the role of potassium in the improvement of …...this project increased our understanding of the...

The Role of Potassium in the Improvement of Growth, Water

Use and Yield of Canola under Varying Soil Water Conditions

Max Bergmann B.Sc. M.Sc. Agricultural Science

M.Sc. Management

This thesis is presented for the degree of Doctor of Philosophy

The University of Western Australia

School of Plant Biology

Faculty of Science

April 2016

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Abstract

Canola production in Western Australia is vulnerable to large variations in rainfall and

frequent droughts. Recent poor seasons and price volatility have contributed to

farmers’ perceptions of risk with canola and it can be observed that the area of canola

grown often declines after periods of drought. Nevertheless, the total area of canola

grown in Western Australia has still increased over the last five years and the

importance of the crop has become even more apparent. An increase in the area of

potassium deficient agricultural sandy soils and the prediction of drier years in the

Western Australian wheat belt has been identified. Therefore, more work is needed to

enhance the current knowledge on plant physiological responses to potassium

deprivation and drought stress particularly for canola.

The first objective was to measure drought tolerance of canola under different

potassium treatments. The second objective was to understand the mechanisms

controlling drought tolerance of canola by comparing the response of varying

potassium treatments during a drying cycle as well as monitoring the ability to recover

after the drought stress. The third objective was to observe any morphological

differences of canola plants exposed to different potassium levels and water stress. In

order to achieve these objectives, a range of climate chamber, glasshouse and field

experiments were conducted growing Trigold canola plants in sandy soils and

hydroponically, mixed with increasing potassium concentrations, which were subjected

to drought stress.

This project increased our understanding of the effect of potassium nutrition on the

drought response of canola. A mild potassium deficiency (40 mg kg-1 soil potassium)

only decreases the dry weight of canola significantly if plants are exposed to a long-

term potassium deprivation. The ability to maintain a similar size over a range of

applied potassium levels could be due to a morphological mechanism that might have

evolved in canola to manipulate root growth towards a smaller shoot:root ratio. The low

K treatments had increased transpiration and stomatal conductance and reduced

relative turgor pressure compared with high K, under drought stress. This was due to

the stomata remaining open in the low k and closed in the high K. Canola with a higher

supply of potassium also had a higher rate of photosynthesis under water stress than

canola with a low level of applied potassium. The opening of the stomata and

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associated higher transpiration rate and stomatal conductance may be caused by an

interaction effect of the plant hormones abscisic acid and ethylene depending on the

level of potassium and water stress that the canola plant is exposed to.

Thus, further work should focus on measuring abscisic acid and ethylene levels under

varying water and potassium treatments in order to understand the mechanisms behind

this response and how best to mitigate the effects of the stress. In addition to that,

research should be done on the interaction between potassium and drought by

studying the canola plants grown continually at different soil moisture and potassium

levels in particular focusing on the 40-60 mg kg-1 soil potassium level where most

changes have been observed in order to determine any effects on plant size, shoot:root

ratio, relative turgor pressure and transpiration per leaf area. Especially to monitor any

changes in shoot:root ratio, which may be a useful morphological trait for selection of

drought tolerance in canola. The interaction between nitrogen and potassium on canola

response to drought also needs further investigation because it may be necessary to

apply higher amounts of potassium fertiliser when applying relatively high nitrogen

rates.

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Table of Contents

Abstract I Acknowledgements XV

Thesis Declaration XIX

List of Abbreviations XX

Chapter 1: General Introduction 1

Chapter 2: Literature Review 5

2.1 The Biology and Agronomy of Canola 5

2.2 The Role of Potassium and the Effect of Drought in Plants 8

2.3 Root Growth, Water and Nutrient Uptake in Plants 9

2.4 Potassium Uptake 11

2.5 Movement of Potassium Ions and Assimilates in Plants 13

2.6 Accumulation Patterns of Potassium and the Effect of Potassium Deficiency

on Plant Morphology 15

2.7 The Effect of Potassium on Stomatal Regulation and Osmotic Adjustment 17

2.8 The Influence of Potassium on Photosynthesis 20

2.9 The Role of Potassium under Drought Conditions and the Impact of

Potassium Starvation on Plant Physiology 20

2.10 Water Dynamics in Plants 22

2.11 Water Use Efficiency 22

2.12 Water Transport in Plants 23

2.13 Leaf Water Potential Measurements in Plants 27

2.14 Conclusion 31

Chapter 3: Effect of Drought and Potassium on Plant Water Relations of Canola and Its Recovery after Watering 33

3.1 Introduction 33

3.2 Methodology 36

3.2.1 Soil and Pot Preparation 36

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3.2.2 Potassium 38

3.2.3 Watering 40

3.2.4 Relative Turgor Pressure 41

3.2.5 Leaf Water Potential in Experiment 2 42

3.2.6 Osmotic Potential 42

3.2.7 Photosynthesis, Stomatal Conductance and Transpiration 42

3.2.8 Leaf Temperature 43

3.2.9 Harvest 44

3.2.10 Experimental Design and Statistical Analysis 44

3.3 Results 44

3.3.1 Environmental Conditions 45

3.3.2 Experiment 1 47

3.3.2.1 Leaf Area 47

3.3.2.2 Shoot and Root Dry Weight 47

3.3.2.3 Leaf Temperature 49

3.3.2.4 Water Use 50

3.3.2.5 Transpiration per Leaf Area 51

3.3.2.6 Transpiration Rate 53

3.3.2.7 Stomatal Conductance 54

3.3.2.8 Rate of Photosynthesis during Two Consecutive Drying Cycles 54

3.3.2.9 Relative Turgor Pressure during Drought Stress 56


3.3.3.1 Leaf Area 57

3.3.3.2 Root and Shoot Dry Weight 58

3.3.3.3 Pod Number 59

3.3.3.4 Leaf Water Potential 60

3.3.3.5 Diurnal Changes of Photosynthesis, Transpiration and Stomatal

Conductance 60

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3.3.3.6 Short Term Recovery 64

3.4 Discussion 67

3.4.1 Plant Morphological Changes to Potassium and Drought Stress 67

3.4.2 Plant Physiological Changes to Potassium and Drought Stress 69

3.5. Conclusion 73

Chapter 4: Short Term Plant Water Relations of Canola Grown in Nutrient Solution and Exposed to Rapid Potassium and Water Stress 75

4.1 Introduction 75

4.2 Methodology 77

4.2.1 Seed and Pot Preparation 77

4.2.2 Transplantation of Seedlings 78

4.2.3 Nutrient Composition and Change of Nutrient Solution 78

4.2.3.1 Potassium Treatments 79

4.2.4 Water Stress 79


4.2.6 Pot Weight and Transpiration per Leaf Area 82

4.2.7 Stomatal Conductance, Transpiration rate and Photosynthesis 82

4.2.8 Harvest and Isotope Analysis 83

4.2.9 Experimental Design and Statistical Analysis 83

4.3 Results 84

4.3.1 Environmental Conditions 84

4.3.2 Leaf Area 85

4.3.3 Shoot and Root Dry Weights 86

4.3.4 Potassium Concentration 87

4.3.5 Isotope Analysis of Shoot Material 88

4.3.6 Transpiration per Leaf Area 89

4.3.7 Transpiration Rate 95

4.3.8 Stomatal Conductance 92

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4.3.9 Photosynthesis 97


4.4 Discussion 97

4.4.1 Changes in Relative Turgor Pressure and Transpiration per Leaf Area

under Water Stress and Different Potassium Levels 97

4.4.2 Morphological Impacts of Potassium on Canola under Drought 98

4.4.3 Potassium Distribution in Water Stressed Canola and the Effect on

Stomatal Conductance and Photosynthesis 99

4.5 Conclusion 101

Chapter 5: The Longer Term Effects of Potassium and Water Stress on Canola Growth and Transpiration Rate 103

5.1 Introduction 103

5.2 Methodology 104

5.2.1 Pot and Soil Preparation 105

5.2.2 Planting 107

5.2.3 Pot Weight and Transpiration per Leaf Area 107


5.2.5 Harvest 108

5.3 Results 109


5.3.1.1 Leaf Area 109

5.3.1.2 Shoot and Root Dry Weight, and Shoot:Root Ratio 110

5.3.1.3 Root Diameter 112

5.3.1.4 Potassium Concentration in Shoot and Root, as well as Shoot:Root

Ratio 112

5.3.1.5 Pot Weights and Transpiration per Leaf Area 114

5.3.1.6 Relative Turgor Pressure 116


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5.3.2.1 Leaf Area 119

5.3.2.2 Shoot and Root Dry Weight, and Shoot:Root Ratio 119

5.3.2.3 Root Diameter 121

5.3.2.4 Potassium Concentration in Shoot and Root, as well as Shoot:Root

Ratio 121

5.3.2.5 Pot Weights and Transpiration per Leaf Area 124

5.3.2.6 Relative Turgor Pressure 128

5.4 Discussion 130

5.4.1 Changes in Dry Weight of Longer Term Water and Potassium

stressed Canola 131

5.4.2 Potassium Concentration 133

5.4.3 Response of Transpiration per Leaf Area and Relative Turgor

Pressure under Drought 134

5.5 Conclusion 136

Chapter 6: The Role of Potassium in Water Relations and Yield of Field Grown Canola in the WA Grain Belt 139

6.1 Introduction 139

6.2 Methodology 140

6.2.1 Experimental Design 140

6.2.2 Soil analysis 141

6.2.3 Measurements 141

6.2.3.1 Biomass Cuts 141

6.2.3.2 Photosynthesis, Stomatal Conductance and Transpiration Rate 142

6.2.3.3 Leaf Temperature 142

6.2.3.4 Leaf Water Potential 142

6.2.3.5 Osmotic Potential 143

6.2.4 Harvest 143

6.2.5 Statistical Analysis 143

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6.3 Results 143

6.3.1 Soil and Climate 143

6.3.2 Biomass Cuts and Grain Yield 144

6.3.3 Shoot Potassium and Nitrogen Concentration 146

6.3.4 Canopy Temperature 147

6.3.5 Transpiration Rate 148

6.3.6 Stomatal Conductance 149

6.3.7 Photosynthesis Rate 149

6.3.8 Leaf Water Potential 150

6.4 Discussion 151

6.4.1 Environmental Growth Conditions and Biomass Production of Field

Grown Canola 151

6.4.2 Shoot Potassium and Nitrogen Concentration 153

6.4.3 The Effect of Potassium and Nitrogen on Transpiration Rate, Stomatal

Conductance and Photosynthesis of Field Grown Canola 154

6.5 Conclusion 155

Chapter 7: General Discussion and Conclusion 157

7.1 Impact of potassium on Growth in Canola Suffering Under Water Stress 157

7.2 Potassium Concentration and the Impact on Transpiration, Stomatal

Conductance, Photosynthesis and Relative Turgor Pressure in Canola

under Drought 159

References 163

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List of Figures

Figure 2.1: BBCH decimal system of Brassica napus growth stages. 7

Figure 2.2: Schematic diagram of the leaf clamp pressure probe. 29

Figure 3.1: Soil water retention curve of experimental soil. 36

Figure 3.2: Light response curve measured for Trigold Canola used in study. 40

Figure 3.3: Temperature (A), relative humidity (B) and ambient light level (C)

during Experiments 1 and 2 lasting from 31 July 2013 to 20

September 2013. 43

Figure 3.4: Mean leaf area per pot of drought stressed (D) and well-watered (C) canola

plants treated with different potassium rates (no K, low K,

high K) measured on the day of harvest. 44

Figure 3.5: Average dry weights of shoot (A) and root (B), and shoot:root ratio (C)

of drought-stressed (D) and control (C) plants treated with three different

potassium rates (no K, low K, high K). 45

Figure 3.6: Difference between leaf and ambient temperature of the drought-

stressed (D) and well-watered (C) canola plants treated with three

different potassium rates (no K, low K, high K). 46

Figure 3.7: Difference between leaf and ambient temperature of the drought-

stressed (D) and well-watered (C) canola plants treated with three

different potassium rates (no K, low K, high K) on days 10 and 13 of

the experiment. 47

Figure 3.8: Pot weights of drought stressed (D) and control (C) samples treated with

three different potassium rates (no K, low K, high K) during two

consecutive drying cycles. 48

Figure 3.9: Transpiration per leaf area of drought-stressed (D) and control (C)

samples treated with three different potassium rates (no K, low K, high

K) during two consecutive drying cycles measured by weight. 49

Figure 3.10: Transpiration rate of drought-stressed (D) and control (C) samples

treated with three different potassium rates (no K, low K, high K)

during two consecutive drying cycles. 50

Figure 3.11: Stomatal conductance of drought-stressed (D) and control (C) samples



Figure 3.12: Photosynthetic rate of drought-stressed (D) and control (C) samples


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Figure 3.13: Examples of Pp measurements on canola subjected to different

potassium treatments. 53

Figure 3.14: Leaf area of drought-stressed (D) and well-watered (C) plants treated

with three different potassium rates (no K, low K, high K). 54

Figure 3.15: Root and shoot dry weight of drought-stressed (D) and well-watered (C)

plants treated with three different potassium rates (no K, low K,

high K). 55

Figure 3.16: Shoot:root ratio of drought-stressed (D) and well-watered (C) plants

treated with three different potassium rates (no K, low K, high K). 55

Figure 3.17: Pod number of drought-stressed (D) and well-watered (C) plants

treated with three different potassium rates (no K, low K, high K). 56

Figure 3.18: Midday leaf water potential of drought-stressed (D) and well-watered

(C) plants treated with three different potassium rates (no K, low K, high

K) measured on day 1 of the experiment. 57

Figure 3.19: Temperature and relative humidity on the day of the diurnal

measurements (18 September 2014). 58

Figure 3.20: Average diurnal transpiration (A), stomatal conductance (B) and

photosynthesis (C) of control plants (n = 5) treated with three different

potassium rates (no K, low K, high K) measured on day 1 of the

experiment. 59

Figure 3.21: Short term recover of transpiration, stomatal conductance and

photosynthesis of plants treated with three different potassium levels

(no K, low K, high K) measured 0-1 h, 1-2 h and 2-3 h after watering. 65

Figure 3.22: Examples of Pp measurements on canola subjected to different K

treatments (no K, low K, high K) and the corresponding ambient

temperature and relative humidity. 66

Figure 4.1: Overview of experimental set up showing canola plants in nutrient

solution on automated balance system. 78

Figure 4.2: Temperature and relative humidity during the experiment starting on

4 March 2013 and ending on 13 March 2013. 85

Figure 4.3: Average leaf areas of the three different drought-stressed

potassium treatments (no K, low K, high K). 86

Figure 4.4: Mean shoot and root dry weights and shoot to root ratio for the three

drought-stressed potassium treatments (no K, low K, high K). 86

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Figure 4.5: Average potassium concentration in shoot and root of drought-

stressed canola plants treated with three different potassium rates (no

K, low K, high K) and shoot to root ratio of potassium concentration. 87

Figure 4.6: Average 13C/12C (A) and 18O/16O (B) stable isotope ratios of the

drought-stressed canola plants treated with three different

potassium rates (no K, low K, high K). 88

Figure 4.7: Transpiration per leaf area of canola plants treated with three different

potassium treatments (no K, low K, high K) undergoing a 3- (A) and

4-day (B) drying cycle, respectively. 90

Figure 4.8: Average transpiration rate of PEG- and non-PEG treated no and high K

fertilized canola plants. 91

Figure 4.9: Average daily stomatal conductance of drought-stressed canola plants


undergoing (A) 3- and (B) 4-day drying cycle, respectively. 92

Figure 4.10: Average photosynthetic rate of PEG- and non-PEG treated no and

high K fertilized canola plants. 93

Figure 4.11: Representative examples of Pp measurements on canola subjected

to different potassium treatments and the corresponding ambient

temperature and relative humidity. 94

Figure 4.12: Representative examples of Pp measurements on drought-stressed

canola subjected to different potassium treatments and the corres-

ponding ambient temperature and relative humidity. 95

Figure 5.1: Average leaf area for canola plants treated with five different

potassium rates. 109

Figure 5.2: Average shoot (A) and root dry weights(B), as well as shoot:root ratio

