincoln, NE, USA) and varied from 350 to 450 mmol m-2 s-1.

Uniformly sized seeds of each genotype were surface sterilized in 70% ethanol for 1

min, followed by soaking in 3% sodium hypochlorite for 5 min, then rinsed three times

with deionised water. Seeds were germinated on filter paper at room temperature for 4

8

days. The seedlings were then transplanted into the PVC containers. At 8–10 days after

transplanting, when three leaves were unfolded, the salt treatment was introduced in

increments of 25 mM NaCl per day. Supplemental Ca2+ was also added as CaCl2 to give

a final Na+: Ca 2+ of 15:1, thereby avoiding Ca2+ deficiency.

Plants were harvested after 42 days and separated into leaves and stems. The fresh and

dry weights were recorded, and the whole shoot moisture content was calculated. Salt

tolerance was assessed as the ratio of shoot dry weight in the salt and control treatments,

expressed as a percentage. The osmotic potential of leaf sap was measured. A disc of

Whatman GF/B glass micro-fibre paper was placed in the barrel of a 2 mL plastic

syringe so that it covered the outlet hole. A fresh leaf was then put in the barrel, the

plunger was re-inserted, and the tip of the syringe was sealed with Blu-Tack. The

syringe was frozen in liquid nitrogen and, still sealed, was thawed to ambient

temperature. When temperature equilibration was complete, the plunger and Blu-Tack

were removed and the barrel of the syringe was placed in a 15 mL centrifuge tube, with

its tip resting inside a 1.5 mL ependorff tube. After centrifugation at 2500 g for 10 min

at 40C, the osmolality of a 10 µl sample was measured in a Vapro pressure osmometer.

The ionic composition of whole dry shoots was estimated by digesting a ground sample

in 40 mL of 4% HNO3 at 95°C for 6 hours in a 54-well HotBlock (Environmental

Express, Mt Pleasant, South Carolina, USA). The concentrations of Na+ and K+ in the

digested samples were measured using a flame photometer (Model 420, Sherwood,

Cambridge, UK). Chloride concentrations of the digested extracts were measured using

a chloride analyser (Model 926, Sherwood Scientific, Cambridge, UK). Recovery of

9

Na+, Cl- and K+ from plant standards (Australasian Soil and Plant Analysis Council)

were 98, 99 and 98% respectively.

2.3 Experiment 2: Field study

Site description, experimental design and agronomic practices

Field experiments were conducted on farmer’s field at Pinery, South Australia (34° 18'

0" South, 138° 27' 0" East) in 2008 and 2009. The climate of this region is

Mediterranean with an annual rainfalls of 351 mm in 2008 and 405 mm in 2009

respectively (Fig 1). The soil at Pinery site is a calcareous loam, classified as

Endohypersodic, Regolithic, Lithocalcic Calcarosol (Isbell 1996) and is the most

extensive soil of the region accounting for 8.7% of Southern South Australia (Hall et al.

2009). The profile texture is gradational with loamy surface grade to clay loamy subsoil.

The topsoil is alkaline and the subsoil is strongly alkaline (pH ≥ 9.5). The topsoil is non-

saline and non-sodic but the soil ECe and ESP increased with depth as did Na+ and Cl-

concentration too (Fig 2).

Figure 1. Mean monthly rainfall (histogram) and mean monthly maximum and minimum air temperatures at experimental site (Pinery) in South Australia in 2008 and 2009.

Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep

Oct

Nov Dec

Rai

nfal

l (m

m)

0

20

40

60

80

10020082009

Jan

Feb

Mar

A

pr

May

Ju

n Ju

l A

ug

Sep

Oct

N

ov

Dec

Tem

pera

ture

(°C

)

0

10

20

30

40

50

20082009

10

The trials were planted in plots 6 m long × 1.5 m wide at a plant density of 25 seeds/ m2

on the 14 May 2008 and 11 May 2009. The trial was a randomised block design with

four replications. Fertiliser was applied at the time of sowing as 150 kg/ha 5:14:0:13 (N,

P, K and S) + 2% Zn in 2008 and as 150kg/ha 5:14:0:7 +2.1% Zn in 2009. Weeds were

controlled with a range of commercially-available herbicides and the plots were sprayed

with insecticides to control insects. All pesticides were applied at the recommended

rates and times and growth and yield of the faba beans were not affected by weed

competition or insect damage.

Figure 2. The selected physical and chemical characteristics of soil at Pinery site. All the analysis were made on soil solution extracted from saturated paste extract. The bars are standard errors of the means (n=10).

pH

7 8 9 10 11 12

Dep

th (c

m)

0

20

40

60

80

100

120Na+ (mg/kg)

0 500 1000 1500

Dep

th (c

m)

0

20

40

60

80

100

120

Cl- (mg/kg)

0 500 1000 1500

ECe (dS/m)

0 2 4 6 8 10

ESP (%)

0 20 40 60 80 100

K+ (mg/kg)

0 100 200 300 400 500

11

Measurements were made on six genotypes (Nura, Farah, Fiord, Fiesta, Cairo and

Manafest) in 2008 and on all 11 genotypes in 2009. At growth stages 51 (first flower

buds visible outside leaves), 65 (full flowering) and 75 (50% of pods have reached final

length) (Meier 2001) ten randomly-selected leaflets from each plot were sampled. The

leaves were rinsed in Milli-Q water (conductivity ≥18.2 MΩ cm-1) three times and the

dry weight, ion concentrations and leaf osmotic potential measured as described for

experiment 1.

The concentrations of compatible solutes were measured in the leaf samples (the same

leaf as sampled for leaf osmotic potential) using high performance liquid

chromatography (HPLC). Immediately following harvest the leaf sap was extracted as

described for osmotic potential measurement in experiment 1. One mL of

methanol:chloroform:water (60:25:15 by vol.) was added to each sample and the

samples were vortexed for 1 min before centrifugation for 10 min at 10000 g at 40C.

The supernatant was removed and the samples were freeze-dried. The samples were

resuspended in 200 µL of milliQ water prior to injection into the HPLC. Samples were

analysed using a Dionex DX 500 system consisting of an AS40 Autosampler, GP40

gradient pump, AD20 UV/Visible absorbance detector, ED40 electrochemical detector

and LC20 chromatography enclosure was used to quantify levels of compatible solutes

in plants. A mixture of standards (glycine betaine, sucrose, glucose, fructose, mannitol,

trigonelline and sorbitol), was prepared in methanol:water (50:50, v:v) at 0.5 µg µL-1 for

glycine betaine and 2.5 µg µL-1 for the remaining solutes. Ten microlitres of the

standard solution was injected into the HPLC while running each batch of samples. The

contribution of organic and inorganic ions to leaf osmotic potential was determined

using the van’t Hoff equation, where the calculated contribution of individual solutes to

12

measured solute potential, Ψs, was based on solute concentration on a molar basis

(Marigo and Peltier 1996).

Ten soil cores were randomly taken from a soil depth of 10–75 cm in September 2009.

Electrical conductivity (ECe), pH, soluble Na+, Ca2+ and Mg2+ were determined in a

saturated paste extract. Exchangeable sodium percentage (ESP) was calculated from the

values of soluble Na+, Ca2+ and Mg2+ according to Rowell (1994). Chloride

concentration was determined using a chloride analyser (Model 926, Sherwood

Scientific, Cambridge, UK). Grain yield was measured by harvesting each plot with a

small plot harvester. The harvest dates were 11 November 2008 for the first year and 16

November 2009 for the second year.

2.4 Statistical analysis

Statistical analyses were performed in R 2.10.1 (R Development Core Team 2006).

Data for growth and ion content were analysed using two-way ANOVA to determine if

significant differences were present among means. Variances were checked by plotting

residual vs. fitted values to confirm the homogeneity of the data. Differences among the

mean values were assessed by Least Significant Differences (LSD). Relationships

between individual variables were examined using simple linear correlations and

regressions which were performed using SigmaPlot (version 10). Spearman’s rank

correlation test (rs) was used to examine consistency in the rankings of genotypes for

salt tolerance and grain yield production between the three experiments.

13

3. Results

3.1 Experiment 1

Genotypic variation in biomass, ion concentration, osmotic potential and salt tolerance

Large genotypic variation was found for the plant biomass under salt stress

(Supplementary Table 1S) and in salt tolerance (Fig 3 a,b). Salt tolerance was greater at

the lower concentration of NaCl and salt tolerance among genotypes ranged from 41%

in Cairo to 93% in 1487/7 when grown at 75 mM NaCl and from 35% in Fiord to 72%

in 1487/7 and Fiesta at 150 mM NaCl. The rankings of genotypes based on their salt

tolerance were significantly correlated in the two salt concentrations (rs = 0.69, P <

0.05).

At 75 mM NaCl, the Na+ concentration in the whole shoot varied over 1.5-fold among

the 11 genotypes (Fig 3c), ranging from 1153 mmol kg-1 DW in line 1512/2 to 1813

mmol kg-1 DW in Manafest. Shoot Na+ concentration under 75mM was negatively

related to the salt tolerance (Fig 3c, r = -0.96, P < 0.001). There was greater variation in

the concentration of Cl-among the genotypes and this ranged from 715 mmol kg-1 DW

in line 1487/7 to 1912 mmol kg-1 DW in Manafest (Fig 3d). The salinity tolerance of

plants at 75mM was also highly correlated with the ability of genotypes to exclude Cl-

(Fig 3d; r = -0.94, P < 0.001). Shoot K+ concentration under 75 mM conditions was

significantly related to salt tolerance (Fig 3e; r = 0.97, P < 0.001) and the observed

variations among the genotypes in K+ concentrations in the whole shoot were negatively

related to the Na+ concentration (r = -0.97, P < 0.001). Leaf osmotic potential

significantly decreased at 75 mM NaCl treatment compared to the control but there was

no significant difference among genotypes in osmotic potential and consequently there

was no relationship to salt tolerance (Fig 3f, P > 0.05).

14

These relationships changed at 150 mM NaCl. Shoot Na+ and Cl- concentration

increased significantly under 150 mM NaCl but the range in concentrations among the

genotypes was smaller and there was no significant relationship with salt tolerance (r =

0.09 for Na+ and r = - 0.17 for Cl- respectively). The concentration of K+ was also lower

at the higher NaCl concentration but there was no relationship between the whole shoot

K+ concentration salt tolerance under 150 mM NaCl (r = -0.01). However, in contrast to

75 mM, significant genotypic variation was observed in leaf osmotic potential among

the 11 genotypes (P < 0.05) and it was highly correlated with salt tolerance (Fig 3f, r = -

0.92, P < 0.001).

3.2 Experiment 2

3.2.1. Weather

In 2008, rainfall during the May to October growing season was 71 mm less than the

long-term average, whereas in 2009, it was just 0.3 mm less than average rainfall. The

average rainfall in 2009 was about 14% higher than 2008, but the May-October rainfall

in 2009 was 35% higher than 2008. In general, air temperatures were less in 2008 than

in 2009 (Fig 2).

3.2.2 Genotypic variation in ion concentration and leaf osmotic potential in relation

to grain yield

In 2008, grain yield of the 6 genotypes ranged from 843 kg ha-1 in Manafest to 1370 kg

ha-1 in Fiord. Significant genotypic variation occurred in Na+ and Cl- and K+

concentrations (Fig 4). Sodium concentrations varied widely, ranging from 105 to 322

mmol kg-1 DW. Chloride concentration varied about 2.5-fold ranging from 188 to 530

mmol kg-1 DW. As well, K+ concentration varied about 1.9-fold ranging from 490 to

15

900 mmol kg-1 DW. Grain yield was negatively and significantly correlated with leaf

Na+ and Cl- concentrations (Fig 4a and b, P < 0.001) and the observed variations among

the genotypes in K+ concentrations were negatively related to the Na+ concentration and

positively related to the grain yield (Fig 4c and d, P < 0.001).

Large genotypic variation was also found among the genotypes in 2009 in grain yield,

their tissue ionic concentrations and leaf osmotic potentials. Grain yield of the 11

genotypes ranged from 2923 kg ha-1 in accession 1477/4 to 3650 kg ha-1 in Nura.

Significant genotypic variation occurred in Na+, Cl- and K+ concentrations (Fig 5).

Sodium concentrations ranged from 195 in accessions 1487/7 and 1512/2 to 445 mmol

kg-1 DW in Cairo and Manafest. Chloride concentration varied about 2.2-fold ranging

from 265 in line 1512/2 to 588 mmol kg-1 DW in Manafest. As well, K+ concentration

varied about 2-fold ranging from 375 to 790 mmol kg-1 DW. Leaf Na+ and Cl-

concentrations were negatively related to the grain yield (Fig. 5a and b, P < 0.001) and

the observed variations among the genotypes in K+ concentrations were negatively

related to the Na+ concentration and positively related to the grain yield. Significant

genotypic variation (2-fold) was observed in leaf osmotic potential among the 11

genotypes (P < 0.01) and it was negatively correlated with grain yield (Fig 5d, r = -0.85,

P < 0.001).

16

Figure 3. The range in salt tolerance among 11 genotypes of faba bean grown at (a) 75

mM NaCl and (b) 150 mM NaCl, and the relationships between salt tolerance and shoot

concentration of (c) Na+ concentration (mmol kg-1 DW), (d) Cl- concentration (mmol kg-

1 DW), (e) K+ (mmol kg-1 DW) and (f) leaf osmotic potential (-MPa) grown at 75 mM

(●) or 150 mM (○) NaCl for 42 days in an hydroponic system. Fitted curves are derived

from linear regression. The vertical bars are LSD at 95%. Values are means (n=4).

(c)

Na+ concentration (mmol kg-1 DW)

300 600 900 1200 1500 1800 2100 2400

Salt

tole

ranc

e (%

)

0

20

40

60

80

100

75 mM NaCl r = -0.96150 mM NaCl r = n.s

(d)

Cl- concentration (mmol kg-1 DW)

300 600 900 1200 1500 1800 2100 2400

75 mM NaCl r = -0.94150 mM NaCl r = n.s

(e)

K+ concentration (mmol kg-1 DW)

200 300 400 500 600 700

Salt

tole

ranc

e (%

)

0

20

40

60

80

100

75 mM NaCl r = 0.97150 mM NaCl r = n.s

(f)

Leaf osmotic potential (-MPa)

1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

75 mM NaCl r = n.s150 mM NaCl r = 0.92

(a)

Cai

ro16

08/2

Man

afes

tFa

rah

1477

/4N

ura

Icar

usFi

ord

Fies

ta15

12/2

1487

/7

Salt

tole

ranc

e (%

)

0

20

40

60

80

100(b)

Fior

dC

airo

1608

/2Fa

rah

1477

/4N

ura

Man

afes

t15

12/2

Icar

us14

87/7

Fies

ta

17

Figure 4. The relationship between grain yield and leaf concentration of (a) Na+ (mmol

kg-1 DW), (b) Cl- (mmol kg-1 DW), (c) K+ (mmol kg-1 DW) and (d) the relationship

between leaf Na+ and K+ concentration of 6 faba bean genotypes grown at Pinery site in

2008. The results are from youngest emerged leaves at full flowering. Fitted curves are

derived from linear regression. The vertical bars are LSD at 95%. Values are means

(n=4).

(a)

Na+ concentration (mmol kg-1 DW)

0 100 200 300 400 500 600

Gra

in y

ield

(kg

ha-1

)

800

900

1000

1100

1200

1300

1400

r = -0.92

(b)

Cl- concentration (mmol kg-1 DW)

0 100 200 300 400 500 600

Gra

in y

ield

(kg

ha-1

)

800

900

1000

1100

1200

1300

1400

r = -0.94

(c)

K+ concentration (mmol kg-1 DW)

400 600 800 1000

Gra

in y

ield

(kg

ha-1

)

800

900

1000

1100

1200

1300

1400

r = 0.90

(d)

Na+ concentration (mmol kg-1 DW)

100 200 300 400

K+ c

once

ntra

tion

(mm

ol k

g-1 D

W)

400

600

800

1000

r = -0.98

18

Figure 5. The relationship between grain yield and leaf concentration of (a) Na+ concentration (mmol kg-1 DW), (b) Cl- concentration (mmol kg-1 DW), (c) K+ (mmol kg-1 DW) and (d) leaf s (-MPa) of 11 faba bean genotypes grown at Pinery site in 2009. The results are from youngest emerged leaves at full flowering. Fitted curves are derived from linear regression. The vertical bars are LSD at 95%. Values are means (n=4).

The variation in the accumulation of compatible solutes was associated with variation in

Na+ and Cl-. Among the 11 genotypes (Table 2), the five varieties that maintained lower

leaf Na+ and Cl- concentrations also exhibited a stronger capacity for accumulation of

organic solute that contributed to total leaf osmotic potential. For example in accessions

(a)

Na+ concentration (mmol kg-1 DW)

100 200 300 400 500 600

Gra

in y

ield

(kg

ha-1

)

1500

2000

2500

3000

3500

4000

r = -0.84

(b)

Cl- concentration (mmol kg-1 DW)

100 200 300 400 500 600

r = -0.91

(c)

K+ concentration (mmol kg-1 DW)

200 400 600 800 1000

Gra

in y

ield

(kg

ha-1

)

1500

2000

2500

3000

3500

4000

r = 0.87

(d)

Leaf osmotic potential (-MPa)

1.0 1.2 1.4 1.6 1.8 2.0 2.2

r = -0.85

19

1487/7, 1512/2, Fiesta, Fiord and Icarus organic solutes contributed 29-37% to total leaf

osmotic potential, while the contribution from inorganic solutes accounted for 55-64%.

However, in Cairo, Manafest and Farah, the major contribution to total leaf osmotic

potential was from the high concentrations of Na+ and Cl- which accounted for about

77-79% (Table 2).

4. Discussion

This study was conducted to assess the variation among faba bean genotypes in

response to salinity and also to examine which physiological traits can be used to assess

salt tolerance among faba bean genotypes under both field and controlled conditions.

Significant genotypic variation for salinity tolerance in faba bean was measured among

the 11 varieties screened in hydroponics (Fig 1, Table 1S). The most tolerant entries

were 1487/7, 1512/2 and Fiesta which are used regularly in the local faba bean breeding

program for introgressing various other tolerance characteristics. A significant result

from the screening is that the importance of Na+ and Cl- exclusion to salt tolerance

varied with the severity of stress: it was important to salinity tolerance at low levels of

salinity (75 mM NaCl) but its importance was diminished at 150 mM NaCl, when

osmotic tolerance became more important (Fig 1). The two mechanisms – ion exclusion

and tolerance to osmotic stress – were independent and altered the relative salt tolerance

of genotypes depending on the severity of stress. For example, Fiesta and 1512/2

display similar levels of Na+ and Cl- exclusion at high and low levels of salinity (Fig 1,

Table 1s). At 75 mM they also have similar levels of salt tolerance, but at 150 mM

Fiesta had 11% greater salt tolerance than 1512/2 which was associated with a lower

leaf osmotic potential suggesting it had also a greater level of osmotic tolerance.

20

Table 2. Estimated contribution of organic and inorganic ions to leaf osmotic potential (s). The contribution of individual solutes to measured s was determined using the van’t Hoff equation, where the calculated Ψs, was based on solute concentration on a fresh weight basis. The percentage value is based on the measured value of leaf s.

s Na+ Cl- K+ Sucrose Glucose Fructose Betaine Proline %Inorganic contribution %Organic contribution %Total

1477/4 -1.88 -0.48 -0.38 -0.42 -0.09 -0.08 -0.09 -0.09 -0.09 68 23 91

1487/7 -1.05 -0.21 -0.18 -0.31 -0.08 -0.05 -0.05 -0.02 -0.11 67 30 96

1512/2 -1.15 -0.15 -0.23 -0.25 -0.11 -0.07 -0.07 -0.05 -0.12 55 37 91

1608/2 -1.75 -0.45 -0.48 -0.39 -0.05 -0.08 -0.05 -0.07 -0.08 75 19 94

Cairo -1.99 -0.55 -0.58 -0.41 -0.02 -0.05 -0.05 -0.09 -0.08 77 15 92

Farah -1.55 -0.49 -0.51 -0.22 -0.05 -0.08 -0.05 -0.08 -0.05 79 20 99

Fiesta -1.25 -0.21 -0.18 -0.31 -0.11 -0.12 -0.11 -0.08 -0.06 59 37 96

Fiord -1.38 -0.21 -0.33 -0.35 -0.05 -0.09 -0.15 -0.05 -0.06 64 29 93

Icarus -1.33 -0.22 -0.28 -0.35 -0.15 -0.12 -0.09 -0.08 -0.02 64 35 98

Manafest -2.01 -0.51 -0.67 -0.35 -0.05 -0.05 -0.08 -0.05 -0.04 76 13 90

Nura -1.33 -0.33 -0.32 -0.33 -0.05 -0.09 -0.07 -0.08 -0.05 74 26 99

Van’t Hoff equation Ψs (MPa) = –csRT, where cs = Osmolarlity (mol L–1), R = 0.0083143 L MPa mol–1 K–1, and T = 293 K were considered. Contribution = (Ψs calculated/Ψs measured) × 100.

21

Salt tolerance reflects the ability of the plant to exclude Na+ and Cl- as well as

mechanisms associated with tolerance of the cells to high osmotic potential. As the

concentration of salt increases, the ability to exclude salt may become less effective in

protecting the plant from salt stress and other mechanisms, such as osmotic tolerance,

become increasingly important. Salinity stress in the field is variable over time and

space and so overall salt tolerance will depend on the relative contribution from the

different mechanisms of salt tolerance, which in turn can vary with the severity of

salinity stress (Tavakkoli et al. 2010b).

Improving the grain yield of crops is always a major target in plant breeding. Therefore,

the evaluation of final grain yield and growth parameters determining grain yield is a

critical aspect of breeding programs. Linear regressions adequately described the

relationship between Na+ and Cl- concentrations in the plant and grain yields of

genotypes, thus variation in yields was attributed to variation in ability of genotypes to

exclude Na+ and Cl- ions (Fig 4 and 5).

A common mechanism in response to salt stress is the accumulation of compatible

solutes which may be interpreted as a symptom of injury caused by stress or some type

of adaptive response (Ashraf and Harris 2004). This poses the question of whether salt-

tolerant genotypes also have a superior ability to accumulate higher concentrations of

compatible solutes. In our study, salt stress caused an increase in ions and organic

solutes in all genotypes, but the more salt tolerant varieties had significantly higher

concentration of soluble sugars (glucose, fructose and sucrose), glycine betaine, proline

and trigonelline. Cram (1976), showed that of the various organic osmotica, sugars

contribute up to 50% of the total osmotic potential in glycophytes subject to saline

22

conditions. Ion accumulation in plants can also play a major role in osmotic adjustment

to high salinities. It would seem, however, from the relationship between ion

accumulation and water status observed here for genotypes that the simple accumulation

of Na+ and Cl- alone can not account for the osmotic behaviour of these varieties (Table

2). While the ability to restrict Na+ and Cl- accumulation could prevent the development

of an internal osmotic imbalance in some genotypes, the concentration of Na+ and Cl-

accumulated when considered together with the reduction in shoot K+ would seem to

necessitate the synthesis of additional osmotically active solute in order to prevent an

osmotic imbalance with respect to the external salt in soil solution (Table 2). The

increases observed in soluble sugars, glycine betaine and proline amount to a total of

~800µmol g-1 DW (data not shown) and could therefore represent an important

component of the shoot osmotic potential (Table 2). Notwithstanding this caveat the

present study suggests that salt exclusion coupled with a synthesis of organic solutes are

important components of salt tolerance in the tolerate genotypes.

In order to assess the consistency in ranking of faba bean genotypes under both field

and controlled condition, the salt tolerance ranking of 11 genotypes in Experiment 1

was compared with grain yield production under field condition in 2009. The rankings

of genotypes based on their salt tolerance in controlled condition at 75 mM NaCl (but

not 150 mM NaCl) and grain yield production in the field (P=0.006, n = 11) (Table 3),

and the crucial parameters of leaf Na+ and Cl- concentration (rs = 0.88; P = 0.002; n =

11, data not shown) were significantly correlated. Moreover, in the hydroponic

experiment the rankings of genotypes based on their salt tolerance in the two salt

concentrations was only just significant (rs = 0.68, P = 0.04). This consistency of the

23

rankings between hydroponics (especially at the lower NaCl concentration) and field

supports the robustness of the overall results.

Table 3. The Spearmans’s rank correlations between grain yield of field-grown plants and salt tolerance of plants grown in the hydroponic (n=11).

Field Hydro 75 mM

Grain yield Salt tolerance

Field Grain yield

Hydro 75 mM Salt tolerance 0.85 (P=0.006)

Hydro 150 mM Salt tolerance 0.47 (P=0.128) 0.68 (P=0.04)

To answer the question of which physiological screening criteria can enable an accurate

ranking of faba bean genotypes under both field and control conditions, the correlation

coefficient between the grain yield and the scores of different physiological parameters

were analysed by simple linear correlation (Table 4). The grain yield production in the

field was significantly associated with the salt tolerance index of plants grown in 75

mM NaCl and the exclusion of Na+ and Cl− in leaves. Interestingly, the results in this

study indicate that K+ content in plants demonstrated a great genotypic variation and

was well correlated with the salt tolerance ranked by using grain yield (Table 4). Under

saline conditions, low osmotic potentials of the soil solution induce water deficit in

plant tissue. As a consequence, the turgor in plants may decrease. Leaf osmotic potential

was significantly correlated with grain yield in the field (Table 4).

24

Table 4. Correlation coefficients (r) between pairs of physiological attributes of salt stressed faba bean plants grown in the field in 2009 and at 75 mM of 150 mM NaCl in hydroponics . *, **, *** = significant at 0.05, 0.01, and 0.001 levels, respectively. n = 11.

Field Field Field Field Field Hydro 75mM Hydro 75mM Hydro 75mM Hydro 75mM Hydro 75mM Grain Yield Na+ K+ Cl- s ST Na+ K+ Cl- s Field (2009) yield Field (2009) Na+ -0.84** Field (2009) K+ 0.87** -0.89** Field (2009) Cl- -0.91*** 0.91*** -0.95 Field (2009) s -0.86** 0.89** -0.93*** 0.94*** Hydro 75mM ST 0.85** -0.95*** 0.86** -0.85** -0.89** Hydro 75mM Na+ -0.84** 0.92*** -0.81** 0.81** 0.84** -0.97*** Hydro 75mM K+ 0.76** -0.91*** 0.84** -0.79** -0.87** 0.97*** -0.92*** Hydro 75mM Cl- -0.79** 0.92*** -0.73** 0.75** 0.78** -0.94*** 0.94*** -0.92*** Hydro 75mM s -0.70* 0.92*** -0.88** 0.84** 0.87** -0.88** 0.88** -0.90** 0.89** Hydro 150mM ST 0.53 -0.43 0.51* -0.44 -0.55* 0.66* -0.63* 0.64* -0.56* -0.51* Hydro 150mM Na+ 0.03 0.21 -0.10 0.17 0.25 -0.15 0.16 -0.09 0.28 0.18 Hydro 150mM K+ 0.20 -0.03 -0.14 0.08 0.09 0.04 -0.22 -0.11 -0.03 0.09 Hydro 150mM Cl- -0.67 0.91 -0.71** 0.80** 0.77** -0.88** 0.85** -0.82** 0.95*** 0.83** Hydro 150mM s -0.40 0.39 -0.43 0.33 0.53* -0.63* 0.62* -0.68* 0.57* 0.52*

25

Because of the spatial and temporal variation in salt stress at field level, plants are

exposed to varying levels of salt stress, whereas much of the past work has focussed on

using one mechanism to improve salt tolerance. The present results suggest mechanisms

of exclusion are important under low-moderate stress and osmotic effects more

important at higher levels of stress. While Na+ and Cl- exclusion is an important primary

mechanism of salt tolerance (Tavakkoli et al. 2010a), the development of more salt-

tolerant germplasm is likely to be accelerated if screening based on ion exclusion also

takes into account genotypic differences in osmotic tolerance. It is also important to

emphasise that while use of hydroponic-based screening in this study was shown to be

well-correlated with results from the field experiment for faba bean, previous studies in

cereals such as barley demonstrated that solution culture may not allow differences in

salt tolerance between genotypes to be discerned and the physiological responses are not

the same when the same materials were grown in soil (Tavakkoli et al. 2010b).