(C) for canola plants treated with five different potassium rates. 101

Figure 5.3: Average root diameter of canola plants treated with five different


Figure 5.4: Average shoot (A) and root (B) potassium content, as well as

shoot:root ratio (C) for canola plants treated with five different


Figure 5.5: Average pot weights of canola plants treated with five different

potassium rates during drying cycle. 114

Figure 5.6: Average transpiration per leaf area of canola plants treated with

five different potassium rates during drying cycle. 115

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Figure 5.7: Average transpiration per leaf area of well-watered canola plants

treated with five different potassium rates. 115

Figure 5.8: Examples of Pp measurements on canola subjected to five

different potassium treatments and the corresponding ambient light

and relative humidity. 117

Figure 5.9: Overshoot examples of no added and high K treatment prior to the

drying cycle. 119

Figure 5.10: Average leaf area for canola plants treated with three different

potassium and two different water rates. 120

Figure 5.11: Average shoot and root dry weights, as well as shoot:root ratio for

canola plants treated with two different potassium and three

different water rates. 121

Figure 5.12: Average root diameter of canola plants treated with two different

potassium and three different water rates. 122

Figure 5.13: Average shoot and root potassium content, as well as shoot:root ratio

for canola plants treated with two different potassium and three

different soil moisture rates. 124

Figure 5.14: Average pot weights of canola plants treated with two different

potassium and three different soil moisture rates (A: 8.5%, B: 17%,

C: 28% soil moisture) during drying cycle. 126

Figure 5.15: Average transpiration per leaf area of canola plants treated with two

different potassium and three different soil moisture rates (A: 8.5%,

B: 17%, C: 28% soil moisture) during drying cycle. 128

Figure 5.16: Pp output pressure of representative canola plants treated with low K

(A) and recommended K (B) grown at 8.5% soil moisture during the

drying cycle. 129


(A) and recommended K (B) grown at 17% soil moisture during the

drying cycle. 130


(A) and recommended K (B) grown at 28% soil moisture during the

drying cycle. 131

Figure 6.1: Average minimum and maximum temperature as well as evaporation

and rainfall over the growing period from 23 May 2013 and 19

November 2013. 144

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Figure 6.2: Average shoot biomass of field grown canola plants treated with two

different potassium (no added and high K) and nitrogen (low and

medium N) rates. 145

Figure 6.3: Average grain yield of field grown canola plants treated with two


medium N) rates. 145

Figure 6.4: Average shoot potassium concentration of field grown canola plants

treated with two different potassium (no added and high K)

and nitrogen (low and medium N) rates. 146

Figure 6.5: Average shoot nitrogen concentration of field grown canola plants

treated with two different potassium (no added and high K) and

nitrogen (low and medium N) rates. 147

Figure 6.6: Difference between leaf and ambient temperature of field grown

canola plants treated with two different potassium (no added and high

K) and nitrogen (low and medium N) rates during bud development and

flowering stage. 148

Figure 6.7: Average transpiration rate of field grown canola plants treated with

two different potassium (no added and high K) and nitrogen (low

and medium N) rates during bud development and flowering stage. 148

Figure 6.8: Average stomatal conductance of field grown canola plants treated

with two different potassium (no added and high K) and nitrogen (low

and medium N) rates during bud development and flowering stage. 149

Figure 6.9: Average photosynthesis of field grown canola plants treated with two


medium N) rates during bud development and flowering stage. 150

Figure 6.10: Average midday leaf water potential of field grown canola plants

treated with two different potassium (no added and high K) and

nitrogen (low and medium N) rates during bud development and

flowering stage. 151

List of Tables

Table 3.1: Nutrient composition of applied fertilizer stock solution. 39

Table 4.1: Nutrient composition of applied fertiliser stock solution. 79

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Table 4.2: Timeline of conducted PEG induced drying cycles 82

Table 5.1: Fertiliser composition 106

Table 5.2: Potassium fertilser rates 106

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Acknowledgements

During the past three and a half years I have learned that completing a PhD does not

only require a lot of work, time, effort, nerves, passion, commitment, motivation,

inspiration, organisation, patience, thinking and communication, to name just a few of

the important qualities a PhD student should have, but also and more importantly

resembles a massive team effort that involves the entire personal and professional

surrounding of a PhD candidate. Without the tireless support, resources and

encouragement of my family, friends, professors, fellow students and the institutions I

collaborated with, this PhD would not have been achievable.

First and foremost I would like to thank my coordinating supervisor Dr Ken Flower. No

matter which problem I faced, Ken was always there for honest advice and inspiring

discussions. With his calm and understanding personality he helped me to see things

from a different perspective and often got me to re-focus on the important things. Many

thanks also to my five other supervisors; every single one of you has been an

invaluable resource to me: Prof Klaus Dittert and Junior Prof Andrea Carminati, with

your knowledge and experience you guided me through my first experiments in

Germany. Thank you both for all that you have done for me; I really enjoyed my time in

Goettingen; Dr Andrew Guzzomi, thanks for your very attentive proof reading and

insightful questions and comments; Dr Craig Scanlan, I really appreciate your support

in organising my field experiment and would like to thank you for being a never ending

source of information on potassium research in the Western Australian wheat belt; and

Kadambot Siddique, thanks for being always available to point me in the right direction

and for helping me to fine-tune my thesis, even after I already thought “Hey, this is

actually not too bad what I have got here!”.

This work was supported by funding from the Australian government, the Australian

Postgraduate Award (APA). Moreover, I would like to acknowledge the Grain Research

and Development Cooperation for their additional funding of my project by providing

me with a GRS top-up scholarship. I would also like to recognise the endless support of

the entire team of the Yara Zim-Plant Technology GmbH, providing me with their

advice and knowledge in relation to the Zim probes. In particular my thanks go out to

Wilhelm Ehrenberger for the many hours spent to smooth out my figures, Prof Ulrich

Zimmermann for supporting me to interpret the probe data and Rebecca Bitter who

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was a great help teaching me how to clamp the probes under the most difficult

conditions. Furthermore, I would like to thank the staff at the Institute of Applied Plant

Nutrition and the University of Goettingen for looking after me so well and enabling me

to carry out my experiments, lending a hand during harvest and the processing of

samples. I am also very thankful to the work of Gavin Sarre from the Department of

Food Western Australia, who helped me with the biomass cuts and sample analysis

during the field experiment.

I would also like to extent my gratitude to the University of Western Australia for

providing me with such a great learning environment and especially to the staff and

students of the School of Plant Biology, who assisted me throughout the period of my

studies; especially Pandy, Barbara, Pauline, April and Natalie, who were always there

to support me. Also, thanks to Bill and Robert down at the growth facilities. There

would not be any research without the endless help of you. Big thanks to Jennie, Araz,

Sonja, Hammad and Renu who managed to help me out during my measurements

compromising on their own projects. Furthermore, thanks to Elizabeth at the soil labs

who was always willing to help and give guidance if need be.

I greatly appreciate the work and support from the team at Student Access who put in a

lot of efforts to enable me to undertake a PhD degree in a science discipline despite my

vision impairment. In particular, thanks to Alistair Miller for his technical support and

drive to always find new ways of making text or images available to me in a different

format. I would also like to thank Elizabeth Sullivan for her open minded and friendly

support throughout the years as without it I could not have managed all the tasks I

encountered.

And of course thank you to my family in particular my parents and parents-in-law who

were always there to cover my back so I did not have to worry about anything else but

my thesis and who also shared with me their views as an outsider, which helped me to

look at problems from a different angle. But more importantly, they tolerated the long

working hours and always believed in me, encouraging me to never give up my

dreams.

Last but not least, I would like to attempt to express my gratitude to my beloved

“Naughty Team”, my wife Ronja and my Seeing Eye dog Forest, who never left my side

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during the entire time. I do not know how they managed to put up with everything that

came with the conduction of my PhD, but they just did and they did it superbly, easily

carrying most of the stress and constraints that come with such an endeavour. Thank

you, Forest, for your safe, reliable and often fearless guiding; whether on the busy

UWA campus, in the USA for a conference or in Germany for conducting experiments,

with you I always safely reached my destination. Besides your guiding skills you are a

perfect mate that always brightens up my day.

Ronja, I cannot say how much I appreciate your never ending support. Without your

help completing this PhD would never have been possible. After your normal 8-hour

work day you spent hours helping me to create figures, add references, format my

thesis and everything else I could not do because of my eye sight. During the

conduction of my experiments, you were the one helping me out with my

measurements driving me out to the fields at 4 o’clock in the morning on the weekend.

You were always there to discuss things over and over again and your efficiency,

perfectionism and constructive criticism inspired and encouraged me to work even

harder. Thank you.

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P a g e | XIX

Thesis Declaration The work presented in this PhD thesis does not contain any material that has been

accepted for award of any other degree or diploma at any other university.

I declare that this thesis is my own work and contains no material previously published

or written by another person except where acknowledgement is made in the text.

Max Bergmann

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List of Abbreviations

Abbreviation Meaning

ABA Abscisic acid

ANOVA Analysis of Variance

ATP Adenosine triphosphate

C Gradient in net water potential

CT Cohesion tension theory

F&S French and Schultz

Fa Attenuation factor

GS Growth stage

Kir Potassium inwardly rectifying channel

LAI Leaf area index

LPCP Leaf patch clamp pressure

n Sample size

P Hydrostatic force

p Osmotic force

Pb Balancing pressure

Pc Relative changes in leaf turgor pressure

Pc Turgor pressure

Pclamp Clamping pressure

PEG Polyethylene glycol

Pp Transfer of pressure

RSA Root system architecture

SE Standard error

SPAC Soil-plant-atmosphere continuum

TPK Tandem-pore potassium channel

WA Western Australia

WUE Water use efficiency

Chapter 1: General Introduction

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Chapter 1 General Introduction

Canola, oil seed rape, is a major crop in various crop rotation systems around the

world. Since the early 1990s it has been grown in Western Australia (WA) in rotation

with barley (Hordeum vulgare L.), spring wheat (Triticum aestivum L.) and lupin

(Lupinus angustifolius L.) (Brennan and Bollard, 2007). The grain belt region of WA has

been traditionally classified as having a Mediterranean-type climate, characterized by

hot, dry summers and cool, wet winters (Farré et al., 2007; To et al., 2011). Canola

production in this region is vulnerable to large variations in rainfall and frequent

droughts. Recent poor seasons and price volatility have contributed to farmers’

perceptions of risk with canola, such that the area sown to canola often declines after

periods of drought (Farré et al., 2007). Nevertheless, the total area sown to canola in

WA has increased over the past five years to 7.6% (0.56 million hectare) of the 18

million hectare arable land and the importance of the crop has become more apparent

(To et al., 2011; Seymour et al., 2012). In this variable climate it is clear that soil water

is the most limiting factor for crop production, especially in low to medium rainfall

regions (Moir and Morris, 2011). Hence, greater water use efficiency of canola would

contribute to improved yields and crop reliability (Farré et al., 2007).

According to a simulation study conducted by Farré and Foster (2010), a higher

frequency of low yield years is forecast in the future for the WA grain belt, as a result of

climate change. The prediction for the coming decades is that south-western Australia

will become drier, and experience higher temperatures and less rainfall, particularly

during spring, and yields of rain-fed crops grown in this area may be adversely

affected. Hence, climate change could have a major impact on canola production in the

future (Moir and Morris, 2011).

Today, about 8% of the Western Australian cropping region is potassium (K) deficient

(Weaver and Wong, 2011). Furthermore, there is an increased risk that most of the

sandy soils, which comprise 75% of the 18 million ha of arable land in WA, will also

become potassium deficient in the coming years, due to leaching and the removal of

potassium from soils with hay and grains (Brennan and Bolland, 2007). After nitrogen

(N), potassium is the mineral element required in the largest quantities for optimal crop

productivity, especially during the reproductive phase of plants. Today, large amounts


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of potassium fertilisers are required for modern crop production (Oosterhuis et al.,

2014). According to Marschner (1995), 2–5% of plant dry weight is required in the form

of potassium for optimal plant growth. Nevertheless, the amount used by the crop

depends on a number of factors which significantly impact the uptake of potassium

(Mullins et al., 1997). These include the availability of potassium in the soil, crop

species (Oosterhuis et al., 2014), applied management practices (Rengel and Damon,

2008; Brennan and Bolland, 2009) and the environmental conditions during the

growing season (Dorlodot et al., 2007).

Canola has a relatively short history of production in the grain belt of WA. Although

there is some knowledge about the relationship between potassium requirements and

yield expectations for canola, there is limited information on potassium requirements

under different growing conditions in WA (e.g. rainfall areas, soil types, row spacing,

time of seeding, interaction with other nutrients) (Brennan and Bolland, 2009; Brennan,

Mason and Walton, 2000; Farré et al., 2007). Furthermore, potassium deficiencies also

occur following removal of crop residues (e.g. for use in the biofuel industry) as well as

different management practices (Rengel and Damon, 2008; Romhel and Kirkby, 2010).

This highlights the importance of proper plant nutrition management practices to avoid

nutrient deficiencies in order to achieve optimal crop productivity as summarised by

Oosterhuis et al. (2014).

There is also evidence of decreased water loss from plants with higher potassium

concentrations in different crops. Brag (1972) showed that the transpiration rate of

wheat (Triticum aestivum L.) and field peas (Pisum sativum L.) could be regulated by

varied potassium concentrations in the nutrient solution, with higher concentrations

decreasing the transpiration rate by up to 50%. Further evidence was provided by

Lösch et al. (1992) who showed, for spring barley (Hordeum distichum L.), that daily

accumulated transpiration water loss was lower in high potassium leaves than in low

potassium leaves and that generally, bulk water relations of the leaves were more

favourable in high potassium plants than low potassium plants. There is little published

information of the effect of potassium nutrition on crop water use in canola. However,

several studies have suggested a similar effect for canola, reporting better growth from

canola plants supplied with sufficient potassium (Brennan and Bolland, 2007).