In conclusion, the tested physiological traits showed significant genotypic variation,

indicating that the traits that have a significant genotypic variation may possibly be used

as screening criteria. The increased production of faba bean under rainfed conditions on

saline-sodic soils highlights the importance of improving salinity tolerance through

breeding. The availability of large and useful genotypic variation as shown in this study,

and the high association of Na+ and Cl- exclusion and K+/Na+ ratio with biomass

indicates that the introduction of low Na+ and Cl- accumulation into modern cultivars

should be possible as part of a faba bean breeding program. This study also clearly

shows that several processes are involved in salt tolerance and that the relative

importance of these traits may differ with the severity of the salt stress. The osmotic

stress was the predominant cause of reduced growth at high levels of salinity, while

26

specific-ion toxicity was more important under mild salinity stress. It is likely that

stress-tolerant faba bean plants accumulate compatible solutes and further field tests of

these plants under stress conditions will help to verify their potential utility in crop-

improvement programs.

Acknowledgements

This work was supported by a grant from the Grains Research and Development

Corporation to ET and by the University of Adelaide. We thank Mr. S. Coventry (The

University of Adelaide) for excellent HPLC guidance and Mr K. James for his support

with the field studies.

27

References

Ashraf M, Harris PJC (2004) Potential biochemical indicators of salinity tolerance in plants. Plant Science 166, 3-16. Bernstein L (1961) Osmotic adjusment of plants to saline media. I. steady state. American journal of botany 48, 909-918. Boyer JS, James RA, Munns R, Condon TAG, Passioura JB (2008) Osmotic adjustment leads to anomalously low estimates of relative water content in wheat and barley. Functional Plant Biology 35, 1172-1182. Cram WJ (1976) Negative feedback regulation of transport in cells. The maintenance of turgor, volume and nutrient supply. In 'Encyclopaedia of Plant Physiology'. (Eds U Luttge and MG Pitman) pp. 284-316. (Springer-Verlag: Berlin) Del Pilar Cordovilla M, Ligero F, Lluch C (1999) Effect of salinity on growth, nodulation and nitrogen assimilation in nodules of faba bean (Vicia faba L.). Applied Soil Ecology 11, 1-7. Duc G, Bao S, Baum M, Redden B, Sadiki M, Suso MJ, Vishniakova M, Zong X (2010) Diversity maintenance and use of Vicia faba L. genetic resources. Field Crops Research 115, 270-278. Epstein E, Norlyn JD, Rush DW, Kingsbury RW, Kelley DB, Cunningham GA (1980) Saline culture of crops: a genetic approach. Science 210, 399-404. Flowers TJ (2004) Improving crop salt tolerance. Journal of Experimental Botany 55, 307-319. Genc Y, McDonald GK, Tester M (2007) Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant, Cell and Environment 30, 1486-1498. Hall J, Maschmedt D, Billing B (2009) 'The soils of Southern South Australia ' (Department of water, land and biodiversity conservation, Government of South Australia) Isbell RF (1996) 'The Australian Soil Classification.' (CSIRO: Melbourne) Jensen ES, Peoples MB, Hauggaard-Nielsen H (2010) Faba bean in cropping systems. Field Crops Research 115, 203-216. Kebebew F, McNeilly T (1996) The genetic basis of variation in salt tolerance in Pearl Millet, Pennisetum americanum (L.) Leeke. J Genet and Breed 50, 129-136. Krishnamurthy L, Serraj R, Hash C, Dakheel A, Reddy B (2007) Screening sorghum genotypes for salinity tolerant biomass production. Euphytica 156, 15-24.

28

Li-juan L, Zhao-hai Y, Zhao-jie Z, Ming-shi X, Han-qing Y (1993) Study and utilization of faba bean germplasm resources. In 'Faba bean in China: State-of-the art review.'. (Eds MC Saxena, S Weigand and L Li-Juan) pp. 51-63. (ICARDA Press) Malhotra RS (1997) Evaluation techniques for abiotic stresses in cool season food legumes. In 'Recent Advances in Pulses Research'. (Eds AN Asttranu and A Masood) pp. 459-473. ( Indian Society of Pulses Research and Development: Kanpur, India) Marigo G, Peltier JP (1996) Analysis of the diurnal change in osmotic potential in leaves of Fraxinus excelsior L. Journal of Experimental Botany 47, 763-769. Meier U (Ed.) (2001) 'Growth stages of mono-and dicotyledonous plants: BBCH Monograph.' (Federal Biological Research Centre for Agriculture and Forestry: Germany) Morgan JM, Rodriguez-Maribona B, Knights EJ (1991) Adaptation to water-deficit in chickpea breeding lines by osmoregulation: relationship to grain yields in the field. 27, 61-70. Munns R, Hare RA, James RA, Rebetzke GJ (2000) Genetic variation for improving the salt tolerance of durum wheat. Aust. J. Agric. Res. 51, 69-74. Munns R, James RA (2003) Screening methods for salinity tolerance: a case study with tetraploid wheat. Plant and soil 253, 201-218. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651-681. Noble CL, Halloran GM, West DW (1984) Identification and selection for salt tolerance in Lucerne (Medicago sativa L.). Australian Journal of Agricultural Research 35, 239-252. Noble CL, Rogers ME (1992) Arguments for the use of physiological criteria for improving the salt tolerance in crops. plant Physiol 146, 99-107. Norlyn JD, Epstein E (1984) Variability in salt tolerance of four tritcale lines at germination and emergence. Crop Sci 24, 1090-1092. Poustini K, Siosemardeh A (2004) Ion distribution in wheat cultivars in response to salinity stress. Field crops research 85, 125-133. R Development Core Team (2006) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Richards RA (1983) Should selection for yield in saline regions be made on saline or non-saline soils? Euphytica 32, 431-438. Rispail N, Kaló P, et al. (2010) Model legumes contribute to faba bean breeding. Field Crops Research 15, 253-269.

29

Robson MC, Fowler SM, Lampkin NH, Leifert C, Leitch M, Robinson D, Watson CA, Litterick AM (2002) The agronomic and economic potential of break crops for ley/arable rotations in temperate organic agriculture. Adv Agron 77, 369-472. Rowell D (1994) 'Soil science : methods and applications.' (Wiley: New York) Tavakkoli E, Rengasamy P, Mcdonald GK (2010a) High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany 61, 4449–4459. Tavakkoli E, Rengasamy P, Mcdonald GK (2010b) The response of barley to salinity stress differs between hydroponics and soil systems. Functional Plant Biology 37, 621-633. Turner NC, Wright GC, Siddique KHM (2001) Adaptation of grain legumes (pulses) to water-limited environments. In 'Advances in Agronomy' pp. 193-231. (Academic Press) Verma OPS, Yadava RBR (1986) Salt tolerance of some oats (Avena sativa L.) varieties at germination and seedling stage. J. Agronomy & Crop Science 156, 123-127. Yeo AR, Flowers TJ (1983) Varietal differences in the toxicity of sodium ions in rice leaves. Physiologia Plantarum 59, 189-195.

30

Table1S. Salt tolerance (ST), shoot dry matter, Na+, K+, Cl- concentration and s of genotypes grown at 75 and 150 mM NaCl for 49 days. Genotypes are arranged in ascending order of salt tolerance.

Genotype Shoot dry matter (g

plant-1)

ST (%) Na+ K+ Cl- s

Control Salt 75 mM NaCl Cairo 4.99 2.02 41 1754 401 1888 -1.51

1608/2 3.47 1.50 45 1658 407 1655 -1.51

Manafest 4.51 2.30 52 1813 401 1911 -1.53

Farah 4.31 2.35 56 1584 453 1233 -1.49

1477/4 3.62 2.15 59 1411 453 1215 -1.51

Nura 3.75 2.22 60 1653 453 1656 -1.50

Icarus 4.12 3.01 73 1565 455 1255 -1.51

Fiord 3.82 2.73 75 1501 553 988 -1.47

Fiesta 4.29 3.48 81 1126 628 858 -1.46

1512/2 3.93 3.28 85 1153 657 834 -1.40

1487/7 3.66 3.34 93 1253 720 730 -1.43

150 mM NaCl Fiord 3.82 1.28 35 1956 446 1188 -2.65

Cairo 4.99 1.94 39 2356 225 1724 -2.55

1608/2 3.47 1.44 43 1741 323 1353 -2.45

Farah 4.31 2.00 48 2019 516 1858 -2.35

1477/4 3.62 1.93 53 2192 686 1939 -2.40

Nura 3.75 2.06 55 2221 432 1699 -2.44

Manafest 4.51 2.58 58 2124 422 1956 -2.35

1512/2 3.93 2.20 61 2105 453 1299 -2.22

Icarus 4.12 2.61 63 2115 455 1205 -2.14

1487/7 3.66 2.76 72 2109 308 1264 -2.05

Fiesta 4.29 3.11 72 1988 358 1258 -1.95

LSD0.05 (genotype×salt) 0.136 2.1 7.2 7.19 11.2 0.047

31

Table 2S. The grain yield (kg ha-1) and leaf Na, Cl and K concentration (mmol kg-1

DW) of 6 faba bean genotypes grown at Pinery site in 2008. Values are averages (n=4).

Yield

(kg/ha) Na K Cl

Cairo 1050.0 309.7 503.7 465.0 Farah 1170.4 195.0 718.7 278.7 Fiesta 1247.2 113.0 887.3 227.0 Fiord 1371.0 105.3 902.0 188.3 Manafest 843.8 322.0 491.0 530.0 Nura 1139.5 219.7 655.0 394.7

32

Table 3S. The grain yield (kg ha-1) and leaf Na, Cl and K concentration (mmol kg-1

DW) of 11 faba bean genotypes grown at Pinery site in 2009. Values are averages

(n=4).

yield Na K Cl

1477/4 2923 378 405 523 1487/7 3575 198 745 277 1512/2 3512 195 788 265 1608/2 2115 425 450 530 Cairo 2055 445 385 544 Farah 2488 385 411 502 Fiesta 3314 295 588 355 Fiord 3350 225 601 311 Ic*As/15/1-20 3150 261 605 291 Manafest 2350 445 374 585 Nura 3650 355 656 305

Chapter 9

70

Chapter 9

General discussion and conclusions

9.1 Introduction

Broadacre cropping in Australia is based on rainfed systems in a semiarid environment,

where the efficient uptake and use of water is the main driver of productivity, but the

presence of subsoil constraints such as salinity and sodicity in many soils reduces the amount

of water and nutrients plants can obtain from the soil (Hochman et al. 2007; Rengasamy

2010a). More than 60% of the 20 million ha of cropping soils in Australia are sodic, which

together with low rainfall and high rates of evapotranspiration have contributed to the

development of transient salinity (Rengasamy 2002).

Salinity stress inhibits plant growth for two reasons (Munns et al. 2006). First, the presence

of salt in the soil solution reduces the ability of the plant to take up water and this leads to

slower growth (osmotic stress). In soil, the effect may be exacerbated by the influence of the

soil matrix on water retention (the soil matric potential). Second, excessive amounts of salt

can accumulate in leaves and this may further reduce growth (ion specific toxicity) (Munns

and Termaat 1986; Munns et al. 2002; Munns et al. 2006). The physiology of plant responses

to salinity and their relation to salinity resistance have been thoroughly researched and

frequently reviewed in recent years (Munns 1993; Flowers and Yeo 1995; Munns 2002;

Tester and Davenport 2003; Flowers 2004; Colmer et al. 2005; Munns et al. 2006; Apse and

Blumwald 2007; Munns and Tester 2008; Dang et al. 2010; Nuttall et al. 2010; Rengasamy

2010b). A common theme is that plants with increased salinity tolerance are expected to

maintain higher rates of growth than less tolerant plants under equivalent levels of salinity,

71

but there is some controversy regarding the importance of the different mechanisms of

salinity tolerance to effect improvements in growth. This controversy reflects the difficulty in

separating osmotic effects from specific ion effects. It was often impossible to assess with

any confidence the relative importance of ion excess and osmotic stress partly because after

the initial osmotic stress of low levels of salinity, osmotic stress and ion specific stress

develop concurrently as salt stress increases (Munns et al. 2006).

Soil is a heterogeneous medium and this presents a number of challenges in developing

strategies to generate salt-tolerant varieties. The severity of salt stress in the field is inherently

variable, both over time and space, and there are also interactions with other soil properties

such as soil texture that influence how plants respond to saline-sodic conditions. Given that

the cause of reductions in growth under salt stress varies with the severity of the stress, it is

difficult to say which is the major cause of yield reductions under salt stress and perhaps

unrealistic to specify a single trait which should be targeted for plant breeding. Additionally,

the development of appropriate strategies for the management of broad acre crops produced

on saline-sodic soils has been hampered by uncertainty regarding the principal mechanisms

by which saline-sodic soils limit crop performance. The physical characteristics of saline

soils affects water holding capacity and soil water availability and the presence of high

concentrations of salt may exacerbate the poor soil-plant water relations of these soils.

Despite the fact that the osmotic effect on growth of the more-tolerant species such as wheat

and barley is much greater than the salt-specific effect, the relative importance of the

mechanisms that regulate the growth are not yet well understood (Munns et al. 2006).

There has also been some recent debate about the importance of soil Cl, and by implication

plant Cl- uptake, as predictors of damage and yield loss, rather than electrical conductivity.

72

Where NaCl is high, increased uptake of Na+ ions will be associated with high uptake of Cl-

ions (Dang et al. 2006a; Dang et al. 2006b; Teakle and Tyerman 2010). Soil Cl- has been

suggested to be a better predictor than Na+ of growth and grain yield of dryland cereal crops

under salt stress in southwest Queensland where there are high levels of gypsum (CaSO4) in

the soil (Dang et al. 2006a). Under such conditions, electrical conductivity is high because of

the high concentrations of CaSO4 in the soil but, Na+ uptake may be relatively low and thus

the uptake of Cl- relative to Na+ may be high. However the soils of southern Australia have

lower concentrations of gypsum and the importance of Cl- to salt stress in annual grain crops

has generally been overlooked. Chloride toxicity is known to be important in some species,

especially perennial plants (Wieneke and Läuchli 1979; Hajrasuliha 1980; White and

Broadley 2001), but there is little information on its impact on most broadacre crops. Little is

known concerning the primary acquisition mechanisms of Cl- by plants, and knowledge about

its subcellular distribution and flux dynamics is scarce (Britto et al. 2004).

Reliable and effective screening techniques for salt tolerance to predict field performances

are important for breeding programmes. While much of the work on salt tolerance has been

conducted under controlled conditions, it is important to verify whether or not the laboratory

conditions can predict responses to field stresses. Studies using solution culture methods have

failed to address the actual plant response under field conditions, where several

environmental factors are also involved. Thus, there is a clear need to characterise the relative

contributions of the various soil and plant factors that reduce growth and yield over the length

of a growing season.

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The studies conducted in this thesis aimed to quantify the effects of salinity on the growth

and yield of barley and faba bean in relation to the mechanisms responsible for these effects.

The main objectives of this research were to:

quantify the relative importance of ion (Na+ and/or Cl-) toxicity and osmotic stress on

growth and yield reduction under different levels of salinity

determine which of the two ions most frequently implicated in salinity, Na+ and Cl-, is

more toxic to barley and faba bean

investigate whether hydroponic and pot experiments under controlled environmental

conditions are useful plausible surrogates for evaluating whole-plant response to

salinity under field conditions.

The value of the research outcomes of this thesis lie in their potential to improve the

understanding of the soil processes that limit crop productivity in saline-sodic soils. The

experiments described in Chapters 3 and 4 were designed to determine the relative

importance of osmotic stress and specific ion toxicity in barley and faba bean. The results of

these experiments were also used to inform the design of subsequent glasshouse experiments

in Chapters 5 and 6, which determined the extent to which high concentrations of Na+ and Cl-

limit the growth of crop plants. In Chapters 7 and 8 the physiological responses of a relatively

large number of barley and faba bean genotypes to salinity were examined under controlled

conditions and the results compared to ion uptake and yield for these genotypes under field

conditions. These results have been used in the interpretations of the results of glasshouse

studies in Chapters 3-6.

In terms of growth conditions of plants in Chapters 5-8 some precautions were taken to

minimise probable, often unrecognised, artefacts that exist in pot experiments, especially

74

those related to the effects of the water relations and oxygen status of the soil on the

functioning of plants. This has been done by using larger pots (10 cm in diameter and 32cm

in height) in Chapters 5-8 compared to the pots (15 cm in diameter and height) used in

Chapters 3 and 4. As well, using a two-layer soil design (saline-subsoil and non-saline

topsoil) and limiting watering to the saline sub-soil allowed the experiments to simulate the

effects of saline subsoil that occur in the field. In this way it was possible to draw conclusions

regarding soil processes responsible for limitations to crop growth and nutrition in saline-

sodic soils and the results of field trials. The implications of the results of the experiments in

Chapters 3-8 are outlined below.

9.2 Relative importance of ion (Na+ and/or Cl-) toxicity and osmotic

effect to growth and yield reduction under different levels of salinity

Salinity stress is dynamic as osmotic and ionic stresses vary over time (Munns 2002).

Consequently the response of plants to salt stress changes as the importance of the different

components of salt stress – osmotic stress and ion toxicity - and the corresponding tolerance

mechanisms varies. This dynamic nature of salt stress is often overlooked in many studies

which focus on one specific mechanism to improve salt tolerance. Moreover, as different

genotypes have different abilities to exclude Na+ and Cl- the relative effects of ionic and

osmotic effects will also differ.

The experiments in Chapters 3 and 4 were designed to investigate the relative importance of

osmotic stress and ion toxicity in genotypes with different abilities to exclude Na+ and Cl-.

The osmotic effect was examined by using concentrated nutrient solution at similar EC as the

salt treatments. In Chapter 4 a non-destructive, real-time method using image capture and

analysis equipment (LemnaTec ‘Scanalyser 3D’) was used to assess the growth of plants

75

during a greenhouse experiment. These measurements were done in parallel with

measurements of plant water use. The results clearly showed that the relative importance of

ion toxicity and osmotic stress varied with: (1) the degree of stress encountered (mild,

moderate, or severe); (2) the variety which is investigated; (3) the duration of the stress and

(4) the time of sampling. These results were consistently observed in both barley and faba

bean and also in Chapters 7 and 8 in which 15 genotypes of barley and 10 genotypes of faba

bean were screened for salt tolerance at different levels of salinity stress. Plant growth was

strongly reduced by salinity, but non-excluding genotypes showed a greater reduction in

growth than excluding genotypes. Under mild salinity stress (ECFC =4.2 dS m-1), growth of

excluding varieties of barley and faba bean (Clipper for barley and Fiesta for faba bean) was

reduced mainly by osmotic stress due to efficient exclusion of Na+ and Cl-. However, the

efficiency of ion exclusion declined at higher levels of salinity (ECFC=8 and 15 dS m-1) and

specific ion toxicity appeared to be the main cause of reduced growth. At high levels of

salinity stress (15 dS m-1), exclusion of Na+ and Cl- in the excluding genotypes, Clipper and

Fiesta, appeared to contribute less to salt tolerance, however, Fiesta showed a high level of

tolerance to osmotic stress which was reflected its ability to maintain a relatively high plant

moisture content uner increasing salt stress. On the other hand, the reduction in growth of

Cairo (faba bean) compared to Fiesta and Sahara (barley), compared to Clipper, was a result

of NaCl treatments rather than pure osmotic treatments at low salinity stress. However, the

osmotic stress became the major cause of growth reduction with increasing levels of soil

salinity.

The studies in Chapter 8 were conducted to assess the variation among faba bean genotypes

in response to salinity and also to examine which physiological traits can be used to assess

salt tolerance among faba bean genotypes under both field and controlled conditions. The

76

results of screening faba bean genotypes in two levels of salinity stress showed that the

importance of Na+ and Cl- exclusion to salt tolerance varied with the severity of stress: it was

important to salinity tolerance at low levels of salinity (75 mM NaCl) but its importance was

diminished at 150 mM NaCl, when osmotic tolerance became more important (Figure 1 of

Chapter 8). The two mechanisms – ion exclusion and tolerance to osmotic stress – were

independent and altered the relative salt tolerance of genotypes depending on the severity of

stress.

In conclusion, an interpretation of the results in Chapters 3, 4, 7 and 8 is that the difference

between the genotypes at low ECFC (~ 4-8 dS m-1) values is an ion-specific effect, while the

rapid decline in growth above EC 8.5 dS m-1 is a result of both osmotic and ion-toxicity

effects. Osmotic stress was the predominant cause of differences in growth between

genotypes at high levels of salinity, while specific-ion toxicity was more important under

mild salinity stress. It was also shown that genotypes which maintained greater whole-plant

tolerance to salinity had two mechanisms such as tissue tolerance and osmotic adjustment

(Chapter 3) or ion exclusion and osmotic adjustment (Chapter 4), compared to sensitive

genotypes. As the importance of different mechanisms to salinity tolerance differs by the

severity of stress, robust levels of salt tolerance may depend on more than one mechanism

and selection for improved salt tolerance therefore needs to be able to identify these.

9.3 Relative importance of Na+ and Cl- toxicity in growth reduction of

barley and faba bean

Despite the fact that most plants accumulate both sodium (Na+) and chloride(Cl-) ions in high

concentration in their shoot tissues when grown in saline soils, most research on salt

tolerance in annual plants has focused on the toxic effects of Na+ accumulation. Chapters 5

77

and 6 were designed to determine the extent to which specific ion toxicities of Na+ and Cl-

reduce the growth of barley and faba bean plants and to clarify the controversy which exists

in the current literature of salinity research concerning the toxic effects of these two ions on

plant growth (Kingsbury and Epstein 1986; Munns et al. 1988; Kinraide 1999; Dang et al.

2006a; Dang et al. 2008; Slabu et al. 2009). A soil-based design was employed using a

combination of different salts to produce soils enhanced with Na+, Cl- and NaCl with similar

soil solution EC and ΨOThis differs from many earlier studies comparing the effects of Na+

and Cl− in grain crops in which plants were grown under salinity stress and have mainly used

short-term hydroponic experiments (ranging from 2-14 days) with a single salt of Na+ or Cl-

and one or two genotypes (Kingsbury and Epstein 1986; Lin and Kao 2001; Tsai et al. 2004;

Luo et al. 2005; Slabu et al. 2009). Sodium gluconate and the Cl- inhibitor DIDS were also

used to separate the effects of Na+ and Cl- on growth.

9.3.1 Barley

In order to determine which of the two ions is more toxic to growth of barley three

experiments were conducted (Chapter 5). A solution culture experiment was conducted to

assess the effect of different concentrations of Na+ and Cl- ions (0-150mM) on the growth of

Barque73 and Tadmor, which differed in Cl- uptake. In a following solution culture

experiment the relative importance of Na+ and Cl- ions to salt toxicity was assessed in 4

genotypes of barley. The effect of Na+ independently of Cl- was examined in three ways: by

using DIDS, which is a non-permeating amino acid that inhibits Cl- transport (Lin 1981; Lin

and Kao 2001); by using Na+-gluconate because gluconate is an anion that is unable to

permeate the cell membrane; and by using the Na+-Hoagland and Cl--Hoagland solutions.

The Na+-dominant and Cl--dominant Hoagland’s solutions were designed to provide

equimolar concentrations of the Na+ and Cl- ions generated from various salts of Na+ and Cl-

78

to avoid increasing particular counteranions/countercations. Using barley varieties with

known genetic variation in salinity tolerance and in Na+ and Cl- uptake also assisted in

distinguishing the toxic effects of Na+ from Cl-. A third soil based experiment was designed

to simulate the responses in field-grown plants. The method employed here maintained a

constant EC and ΨObut used a combination of different salts to produce soils enhanced with

Na+, Cl- and NaCl.

The results of these studies and also the results from Chapters 3 and 7 indicated that growth

of barley under NaCl stress was caused by the additive effect of the reductions due to Na+

and Cl-. Moreover, the responses to Na+ and Cl- stress among the genotypes were

independent and so similar levels of tolerance to NaCl could be achieved by different

combinations of responses to Na+ and Cl- (Tables 4, 5 and 6 of Chapter 5). This result is

clearly at odds with previous studies that have dismissed Cl- toxicity as a contributing factor

to salt damage (Kingsbury and Epstein 1986; Kinraide 1999; Lin and Kao 2001; Tsai et al.

2004). These previous experiments either used sole counter-anions such as 120 mM nitrate

(Kingsbury and Epstein 1986), which at high concentrations can be phytotoxic (Chen et al.

2004), or were short-term studies lasting less than a week (Kinraide 1999). On the basis of

two-phase model of plant response to salinity stress (Munns et al. 1995) the specific ion

toxicity develops over several weeks when the accumulated Na+ and Cl- reach the toxic

concentration and therefore any interpretation on the basis of short-term experiments may not

be valid.

9.3.2 Faba bean

To assess the relative importance of toxicity of Na+ versus Cl− in faba bean an experiment

was conducted in a field soil using two varieties of faba bean, Nura and line 1487/7 differing

in their ion exclusion mechanism and salt tolerance (Chapter 6). Salinity reduced biomass

79

production and water uptake of faba bean plants (Figure 1, Table 2 of Chapter 5) and from

the results it was clear that plants were more sensitive to Cl- than to Na+. However, the data

also show that when leaf Cl- concentration is high, the presence of Na+ as the dominant cation

exacerbates the severity of the effects (Figure 1 of Chapter 6 and Figures 1 and 2 of Chapter

4).

According to the results of the experiments, it is concluded that concentrations of Na+ and Cl-

have an additive (barley) and/or an interactive (faba bean) effect and high concentrations of

both ions can be harmful for plant growth in saline soils. The results also demonstrated that

Na+ and Cl- exclusion are independent mechanisms, and different genotypes expressed

different combinations of the two mechanisms. Faba bean was more sensitive to Cl- toxicity

than Na+ but the presence of Na+ as dominant cation exacerbates the severity of the

alterations.