Moreover, Gunasekera et al.

(2009) pointed out that a greater capacity for osmotic


P a g e | 3

adjustment may enable canola to produce more dry matter, and potentially to partition it

more efficiently under severe soil water deficits.

Farmers use significantly more commercial fertiliser inputs now than in the past. The

annual estimated increase in potassium fertilisers is 3.5% or 2 million tons world-wide

(Oosterhuis et al., 2014). However, globally the amount of potassium per 100 kg of N

applied has decreased i.e. the N:K ratio in the fertiliser has substantially increased

from 100:63 in 1960 to 100:27 in 2000 (Maene, 2001). As a consequence, there has

been renewed emphasis on potassium management, focusing in particular on

understanding potassium fertiliser requirements and use by crops. Hence, further

research on the role of potassium in plant metabolism and yield formation is needed

(Oosterhuis et al., 2014).

The general objective of this study was, therefore, to understand how potassium

influences the drought tolerance of canola. In particular, its effect on regulating

stomatal conductance under water stress and how this may lead to higher growth and

yield potential. The overall hypothesis was that plants well-supplied with potassium are

better able to regulate their stomata, maintain a higher turgor pressure and rate of

photosynthesis, leading to increased yields, compared with those with low or deficient

levels of potassium.

P a g e | 4

Chapter 2: Literature Review

P a g e | 5

Chapter 2 Literature Review

This chapter outlines the biology of canola and reviews the role of potassium in plant

growth and water relations, providing a contextual background to this research project.

First, the biology of canola and some general insights on its growth and production in

Western Australia are explained. Then the plant physiological mechanisms during

drought stress and the influence of potassium as an essential macronutrient in plants

are presented. Finally, the current knowledge on water and nutrient transport in plants

is outlined and different methods of measuring leaf water potential and calculating

water use efficiency are discussed.

2.1 The Biology and Agronomy of Canola

Canola, also known as rape seed, belongs to the Brassicaceae family, formerly

Cruciferae, which consists of about 375 genera and 3200 plant species. Of these,

approximately 52 genera and 160 species occur in Australia (Jessop and Toelken,

1986). Rape seed was first grown by ancient civilisations in the Mediterranean Basin

and Asia. Its usage, primarily as oil for lamps, has been reported in India as early as

2000 BC and it was planted in Europe dating back to the 13th century (Colton and

Sykes, 1992). Rape seed was first cultivated commercially in Canada in 1942 and used

as a lubricant in war ships (Canola Council of Canada, 2011). Due to the presence of

two naturally-occurring toxins, glucosinolates and erucic acid, it was was unsuitable as

a food source for both animals and humans. However during the 1970s, a number of

intensive breeding programs in various countries, including Australia, selected high

quality varieties, such as Brassica napus, Brassica rapa and Brassica juncea, that

were significantly lower in these two toxins. Thus, the term “canola” (Canadian oil, low

acid), also often referred to as “double low varieties”, describes those varieties of rape

seed that contain meal low in glucosinolates (total glucosinolates of <30 μmoles g-1

toasted oil-free meal) and oil with <2% erucic acid (CODEX, 1999). In 1956, the first

edible oil was extracted in Canada (Colton and Potter, 1999).

Today, canola is primarily cultivated for its seeds which contain between 35% to 45%

oil, of which approximately 62% is oleic acid, 20% linoleic acid and 9% linolenic acid

(McCaffery et al., 2009). The main use of canola oil is in the food industry;

approximately two-thirds are used in the commercial food service sector and around


P a g e | 6

one-third in spreads and cooking oil. However, because of its high level of

polyunsaturated fatty acids, making it prone to oxidation, canola oil is not ideal for deep

frying purposes and it is, hence, often blended with other oils, for example olive oil, to

enhance its stability and flavour (Salisbury et al., 1999). Canola was first commercially

cultivated in Australia in 1969 (Smith and Baxter, 2002). Today, Australia exports about

20–25% of the extracted canola oil. Canola seed meal, as the main by-product after oil

extraction, is used as a high protein animal feed mainly in intensive livestock

operations such as the poultry, pig and dairy industries (McCaffery et al., 2009).

Canola is one of the most profitable crops for grain growers in southern Australia as it

is suited to local conditions, which are generally regarded as Mediterranean type with

hot, dry summers and cool, wet winters (Farré et al., 2007; Brennan and Bollard, 2007).

According to Seymour et al. (2012), 7.6% (0.56 million ha) of the 18 million ha arable

land in the Western Australian wheat belt was sown to canola (Brassica napus L.) in

the 2010/2011 growing season. Canola provides farmers the opportunity to increase

diversity in their crop rotations, which are currently dominated by cereals, as it has

positive effects on the following crops because their root systems improve soil structure

and soil water infiltration (Smith and Baxter, 2002; Duff et al., 2006; Kirkegaard et al.,

2008; McCaffery et al., 2009). This in turn ultimately leads to higher protein levels and

yield in the following cereal crop (Smith and Baxter, 2002; Canola Council of Canada,

2011). Seymour et al. (2012) estimated the break-crop benefit for wheat was 0.4 t ha-1

following canola. Moreover, canola root exudates have biofumigation effects on fungal

pathogens through the release of isothiocyanates (Kirkegaard et al., 1994; Kirkegaard

et al., 1998). About 8% of the Western Australian cropping region is already potassium

(K) deficient with another 75% of that region, comprised of sandy soils, under

increasing risk of potassium deficiency due to the removal of crop residues and

potassium leaching (Brennan and Bolland, 2007; Weaver and Wong, 2011). Using the

Colwell method for potassium, a concentration of 50 mg K kg-1 soil is considered as

sufficient potassium for adequate yield, 40 to 50 is marginal, 30-40 is mildly deficient

and less than 30 mg K kg-1 is severely deficient in soils (Brennan et al., 2013).

In Australia, only spring-type canola varieties are grown between 30oS and 38oS,

mostly in environments like the south western region of Western Australia with winter-

dominant precipitation (Smith and Baxter, 2002; Duff et al., 2006; Farré et al., 2007).

These canola varieties are grown as a winter annual for about 6 months but, unlike


P a g e | 7

European winter varieties, they do not need vernalisation to induce flowering, even

though winter chilling usually speeds up flowering (McCaffery et al., 2009). This is in

contrast to the considerably longer growing periods in Europe of nearly 12 months,

where mainly winter varieties with vernalisation requirements are sown, and the short

growing season in Canada (less than 4 months) where spring-type varieties with a long

day length and warm temperature requirement are cultivated (McCaffery et al., 2009;

Canola Council of Canada, 2011).

Canola plant development can be separated into easily recognisable growth stages.

One simplified and accurate approach to describe the different canola growth stages is

provided by a standardised growth stage scale developed by BASF, Bayer, Ciba-Geigy

and Hoechst named the BBCH decimal system (Figure 2.1).

Figure 2.1: BBCH decimal system of Brassica napus principal growth stages (Canola

Council of Canada, 2011)

Seeding typically commences in Western Australia with the start of the winter rains in

April/May. After a growing period of 2 to 3 months, canola varieties flower for about 6

weeks. Crops ripen towards the end of spring or beginning of summer in

October/November. The average plant population for canola in Australia is

approximately 50 to 70 plants m-2 achieved with a sowing rate between 3 and 6 kg

ha−1, with hybrid varieties sown at 3 kg ha−1 (McCaffery et al., 2009). Under dry soil

moisture conditions and high soil temperatures, canola seed is sown at depths up to 6


P a g e | 8

cm, where there is sometimes more soil moisture (Smith and Baxter, 2002; Duff et al.,

2006; McCaffery et al., 2009). However if there is good soil moisture for germination,

canola seed is sown at a depth of 2 to 4 cm, allowing for rapid emergence (Duff et al.,

2006; McCaffery et al., 2009). Fully-grown canola plants reach a height of about 75 to

175 cm. Plant height is influenced by seeding date, variety, available moisture, soil

plant population and fertility (Duff et al., 2006; McCaffery et al., 2009). A typical canola

plant has a distinct main stem with few secondary branches. Crops with a normal

uniform canopy will average approximately four to six branches per plant. However,

individual plants can range from about two to nine branches. Plants in high-density

crops are normally thinner with fewer branches and more prone to lodging than plants

in low-density crops which usually have thicker stems with more branches and are

more resistant to lodging (Smith and Baxter, 2002; Canola Council of Canada, 2011).

In Western Australia soil water is the main limitation to crop production with terminal

drought common in spring (Duff et al., 2006; Farré et al., 2007). Canola yields in

Australia are mostly about 1 to 2 t ha−1 but can be about 5 t ha−1 in ideal growing

situations (i.e. with adequate moisture and long, cool growing seasons) (Duff et al.,

2006; McCaffery et al., 2009). In order to increase the drying rate of the mature crop,

while reducing the possibility of seed losses from wind or hail, and to ensure even

ripening, most canola crops in Australia are windrowed or swathed. That is, the crop is

cut and then placed in rows for drying, which normally occurs within 7 to 10 days after

swathing when seed moisture is less than 8.5%. The windrows are picked up by a

combine harvester and threshed (McCaffery et al., 2009).

2.2 The Role of Potassium and the Effect of Drought in Plants

Following on from the general biology of canola and its importance to Western

Australian producers, the next section provides more detail about typical plant

physiological and morphological characteristics involved in nutrient uptake and drought

adaptation, with particular emphasis on the role of potassium. Furthermore, the impact

of surrounding environmental growth conditions such as soil type and water availability

is discussed.


P a g e | 9

2.3 Root Growth, Water and Nutrient Uptake in Plants

Initially, this section discusses general root growth strategies and water and nutrient

uptake mechanisms of plants, highlighting the impact of abiotic factors. Subsequently,

more detail on the role and impact of potassium is described.

Root growth and development of crop plants is often restricted by the physical,

biological and chemical properties of the surrounding soil. Referring to the physical

restrictions of root growth, the major causes of poor root system growth and

development are anoxia or hypoxia (too little oxygen), water stress (not enough water

for root growth) and mechanical impedance (roots cannot penetrate rapidly because

the soil is too hard). There is a significant interplay between soil water content and soil

strength. During soil drying, capillary forces make matric potentials more negative,

which often cause soil strength to rapidly increase (Dexter, 1986; Whalley et al., 2005a;

Whitmore and Whalley, 2009). Moreover, due to the direct signalling between roots

and shoots, various studies have shown that leaf expansion decreases in hard soils,

which is often associated with mechanical impedance (Masle and Passioura, 1987;

Young et al., 1997). It is generally regarded that a penetrometer cone resistance of 2

MPa is an indicator of a soil where mechanical impedance might have significant

influence on root elongation, unless a network of fissures or channels exists for the

roots to exploit (DaSilva et al., 1994; Bengough et al., 2006). Nevertheless, water and

nutrient uptake may be limited to areas where channels bypass the hard soil, leading to

clustered root growth in these areas (Passioura, 1991).

De Dorlodot et al. (2007) outlined in their study that one way to reduce the adverse

effect of abiotic environmental impacts on yield, is to manipulate root system

architecture (RSA) towards a better distribution of root systems in the soil medium,

optimising nutrient and water uptake. Potassium could, therefore, be significantly

involved because the mechanism that drives root elongation through the soil depends

on the osmotic potential to create the required turgor pressure in the root tip

(Bengough et al., 2006). This was also noted by Damon et al. (2007) who concluded

from their study that root distribution and potassium uptake efficiency in canola can be

influenced by different genotypes. Numerous other studies have also concluded that,

for grain crops, root architecture and distribution varies with genotype (Liao, Palta and

Fillery, 2006; Manschadi et al., 2006; Palta et al., 2011; Siddique, Belford and Tennant,

1990).


P a g e | 10

For instance the study conducted by Dexter (1986), developed a model to estimate the

rate of root tip elongation considering the balance of pressures that act on plant roots.

Differentials of this model provide expressions for the changes in root elongation rate

with respect to different soil constraints i.e. soil mechanical resistance and soil water

potential. In his paper, Dexter (1986) predicts that root cells osmoregulate against both

soil mechanical resistance and water stress with similar efficiencies which are less than

100%. His conducted analysis of data available in the current literature led him to

conclude that osmoregulation in root tips of pea (Pisum sativum L.) has an efficiency of

about 70%.

Most nutrient and water uptake occurs in the root hair zone where roots are most

permeable and active (Segal et al., 2008). Individual root hair development takes place

in the maturation zone of the root tip and growth is rapid (1 μm min−1) but short lived

(vital staining 1–3 weeks, cytoplasmic disintegration 2–3 days) (Grierson and

Schiefelbein, 2002; Johnson et al., 2001; Taiz and Zeiger, 2006). Root hairs and

functional roots are generally those in the most distal regions of the root system (Hofer,

1996). Both Gao et al. (1998) and Taiz and Zeiger (2006) suggest that the older

proximal root tissues lose function due to suberisation of roots leading to a decline in

nutrient uptake per unit of root length with root age, while younger root sections more

effectively take up water and nutrients. In nutrient-restricted environments, the

dysfunction of root sections in the proximal regions is considered an important survival

mechanism for plants (Gao et al., 1998; Johnson et al., 2001; Taiz and Zeiger, 2006).

Assuming that roots in the distal and proximal regions were equally permeable to water

in a nutrient deficient but water-abundant environment, distal region roots would be

“short circuited” and hence lose the ability to take up nutrients and water (Taiz and

Zeiger, 2006).

It is, therefore, essential to know what soil properties reduce root growth, for how long

and under which associated soil water and weather conditions in order to manage soil

in a way that maximises crop production (Bengough et al., 2010). Moreover, further

knowledge about the effect of nutrient availability, e.g. potassium on root functioning, is

needed. The study by Ma et al. (2013) described the effect of different potassium rates

on root and shoot growth in wheat highlighting that root growth is more depressed than

shoot growth under potassium deprivation. However, there is little information on the

effect of potassium on the shoot:root ratio in canola (Rose et al., 2007).


P a g e | 11

2.4 Potassium Uptake

As discussed in the previous section, there are a number of plant specific and external

environmental factors influencing the ability of plants to grow well and take up water

and nutrients. This section focuses on the potassium (K) element, with an overview on

potassium uptake from the soil medium, potassium transport and its role in plants

emphasising the relevance for the Western Australian wheat belt. It also highlights

recent areas of research that require further investigation and are relevant for the

underlying study.