The work also showed that the toxic effects of Na+ and Cl- operated through different

mechanisms. A significant correlation between reduced leaf chlorophyll content and the

parameters of chlorophyll fluorescence occurred with increasing Cl- concentration but not

with Na+ concentrations. Using simultaneous measurements of leaf gas exchange and

chlorophyll fluorescence it was shown that there was a significant reduction in both the

efficiency of light harvesting of PSII (F′v/F′m) and actual quantum efficiency of PSII (PSII)

due to high concentrations of Cl and NaCl in soil solution. Moreover, while a significant

reduction in F′v/F′m, PSII and qP of sensitive genotypes under Cl- and NaCl stress was

indicated, the Cl- excluding varieties maintained a higher capacity of PSII system. These

results together with findings in Chapter 3 and 4 on the effects of CaCl2 treatment in growth

dynamics and gas exchange of barley and faba bean, suggest that a high concentration of Cl-

is damaging the photosynthetic apparatus, and that Cl- exclusion is an important mechanism

80

under saline conditions to maintain the function of the chloroplasts. In contrast, Na+ toxicity

operated mainly through stomatal effects and leaf chlorophyll content was not affected or

increased.

The interpretation of these results in relation to research on salt tolerance of crops is that the

toxicity of NaCl is not merely the result of uptake of excess Na+, a belief that lies behind

many attempts to select for salt tolerance on the basis of tissue Na+ levels in grain crops. The

high concentrations of Cl- also can be damaging to crop growth and needs to be taken into

account for the understanding of salt damage and manipulation of salt tolerance.

9.4 Evaluation of crop salt tolerance in solution and soil cultures under controlled

environmental conditions: Are they good surrogates for evaluating whole-plant

response to salinity under field conditions?

Many studies on the mechanisms of salt tolerance have been conducted in nutrient solution or

supported hydroponics (sand or inert growth media flushed with nutrient solution several

times a day) (Munns et al. 2002; Genc et al. 2007). While such systems facilitate the

selection and maintenance of plants, the results do not cover all processes relevant under field

conditions (Vetterlein et al. 2004). In fact there has been little or no work that has directly

compared the responses of different varieties to salt in different growth media or under

controlled conditions and the field. The experiments described in Chapters 3, 7 and 8

compared the responses to salinity in hydroponics and in soil in several varieties of barley

and faba bean known to differ in their salt tolerance and ability to exclude Na+ and to assess

the importance of different mechanisms of salinity tolerance in the two systems under both

controlled and field conditions.

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9.4.1 Barley

Genetic differences in Na+ exclusion, Na+: K+ discrimination and tissue tolerance between

two barley genotypes, Clipper and Sahara were not expressed at any EC level in hydroponics

(Chapter 3). Similar results were obtained in another screening experiment using supported

hydroponics where no significant differences in Na+ exclusion were found between Clipper

(shoot Na+ concentration = 2258 mmol kg-1) and Sahara (shoot Na+ concentration = 2364

mmol kg-1) (Chapter 7). In soil, however, genetic differences in Na+ exclusion between these

two varieties were expressed. Genetic differences in Na+ exclusion have been previously

demonstrated in hydroponics studies (Schachtman et al. 1991; Munns et al. 2006; Genc et al.

2007), but the different results for selected genotypes in this study (Chapters 3 and 7) suggest

that the range in Na+ and Cl- concentration was less in hydroponics than in soil.

The results in Chapters 3 and 7 confirmed the hypothesis that there are differences in the

responses to salinity between plants grown in hydroponic and soil systems, a result that has

important implications for the development of salt tolerant germplasm and for elucidating the

relative importance of the mechanisms of salt tolerance in the field (Chapters 7). These

studies on salt tolerance of barley demonstrated that the genotypic variation observed in

solution culture for differences in salt tolerance, Na+ and Cl- do not reflect those in soil and

the physiological responses are not the same when the same materials were grown in soil in

both pot experiments under controlled conditions and in the field.

While the majority of salt tolerance experiments were conducted in hydroponics and sand

cultures in solution with pH ranges of of 6-7 (Munns 2002; Munns et al. 2006; Genc et al.

2007; Genc et al. 2010a; Genc et al. 2010b), the interactions between root-zone environments

and plant responses to increased osmotic pressure or specific ion concentrations in the field

are complicated by many soil processes such as soil water dynamics, soil structural stability,

82

and solubility of compounds in relation to pH and pE (electron concentration related to redox

potential) (Rengasamy 2010b). However, the effect of pH on salt tolerance and growth of

crops in salt-affected soils has not well understood. Under field conditions, the topsoil can be

sodic while the subsoil is alkaline saline–sodic (Chapter 7). There were differences in pH

among the three methods of assessment but specially between hydroponics and soil and the

effect of this on the consistency between hydroponics and soil is not known. When a salt

tolerant wheat variety was grown in this type of saline-sodic soil, the yield was similar to that

of a less salt-tolerant variety. On further investigation it was found that topsoil sodicity and

subsoil alkaline pH (9.6) prevented the roots from reaching the saline subsoil layer (Cooper

2004; Rengasamy 2010b), salt tolerance character of the wheat variety being not utilised. As

the pH of the soil increases above 8, soil becomes alkaline and carbonates dominate the

anions. Thus, salinity affect plants through adverse soil properties of alkalinity and sodicity,

properties imposed on the soil by mobile salts. Further, alkaline pH induces severe soil

structural problems than neutral sodic soils at comparable sodicity (SAR) levels. A

preliminary investigation showed also that chemistry of aluminium and carbonates in soils is

completely different when the soil pH is above 9.5 compared with pH between 8.2 and 9.5

(Rengasamy 2010b). Further research is needed to assess how pH affects the productivity of

crop growth in saline soils, an area which has been underestimated in current literature.

As well, measuring the biomass production at 70 days after germination has provided an

accurate screen for tolerance of the relative biomass production under saline conditions, and

has revealed substantial variation among genotypes at both levels of salinity stress which was

also predictive of grain yield production in the field (Table 3 of Chapter 7). However, there

was no significant relationship between relative shoot growth of different genotypes and their

salt tolerance at earlier harvests. This finding indicated the unsuitability of using an early

assessment of salinity tolerance at the seedling stage.

83

9.4.2 Faba bean

The studies in Chapter 8 were conducted to assess the variation among faba bean genotypes

in response to salinity and also to examine which physiological traits can be used to assess

salt tolerance among faba bean genotypes under both field and controlled conditions using

supported hydroponics system. The ranking of 11 genotypes of faba bean under both

hydroponics and field conditions was compared with grain yield production under field

condition in 2008 and 2009. The rankings of genotypes based on their salt tolerance in

controlled condition at 75 mM NaCl and grain yield production in the field (rs = 0.85,

P=0.006, n = 11) (Table 3 of Chapter 8), and the crucial parameters of leaf Na+ and Cl-

concentration (rs = 0.88; P = 0.002; n = 11) were significantly correlated. The consistency of

results obtained for physiological responses to salinity stress in hydroponics and soil in faba

bean (Chapters 8) is in contrast to the poor relationship in barley (Chapter 7) and highlights

that the most appropriate screening methods may need to vary with different crop species.

There has been the hope that a better understanding of the physiological basis of salinity

tolerance will also result in the identification of critical genes for which breeders might

select, or new genetic resources that could be manipulated by the tools of molecular biology,

but intensive research have not yet identified a single gene or genetic resource that has been

used by breeders to improve salinity tolerance. Research has been fruitful in identifying

characteristics that are important in accounting for differences in salt tolerance between and

within species (Rawson et al. 1988; Munns and Tester 2008) but to date these have not been

routinely used in breeding to improve salt tolerance. There are fundamental and major

differences between experiments in the glasshouse and/or growth chambers and those in the

84

field as shown for barley in this study. For example plants are or can be subjected to various

stresses, but rarely to several of these at the same time when grown under controlled

conditions. This will of course be the case in nature, where a combination of abiotic stresses

affects overall plant growth. In this respect, although uniform growth conditions are

important to compare results, it is equally or even more important to determine if growth

differences are also apparent under various conditions.

9.5 Conclusions

The research reported in this thesis investigated how the osmotic and specific toxic effects of

salinity interact to reduce plant growth and soil water extraction. By better understanding the

mechanisms by which salinity affects plants and the interactions between soil, plant and

water under saline conditions, improved management of saline soils will be possible. The

results presented here allow greater understanding of the osmotic and toxic components of

salinity, and the influences of environmental conditions which allow further development of

the two phase salinity model of growth crops under salt stress (Munns et al. 1995, Munns and

Tester 2008). In conclusion, the results of this study indicated that:

1. The relative effects of osmotic stress and ion toxicity and the genetic tolerance to

these stresses change as the severity of salinity stress varies. Osmotic stress was the

predominant cause of reduced growth at high levels of salinity, while specific-ion

toxicity was more important under mild salinity stress. This has important

implications for interpreting responses in the field and for the development of

screening techniques because robust levels of salt tolerance may depend on more than

one mechanism and so selection for improved salt tolerance therefore needs to be able

to identify these.

85

2. It was shown that the contribution of Cl- toxicity to salinity stress may have been

underestimated in barley and faba bean and tolerance to high concentrations of Na+

and Cl- are independently controlled. High Na+ interferes with K+ and Ca2+ nutrition

and stomatal regulation, while high Cl- concentration reduces the photosynthetic

capacity due to chlorophyll degradation.

3. In barley, it was indicated that responses to salinity in hydroponic screening are

fundamentally different to those observed in soil. The diverse genotypic variation in

ion exclusion and salinity tolerance found in hydroponics did not correlate with the

result so of pot and field experiments. Also the unsuitability of using early assessment

of salinity tolerance at seedling stage was demonstrated.

4. In contrast to barley, the study of screening faba bean genotypes in hydroponics (at 75

mM NaCl) was shown to be predictive of responses in the field. These results clearly

show that the suitability of a screening method for salinity tolerance differs with the

crop species.

5. In both barley and faba bean, the exclusion of Na+ and Cl- significantly contributed to

salt tolerance and grain yield production in pot and field studies but at high

(ECFC>10dS m-1) levels of salinity the capacity of exclusion alone to maintain salt

tolerance was reduced.

6. It was indicated that salt exclusion coupled with a synthesis of organic solutes are

important components of salt tolerance in the tolerant genotypes and further field tests

86

of these plants under stress conditions will help to verify their potential utility in crop-

improvement programs.

9.6 Recommended future research

The experiments described in this thesis have determined soil processes that limit the growth

of crops in saline-sodic soils. The difference in Na+ and Cl- accumulation between the high

and low Na+ and/or Cl- genotypes was obvious; however, there was no beneficial effect of the

low Na+ and Cl- trait at the highest salinity level (150 mM NaCl or ECFC > 10 dS m-1). To

increase yield at such high salinity, it may be necessary to have additional traits for

adaptation to the osmotic stress such as those resulting in increased water use efficiency to

conserve soil moisture and minimise transient increases in salinity in saline subsoils.

Pyramiding of different traits should result in further increments of salt tolerance.

The thesis has highlighted the lack of information on the contribution of Cl- to salt tolerance

mainly because more is known about Na+ transport mechanisms compared with Cl-. It was

demonstrated that tolerance to soil salinity is complex and strongly linked to Na+ and Cl-

transport, therefore reaffirming the importance of studying anion and cation transport in

parallel. Plant responses to salinity and Cl- transport processes associated with salt tolerance

will vary depending on the species, and even the genotype within a species, as we have

shown for barley and faba bean. This highlights the need for genotypic comparisons of ion

transport processes at the whole plant, organ and cellular level. More accurate measurements

of Cl- concentrations in the vacuole versus cytoplasm in genotypes that vary in salt tolerance

may help identify Cl- transport processes important for salt tolerance. The results also suggest

that salt exclusion coupled with a synthesis of organic solutes are important components of

87

salt tolerance in the tolerant genotypes and further field tests of these plants under stress

conditions will help to verify their potential utility in crop-improvement programs.

Current knowledge of salt tolerance of plants is based on saturation water contents of soils.

However, in dryland conditions, soils are never saturated with water and the field soil water

content changes with seasonal weather conditions. Thus, it is essential to develop new

guidelines on salt tolerance of plant species taking into account soil water dynamics. There is

also a gap in our knowledge in identifying the predominant, or a common, factor when

different issues cause constraints to plant growth in different soil layers. The uncertainty in

our ability to separate effects of these factors will need to be overcome for developing

varieties adapted to various physicochemical constraints, in addition to salinity of soil layers.

Most (>85%) saline-sodic soils in Australia have dense subsoils with an alkaline pH which

alter the electron and proton activities (pE and pH) leading to nutrient ion transformations

which render them unavailable for plant uptake. Rengasamy (2010) proposed a scheme for

classifying salt affected soils based upon key soil properties, namely sodium adsorption ratio

(SAR), electrical conductivity (EC) and pH. Although the impacts of SAR and EC on the

structural behaviour and surface properties of saline-sodic soils have long been recognised,

research on pH-plant salt tolerance related constraints has received little attention. Future

research is needed to identify and quantify the influence of pH on crop salt tolerance.

The discussion on salt tolerance in this thesis clearly shows that a multitude of processes is

involved and this would make it a polygenic trait. Despite the many studies investigating the

mechanisms of salt tolerance, this issue is far from resolved. If multiple salt tolerance

mechanisms exist in plants, they would presumably be encoded by different genes, and not all

88

sources of salt tolerance might be identified with one screening method. Thus, using a single

method to identify salt tolerant accessions could be misleading. Therefore, future work on

salt tolerance needs to focus on using a combination of soil-based screening and field

evaluation to identify salt tolerant genotypes possessing multiple salt tolerance mechanisms.