Potassium is the most abundant cation in plant cells, with high mobility between

neighbouring tissues as well as individual cells during short-distance transport and

during long-distance translocation through phloem and xylem vessels (Marschner,

1995; Oosterhuis et al., 2013). The pool of soil solution, which is in dynamic equilibrium

with the exchangeable and, to a minor extent, non-exchangeable pool, is the exclusive

source for potassium uptake in plants. In order to replace the potassium depleted soil

solution, exchangeable is rapidly released from exchange sites on the surfaces of

organic matter and clay minerals (Steingrobe and Claassen, 2000; Römheld and

Kirkby, 2010). It is, therefore, important to balance the removal of potassium by regular

potassium fertilisation with mineral fertilisers or organic manures as well as by

adequate management of crop residues (Ma et al., 2013). Otherwise, there is the risk

that rapid potassium depletion will occur within a relatively short period of time.

Brennan and Bell (2013) analysed the soil in 71 wheat trials across Australia and found

that, depending on soil type, the critical Colwell-K value in the 0 to 10 cm soil layer at

90% of maximum yield ranged from 40 to 64 mg K kg−1.

The uptake of potassium into the plant occurs against its concentration gradient

through K+

channels and transporters embedded in the plasma membrane of root cells

(Ashley et al., 2006). Plants have developed two independently-functioning transport

mechanisms to aid potassium uptake. A high-affinity, selective pathway involving

plasma membrane proton-pumping ATPase complexes enables active uptake of

potassium at concentrations lower than 1 mM. At external potassium concentration

levels greater than 1 mM, the second passively functioning low-affinity channel-

mediated pathway becomes the predominant transporter (Epstein et al., 1963; Britto

and Kronzucker, 2008).


P a g e | 12

Shaker, tandem-pore (TPK) and inwardly rectifying (Kir) are the three channel families

that are mainly responsible for potassium uptake and transport throughout the plant

(Broadley et al., 2001; Cherel, 2004; Amtmann and Armengaud, 2009; Wang and Wu,

2010). Shaker-type channels appear mainly in the plasma membrane and have high

selectivity for potassium ions. The activation of channel gating occurs due to changes

in membrane potential. A depolarisation of membranes leads to outward-rectifying

potassium channels i.e. potassium flow is directed out of the cytosol. In contrast, the

flow of potassium into the cytosol results from inward-rectifying potassium channels

caused by hyperpolarisation of membranes (Oosterhuis et al., 2014). Accordingly,

Shaker channels are involved in regulating the release of potassium, long-distance

potassium transport and potassium uptake in plants, and they are the main conduit for

potassium during cellular movements e.g. regulation of stomatal aperture (Cherel,

2004; Amtmann and Armengaud, 2009; Wang and Wu, 2010; Oosterhuis et al., 2013;

Oosterhuis et al., 2014). TPK channels are part of potassium homeostasis and have

been associated with the regulation of membrane potential. However, in contrast to

Shaker channels they are less, if at all, affected by changes in membrane potential

(Gobert et al., 2007). Nevertheless, further research is required to characterise them in

greater detail.

In addition, there are three transporter families with low and high-affinity properties of

potassium, being KUP/HAK/KT (K+/H+

symporters), HKT (K+/H+

or K+/Na+

transporters)

and CPA (cation/H+

antiporters) KUP/HAK/KT (K+/H+

symporters) (Ahn et al., 2004;

Rodriguez-Navarro and Rubio, 2006). Potassium deficiency causes increased

expression of these transporters (Armengaud et al., 2004; Gierth et al., 2005). In soil,

they are involved in high-affinity potassium

uptake and play a role during turgor-driven

growth, intracellular distribution and general potassium homeostasis (Rigas et al.,

2001; Elumalai et al., 2002; Rodriguez-Navarro and Rubio, 2006).

Under low external, potassium conditions,

the uptake mechanism into the plant

requires additional energy provided through H+

coupled systems, which operate in

plasma membranes of roots (Armengaud et al., 2004). K+:H+

symports can, therefore,

power a 106

fold potassium ion accumulation with a reported coupling stoichiometry of

1:1 (Maathuis and Sanders, 1996). Considerable research has been conducted on

potassium transporter and channel families especially in Arabidopsis, however, only

limited information exists on field crops, for instance wheat (Triticum aestivum L) and


P a g e | 13

barley (Hordeum vulgare L.) with no references referring specifically to potassium

channels and transporters in canola (Brasica napus L) (Ashley et al., 2006; Szczerba

et al., 2009).

2.5 Movement of Potassium Ions and Assimilates in Plants

The transport of ions from the root to the shoot occurs mainly by xylem flow, although it

is not yet fully understood whether transpiration or osmotic water uptake plays the

major role in plant mineral nutrition (Lez et al., 2009). For a long time it was assumed

that solute flow from the root to the shoot was related to transpired water flow (Fischer,

1958; Epstein, 1972; Kramer, 1983). However, most research on whole plants has

failed to identify a direct relationship between transpiration and either the long-distance

transport of solutes, including K+

(Muenscher, 1922; Schulze and Bloom, 1984), or root

uptake of ions present in low concentrations in the external medium (Russell and

Shorrocks, 1959; Blom-Zandstra and Jupijn, 1987; Pitman, 1988). Hence, the results

obtained on the effect of nutrient availability in growth media on the regulation of

transpiration are still contradictory.

Nevertheless, the current review by Oosterhuis et al. (2014) states that transpiration

may influence the translocation of nitrogen and carbon compounds from production

sites to sinks. Through its effect on stomatal regulation, potassium can be directly

linked to the control of transpiration rates and, as a result, to root–shoot transport of

mineral salts and other nitrogen compounds such as amino acids and nitrate (Ben-

Zioni et al., 1971; Blatt, 1988; Marschner, 1995; Schobert et al., 1998). Furthermore,

the translocation of carbohydrates depends mainly on plant potassium levels with

numerous studies reporting that reduced potassium rates lead to an accumulation of

carbohydrates at production sites in several plant species (Haeder et al., 1973; Mengel

and Viro, 1974; Geiger and Conti, 1983; Cakmak et al., 1994a,b; Bednarz and

Oosterhuis, 1999; Pettigrew, 1999; Zhao et al., 2001; Amtmann et al., 2008; Amtmann

and Armengaud, 2009). For efficient apoplasmic phloem loading of sucrose in source

leaves, high potassium concentrations are required to create the steep transmembrane

pH gradient that mediates the proton–sucrose co-transport (Oosterhuis et al., 2014).

The concentration of potassium, thereby, regulates the membrane potential of the

sieve tube/companion cell complex (Wright and Fischer, 1981).


P a g e | 14

Potassium is not only involved in maintaining pH and osmotic gradients between

parenchyma and phloem cells within sieve tubes that are needed for phloem loading

and assimilate transport, but it powers the transmembrane phloem re-loading

processes with the required energy (Marschner, 1995; Gajdanowicz et al., 2011).

Furthermore, during the phloem-loading process, potassium also controls the activation

and function of invertase in sink organs as well as encouraging the efflux of assimilates

into the apoplast pryer to phloem loading (Mengel and Haeder, 1977; Doman and

Geiger, 1979; Oparka, 1990). Although it has been reported that the rate of

transpiration influences the translocation rate, findings in the literature remain

contradictory (Oosterhuis et al., 2014). For example, Bednarz et al. (1998) reported

that potassium deficient conditions increased the transpiration rate while the opposite

was described by Zhao et al. (2001) and Pervez et al. (2004). More research in this

area is, therefore, required to fully understand the role of potassium in regulating

transpiration.

The forms of potassium have a significant role in the physiology of plants and their

response to abiotic stress. Potassium is mainly present in the cationic form, because it

is not metabolised into organic compounds after uptake into the plant (Wyn Jones et

al., 1979). The most common forms of potassium salts in plant cells are KNO3, KCl and

K-malate. Their storage occurs in both the cell vacuole and cytoplasm. These

properties and characteristics determine the major function of potassium and make it

responsible for many physiological and biochemical processes, including maintaining

adequate turgor pressure, photosynthesis, translocation of photosynthates, control of

ionic balance, cell osmotic potential, regulation, of soluble and insoluble molecular

anions, neutralisation, cell pH stabilisation, protein synthesis, activation of plant

enzymes, regulation of plant stomata, leaf heliotropic movements and water use which

are all driven or influenced by potassium as an essential macronutrient for plant growth

and development (Hsiao and Lauchi, 1986; MacRobbie, 1977; Marschner, 1995;

Reddya, Chaitanya and Vivekanandanb, 2004).

Potassium has been described as the most important vacuolar solute regulating

osmoregulatory processes in plant cells (Mengel and Arneke, 1982; Beringer et al.,

1986; Morgan, 1992; Marschner, 1995; Fanaeia et al., 2009; Oosterhuis et al., 2014).

The potassium concentration in the cell vacuole regulates the “biophysical” functions

(Leigh and Wyn Jones, 1984). Potassium content in the vacuole mainly affects water


P a g e | 15

potential, while the synthesis of organic compounds is regulated by the potassium

concentration in the cytoplasm (Hsiao and Lauchi, 1986). For instance, one study

conducted by Morgan (1992) reported that only 22% of organic solutes were attributed

to osmotic adjustment in drought-stressed wheat (Triticum aestivum L.) plants,

whereas 78% was related to potassium. The osmotic adjustment through ion uptake

such as K+ is considered more energy efficient in plants suffering water deficit. Hence,

the accumulation of K+ may be more important than the production of organic solutes

during the initial adjustment phase (Hsiao, 1973). Hence, if the availability of potassium

in plants is low then potassium stored in the vacuole can be replaced by other solutes,

for instance Na (Bednarz and Oosterhuis, 1999), magnesium and amino acids (Leigh

and Wyn Jones, 1984) in order to maintain water potential, which is in contrast with the

cytoplasmic pool (Oosterhuis et al., 2014).

The vacuolar potassium content as a primary osmotic regulator can vary, ranging from

9 to 174 mM, influenced by the growing medium and plant species (Hsiao and Lauchi,

1986; Fanaeia et al., 2009). Thus, water uptake along a soil–plant gradient for crops

growing under drought conditions might be strongly dependent on their ability to

accumulate abundant K+ in their tissues. If the potassium concentration reaches the

critical minimum level in the vacuole (about 10–20 mM), it can have a substantial

impact on vital metabolic functions in the cytoplasmic potassium pool leading, for

example, to disruption of protein synthesis and enzyme activation (Leigh and Wyn

Jones, 1984).

2.6 Accumulation Patterns of Potassium and the Effect of Potassium Deficiency on Plant Morphology

Accumulation of K+ ions occurs under both salinity and/or water stress in a variety of

crops, for example sunflower (Helianthus annuus, L.) (Jones et al., 1980), sorghum

(Sorghum bicolor) (Weimberg et al., 1982), annual clovers (Trifolium sp.) (Lannucci et

al., 2002) and beans (Vicia faba L.) (Mengel and Arneke, 1982). Generally, more Na+

accumulates under saline conditions, while K+ accumulates as a response to soil water

deficits (Glenn et al., 1996). However, as pointed out by Rose et al. (2007), the

seasonal potassium accumulation patterns and distribution within canola plants are still

to be thoroughly investigated in contrast to sulphur (S) (Zhao et al., 1993) and nitrogen

(N) (Rossato et al., 2001). Those information, however, are essential in order to better


P a g e | 16

align the timing of demand and supply of potassium applications and it will

subsequently lead to more efficient use of potassium fertilisers.

It has been documented that canola has a higher demand for potassium than wheat

(Grant and Bailey, 1993; Rose et al., 2007). One study by Barraclough (1989) in the

UK reported a peak of potassium accumulation during the later stages of silique filling,

while numerous other studies conducted in Europe showed large differences in the

time of maximum potassium uptake ranging from mid-flowering to maturity, as

summarised by Holmes (1980). These findings can lead to the conclusion that

seasonal conditions and/or cultivar as well as soil type may influence the accumulation

pattern of potassium. For instance, in sandy soils, potassium leaching is often high and

significant amounts of potassium can be lost, such as those often found in the Western

Australian wheat belt or in acid lateritic soils that contain higher levels of kaolinitic clay

minerals with a low cation exchange capacity (Wulff et al., 1998; Sitthaphanit et al.,

2009).

Rose et al. (2007) suggest that access to potassium during vegetative growth in canola

appears more critical than access during reproductive growth phases, as no apparent

uptake of potassium occurred after GS 5 (Figure 2.1). In addition, they observed no

differences in potassium accumulation patterns between canola cultivars varying in

potassium-use efficiency. The authors further went on to compare canola to wheat

cultivars indicating that the availability of soil potassium post-flowering would be of

more benefit to canola than to wheat, which, in turn, confirms the results of Grant and

Bailey (1993). In that study, the maximum potassium accumulation in canola cultivars

Tribune and Trigold was measured 73 days after sowing (GS 4.8), but the uptake rate

peaked earlier, 61 days after sowing (Figure 2.1) (Rose et al., 2007). However, the

precise growth stage determination of maximum potassium uptake and accumulation in

canola requires further investigation as the findings presented in the literature are

variable and at present there are no further reports in the literature on potassium

accumulation patterns or amounts of potassium required to enhance drought tolerance

(Holmes, 1980).

Leaf expansion in plants is dependent on the mechanism that drives cell expansion

through an increase in turgor pressure, which requires a sufficient supply of potassium

to lower osmotic potential (Dhindsa et al., 1975; Mengel and Arneke, 1982). For


P a g e | 17

example, in maize, smaller individual leaves were related to a reduced leaf area

expansion (Jordan-Meille and Pellerin, 2004). This was also observed in soybean

(Huber, 1985). Consequently, solar radiation interception has a direct effect on

photosynthesis per unit leaf area (Wolf et al., 1976; Longstreth and Nobel, 1980;

Huber, 1985; Pier and Berkowitz, 1987; Cassman et al., 1989; Heckman and

Kamprath, 1992; Pettigrew and Meredith, 1997; Bednarz et al., 1998; Ebelhar and

Varsa, 2000). This influences the pool of photoassimilates available for plant growth

and productivity. Subsequently, a smaller plant stature leads to an overall reduced leaf

area index (LAI) for the crop canopy (Kimbrough et al., 1971; Pettigrew and Meredith,

1997; Jordan-Meille and Pellerin, 2004). The reduced LAI in potassium starved plants

was observed in maize and soy beans due to reduced leaf size as well as the overall

reduction in the number of leaves produced (Huber, 1985; Jordan-Meille and Pellerin,

2004; Oosterhuis et al., 2014).

2.7 The Effect of Potassium on Stomatal Regulation and Osmotic Adjustment

The ability of plants to regulate the opening and closing of stomata cells is crucial to

obtain sufficient CO2 required for photosynthesis and to control transpirational water

loss for improved productivity (Oosterhuis et al., 2014). Marschner (1995) explained

that regulation of stomatal movement occurs through the adjustment of turgor

pressure, which is substantially influenced by the concentration of potassium ions.

Stomatal guard cells release and accumulate potassium, which triggers stomatal

closing and opening, respectively, leading to changes in turgor pressure (Fischer and

Hsiao, 1968). Depending on the plant species, the concentration of potassium in guard

cells around stomates can increase about 8-fold from 100 mM when closed to 800 mM

when open (Taiz and Zeiger, 2010).

There are three relevant metabolic processes involved in the opening of stomates:

solute uptake, proton pump ATPase and organic solute synthesis. Light radiation into

the plant cell triggers the activation of the H+ proton-pumping ATPase, thereby creating

an electrochemical potential that enables the uptake of potassium and the companion

anions Cl-

and/or malate as summarised in the review by Oosterhuis et al. (2014). The

influx of these sucrose and solutes into the guard cell vacuole then leads to a reduction

in osmotic potential, which in turn causes water uptake. This eventually increases the

turgor pressure of guard cells, opening the stomates (Taiz and Zeiger, 2010).


P a g e | 18

The closure of stomates, on the other hand, occurs through a reduction in turgor

pressure, which results from high osmotic potential due to a low concentration of

intercellular solutes. This process is mainly driven by increased abscisic acid (ABA)

activity that stimulates calcium uptake into the cell, blocking potassium

channels and

benefiting the extrusion of Cl- anions into the cell apoplast (Taiz and Zeiger, 2010). As

the amount of intercellular calcium increases, the H+ pump ATPase is inhibited leading

to depolarisation in the cell membrane. This then triggers the extrusion of cytoplasmic

and vacuolar potassium

to the cell apoplast causing a subsequent loss of intracellular

water and, hence, turgor pressure (Oosterhuis et al., 2014). Increased ABA

concentrations can induce stomatal closure in water-stressed plants by releasing

potassium from guard cells (Assmann and Shimazaki, 1999).

It might, thus, seem reasonable to assume that potassium starved plants would have

inhibited closing of their stomata, since potassium is substantially involved in the

stomatal opening mechanism. A lack of potassium would subsequently be expected to

decrease plant stomatal conductivity. However, numerous studies have reported that

the opening of stomata cells and an increase in transpiration is enhanced through

potassium deficiency (Brag, 1972; Sudama et al., 1998; Bednarz et al., 1998;

Cabanero and Carvajal, 2007). This was recently confirmed by another study

conducted by Benlloch-Gonzalez et al. (2008), which investigated the role of potassium

in plant stomatal regulation, concluding that potassium deficient plants would also

increase stomatal conductivity under drought stress. These findings also support the

results of another study (Founier et al., 2005) that reported increased water uptake and

decreased water use efficiency under potassium deprivation. In order to explain the

observed behaviour, Benlloch-González et al. (2010) proposed an interaction effect

between ethylene and ABA, since stomatal closure is known to be regulated by ABA

and the synthesis of ethylene increases under potassium starvation. Furthermore, it

has been documented that in ABA-active plants, stomatal closure can be inhibited

through the interference of ethylene (Tanaka et al., 2005). While this effect could not

be confirmed by the observations made by Benlloch-Gonzalez et al. (2010), they

reported that an increase in stomatal conductance in potassium deficient plants under

drought stress disappeared with the application of an ethylene synthesis inhibitor. The

conclusion drawn from that study was that a mechanism might have evolved in plants

to avoid potassium deficiency by increasing the stomatal conductance of potassium


P a g e | 19

starved plants in order to increase xylem sap movement in the plant (Benlloch-

Gonzalez et al., 2010; Oosterhuis et al., 2014).

Moreover, several other studies have suggested that potassium deprivation favours

osmotic water absorption by the root and potassium flux in the xylem (Kochian and

Lucas, 1982; De la Guardia et al. 1985; Siddiqi and Glass, 1987; Benlloch-Gonzalez et

al., 1989; Quintero et al., 1998; Schraut et al., 2005). A low potassium status of the

plant, therefore, triggers the expression of transporters involved in xylem and phloem

loading and unloading as well as the expression of transporters involved in potassium

uptake (Pilot et al., 2003; Gierth et al., 2005; Ashley et al., 2006; Gierth and Maser,

2007). Long-distance xylem transport of potassium may be regulated by the

recirculation of potassium within the plant via phloem from the shoot to the root

(Marschner, 1995).

Overall, the results presented in the literature on plant nutrient status in relation to root

hydraulic conductivity have mainly focused on the deprivation of one element in the

growth medium and water transport through the roots, are contradictory and are

influenced by the missing type of ion (Lez et al., 2009). For instance, the removal of

nitrate, phosphate, sulphate and Ca2+ from the growth medium inhibited root hydraulic

conductivity (Radin and Ackerson, 1981; Radin and Eidenbock, 1984; Chapin et al.,

1988; Radin and Matthews, 1989; Karmoker et al., 1991; Barthes et al., 1996; Carvajal

et al., 1996; Quintero et al., 1999; Cabanero and Carvajal, 2007; Gorska et al., 2008

and Gloser et al., 2009), whereas, the removal of Mg2+ stimulated root hydraulic

conductivity (Cabanero and Carvajal, 2007; Donovan, 2007). Furthermore, it has been

suggested that osmotic water absorption by the root, in an environment with a reduced

transpiration rate, might be sufficient for long-distance nutrient supply to the plant shoot

(Tanner and Beevers, 1990; Tanner and Beevers, 2001). Nevertheless, plants have

probably developed mechanisms to detect potassium fluctuations in the plant itself and

in the external medium as well as a transductional process in order to coordinate

potassium and water flows between the various parts of the plant. Despite the

significance of water and potassium transport in the xylem for plant growth and

agricultural productivity, further research is required to understand these processes

especially in canola (Lez et al., 2009).


P a g e | 20

2.8 The Influence of Potassium on Photosynthesis

Photosynthesis is influenced by potassium in both non-stomatal and stomatal aspects

of the photosynthetic mechanism. The effect of the potassium concentration on non-

stomatal factors and, thus, on the photosynthetic rate are usually associated with the

role that potassium plays in photophosphorylation and not so much with the effect that

it has on enzymes involved in carbon assimilation (Huber, 1985). This observed

reduction in photophosphorylation, caused by a low supply of potassium, is related to

an inner chloroplast membrane ATPase that maintains high stromal pH. This is

required for the efficient conversion from light energy to chemical energy by pumping

protons out of the stroma into the cytosol, thereby enabling the flux of potassium into

the stroma (Berkowitz and Peters, 1993; Shingles and McCarty, 1994). The level of

CO2

available at the reaction site is, on the other hand, controlled by stomatal aperture

regulation adjusting the flow of CO2

into and the flow of H2O vapor out of intercellular

spaces (Oosterhuis et al., 2014).

Furthermore, increased production of reactive oxygen species (ROS) can occur in

photosynthetic tissue under potassium starved conditions triggering photooxidative

damage under higher light intensities (Cakmak, 2005). Even though the available level

of potassium influences both non-stomatal and stomatal photosynthetic factors, the

extent of any potassium deprivation, timing and development will eventually determine

which the predominant factor impacting photosynthetic production is. It has been

reported, for instance, that during the onset of potassium deficiency, the principle

component regulating photosynthesis is stomatal conductance (Bednarz et al., 1998).

However, once potassium starvation becomes more pronounced, biochemical or non-

stomatal factors evolve as the overriding factors explaining the reduction in

photosynthesis and consequently leading to lower yields and quality of the crops

(Pettigrew, 2008).

2.9 The Role of Potassium under Drought Conditions and the Impact of Potassium Starvation on Plant Physiology

After highlighting the involvement of potassium in various physiological processes, this

section describes how water-limiting conditions and other abiotic and biotic factors can

change and impact plant physiology, and how the level of available potassium might

interact and improve the ability of plants to withstand the stress.


P a g e | 21

Drought is one of the most severe abiotic environmental stresses for plant growth and

productivity and has several unique aspects. For example, it combines low

photosynthesis with high respiration, closed stomata and often high leaf temperatures

resulting in metabolic impairments where activities of photosynthetic enzymes, for

instance ribulose bisphosphate carboxylase/oxygenase and ribulose bisphosphate are

disturbed (Gimenez et al., 1992; Medrano et al., 1997; Tezara et al., 1999; Lawlor and

Cornic, 2002; Mittler, 2006; Fanaeia et al., 2009). These changes often result in tissue

dehydration (Kacperska, 2004; Dobra et al., 2010), which usually occurs during an

imbalance between leaf transpiration and root water uptake (Aroca et al., 2001;

Jackson et al., 2003). As a consequence of the mitigating effects of potassium on

dehydration and chloroplast stromal pH balance, sufficient potassium levels have been

associated with the maintenance of CO2

assimilation rates by controlling stomatal

aperture as well as cell water relations, resulting in more efficient photosynthetic

activity. This has been described for a number of crops such as spinach (Spinacia

oleracea L.), wheat (Triticum aestivum L.), mung beans (Vigna radiata L.) and cowpea

(Vigna unguiculata L.) (Berkowitz and Whalen, 1985; Pier and Berkowitz, 1987; Sen

Gupta et al., 1989; Cakmak and Engels, 1999; Sangakkara et al., 2000; Mengel et al.,

2001), but there is no research in this regard on canola.

Both Andersen et al. (1992) and Sangakkara et al. (2001) found that the application of

potassium fertilisers mitigated the adverse effects of drought stress on crop growth.

Reduced water loss of plants supplied with potassium has been explained by Fusheing

(2006). A reduction in transpiration not only depends on the osmotic potential of

mesophyll cells but is also controlled to a major extent by the closing and opening of

stomata. Furthermore, osmotic adjustments have been closely associated to stomatal

conductance and canopy temperature in some Brassica species (Singh et al., 1985;

Kumar and Singh, 1998). Fanaeia et al., (2009) also stated that potassium application

has the potential to ameliorate negative effects of drought stress on physiological

properties and grain yield in wheat.

In conclusion, adequate potassium in plants is crucial for many physiological processes

and, if reduced, has an adverse effect during abiotic environmental stresses. Prasad et

al. (2008) suggested, in their review, that further studies are required to better

understand the detailed interactions of drought and other stresses on various

physiological plant properties in field crops. This will enhance our current knowledge


P a g e | 22

and may improve crop models. Despite an increase in potassium deficient agricultural

sandy soils in the Western Australian wheat belt and the prediction of drier years for

the future, there is still insufficient knowledge on plant physiological responses to

potassium deprivation and drought stress, particularly for canola (Brennan and Bolland,

2004; Farré et al., 2007).

2.10 Water Dynamics in Plants

There are a number of plant physiological responses and adaptation strategies that

improve the tolerance of the plant to drought stress, many of which are impacted by the

level of potassium available to the plant. Nevertheless, mechanisms have evolved in

plants allowing them to counteract water-limiting environmental conditions, which

subsequently improve the water use efficiency of the plant. The following section

highlights some of these strategies and the current knowledge on estimating water use

efficiency in crops. In addition, the uptake and transport mechanisms for water are

explained and the relevant theories for this research project are discussed.

Furthermore, different methods and technologies that are commonly used to measure

leaf water potential in plant physiological research are presented in order to identify

new methods that could benefit the underlying study.

2.11 Water Use Efficiency

Under Mediterranean-type environments, rain-fed crops usually develop a vigorous

root system for increased growth and yield. However, this strategy can be at risk if

plants deplete available soil moisture well before the crop has completed grain filling

(Palta et al., 2011). Passioura (1983), Lambers et al. (2002) and Song et al. (2009)

emphasised lower yields for crops which are primarily grown for grain, such as canola

and wheat, due to the high metabolic costs associated with the development of a larger

root system. The seed yield of three Brassica species (B. napus, B. juncea and B.rapa)

also decreased due to water stress (Jensen et al., 1996).

A widely-used approach for predicting potential grain yield is based on the work of

French and Schultz (F&S) (French and Schultz, 1984a,b). The defined linear boundary

function is an arbitrary line drawn to encompass the maximum yields of a number of

trials against rainfall. It outlines the attainable yield per unit of water use for wheat.

Despite some criticism, the F&S calculation remains the most widely-accepted

approach among consultants and farm managers as it can be readily applied by


P a g e | 23

farmers and advisors. Researchers such as Hancock (2007), for instance, have

adapted the F&S method to extend the applicability of the approach from the original

275 to 450 mm average annual rainfall zone to other rainfall zones or to other crop

species, such as canola (Robertson and Kirkegaard, 2005). Furthermore, the x-

intercept has been re-interpreted by analysing the structure and size of rainfall events

(Sadras and Rodriguez, 2007). Additionally, Oliver et al. (2009) showed that

enhancements to the predictability of the F&S formula are possible for Mediterranean-

type climates by adjusting the factors that have the largest impacts on the

improvements e.g. accounting for growing season, soil type, rainfall, as well as their

interactions. At the same time, the ease of use of the formula for applicability in the

field for growers and advisors has been maintained. They also stated that the validity of

such proposed improvements in other grain-growing environments requires further

testing.

Robertson and Kirkegaard (2005) concluded from their research that April to October

rainfall and in-crop rainfall were the poorest predictors of yield for canola (R2<0.5). This

conclusion was based on the relationships obtained from measurements such as

rainfall, soil moisture and evapotranspiration, and grain yield in 42 experimental canola

crops grown in southern New South Wales under optimal conditions i.e. sufficient

nutrient supply and free of weeds, with a yield range from 0.5 and 5.4 t ha–1. By

modifying in-crop rainfall to account for stored soil moisture at sowing as well as that

remaining at the end of a season at harvest, 68% of the estimated variance in yield

was explained. In addition, 73–82% of the variance could be explained when the

values were calculated using the APSIM-canola simulation model or simulated totals of

evapotranspiration. The water use efficiency (WUE) presented for grain production was

11 kg ha–1 mm above an intercept of 120 mm. The WUE findings varied from 4 to 18 kg

ha–1 mm. In general, WUE had high variability due to the rainfall distribution and a

decline in WUE by about one-third between different sowings in early April and early

July (Robertson and Kirkegaard, 2005).

2.12 Water Transport in Plants

The WUE of a plant depends on a range of factors as described above. The next

section discusses water transport in plants and describes the different theories

presented in the literature, which are fundamental for this study.


P a g e | 24

There are two main forces which regulate the rate of root water uptake, namely

hydrostatic (P) and osmotic forces (p). These two pressures create the net water

potential which drives water movement in a plant through gradients (C) in water

potential and is expressed in units of pressure, usually MPa, as a measure of the free

energy associated with water (Passioura, 1982; Boyer, 1985). The transpiration stream

generates P leading to external or internal pressures in the plant. As a result of the

rigidity of plant cell walls, cells can generate a positive internal pressure called ‘turgor’,

which also accounts to P (Tyree and Jarvis, 1982). On the other hand, p is controlled

by root pressure (active transport of biosynthates or solutes of new osmolytes) and

depends on the concentration of solutes in a solution (Doussan et al., 1998; Knipfer

and Fricke, 2011).