89

References for literature review and general discussion Almansouri M, Kinet JM,Lutts S (2001). Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.). Plant and Soil 231(243-254), 547-550. Apse MP,Blumwald E (2007). Na+ transport in plants. FEBS Letters 581, 2247-2254. Asch F, Dingkuhn M, Dörffling K,Miezan K (2000). Leaf K/Na ratio predicts salinity induced yield loss in irrigated rice. Euphytica 113(2), 109-118. Ashraf M (1994). Breeding for salinity tolerance in plant. Crit. Rev. Plant Sci. 13, 17-42. Ashraf M (2001). Relationships between growth and gas exchange characteristics in some salt-tolerant amphidiploid Brassica species in relation to their diploid parents. Environmental and Experimental Botany 45(2), 155-163. Ashraf M (2004). Some important physiological selection criteria for salt tolerance in plants. Flora 199, 361-376. Ashraf M,Harris PJC (2004). Potential biochemical indicators of salinity tolerance in plants. Plant Science 166(1), 3-16. Ashraf M,McNeilly T (1988). Variability in salt tolerance of nine spring wheat cultivars. J Agron Crop Sci 160, 14-21. Aslam M, Muhammad N, Qureshi RH, Ahmad Z, Nawaz S,Akhtar J (2003). Calcium and salt-tolerance of rice. Communications in Soil Science and Plant Analysis 34(19/20), 3013-3031. Aslam M, Qureshi RH,Ahmed N (1993). A rapid screening technique for salt tolerance in rice (Oryza sativa L.). Plant and Soil 150, 99-107. Aslam Z, Jeschke WD, Barrett-Lennard EG, Greenway H, Setter TL,Watkin E (1986). Effects of external NaCl on the growth of Atriplex amnicola and the ion relations and carbohydrate status of the leaves. Plant Cell and Environment 9, 571-580. Babourina O, Leonova T, Shabala S,Newman I (2000). Effect of sudden salt stress on ion fluxes in intact wheat suspension cells. Annals of Botany 85, 759-767. Bains SS,Fireman M (1964). Effect of exchangeable sodium percentage on the growth and absorption of essential nutrients and sodium by five crop plants. Agronomy Journal 56, 432-435. Barrett-Lennard EG (1986). Wheat growth on saline waterlogged soils. Department of Agriculture, Western Australia Journal of Agriculture 27, 118-119. Barrett-Lennard EG (2003). The interaction between waterlogging and salinity in higher plants: causes, consequences and implications. Plant and Soil 253, 35-54. Bartels D,Sunkar R (2005). Drought and salt tolerance in plants. Critical reviews in plant sciences 24, 23-58.

90

Belkhodja R, Morales F, Abadia A, Gomez-Aparisi J,Abadia J (1994). Chlorophyll fluorescence as a possible tool for salinity tolerance screening in barley (Hordeum vulgare L.). Plant Physiol. 104(2), 667-673. Ben-Hayyim G, Kafkafi U,Ganmore-Neumann R (1987). Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl2 Concentrations. Plant Physiol. 85(2), 434-439. Bernstein L (1975). Effects of salinity and sodicity on plant growth. Annu. Rev. Phytopathol. 13, 295-312. Bernstein L,Pearson GA (1956). Influence of exchangeable sodium on the yield and chemical composition of plants 1. Soil Science 82, 247-258. Berry JA,Downton WJS (1982). Environmental regulation of photosynthesis. Photosynthesis: development, carbon metabolism, and plant productivity. Govindjee. New York Londo, Academic Press. 2: 263-343. Bhandal IS,Malik CP (1988). Potassium estimation, uptake, and its role in the physiology and metabolism of flowering plants. International Review of Cytology 110, 205-254. Blaha G, Stelzl U, Spahn CMT, Agrawal RK, Frank J,Nierhaus KH (2000). Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy. Rna-Ligand Interactions Pt A. 317: 292-309. Bond WJ (1998). Effluent irrigation – an environmental challenge for soil science. Aust. J. Soil Res 36, 643-555. Bongi G,Loreto F (1989). Gas exchange properties of salt-Stressed olive (Olea europea L.) leaves. Plant Physiol. 90(4), 1408-1416. Boyer JS (1976). Photosynthesis at low water potentials. Philosophical transactions of the royal society 273, 501-512. Bradford MJ, Ferris JE,Remley PA (1987). Interrill soil erosion processes: I. Effect of surface sealing on infiltration, runoff and soil splash detachment. Soil Sci Soc Am J 51, 1566-1571. Bresler E, McNeal BL,Carter DL (1982). Saline and sodic soils. Principles - dynamics - modeling. New York, Springer-Verlag. Britto DT, Ruth TJ, Lapi S,Kronzucker HJ (2004). Cellular and whole-plant chloride dynamics in barley: insights into chloride-nitrogen interactions and salinity responses. Planta 218, 615-622. Campbell GS (1988). Soil water potential measurement: An overview. Irrigation Sciemce 9, 265-273. Castillo EG, Tuong TP, Ismail AM,Inubushi K (2007). Response to salinity in rice: comparative effects of osmotic and ionic stresses. Plant Production Science 10, 159-170. Chauhan R,Chauhan C (1985). Effect of P fertiliser on alleviating chloride toxicity in wheat. Fertiliser Research 6, 171-176. Chavan PD,Karadge BA (1980). Influence of sodium-chloride and sodium-sulfate salinities on photosynthetic carbon assimilation in peanut. Plant and Soil 56, 201-207.

91

Chaves MM, Flexas J,Pinheiro C (2009). Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot 103(4), 551-560. Cheeseman JM (1988). Mechanisms of Salinity Tolerance in Plants. Plant Physiol. 87(3), 547-550. Chen BM, Wang ZH, Li SX, Wang GX, Song HX,Wang XN (2004). Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Science 167(3), 635-643. Chen S, Li J, Wang T, Wang S, Polle A,Hüttermann A (2003). Osmotic stress and ion-specific effects on xylem abscisic acid and the relevance to salinity tolerance in Poplar. Journal of Plant Growth Regulation 21(3), 224-233. Chen Z, Zhou M, Newman IA, Mendham NJ, Zhang G,Shabala S (2007). Potassium and sodium relations in salinised barley tissues as a basis of differential salt tolerance. Functional Plant Biology 34, 150-162. Chinnusamy V, Jagendorf A,Zhu J-K (2005). Understanding and Improving Salt Tolerance in Plants. Crop Sci 45(2), 437-448. Colmer TD, Munns R,Flowers TJ (2005). Improving salt tolerance of wheat and barley: future prospects. Australian Journal of Experimental Agriculture 45, 1425-1443. Cooper DS (2004). Genetics and agronomy of transient salinity in Triticum durum and T.aestivum. PhD Thesis, The University of Adelaide, Australia. Cram WJ (1976). Negative feedback regulation of transport in cells. The maintenance of turgor, volume and nutrient supply. Encyclopaedia of Plant Physiology. U Luttge,MG Pitman. Berlin, Springer-Verlag. 2: 284-316. Cramer GR (1992). Kinetics of maize leaf elongation. 2. Responses of a Na+-excluding cultivar and a Na+-including cultivar to varying Na+ / Ca2+ salinities. Journal of Experimental Botany 43, 857-864. Cramer GR (2002). Response of abscisic acid mutants of Arabidopsis to salinity. Functional Plant Biology 29, 561-567. Cramer GR, Alberico GJ,Schmidt C (1994). Salt tolerance is not associated with the sodium accumulation of two maize hybrids. Functional Plant Biology 21(5), 675-692. Cramer GR, Epstein E,Lauchli A (1989). Na-Ca interactions in barley seedlings: relationship to ion transport and growth. Plant, Cell and Environment 12(5), 551-558. Cramer GR,Lauchli A (1986). Ion Activities in Solution in Relation to Na+ -Ca2+Interactions at the Plasmalemma. J. Exp. Bot. 37(3), 321-330. Cramer GR, Lauchli A,Epstein E (1986). Effects of NaCl and CaCl2 on Ion Activities in Complex Nutrient Solutions and Root Growth of Cotton. Plant Physiol. 81(3), 792-797. Cramer GR, Lauchli A,Polito VS (1985). Displacement of Ca2+ by Na+ from the Plasmalemma of Root Cells : A Primary Response to Salt Stress? Plant Physiology. 79(1), 207-211. Cramer GR, Lynch J, Lauchli A,Epstein E (1987). Influx of Na+, K+, and Ca2+ into Roots of Salt-Stressed Cotton Seedlings : Effects of Supplemental Ca2+. Plant Physiol. 83(3), 510-516.

92

Cresswell HP, Green TW,McKenzie NJ (2008). The adequacy of pressure plate apparatus for determining soil water retention. Soil Sci Soc Am J 72(1), 41-49. Cruz-Romero G,Coleman NT (1975). Reactions responsible for high pH of Na-saturated soils and clays. Soil Science 25, 196-175. Dang Y, McDonald M, Routley R, Dalal RC, Singh D, Orange D,Mann M (2006a). High chloride in subsoil: a key indicator for potential grain yield losses in southwest Queensland. The Australian society of agriculture. Dang YP, Dalal RC, Buck SR, Harms B, Kelly R, Hochman Z, Schwenke GD, Biggs W, Ferguson NJ, Norrish S, Routley R, McDonald M, Hall C, Singh DK, Daniells IG, Farquharson R, Manning W, Speirs S, Grewal HS, Cornish P, N. B,Orange D (2010). Diagnosis, extent, impacts, and management of subsoil constraints in the northern grains cropping region of Australia. Australian Journal of Soil Research 48(2), 105-119. Dang YP, Dalal RC, Mayer DG, McDonald M, Routley R, Schwenke GD, Buck SR, Daniells IG, Singh DK, Manning W,Ferguson N (2008). High subsoil chloride concentrations reduce soil water extraction and crop yield on Vertisols in north-eastern Australia. Australian Journal of Agricultural Research 59, 321-330. Dang YP, Dalal RC, Routley R, Schwenke GD,Daniells I (2006b). Subsoil constraints to grain production in the cropping soils of the north-eastern region of Australia: an overview. Australian Journal of Experimental Agriculture 46(1), 19-35. Del Pilar Cordovilla M, Ligero F,Lluch C (1999). Effect of salinity on growth, nodulation and nitrogen assimilation in nodules of faba bean (Vicia faba L.). Applied Soil Ecology 11(1), 1-7. Devitt D, Stolzy LH,Jarrell WM (1984). Response of sorghum and wheat to different K+/Na+ ratios at varying osmotic potentials. Agronomy Journal 76, 681-688. Diego HS, Felix L, Henning R, Matthew AH, Alexander E, Ute K, Joachim K,Michael KU (2008). Integrative functional genomics of salt acclimatization in the model legume Lotus japonicus. The Plant Journal 53(6), 973-987. Downton WJS (1977). Photosynthesis in salt-stressed grapevines. Aust. J. Plant Physiol. 4, 183-192. Drew MC,Lauchli A (1985). Oxygen-dependent exclusion of sodium ions from shoots by roots of Zea mays (cv Pioneer 3906) in relation to salinity damage. Plant Physiol. 79(1), 171-176. Drew MC,Saker LR (1984). Uptake and long-distance transport of phosphate, potassium and chloride in relation to internal ion concentrations in barley: evidence of non-allosteric regulation. 160(6), 500-507. Dubcovsky J, Santa Maria G, Epstein E, Luo MC,Dvoøák J (1996). Mapping of the K+/Na+ discrimination locus Kna1 in wheat. Theoretical and Applied Genetics 92, 448-454. Duc G, Bao S, Baum M, Redden B, Sadiki M, Suso MJ, Vishniakova M,Zong X (2010). Diversity maintenance and use of Vicia faba L. genetic resources. Field Crops Research 115(3), 270-278. Dudley LM (1994). Salinity in the environment. Handbook of plant and crop stress. M Pessarakli. New York, USA, Marcel Dekker, Inc: 13-30.

93

Dvořak J, Noaman MM, Goyal S,Gorham J (1994). Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination. TAG Theoretical and Applied Genetics 87(7), 872-877. Dyer CL, Kopittke PM, Sheldon AR,Menzies NW (2008). Influence of Soil Moisture Content on Soil Solution Composition. Soil Sci Soc Am J 72(2), 355-361. Eaton FM (1942). Toxicity and accumulation of chloride and sulfate salts in plants. Agricultural research 64(7), 357-399. Eaton FM (1966). Chlorine. Diagnostic criteria for plants and soils. HD Chapman. Riverside, University of California Division of agricultural sciences: 98-135. El-hendawy SE, Hu Y,Schmidhalter U (2005). Growth, ion content, gas exchange, and water relations of wheat genotypes differing in salt tolerances. Australian Journal of Agricultural Research 56, 123-134. Epstein E (1961). The essential role of calcium in selective cation transport by plant cells. Plant Physiology 36, 434-444. Epstein E (1966). Dual pattern of ion absorption by plant cells and by plants. Nature 212, 1324-1327. Epstein E, Norlyn JD, Rush DW, Kingsbury RW, Kelley DB,Cunningham GA (1980). Saline culture of crops: a genetic approach. Science 210, 399-404. Ezlit YD, Smith RJ,Raine SR (2010). A Review of Salinity and Sodicity in Irrigation. CRC for Irrigation Futures Irrigation Matters Series No. 01/10, University of Southern Queensland. Farquhar GD,Sharkey TD (1982). Stomatal conductance and photosynthesis. Annu. Rev. Plant Physiol. 33, 317-345. Felle H (1994). The H+/Cl- symporter in root-hair cells of Sinapis alba. An electrophysiological study using ion-selective microelectrodes. Plant Physiol. 106, 1131-1136. Fitzpatrick RW, Boucher SC, Naidu R,Fritsch E (1994). Environmental consequences of soil sodicity. Aust. J. Soil Res 32, 1069-1093. Flexas J, Bota J, Escalona JM, Sampol B,Medrano H (2002). Effects of drought on photosynthesis in grapevines under field conditions: an evaluation of stomatal and mesophyll limitations. Functional Plant Biology 29, 461-471. Flexas J, Bota J, Loreto F, Cornic G,Sharkey TD (2004). Diffusive and metabolic limitations to photosynthesis under drought and salinity in C3 plants. Plant Biology 6(3), 269-279. Flexas J, Diaz-Espejo A, Galmes J, Kaldenhoff R, Medrano H,Ribas-Carbo M (2007). Rapid variations of mesophyll conductance in response to changes in CO2 concentration around leaves. Plant, Cell & Environment 30, 1284-1298. Flowers TJ (1988). Chloride as a nutrient and as an osmoticum. Adv Plant Nutr 3, 55-78. Flowers TJ (2004). Improving crop salt tolerance. Journal of Experimental Botany 55(396), 307-319. Flowers TJ, Garcia A, Koyama M,Yeo AR (1997). Breeding for salt tolerance in crop plant- the role of molecular biology. Acta physiologiae plantarum 19(4), 427-433.