Root water transport can be divided into axial and radial transport. The axial transport

does not contribute significantly to the resistance of water transport through the whole

plant and consists of water moving along the xylem vessels to aerial parts of the plant

(Doussan et al., 1998; Knipfer and Fricke, 2011). Because of cavitation events in

woody plants, this axial transport can function as an important determinant of drought

resistance (Dalla-Salda et al., 2009). On the other hand, radial water flow from the soil

solution into root xylem vessels is resistant to root water transport and involves three

paths: apoplastic, symplastic and transmembrane, which are dynamically

exchangeable (Steudle and Peterson, 1998). The apoplastic path is described as water

moving through the pores in between the fibrils of the cell wall and through intercellular

spaces. The water flow moving through the cytoplasm and through plasmodesmata

between different cells is the symplastic path. The transmembrane path comprises

water moving through the vacuoles and the cytoplasm where it has to cross the plasma

and vacuolar membranes. The symplastic and transcellular paths together are also

known as the cell-to-cell path since they cannot be experimentally discriminated

(Steudle and Peterson, 1998).

The main drivers of water transport through the soil–plant–atmosphere continuum

(SPAC) are gradients in water potential since water always passively moves down a

gradient, from high to low energy, until reaching equilibrium. This process can partly be

influenced by plants through adjusting their accumulation of osmolates or transpiration

rate and manipulating its C (Baker, 1989). K+ generates the water potential gradient

from the root to the external medium, thereby enabling osmotic water absorption by the


P a g e | 25

root, and is mainly responsible for lowering the solute potential in root xylem vessels

(Lauchli, 1984). This is an important mechanism for plant re-hydration and growth at

night (Kramer, 1983). According to Baker (1989), the movement of water along

gradients through the SPAC occurs in two phases. The first is the liquid phase by bulk

flow through the soil and plant, and the second is the gas phase through the stomatal

region, where water leaves the plant.

The cohesion-tension (CT) theory, introduced by Dixon and Joly (1894, 1895) and

Askenasy (1895), is the traditional theory often used to describe the ascent of water

through the SPAC, although the theory is still vigorously debated and challenged

(Meinzer et al., 2001; Steudle, 2001; Zimmermann et al., 1993, Zimmermann et al.

2004; Angeles et al., 2004). This theory proposes that, during the day, hydrostatic

pressure gradients are generated by the transpiration stream, drawing water into the

roots and through the xylem of the plant. The surface tension that develops at the air–

water interface in the leaves generates a hydrostatic pressure gradient. This is then

transmitted as a negative pressure throughout the entire water column of the plant,

lowering the water potential of the roots below the water potential of the surrounding

soil (Tyree, 1997). This negative pressure is equivalent to a pulling force or tension that

draws water and solutes upward (Tyree, 1997). With increasing transpiration rate

and/or decreasing availability of soil water, the tension within the xylem increases

(Tyree and Sperry, 1988). In order to reduce the amount of cavitation, plants can adjust

the size of the stomatal apertures, controlling the rate of transpiration and hence the

hydrostatic force. Nevertheless, in some cases, plants must compromise between

closing their stomata, which inhibits photosynthesis due to a reduction in CO2

uptake,

and opening their stomata, which increases water loss, for instance, under drought

stress conditions (Fanaeia et al., 2009; Mittler, 2006).

Osmotic pressure is the second force that contributes to water uptake and is based on

the accumulation of solutes across semi-permeable membranes. This also creates a

gradient in water potential generating root pressure, which can cause xylem sap to

exude from a de-topped root system or cut shoot (Oertli, 1991; Zholkevich, 1991).

Current research suggests that the net efflux of solutes from the stem is probably

prevented by the endodermis forming a semi-permeable barrier as the solutes secreted

into the xylem lower the water potential and trigger water flux through the roots. In this

way, more water is drawn into the xylem resulting in an upward flow of solution


P a g e | 26

(Bramley et al., 2007). The driving force of osmotic gradients, however, is only

important during periods of low or inhibited transpiration (Kramer, 1983). Thus, the

osmotic component of the water potential becomes negligible once the solutes in the

xylem sap are slowly diluted by the rising mass flow of water making P the

predominant force controlling the uptake of water within the xylem (Bramley et al.,

2007). In general, as reviewed by Kramer (1983), the transpiration rate can create a

much steeper gradient in the water potential, from the soil to the roots, than the

outlined osmotic mechanism. Nevertheless, when plants experience saline conditions

or reduced water availability during drought stress their water uptake might still be

limited, as the plant cannot lower its water potential sufficiently to create a large

enough gradient between the soil and roots.

Despite the introduction of the CT theory, vigorous debate among scientists in the 19th

century about the mechanism of anti-gravitational water ascent continued as several

scientists had great difficulty understanding the principles behind the theory (Canny,

1995a; Zimmermann et al., 2004). As outlined above, the CT theory considers the

existence of continuous water columns from the roots to the foliage. Through these

columns, water is drawn entirely by transpiration-induced negative pressure gradients

of several MPa. Canny, 1995a and Zimmermann et al. (2004), however, claimed that

they cannot imagine how stable water flow against gravity can occur. Since water

under such negative pressures is extremely unstable, i.e. in a metastable state like

superheated water, especially considering the hydrophobicity of the sap composition

and the vulnerable inner walls of the xylem e.g. proteins, lipids, mucopolysaccharides.

It seems that these would inhibit the development of stable negative pressures larger

than about −1 MPa. Balling and Zimmermann (1990) obtained data using the

minimally-invasive xylem pressure probe technique, which allowed them to directly

measure the pressure in a single xylem vessel of an intact higher plant. This approach

provided clear evidence that moderate negative pressures can exist in the xylem of a

broad variety of higher plant species including well-hydrated leaves in tall trees and

salt-tolerant trees. These studies revealed that xylem tension values are frequently too

low to be consistent with the CT theory (Zimmermann et al., 1994a, b). Moreover, the

basic assumption of a continuous flow through the xylem elements does not

necessarily apply for branches of tall trees, not even under predawn conditions, as

measurements with advanced NMR imaging technology have indicated (Zimmermann

et al., 2002c).


P a g e | 27

A second recognised theory on how water can be lifted and transported in higher

plants is the ‘Multi-Force’ or ‘Water-gate’ Theory, which refers to the analogy during the

lifting of ships by consecutive water-gates. This theory is mainly based on a finely-

tuned interplay of several forces acting in multiple phases of the tissue, in the xylem

and at liquid/gas interfaces. Moreover, in order to keep the various forces, including

tension, at moderate values, an additional strategy of tall trees seems to be the support

of occasional axial barriers in the xylem or adherence to gel matrices for lifted water

volumes.

2.13 Leaf Water Potential Measurements in Plants

The previous sections addressed the mechanisms of how water is transported through

plants and discussed the different strategies of plants to conserve water under drought

stress. This section introduces a range of measurement techniques to determine leaf

water relations, which are relevant to this study.

The pressure bomb technique, pioneered by Scholander et al. (1965), is a widely

referenced and accepted method to measure the leaf water status in agricultural and

plant physiological research (Tyree and Hammel, 1971; Wei and Tyree, 1999; Wei et

al., 1999). This technique, determines the balancing pressure (Pb) values by recording

the external pressure at which water appears at the cut end of a leafy twig or leaf kept

at atmospheric pressure. The Pb values are usually around 1 MPa (Tyree and

Hammel, 1971). The main disadvantages of this technique are that it is destructive,

only gives spot/single time measurements and is unsuitable for automation, and is,

therefore, labour intensive to monitor multiple plants simultaneously (Zimmermann,

2008). Furthermore, a large scatter of balancing pressure values (Pb) is often reported

when multiple twigs or leaves are sampled at the same time (Rueger et al., 2010).

Zimmermann et al. (2004) stated that the microclimate at the measuring site also

influences the obtained values. Moreover, interpreting the data is a matter of debate as

it is not clear whether the recorded balancing pressure is a valid measurement for the

suction tension present in the xylem vessel before cutting the transpiring leaf and

subsequently exposing it to the gas pressure. The many processes that contribute to

the obtained Pb values are not predictable and probably highly environment and

species dependent (Finn, 1989; Wei and Tyree, 1999; Zimmermann et al., 2004,

2007).


P a g e | 28

Another technology for measuring xylem and turgor pressure in individual cells and

xylem vessels, respectively, is the pressure probe technique, pioneered by

Zimmermann et al. (1969), Tomos (1988), Balling and Zimmermann (1990) and

Zimmermann et al. (2004). This technique requires sophisticated equipment and a high

level of technical skills and is not suitable for automation (Ehrenberger et al., 2011).

As a non-invasive measurement approach, leaf thickness is sometimes used as an

indicator for water stress (Burquez, 1987; McBurney, 1992; Malone, 1993). Appropriate

devices to monitor leaf thickness are commercially available and can be used for online

measurements i.e. continuous measurement using data loggers. Since small changes

in water status are not reflected by changes in leaf thickness, this method has major

disadvantages (Zimmermann, 2008). Another non-invasive approach is called ball

tonometry, which enables the monitoring of cell turgor pressure by applying an external

pressure. This technique, however, requires significant technical skills and equipment

(Lintilhac et al., 2000; Geitmann, 2006).

Numerous authors have suggested a range of non-invasive methods to determine

plant water status, mainly to establish feasible indicators for optimal irrigation i.e. plant-

based sensors that measure diurnal changes in trunk diameter, relative changes in leaf

turgor pressure, sap flow, leaf and stem water potential, stomatal conductance, canopy

temperature and/or time domain reflectometry (Scholander et al., 1965; Boyer, 1967;

McBurney, 1988; Cardon et al., 1994; Smith and Allen, 1996; Burgess et al., 2000;

Zweifel et al., 2000; Zweifel et al., 2001; Fernández et al., 2001, 2006; Goldhamer and

Fereres, 2001; Green et al., 2003; Nadler et al., 2003, 2006; Naor et al., 2008;

Zimmermann et al., 2008). However, for technical and practical reasons, the routine

implementation of most of these approaches in crop fields, forests and orchards failed

(Blank et al., 1995; Jones, 2004). In contrast, the leaf patch clamp pressure (LPCP)

probe, introduced by Zimmermann et al. (2008), measures relative changes in leaf

turgor pressure (pc), is non-invasive technique, and leaf water status can be monitored

precisely in real time (Figure 2.2).


P a g e | 29

Figure 2.2: Schematic diagram of the leaf clamp pressure probe (Zimmermann, 2008).

According to Zimmermann et al. (2008), either springs or magnets on the LPCP-probe

generate the clamp pressure. Where leaf-specific structural features influence its

magnitude, the pressure experienced by the cells is lower than the applied clamping

pressure, because of the compressibility of the structural leaf elements, for instance

the walls, cuticle and intercellular air spaces (Rueger et al., 2010; Ehrenberger et al.,

2011). This is accounted for by the attenuation factor (Fa), which measures the gradual

loss of flux through the plant. Ehrenberger et al. (2011) reports that olive leaves, for

example, usually have an attenuation factor of about 0.2–0.3 that is assumed to remain

constant in the turgescent range of the leaf. This means that the transfer of pressure

(Pp) through a leaf patch is mainly dictated by turgor pressure (Pc). Both parameters

are inversely related as indicated from studies on grapevines, olives, lianas and

banana (Westhoff et al., 2009; Zimmermann et al., 2008, 2010; Ehrenberger et al.,

2011). A low output pressure (Pp) measured by the probe corresponds to a high turgor

pressure, preventing pressure transfer through the leaf (Rueger et al., 2010). If the

turgor pressure is very low, the applied clamp pressure is predominantly transferred to

the sensor of the LPCP-probe and the transfer function assumes values close to unity

i.e. Pp assumes a maximum value of the recommended measuring range of the probe


P a g e | 30

(Pp values up to 250 kPa) (Zimmermann et al., 2008). In comparison to the values of

the balancing pressure, i.e. the measurable range of the probes, Pp values are

significantly lower ranging between a maximum turgor pressure of 5 kPa and a

minimum turgor pressure of 200 kPa (Rueger et al., 2010; Ehrenberger et al., 2011).

A number of case studies on several fruit trees and crops under drought have shown

that it is increasingly difficult for plants to compensate for losses of turgor pressure by

water uptake, as indicated by a characteristic change of the profile of the diurnal curves

derived from the output signals of the LPCP-probe (Zimmermann et al., 2008; Ben-

Gala et al., 2010; Rueger et al., 2010; Fernandez et al., 2011; Ehrenberger et al.,

2011). For instance, a typical trend of the Pp curve observed in plants suffering drought

stress usually reaches a low Pp output value during the night relating to a maximum Pc

value. The rise time of the Pp value in the morning then illustrates a Pc decrease,

normally followed by a peak of the Pp value at noon corresponding to a maximum Pc

loss and finally a decline in the Pp value during the afternoon indicating the recovery

phase of Pc. These various parameters are influenced by drought stress and can be

considered as precise measurements for irrigation (Ben-Gala et al., 2010; Fernandez

et al., 2011; Ehrenberger et al., 2011). At noon, when the Pc values decrease to about

50 kPa, the recorded Pp peak values quite often increase up to 250 kPa. At this point

they exceed the recommended measuring range of the LPCP-probe as mentioned

above (Zimmermann et al., 2008; Ehrenberger et al., 2011). Ehrenberger et al. (2011)

suggested that, based on the current theory, the described increase in Pp values can

be used as a clear-cut indication for severe drought stress considering the other Pp

parameters.

A reversal of the diurnal Pp curves, upon approaching the plasmolytic point, occurred

for olive trees under field conditions i.e. peak Pp values occurred during the night,

whereas minimum Pp values occurred at noon (Ben-Gal et al., 2010; Fernandez et al.,

2011). Upon watering the olive trees after a long period of drought, the Pp curves were,

interestingly, completely reversible and the diurnal Pp changes, normally recorded on

leaves, re-established within a relatively short time (Ehrenberger et al., 2011).

Considering the current theory of the LPCP-probe, which suggests that the Pp signal is

exclusively influenced by changes in Pc values, the Pp curves cannot explicitly be

explained (Zimmermann et al., 2008; Westhoff et al., 2009; Rueger et al., 2010a,b).

However, in this context, the study by Ehrenberger et al. (2011) showed that the


P a g e | 31

reversal of the Pp curves, observed in olive trees, may be explained by assuming that,

for Pc values close to zero, Fa is no longer constant due to an unfavourable ratio of

water to air. Therefore, the Pp signals, at very low Pc values, are dominantly influenced

by Fa instead of the Pc factor. The above findings highlight that the LPCP-probe can

detect changes in leaf water relations as long as its limitations under extremely low Pc

values are considered. At present, no work has been conducted on drought-stressed

canola plants using this technology.

2.14 Conclusion

This literature review has clearly shown the significance of canola to the agricultural

industry of Western Australia. An increase in the area of potassium deficient

agricultural sandy soils and the prediction of drier years in the Western Australian

wheat belt has been identified. Therefore, more work is needed to enhance the current

knowledge on plant physiological responses to potassium deprivation and drought

stress particularly for canola. This review has further highlighted the importance of

potassium for many plant physiological and morphological processes, identifying the

need for further research to better understand the effects of soil properties, associated

soil water and weather conditions, and nutrient availability e.g. potassium on root

growth and functioning. The effect of different potassium rates on root and shoot

growth in wheat—root growth is more depressed than shoot growth under reduced

potassium availability—has been investigated. However, more specific knowledge for

canola including estimates for potassium-use efficiency and the effect on shoot to root

ratios is needed.

Moreover, the role of potassium in regulating transpiration rate in canola is an

important research gap as contradictory data exists in the literature for other species

and no information is available on canola. As a consequence, the influence of

potassium on stomatal aperture and the closely-linked rate of photosynthesis needs

further investigation in canola; previous studies have suggested that potassium

deficient plants would increase their transpiration rate to compensate for the increased

rate of potassium uptake by increased xylem sap movement. The leaf clamp pressure

probe has been identified as a suitable method to frequently measure changes in leaf

water potential and might, in combination with other data, shed new light on previous

studies. The underlying project aims to specifically identify the role of potassium on

growth and water use in canola under limiting soil moisture availability by focusing on

plant strategies and mechanisms that impact leaf water content.

P a g e | 32

Chapter 3: Effect of Drought and Potassium on Plant Water Relations of Canola

P a g e | 33

Chapter 3 Effect of Drought and Potassium on Plant Water Relations of

Canola and Its Recovery after Watering

3.1 Introduction

According to simulations conducted by Farré and Foster (2010), 5-10% less rain is

predicted in Western Australia. Therefore, an increasing frequency of low yield years

with grain losses up to 24%, depending on soil type, is expected. With potassium as an

essential macronutrient for plant growth and development, many mechanisms such as

photosynthesis and stomatal regulation, are influenced by the level of potassium

supply (Marschner, 1995; Reddya, Chaitanya and Vivekanandanb, 2004).

Furthermore, Yuncai and Schmidhalter (2005) have outlined that drought stress

reduces both nutrient uptake by the roots as well as transport from roots to the shoots,

leading to limited transpiration rates and impaired active transport as well as decreased

membrane permeability.

Barranco and Benlloch (2006) have highlighted that most studies researching the role

of a specific plant nutrient often only focus on the extreme differences visible under

either a strong deficiency or over-supply of that element. Such studies do not account

for the fact that a mild deficiency, which might occur much faster in the field, can

already impact the physiology of the crop (Barranco and Benlloch, 2006). Hence, there

is a significant economic interest to stabilise and/or increase yields and the value of

crops by improving the regulation of transpiration, to optimise water use efficiency and

enhance survival of plants under environmental stress conditions (Lindhauer, 1989).

This is of particular importance for the potassium deficient sandy soils in the low rainfall

areas of the Western Australian Grain Belt (Farré et al., 2007; Brennan and Bolland,

2007). Furthermore, the accumulation of available potassium prior to the onset of

drought stress has been suggested as an insurance strategy of plants to enhance their

response mechanisms to survive sudden abiotic stress conditions rather than luxury

consumption (Kafkafi, 1990; Kant and Kafkafi, 2002). Potassium is well known to

improve the root hydraulic conductivity, enhancing the water uptake from the soil into

the plant (El-Hadi et al., 1997). Several studies have reported a better growth from

canola plants supplied with sufficient potassium (Brennan and Bolland, 2007).

Moreover, Gunasekera et al. (2009) have pointed out that a

greater capacity for


P a g e | 34

osmotic adjustment could enable canola to produce more dry matter, and potentially to

partition it more efficiently under severe soil water deficits.

There are a number of studies reporting that an adequate supply of potassium plays an

important role in regulating the water relations of plants (Brag, 1972; Hsiao and

Lauchli, 1985; Lösch et al., 1992; Kacperska, 2004; Mittler, 2006; Fanaeia et al., 2009;

Dobra et al., 2010). The resistance of plants to drought could result from an inherent

ability to tolerate internal water stress in the tissue (Samarappuli et al., 1994).

Alternatively, plants might also have developed an osmoregulatory mechanism that

involves potassium, enabling them to actively reduce their transpiration rate during

periods of drought stress (Singh et al., 1985; Kumar and Singh, 1998; Gunasekera et

al., 2009). For instance, one important physiological characteristic allowing plants to

conserve soil water and survive soil moisture deficits, is the ability to decrease cuticular

and stomatal transpirational water loss (Samarappuli et al., 1994). Stomatal closure in

response to moisture stress has, thereby, been highlighted as one of the most rapid

and effective mechanisms for plants to limit stomatal transpirational water loss, which is

mainly impacted by the level of available potassium in the plant (Samarappuli et al.,

1994; Mittler, 2006; Fanaeia et al., 2009).

Moreover, the ability of plants to recover and adjust important physiological functions

such as their stomatal conductance, turgor pressure and photosynthesis quickly after

an imposed drought stress and the interaction of potassium on those mechanisms is

also considered an important trait for survival (Barranco and Benlloch, 2006; Blackman

et al., 2009; Farooq et al., 2008; Lindhauer, 1989; Samarappuli et al., 1994).

Nevertheless, there is little information in the literature on different recovery strategies

in crop plants and no specific information on canola (Blackman et al., 2009).

There is evidence of decreased water loss from plants with higher K concentrations.

For example, Brag (1972) showed that the transpiration rate of wheat (Triticum

aestivum L.) and field peas (Pisum sativum L.) could be regulated by varied K

concentrations in the nutrient solution, with higher concentrations decreasing the

transpiration rate by up to 50%. Further evidence was provided by Lösch et al. (1992)

who showed for spring barley (Hordeum distichum L.) that daily accumulated

transpiration water loss was lower in leaves with high K concentrations than in low K


P a g e | 35

leaves and that, generally, the bulk water relations of the leaves were more favourable

in high K plants i.e. 200 kg ha-1 than in low K plants i.e. 50 kg ha-1, applied as KCl.

A low amount of available potassium in the plant tissue considerably reduces the ability

of the plant to regulate water loss by opening and closing the plant’s stomatal guard

cells (Fischer and Hsiao, 1968; Mengel and Kirkby, 1980; Samarappuli et al., 1994).

Rose et al. (2007) have investigated seasonal potassium accumulation patterns and

distribution within canola plants. Nevertheless, there has been no research conducted

on the physiological effects of a mild deficiency or excess of potassium in canola. In

addition to that, there is no knowledge on the effect of potassium on the diurnal

changes of photosynthesis, stomatal conductance and transpiration rate in canola

although an effect has been described for wheat (triticum eastivum L.) (Guan-He,

2004).

Despite the importance of canola oil for various industries including food, fuel and

livestock sectors, there is little published information of the effect of potassium nutrition

on canola water use, or the optimum rates for grain yield (Smith and Baxter, 2002).

This may be due to a relative short history of canola production in the Grain Belt of WA.

Information on the potassium requirements for different growing environments (rainfall

areas, soil types, row spacing, time of seeding and interaction with other nutrients) is

also scarce (Brennan and Bolland, 2009; Brennan, Mason and Walton, 2000; Farré et

al., 2007).

The aim of this chapter is, therefore, to study the effect of potassium on the water

dynamics of canola. The main hypothesis is that an adequate level of potassium supply

will improve the drought tolerance of canola plants. In particular, it is expected that

plants with a high rate of potassium will better regulate their stomatal conductance

leading to an enhanced rate of photosynthesis and higher relative turgor pressure, by

minimising the overall water loss. This consequently results in a larger plant dry weight

as an indication of the yield potential. In turn, a high potassium level allows the plants

to recover faster at the end of a drying cycle compared to plants that have only

received a low rate or no additional potassium, other than that available in the soil.


P a g e | 36

In order to test this hypothesis, two experiments were conducted. The first focused on

measuring the physiological changes of canola during two consecutive drying cycles

and the sub hypotheses for this experiment were:

I. Canola plants that are fertilised with a high rate of potassium actively reduce

transpirational water loss during drought, by minimising their stomata opening,

while maintaining a higher relative turgor pressure and a relative higher

photosynthetic rate compared to potassium deficient plants.

II. Canola plants are able to adapt to multiple drying cycles by reducing their

stomatal conductance and transpiration rate, which consequently leads to a

reduction of photosynthesis after multiple drying cycles.

The second experiment tested the effect of different potassium fertilizer rates on

diurnal changes in photosynthesis, stomatal conductance and transpiration.

Differences in the rate of recovery between the potassium treatments immediately after

re-watering the stressed plants were specifically monitored. The sub-hypotheses tested

were:

I. The rate of potassium influences the diurnal changes of photosynthesis,

stomatal conductance and transpiration rate of canola plants, whereby, higher

potassium levels lead to a higher rate of photosynthesis compared to lower

levels of potassium.

II. Canola plants that are well supplied with potassium are able to maintain a

higher leaf water potential under drought stress.

III. At the end of a drying cycle, the recovery of the canola plants of the high

potassium treatment will be more rapid as the stomatal conductance and,

subsequently, transpiration rate and relative turgor pressure can be adjusted

faster to changing soil moisture conditions.

3.2 Methodology

The following section will first outline the growing conditions of the two conducted

experiments, before describing the structure of each experiment. After this, the soil and

pot preparation are explained. Subsequently, the conducted measurements and the

harvest procedure is outlined and lastly the statistical analysis presented.


P a g e | 37

The methodology described below applies to both experiments, unless indicated

otherwise. Both experiments were conducted in a glasshouse during the winter months

of 2013 on the Crawley campus of the University of Western Australia in Perth (31.89°

S, 115.81° E), 20 m above sea level, which also represents the main growing season

for the region (Duff et al., 2006). The growing period of both experiments was almost

concurrent, with Experiment 1 being planted on 17 June 2013 and harvested on 23

August 2013, and Experiment 2 being planted on 2 July 2013 and harvested on 20

September 2013.

Experiment 1 comprised 12 well-watered Trigold canola plants and another 12 Trigold

canola plants that were subjected to two consecutive drying cycles. Within the two

water treatments, three different potassium fertilizer levels were established (no added,

low and high K) with four replicates for each potassium rate. Experiment 2 consisted of

30 well-watered and 30 drought-stressed canola plants that were exposed to two

consecutive drying cycles. Again, plants were subjected to the same three different

potassium levels with 10 plants per treatment to have sufficient replications (four) for

destructive measurements (.e.g. pressure bomb measurements).

Sunlight in the glasshouse was recorded every 30 minutes by a roof mounted sensor.

In addition, ambient humidity and air temperature were measured at 5-minute intervals

by two probes positioned adjacent to the samples inside the glasshouse.

3.2.1 Soil and Pot Preparation

Sandy soil was collected from an agricultural field from Grass Valley (S31° 36.382,

E116° 50.068). The soil was taken from a depth of 10 to 40 cm and spread out and air

dried in a drying room for 14 days. Once dry, a sample was taken and passed through

a 2 mm sieve to determine the soil properties. It was subsequently analysed for

potassium, using the Colwell method (1963), and pH (0.01 M CaCl2), as described by

Rayment and Lyons (2010). The analysis showed that the soil was composed of 98%

sand and 2% clay and had an average potassium concentration of 41.6 mg kg-1 and a

pH of 4.9.

The water holding capacity of the soil was analysed according to the method described

by Somasegaran and Hoben (1985). Sieved soil was filled into a cylinder and water

was added from the top until two thirds of the soil column in the cylinder was moist.


P a g e | 38

After 48 hours, a core of the soil in the centre of the cylinder was removed and the wet

weight recorded. The sample was then dried at 105°C until it reached a constant

weight and the water holding capacity or field capacity of the soil was established by

the difference between wet and dry weight. In addition, the water retention curve was

determined using the pressure plate method described by Dane and Topp (2002). The

soil contained 5.6% water by weight when air dried and 15.8% at field capacity. The

soil water content decreased rapidly from 27% at -0.1 kPa to 2.8% at -300 kPa before

staying relatively constant and reaching the permanent wilting point (1500 kPa) at 2.6%

water content (Figure 3.1).

Figure 3.1: Soil water retention curve of experimental soil.

Once all soil properties had been analysed, the air dried soil was passed through a 6

mm sieve in order to remove any gravel or crop residues. Stock solutions were

prepared for each nutrient in a way that 10 ml would supply the amounts of nutrients

listed in Table 3.1. For each pot, 5.5 kg of soil was thoroughly mixed with 10 ml of each

stock solution and 2 g of dry CaCO3 to increase the pH above 5.5. In addition, the soil

received another application of 0.142 g nitrogen kg-1 soil before the drying cycle to

ensure that the nitrogen supply was not limiting growth.

0

5

10

15

20

25

30

0.1 100 300 1500

Gra

vim

etric

Wat

er C

onte

nt (%

)

Suction (kPa)

Permanent wilting point


P a g e | 39

Table 3.1: Nutrient composition of applied fertiliser stock solution.

Nutrient Concentration of nutrient (mg kg-1) In form of

N 142 NH4NO3

P 53 NaH2PO4.2H2O

Mg 81 MgSO4

Ca 81 Ca(H2PO4)2

B 0.773 H3BO3

Mn 0.169 MnSO4.7H2O

Zn 0.288 ZnSO4.7H2O

Cu 0.063 CuSO4.5H2O

Mo 0.041 NaMoO4

Fe 0.05 Fe EDTA

For Experiment 1, the prepared soil was subsequently filled into 4.3 L sealed plastic

pots, which were 19 cm in height and diameter at the top and 16 cm diameter at the

bottom. The pots were placed randomly in the glasshouse and watered to field

capacity. Forty eight hours after watering, 10 Trigold Canola seeds were placed in

each pot at a depth of 0.5 cm to ensure the growth of at least one seedling per pot.

Two weeks after planting, the seedlings were thinned to only one plant per pot.

Additionally, three further pots were filled with the substrate and also watered to field

capacity. These pots were covered with plastic beads and weighed at regular intervals

to monitor soil water evaporation potential.

Pot preparation was similar for Experiment 2, except two 14-day old pre-germinated

canola seedlings were transplanted from a seedling tray into each pot and this was

done forty eight hours after each pot was watered to field capacity. After a further

week, these plants were subsequently thinned to only one plant per pot. Before the

water stress was imposed, all pots were kept at field capacity to allow even canola

growth. Once the seedlings had reached the four leaf stage, 200 g of plastic beads

were added to cover the soil surface of each pot in order to minimise water loss by

evaporation. One week prior to the start of the drying cycle the three potassium

treatments were established, as described in the next section.


P a g e | 40

3.2.2 Potassium

During the initial growing period no additional potassium fertiliser was added to the

pots. At the 6-8-leaf stage, one week prior to the start of the drying cycle, three

different potassium levels were implemented by adding potassium in form of KCl to the

irrigation water. The first treatment did not receive any additional potassium (no added

K: 41 mg kg-1 soil), the second received 15 mg kg-1 soil of potassium (low K: 56 mg kg-1

soil) and the third treatment received 61 mg kg-1 soil of potassium (high K: 102 mg kg-1

soil).