94

Flowers TJ, Hajibagheri MA,Yeo AR (1991). Ion accumulation in the cell walls of rice plants growing under saline conditions – evidence for the Öertli hypothesis. Plant, Cell and Environment 14, 319-325. Flowers TJ, Troke PF,Yeo AR (1977). Mechanism of salt tolerance in halophytes. Annual Review of Plant Physiology 28, 89-121. Flowers TJ,Yeo AR (1995). Breeding for Salinity Resistance in Crop Plants: Where Next? Functional Plant Biology 22(6), 875-884. Forster BP, Ellis RP, Thomas WTB, Newton AC, Tuberosa R, This D, El-Enein RA, Bahri MH,Ben Salem M (2000). The development and application of molecular markers for abiotic stress tolerance in barley. J. Exp. Bot. 51(342), 19-27. Forster BP, Russell JR, Ellis RP, Handley LL, Robinson D, Hackett CA, Nevo E, Waugh R, Gordon DC, Keith RP,Powell W (1997). Locating genotypes and genes for abiotic stress tolerance in barley: a strategy using maps, markers and the wild species. New Phytologist 137, 141-147. Fortmeier R,Schubert S (1995). Salt tolerance of maize (Zea mays L.): the role of sodium exclusion. Plant, Cell and Environment 18(9), 1041-1047. Francois LE, Grieve CM, Maas EV, Donovan TJ,Lesch SM (1994). Time of salt stress affects growth and yield components of irrigated wheat. Agronomy Journal 86, 100-107. Francois LE,Maas EV (1999). Crop response and management of salt-affected soils. Handbook of Plant and Crop Stress. M Pessarakli. New York, Marcel-Dekker. 2nd edn: 169-201. Garcia A, Senadhira D, Flowers TJ,Yeo AR (1995). The effects of selection for sodium transport and of selection for agronomic characteristics upon salt resistance in rice (Oryza sativa L.). TAG Theoretical and Applied Genetics 90(7), 1106-1111. Genc Y, McDonald GK,Tester M (2007). Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant, Cell and Environment 30(11), 1486-1498. Genc Y, Oldach K, Verbyla AP, Lott G, Hassan M, Tester M, Wallwork H,McDonald GK (2010a). Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress. Theor Appl Genet 121, 877-894. Genc Y, Tester M,Mcdonald GK (2010b). Calcium requirement of wheat in saline and non-saline conditions. Plant & Soil 327, 331-345. Genty B, Briantais JM,Baker NR (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87-92. Ghassemi F, Jakeman AJ,Nix HA (1995). Salinization of land and water resources. Human causes, extent, management and case studies. Sydney: University of New South Wales Press Ltd. Ghoulam C, Foursy A,Fares K (2002). Effects of salt stress on growth, inorganic ions and proline accumulation in relation to osmotic adjustment in five sugar beet cultivars. Environ. Exp. Bot. 47, 39-50.

95

Glen EP,Brown JJ (1998). Effects of soil salt levels on the growth and water use efficiency of Atriplex canescens (Chenopodiaceae) varieties in drying soil. Am. J. Bot 85, 10-16. Gorham J, Jones RGW,Bristol A (1990). Partial Characterization of the Trait for Enhanced K+-Na+ Discrimination in the D-Genome of Wheat. Planta 180(4), 590-597. Gorham J, Papa R,Aloy-Lleonart M (1994). Varietal differences in sodium uptake in barley cultivars exposed to soil salinity or salt spray. Journal of Experimental Botany 45(7), 895-901. Greenway H,Munns R (1980). Mechanisms of salt tolerance in nonhalophytes. Ann. Rev. Plant Physiol. 31, 149-190. Groenevelt PH, Grant CD,Murray RS (2004). On water availability in saline soils. Australian Journal of Soil Research 42, 833-840. Hajrasuliha S (1980). Accumulation and toxicity of chloride in bean plants. Plant and soil 55, 133-138. Hasegawa PM, Bressan RA, Zhu J-K,Bohnert HJ (2000). Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51(1), 463-499. Hochman Z, Dang YP, Schwenke GD, Dalgliesh NP, Routley R, McDonald M, Daniells IG, Manning W,Poulton PL (2007). Simulating the effects of saline and sodic subsoils on wheat crops growing on Vertosols. Australian Journal of Agricultural Research 58(8), 802-810. Huang J,Redmann RE (1995). Responses of growth, morphology and anatomy to salinity and calcium supply in cultivated and wild barley. Canadian Journal of Botany 73, 1859-1866. Husain S, Munns R,Condon AG (2003). Effect of sodium exclusion trait on chlorophyll retention and growth of durum wheat in saline soil. Australian Journal of Agriculture Research 54, 589-597. Hutson JL (1990). Modelling transient root zone salinity. Agricultural salinity assessment and management. KK Tanji. New York, American Society of Civil Engineers: 482–503. Isbell RF (1996). The Australian Soil Classification. Melbourne, CSIRO. Isla R, Aragüés R,Royo A (1998). Validity of various physiological traits as screening criteria for salt tolerance in barley. Field Crops Research 58(2), 97-107. Iwata S, Tabuchi T,Warkentin BP (1994). Soil-water interactions: mechanism and applications. New York, Marcel Dekker. James R, Rivelli AR, Munns R,Caemmere SV (2002). Factors affecting CO2 assimilation, leaf injury and growth in salt-stressed durum wheat. Functional Plant Biology 29, 1393-1403. James RA, Caemmerer SV, Condon AGT, Zwart AB,Munns R (2008). Genetic variation in tolerance to the osmotic stress component of salinity stress in durum wheat. Functional Plant Biology 35(2), 111-123. James RA, Davenport RJ,Munns R (2006). Physiological charaterisation of two genes for Na+ exclusion in durum wheat, NAx1 and Nax2. Plant Physiol. 142, 1537-1547. Jayawardane NS,Chan KY (1994). The management of soil physical properties limiting crop production in Australian sodic soils: a review. Australian Journal of Soil Research 32, 13-44.

96

Jensen ES, Peoples MB,Hauggaard-Nielsen H (2010). Faba bean in cropping systems. Field Crops Research 115(3), 203-216. Jeschke WD,Pate JS (1991). Cation and chloride partitioning through xylem and phloem within the whole plant of Ricinus communis L. under conditions of salt stress. J. Exp. Bot. 42(9), 1105-1116. Jones HG (1976). Moderate-term water stresses and associated changes in some photosynthetic parameters in cotton. New Phytologist 72, 1095-1105. Kafkafi U, Siddiqi MY, Ritchie RJ, Glass ADM,Ruth TL (1992). Reduction of nitrate (13NO3) influx and nitrogen (13N) translocation by tomato and melon varieties after short exposure to calcium and potassium chloride salts. Plant Nutr 15, 959-975. Kapulnik Y, Tueber LR,Phillips DA (1989). Lucerne (Medicago sativa L.) selected for vigor in a nonsaline environment maintained growth under salt stress. Australian Journal of Agricultural Research 40, 1253-1259. Kazman Z, Shainberg I,Gal M (1983). Effects of low levels of exchangable Na and applied phosphogypsum on the infiltration rate of various soils. Soil Science 35, 184-192. Kelly J,Rengasamy P (2006). Diagnosis and management of soil constraints: transient salinity, sodicity and alkalinity. The University of Adelaide and Grain Research and Development Corporation, Australia. Available at http://www.arris.com.au/index.php?id=39 [Verified 24 May 2010]. Kendall HW,Pimentel D (1994). Constraints on the expansion of the global food supply. Ambio 23, 198-205. Kent IM,Lauchli A (1985). Germination and seedling growth of cotton: salinity-calcium interactions. Plant, Cell and Environment 8, 155-159. Kingsbury R,Epstein E (1986). Salt Sensitivity in Wheat. A case for specific ion toxicity. Plant Physiology 80, 651-654. Kingsbury R, Epstein E,Pearcy RW (1984). Physiological responses to salinity in selected lines of wheat. Plant Physiology 74, 417-423. Kinraide TB (1998). Three Mechanisms for the Calcium Alleviation of Mineral Toxicities. Plant Physiol. 118(2), 513-520. Kinraide TB (1999). Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects. Journal of Experimental Botany 50(338), 1495-1505. Krishnamurthy L, Serraj R, Hash C, Dakheel A,Reddy B (2007). Screening sorghum genotypes for salinity tolerant biomass production. Euphytica 156(1), 15-24. Kurth E, Cramer GR, Lauchli A,Epstein E (1986). Effects of NaCl and CaCl2 on Cell Enlargement and Cell Production in Cotton Roots. Plant Physiol. 82(4), 1102-1106. Lauchli A,Wieneke JZ (1979 ). Studies on growth and distribution of Na, K and CI in soybean varieties differing in salt tolerance. Pflanzenernaehr. Bodenkd 142, 3-13.

97

Lawlor DW (1970). Absorption of polyethylene glycols by plants and their effects on plant growth. New Phytologist 69, 501-513. Lawlor DW,Cornic G (2002). Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant, Cell & Environment 25, 275-294. Lea-Cox JD,Syvertsen JP (1993). Salinity Reduces Water Use and Nitrate-N-use Efficiency of Citrus. Ann Bot 72(1), 47-54. Li-juan L, Zhao-hai Y, Zhao-jie Z, Ming-shi X,Han-qing Y (1993). Study and utilization of faba bean germplasm resources. Faba bean in China: State-of-the art review. MC Saxena, S Weigand,L Li-Juan, ICARDA Press: 51-63. Lin CC,Kao CH (2001). Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings. Plant and soil 237(1), 165-171. Lin W (1981). Inhibition of anion transport in corn root protoplast. Plant Physiol 68, 435-438. Longstreth DJ,Nobel PS (1979). Salinity effects on leaf anatomy. Consequences for photosynthesis. Plant Physiol 63, 700-703. Luo Q, Yu B,Liu Y (2005). Differential sensitivity to chloride and sodium ions in seedlings of Glycine max and G. soja under NaCl stress. Journal of Plant Physiology 162(9), 1003-1012. Maas EV,Grieve CM (1987). Sodium-induced calcium deficiency in salt-stressed corn. Plant, Cell & Environment 10(7), 559-564. Maas EV,Hoffman GJ (1977). Crop salt tolerance-current assessment. J. Irrig. Drain. Div. ASCE 103, 115-134. Malhotra RS (1997). Evaluation techniques for abiotic stresses in cool season food legumes. Recent Advances in Pulses Research. AN Asttranu,A Masood. Kanpur, India, Indian Society of Pulses Research and Development: 459-473. Manchanda HR,Sharma SK (1989). Tolerance of chloride and sulfate salinity in chickpea (Cicer arietinum). Journal of Agricultural Science 113, 407-410. Marschner H (1995). Mineral nutrition of Higher Plants, Academic Press. McCown RL, Murtha GG,Smith GD (1976). Assessment of available water storage capacof soils with restricted permeability. Water Resource Research 12, 1255-1259. McCree K,Richardson S (1987). Salt increases the water use efficiency in water stressed plants. Crop Sci 27, 543-547. McIntyre DS (1979). Exchangeable sodium, subplasticity and hydraulic conductivity of someAustralian soils. Australian Journal of Soil Research 17, 115-120. Melesse T,Caesar K (1992). Stomatal and non-stomatal effects of salinity on photosynthesis in Faba Beans (Vicia faba L.). Journal of Agronomy and Crop Science 168(5), 345-353. Mittler R,Blumwald E (2010). Genetic engineering for modern agriculture: challenges and perspectives. Annu. Rev. Plant Biol 61, 443-462.

98

Moller IS, Gilliham M, Jha D, Mayo GM, Roy SJ, Coates JC, Haseloff J,Tester M (2009). Shoot Na+ exclusion and increased salinity tolerance engineered by cell Type-Specific alteration of Na+ transport in Arabidopsis. Plant Cell, tpc.108.064568. Morgan JM, Rodriguez-Maribona B,Knights EJ (1991). Adaptation to water-deficit in chickpea breeding lines by osmoregulation: relationship to grain yields in the field. 27, 61-70. Munns R (1985). Na+, K+ and Cl- in xylem sap flowing to shoots of NaCl-treated barley. Journal of Experimental Botany 36(168), 1032-1042. Munns R (1993). Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant cell environment 16, 15-24. Munns R (2002). Comparative physiology of salt and water stress. Plant, Cell and Environment 25, 239-250. Munns R (2007). Utilizing genetic resources to enhance productivity of saltprone land: published as part of a theme on salt-prone land resources. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2, 1-11. Munns R, Gardner PA, Tonnet ML,Rawson HM (1988). Growth and development in NaCl-Treated plants. II. Do Na+ or Cl- concentrations in dividing or expanding tissues determine growth in barley? Functional Plant Biology 15(4), 529-540. Munns R, Hare RA, James RA,Rebetzke GJ (2000a). Genetic variation for improving the salt tolerance of durum wheat. Aust. J. Agric. Res. 51, 69-74. Munns R, Husain S, Rivelli AR, James R, Condon AG, Lindsay M, Lagudah ES, Schachtman DP,Hare RA (2002). Avenues for increasing salt tolerance of crops, and the role of physiologically based selection traits. Plant and Soil 247, 93-105. Munns R,James RA (2003). Screening methods for salinity tolerance: a case study with tetraploid wheat. Plant and soil 253, 201-218. Munns R, James RA,Lauchli A (2006). Approaches to increasing the salt tolerance of wheat and other cereals. Journal of Experimental Botany 57(5), 1025-1043. Munns R, Passioura JB, Guo J, Chazen O,Cramer GR (2000b). Water relations and leaf expansion: importance of time scale. J. Exp. Bot. 51(350), 1495-1504. Munns R, Rebetzke GJ, Husain S, James R,Hare R (2003). Genetic control of sodium exclusion in durum wheat. Australian Journal of Agriculture Research 54, 627-635. Munns R, Schachtman DP,Condon AG (1995). The significance of a two-phase growth response to salinity in wheat and barley. Australian Journal of Agricultural Research 22, 561-569. Munns R,Termaat A (1986). Whole-plant responses to salinity. Australian Journal of Agricultural Research 13, 143-160. Munns R,Tester M (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651-681.

99

Murillo-Amador B, Lopez-Aguilar R, Kaya C, Larrinaga-Mayoral J,Flores-Hernandez A (2002). Comparative effects of NaCl and polyethylene glycol on germination, emergence and seedling growth of cowpea. Journal of Agronomy and Crop Science 188, 235-247. Nature E (2010). How to feed a hungry world. Nature 466(7306), 531-532. Netondo GW, Onyango JC,Beck E (2004). Sorghum and Salinity: II. Gas Exchange and Chlorophyll Fluorescence of Sorghum under Salt Stress. Crop Sci 44(3), 806-811. Nonami H,Boyer JS (1990). Primary events regulating stem growth at low water potentials. Plant Physiol. 93(4), 1601-1609. Norlyn JD,Epstein E (1982). Barley production: irrigation with seawater on coastal soil. Biosaline research: a look to the future. A San Pietro. New York, Plenum: 525-529. Nuttall JG, Armstrong RD,Connor DJ (2003). Evaluating physicochemical constraints of Calcarosols on wheat yield in the Victorian southern Mallee. Australian Journal of Agricultural Research 54, 487-497. Nuttall JG, Armstrong RD,Connor DJ (2006). Early growth of wheat is more sensitive to salinity than boron at levels encountered in alkaline soils of south-eastern Australia. Australian Journal of Experimental Agriculture 46, 1507-1514. Nuttall JG, Hobson KB, Materne M, Moody DB, Munns R,Armstrong RD (2010). Use of genetic tolerance in grain crops to overcome subsoil constraints in alkaline cropping soils. Australian Journal of Soil Research 48, 188-199. Öertli JJ (1968). Extracellular salt accumulation, a possible mechanism of salt injury in plants. Agrochimica 12, 461-469. Pessarakli M (1991). Formation of saline and sodic soils and their reclamation. J. Envriron. Sci. Health 26, 1303-1320. Pimentel D, Harman R, Pacenza M, Pecarsky J,Pimentel M (1994). Natural resources and an optimum human population. Population and Environment 15, 347-369. Pimentel D, Hurd LE, Bellotti AC, Forster MJ, Oka IN, Sholes OD,Whitman RJ (1973). Food production and the energy crisis. Science 182(4111), 443-449. Pooviah BW,Reddy ASN (1987). Calcium messenger systems in plants. CRC Critical reviews in Plant Science 6, 47-103. Qadir M,Schubert S (2002). Degradation processes and nutrient constraints in sodic soils. Land Degradation & Development 13, 275-294. Quirk JP (2001). The significance of the threshold and turbidity concentrations in relation to sodicity and microstructure. Australian Journal of Soil Research 39, 1185-1217. Quirk JP,Schofield RK (1955). The effect of electrolyte concentration on soil permeability. Journal of Soil Science 6, 163-178. Rajendran K, Tester M,Roy SJ (2009). Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment 32(32), 237.

100

Rawson HM,Munns R (1984). Leaf expansion in sunflower as influenced by salinity and short-term changes in carbon fixation. Plant Cell Environ 7, 207-213. Rawson HM, Richards RA,Munns R (1988). An examination of selection criteria for salt tolerance in wheat, barley and triticale genotypes. Australian Journal of Agricultural Research 39, 759-772. Reid JR,Smith FA (2000). The limits of sodium/calcium interactions in plant growth. Australian Journal of Plant Physiology 27, 709-715. Rengasamy P (2002). Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. Australian Journal of Experimental Agriculture 42, 351-361. Rengasamy P (2006). World salinization with emphasis on Australia. Journal of Experimental Botany 57(5), 1017-1023. Rengasamy P (2010a). Osmotic and ionic effects of various electrolytes on the growth of wheat. Australian Journal of Soil Research 48 120-124. Rengasamy P (2010b). Soil processes affecting crop production in salt-affected soils. Functional Plant Biology 37, 613-620. Rengasamy P,Marchuk A (2010). Cation ratio of soil structural stability (CROSS). Australian Journal of Soil Research 49, 1-8. Rengasamy P,Olsson KA (1991). Sodicity and soil structure. Australian Journal of Soil Research 29, 935-952. Rengasamy P,Sumner ME (1998). Processes involved in sodic behaviour. Sodic soils: distribution, properties, management and environmental consequences. ME Sumner,R Naidu, Oxford University. Rhoades JD, Manteghi NA, Shouse PJ,Alves WJ (1989). Soil electrical conductivity and soil salinity: New formulations and calibrations. Soil Sci. Soc. Am. J. 53(2), 433-439. Richards RA (1983). Should selection for yield in saline regions be made on saline or non-saline soils? Euphytica 32(2), 431-438. Richards RA, Dennett CW, Qualset CO, Epstein E, Norlyn JD,Winslow MD (1987). Variation in yield of grain and biomass in wheat, barley, and triticale in a salt-affected field. Field Crops Research 15(3-4), 277-287. Rispail N, Kaló P, Kiss GB, Ellis THN, Gallardo K, Thompson RD, Prats E, Larrainzar E, Ladrera R, González EM, Arrese-Igor C, Ferguson BJ, Gresshoff PM,Rubiales D (2010). Model legumes contribute to faba bean breeding. Field Crops Research 15(3), 253-269. Rogers ME,Noble CL (1992). Variation in growth and ion accumulation between two selected populations of Trifolium repens L. differing in salt tolerance. Plant and soil 146(1), 131-136. Rogers ME, Noble CL, Halloran GM,Nicolas ME (1997). Selecting for salt tolerance in white clover (Trifolium repens): chloride ion exclusion and its heritability. New Phytologist 135, 645-654. Royo A,Aragüés R (1999). Salinity-yield response functions of barley genotypes assessed with a triple line source sprinkler system. Plant and soil 209(1), 9-20.

101

Sanders D (1980). The mechanism of Cl- transport at the plasma-membrane of characorallina. 1. Cotransport with H+. J. Membr. Biol. 53, 129-141. Schachtman DP, Munns R,Whitecross MI (1991). Variation in sodium exclusion and salt tolerance in Triticum tauschii. Crop Sci 31(4), 992-997. Serrano R, Mulet J, Rios G, Marquez J, de Larrinoa I, Leube M, Mendizabal I, Pascual-Ahuir A, Proft M, Ros R,Montesinos C (1999). A glimpse of the mechanisms of ion homeostasis during salt stress. J. Exp. Bot. 50(suppl_1), 1023-1036. Shabala S, Shabala L,Volkenburgh EV (2003). Effect of calcium on root development and root ion fluxes in salinised barley seedlings. Functional Plant Biology 30(5), 507-514. Shainberg I,Caiserman A (1971). Studies on Na/Ca montmorillonite systems. II. The hydraulic conductivity. Soil Sci. 11, 111: 276. Shalhevet J (1993). Plants under water and salt stress. Plant adaptation to environmental stress. L Fowden, T Mansfield,J Stoddart. New York, Chapman and Hall: 133-154. Shannon MC (1997). Adaptation of plants to salinity. Adv. Agron. 60, 75-120. Sharkey TD (1990). Water stress effects on photosynthesis. Photosynthetica 24, 651-658. Shavrukov Y, Gupta N, Miyazaki J, Baho M, Chalmers K, Tester M, Langridge P,Collins N (2010). HvNax3—a locus controlling shoot sodium exclusion derived from wild barley (Hordeum vulgare ssp. spontaneum). Functional &amp; Integrative Genomics 10(2), 277-291. Shomer-Ilan a, Jones GP,Paleg LG (1991). In vitro Thermal and Salt Stability of Pyruvate Kinase Are Increased by Proline Analogues and Trigonelline. Australian Journal of Plant Physiology 18, 279-286. Singh S,Singh M (2000). Genotypic basis of response to saliniity stress in some crosses of spring wheat Triticum aestivum L. Euphytica 115(3), 209-214. Skerrett M,Tyerman SD (1994). A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots. Planta 192, 295-305. Slabu C, Zörb C, Steffens D,Schubert S (2009). Is salt stress of faba bean (Vicia faba) caused by Na+ or Cl- toxicity? J. Plant Nutr. Soil Sci. 172, 644-650. Sohan D, Jasoni R,Zajicek J (1999). Plant-water relations of NaCl and calcium-treated sunflower plants. Envriron. Exp. Bot 42, 105-111. Srivastava JP,Jana S (1984). Screening wheat and barley germplasm for salt tolerance. Salinity tolerance in plants: strategies for crop improvement. RC Staples,GH Toenniessen. New York, Wiley: 273-283. Stoddard FL, Balko C, Erskin W, Khan HR, Link W,Sarkar A (2006). Screening techniques and sources of resistance to abiotic stresses in cool-season food legumes. Euphytica 147, 167-186. Storey R,Walker RR (1999). Citrus and salinity. Sci. Hort. 78, 39-81. Sumner ME (1993). Sodic soils - new perspectives. Australian Journal of Soil Research 31(6), 683-750.

102

Sumner ME (1995). Sodic soils: new perspectives. Australian Sodic Soils: Distribution, Properties and Management. ME Naidu,P Rengasamy. Australia, CSIRO. Szabolcs I (1994). Soils and salinization. Handbook of Plant and Crop stress. M Pessarakli. New York, Marcel Dekker: 3-11. Tanji KK (1990). Nature and extent of agriculture salinity. Agricultural salinity assessment and management, Manual and Report on Engineering Practice. KK Tanji. New York, American Society of Civil Engineers: 1-17. Teakle NL,Tyerman SD (2010). Mechanisms of Cl- transport contributing to salt tolerance. Plant, Cell & Environment 33, 566-589. Tester M,Davenport R (2003). Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91, 503-527. Tsai YC, Hong C-Y, Liu L-F,Kao H (2004). Relative importnace of Na+ and Cl- in NaCl-induced antioxidant systems in roots of rice seedlings. Physiologia Plantarum 122, 86-94. Turner NC, Wright GC,Siddique KHM (2001). Adaptation of grain legumes (pulses) to water-limited environments. Advances in Agronomy, Academic Press. Volume 71: 193-231. Ueda A, Kathiresan A, Inada M, Narita Y, Nakamura T, Shi W, Takabe T,Bennett J (2004). Osmotic stress in barley regulates expression of a different set of genes than salt stress does. J. Exp. Bot. 55(406), 2213-2218. USSL (1954). Diagnosis and improvement of saline and alkali soils. Washington DC, USDA. Vetterlein D, Kuhn K, Schubert S,Jahn R (2004). Consequences of sodium exclusion on the osmotic potential in the rhizosphere- Comparing of two maize cultivars differing in Na+ uptake. Plant Nutrition and Soil Science 167, 337-344. Weimberg R (1983). Induction of solute release from nicotiana tabacum tissue cell suspensions by polymyxin and EDTA. Journal of Experimental Botany 34(147), 1333-1346. Westgate ME,Boyer JS (1985). Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize. Planta 164, 540-549. Wetson AM,Flowers TJ (2010). The effect of saline hypoxia on growth and ion uptake in Suaeda maritima. Functional Plant Biology 37, 646-655. White PJ,Broadley MR (2001). Chloride in Soils and its Uptake and Movement within the Plant: A Review. Annals of Botany 88(6), 967-988. Wieneke J,Läuchli A (1979). Short-term studies on the uptake and transport of Cl- by soybean cultivars differing in salt tolerance. Plant Nutrition and Soil Science 142(6), 799-814. Wright D,Raiper I (2000). An assessment of the relative effects of adverse physical and chemical properties of sodic soil on the growth and yield of wheat (Triticum aestivum L.). Plant and soil 223, 277-285. Xu G, Magen H, Tarchitzky J,Kafkafi U (2000). Advances in chloride nutrition of plants. Advances in Agronomy 68, 97-150.

103

Yamashita K, Kasai M, Yamamoto Y,Matsumoto H (1994). Stimulation of plasmamembrane H+-transport activity in barley roots by salt stress – possible role of increase in chloride permeability. Soil Sci. Plant Nutr. 40, 555-563. Yeo A (1998). Molecular biology of salt tolerance in the context of whole-plant physiology. J. Exp. Bot. 49(323), 915-929. Yeo AR, Lee S, Izard P, Boursier PJ,Flowers TJ (1991). Short- and Long-Term Effects of Salinity on Leaf Growth in Rice (Oryza sativa L.). J. Exp. Bot. 42(7), 881-889. Yermiyahu U, Nir S, Ben-Hayyim G,Kafkafi U (1994). Quantitative competition of calcium with sodium or magnesium for sorption sites on plasma membrane vesicles of melon (Cucumis melo L.) root cells. Journal of Membrane Biology 138, 55-63. Yermiyahu U, Nir S, Ben-Hayyim G, Kafkafi U,Kinraide TB (1997). Root elongation in saline solution related to calcium binding to root cell plasma membranes Plant and soil 191(10), 67-76. Ziska LH, Seemann JR,DeJong TM (1990). Salinity induced limitations on photosynthesis in Prunus salicina, a deciduous tree species. Plant Physiology 93(3), 864-870.