3.2.3 Watering

During the growing period of both experiments each pot was placed on a balance

every two days and deionised water was added until field capacity (pot weight of 6275

g) was reached. At bud development stage during Experiment 1 half of the plants of

each potassium treatment were exposed to a 10-day drying cycle lasting from 31 July

to 10 August 2013. The aim of the drought period was to generate a continuing stress

level which increased over time. In order to sustain the drying cycle for this relatively

long period on the sandy soil, the amount of irrigation water for all stressed treatments

was reduced in increments of 60 ml per day until the samples reached 64.5% field

capacity. The plants were then kept at 64.5% field capacity for the rest of the drying

cycle to prevent premature death. This drought period was followed by a 2-day

recovery period where samples were irrigated daily to field capacity. A second 9-day

drying cycle was initiated on 12 August 2013 ending on 20 August 2013 following the

same approach as during the first drying cycle. Control plants were kept at field

capacity throughout the experiment by watering them at least every second day.

Experiment 2 was conducted in a similar way. Half of the plants were exposed to two

consecutive drying cycles; however, this time no additional water was added to sustain

the drying cycle for an extended time frame. Thus, both drying cycles in the second

experiment were much shorter than for Experiment 1. The first drying cycle lasted for

three days (10 - 12 September 2013) and was followed by a 3-day recovery period.

The second drying was imposed for four days (16 – 19 September 2013) before all

plants were watered back to field capacity. As during the first experiment, all control

samples were kept at field capacity throughout the whole experiment.


P a g e | 41

During both experiments the pot weight of each sample was recorded four times a day,

at 08:00, 10:30, 13:30 and 16:30 hrs, during the two consecutive drying cycles and the

subsequent recovery phase to monitor diurnal changes and the overall loss of water

throughout the experiment.

3.2.4 Relative Turgor Pressure

The relative turgor pressure was monitored using leaf patch clamp pressure (LPCP)

probes (Yara ZIM plant technology, Hennigsdorf, Germany) as described by

Zimmermann (2008). For both experiments, twelve canola plants were monitored using

the LPCP-probes, with one LPCP-probe clamped onto the fourth fully expanded leaf of

each plant using the 2.5 mm magnet in order to prevent any damage to the fragile

canola leaves. This approach ensured comparable probe readings as the leaves were

fully developed, but not too old to be the next senesced leaf by the plant. Six of the

plants from the no added K treatment and six from the high K treatment were clamped.

Three plants of each treatment were water-stressed samples and the other three were

control samples.

The clamping occurred in the morning when the plants had a high turgor pressure. The

applied clamping pressure (PClamp) ranged from 20 to 40 kPa. For Experiment 1, the

relative turgor pressure was logged every five minutes throughout the experiment.

During the recovery phase of Experiment 2 the relative turgor logging interval was

decreased to 2 seconds to achieve increased resolution and allow monitoring of

potential time differences in water uptake between the three potassium treatments.

Note that the output from the pressure probes (Pp) reflects the inverse of the relative

turgor pressure (Pc) i.e. an increase in Pp equals a decrease in Pc. The baseline Pp is

the linear regression of all minima Pp readings, illustrating the trend of relative turgor

pressure (Rueger et al., 2010). In order to enable comparison of the diurnal LPCP-

probe output pressure readings (Pp), Pp data for each plant was normalised to the

readings before the treatment was imposed, using the formula:

lamp – p min nd day after clamping

p ma nd day after clamping


P a g e | 42

3.2.5 Leaf Water Potential in Experiment 2

The midday leaf water potential of droughted and control canola samples was obtained

using a PMS 1000 pressure bomb from PMS Instruments (Albany, USA). One leaf was

cut from each plant and immediately placed in the pressure chamber, which was then

pressurised until plant sap was visible at the cut end. At this point the pressure was

noted and used as the turgor pressure of the measured sample as described by

Scholander et al. (1964). In order to minimise any evaporational water losses, each

leaf was put in a plastic bag once cut off the canola plant.

3.2.6 Osmotic Potential

Immediately before the harvest of both experiments, the osmotic and leaf water

potential of six samples per potassium treatment, including three drought-stressed and

three control samples, were determined according to the method described by Callister

et al. (2006) using a Fiske 110 osmometer (Norwood, USA). Thereby, leaf sap was

extracted from a single leaf of each tested plant using a plant sap press and pipetted

into the osmometer cell for analysis.

3.2.7 Photosynthesis, Stomatal Conductance and Transpiration

In order to monitor any differences in photosynthesis, stomatal conductance and

transpiration between the three potassium treatments, gas exchange measurements

were conducted for both experiments using a LI-6400 portable photosynthesis system

from Licor (Lincoln, USA). In preparation for the analysis a light response curve was

obtained with the LI-6400 in order to determine appropriate light settings for the system

(Figure 3.2). Based on the resulting curve, the light intensity was set to 1000 µmol m-2

s-1 and the rate of CO2 in the cuvette was kept at 380 µmol m-2 s-1 reflecting CO2 in the

ambient air.


P a g e | 43

Figure 3.2: Light response curve measured for Trigold Canola used in study.

During Experiment 1, three samples of each potassium and water stress treatment

were monitored daily between 9:00 hrs and 12:00 hrs always using the third youngest,

fully developed leaf. For Experiment 2 two different measurements were conducted.

Firstly, the diurnal changes of photosynthesis, stomatal conductance and transpiration

rate of the three control potassium treatments were monitored to observe differences in

diurnal adjustment strategies between the different treatments. Samples were

measured every 90 minutes between 9.00 hrs and 17.30 hrs with the data being

continuously logged for 30 minutes per sample. The second part of Experiment 2

investigated the short-term recovery phase of the drought-stressed canola samples by

measuring photosynthesis, stomatal conductance and transpiration immediately after

watering back to field capacity and then again about 60 and 150 minutes after

irrigation.

3.2.8 Leaf Temperature

For Experiment 1, the leaf temperature was measured with a TiR32 infrared camera

from Fluke (Everett, USA). Three replicates of one drought stressed and one control

sample of each potassium treatment were positioned next to each other and three

images were taken at a height of 1.5 m above and an angle of 45°. The images were

taken every 3 to 5 days during the two consecutive drying cycles and the recovery

periods aiming to have similar light conditions for each recording. On each measuring

day the images were always taken between 11:00 and 13:00 hrs to coincide with high

-9.4

0.0

9.4

18.8

28.1

37.5

0 200 400 500 600 700 800 900 1000 1100 1200

Phot

osyn

thes

is (μ

mol

m-2

s-1

)

Light Intensity (μmol m-2 s-1)


P a g e | 44

levels of solar radiation. Furthermore, the ambient temperature was also recorded by a

temperature sensor that was positioned between the two samples each time an image

was taken as well as the temperature of a metal plate probe that was always included

in the image, which served as a reference point for calibration. For the analysis of the

images, the calibrated temperature of three leaves per sample was determined and the

mean leaf temperature per sample and per treatment calculated using the software

provided with the camera called ‘Smartview’ (Fluke, Everett, USA).

3.2.9 Harvest

The samples were harvested at the end of the two consecutive drying cycles. By that

time, 70% of all samples had reached the flowering stage. During the harvest, all

leaves were cut off each plant and the leaf area determined using an LI-3000C

portable area meter (Licor, Lincoln, USA), then the shoots were separated from the

roots. The complete shoot material (leaves and stems) as well as the roots of each

sample were then dried in an oven at 70°C for 48 hours to determine the dry weight of

each plant. For Experiment 2, only four of the ten replicates of each treatment were

harvested to allow better comparison to Experiment 1. Additionally, the number of pods

of all canola plants was counted in order to estimate the yield potential of each

potassium treatment.

3.2.10 Experimental Design and Statistical Analysis

The experiment was set up in a complete randomised block design with four replicates

of six treatments. The treatments had a factorial arrangement of three potassium

treatments (no added K, low K and high K) by two water treatments (stressed and well

watered control). To ensure similar environmental growth conditions, all samples were

regularly relocated in the glasshouse. The obtained data were statistically analysed

with Microsoft Office Excel 2008 (Microsoft, Redmond, USA), Origin V from OriginLab

(Northampton, MA) and SAS/STAT (SAS Institute Inc., Cary, USA) to calculate mean

values, standard error and, where appropriate, an ANOVA with a Tukey-test as a

posthoc test, with differences at p ≤ 0.05 being regarded as significant.

3.3 Results

The following section will outline the relevant obtained findings for the two conducted

experiments. First the environmental growing conditions for both experimental periods


P a g e | 45

are presented. After that, the morphological measurements of Experiment 1 e.g. leaf

area, shoot and root dry weight will be shown, before any physiological measurements

such as transpiration per leaf area, stomatal conductance, photosynthesis and relative

turgor pressure are described and depicted. The second part of the result section will

then also first describe the morphology of Experiment 2, before the physiological

measurements are explained starting with the conducted leaf water potential

measurements under drought-stress and then illustrating the diurnal changes of the

control samples of the three potassium rates. At the end, the focus will be on the short

term recovery after water limiting conditions specifically looking at the response of

transpiration rate, stomatal conductance, photosynthesis and relative turgor pressure.

3.3.1 Environmental Conditions

Figure 3.3 shows the environmental conditions for both conducted experiments with

Experiment 1 lasting from 31 July 2013 until 23 August 2013 and Experiment 2

commencing on 8 September 2013 and finishing on 20 September 2013. The climatic

conditions for both experiments were very similar with temperature ranging from 12°C

to 34°C during the day, with an average temperature of 20°C. The coldest days were

observed on days 5 and 7 for Experiment 1, showing temperature below 23°C, and

days 40, 41 and 44 for Experiment 2, ranging between 21 and 26°C. A series of warm

days was noticeable towards the end of Experiment 1 (days 16 – 23) with temperatures

reaching 31 to 33°C. For Experiment 2 the hottest days were noticeable at the start of

each of the two drying cycles. The relative humidity showed a minimum of 18% and a

maximum of 94%, averaging 67% during the measurement phases of both

experiments.

The ambient light level during Experiment 1 was on average 382 µE m-2 s-1 with a

maximum of 741 µE m-2 s-1. Day 5 showed the lowest daily maximum of 245 µE m-2 s-1

(Figure 3.3). The ambient light level during Experiment 2 was generally higher than

during Experiment 1 with an average of 663 µE m-2 s-1. Overall the ambient light level

was fairly constant during the measurement period of Experiment 2, with values around

1400 µE m-2 s-1, except for day 40 where only 580 µE m-2 s-1 was reached and day 48

with a maximum of 1667 µE m-2 s-1 (Figure 3.3).


P a g e | 46

Figure 3.3: Temperature (A), relative humidity (B) and ambient light level (C) during

Experiments 1 and 2 lasting from 31 July 2013 to 20 September 2013. Black arrows

indicate initiation of drying cycles; white arrows indicate watering of plants.

Experiment 2

0

5

10

15

20

25

30

35

40

1 2 3 4 5 10

Tem

pera

ture

(°C

)

A

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 10

Rel

ativ

e H

umid

ity (%

)

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0

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400

600

800

1,000

1,200

1,400

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1 4 6 9 10 13 16 19 23 26 29 32 35 38 41 44 47 50

Am

bien

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ht L

evel

(μE

m-2

s-1

)

Days after 30 July 2013

C

Experiment 1


P a g e | 47

3.3.2 Experiment 1

The following part outlines the main findings of Experiment 1. First the harvest results

of the canola plants are shown, followed by the physiological measurements during the

experimental period.

3.3.2.1 Leaf Area

Figure 3.4 shows the mean leaf area per pot of all potassium treatments at the end of

the experiment. The control samples had significantly larger leaf areas than the

drought-stressed plants (P=0.007). However, there was no significant difference

between the three potassium treatments within each water treatment. Nonetheless, it is

interesting that, for both, control and drought-stressed samples, the low K treatment

had on average the smallest leaf area (295 and 381 cm2, respectively), whereas the

high K treatment showed the largest leaf area with 315 and 360 cm2, respectively.

Figure 3.4: Mean leaf area per pot of drought stressed (D) and well-watered (C) canola

plants treated with different potassium fertiliser rates (no K, low K, high K) measured on the

day of harvest (23/08/2013). Error bars show ± SE (n=4). Different letters above the bars

indicate significance at ≤0.05 (potassium main effect P=0.76, water main effect P=0.04,

interaction P=0.91).

3.3.2.2 Shoot and Root Dry Weight

The well-watered samples had significantly higher shoot and root dry weights and a

significantly smaller shoot:root ratio than the water-stressed samples (P=0.001,

P<0.001 and P=0.03, respectively) (Figures 3.5). There were no significant differences

between the potassium treatments for these measurements.

0

150

300

450

600

No K Low K High K No K Control Low K Control High K Control

Le

af

Are

a (

cm

2)

Potassium Rate and Water Treatment

No K (D) Low K (D) High K (D) No K (C) Low K (C) High K

a

b

a a


P a g e | 48

0

5

10

15

20

25

30


Shoo

t Dry

Wei

ght (

g) a

a a b b b

0

1

2

3

4

5

6

7

8

9


Roo

t Dry

Wei

ght (

g) a

a a

b b b

0

0.5

1

1.5

2

2.5

3

3.5

4


Shoo

t:Roo

t Rat

io


a a a b b b

Figure 3.5: Average dry weights of shoot (A) and root (B), and shoot to root ratio (C) of

drought-stressed (D) and control plants (C) treated with three different potassium rates (no

K, low K, high K). Error bars show ±SE (n=4). Different letters above the bars indicate

significance at ≤0.05 (potassium main effect A: P=0.30, B: P=0.85, C: P=0.85; water main

effect A: P=0.001, B: P<0.001, C: P=0.03; interaction A: P=0.62, B: P=0.78, C: P=0.78).

A

B

C

A


P a g e | 49

3.3.2.3 Leaf Temperature

There was a significant difference between the leaf temperatures of the control and the

drought-stressed samples (P=0.001). Generally the leaves of the control samples were

cooler than ambient temperatures, when measured with an infrared camera, whereas

the drought-stressed samples showed leaf temperatures higher than the ambient

temperature (Figure 3.6).

Figure 3.6: Difference between leaf and ambient temperature of the drought-stressed

(D) and well-watered (C) canola plants treated with three different potassium rates (no

K, low K, high K). ‘Watered*’ indicates watering back to 64.5% available soil water to

prevent premature dying of plants.

Focusing on the last day of the first drying cycle (day 10), significant differences

between the no added K and the high K treatment of both the drought-stressed and the

control samples were measured (P=0.02) (Figure 3.7). On the last day of the recovery

period (day 13) the low K treatment of the drought-stressed and control samples had

the lowest leaf temperature, being significantly lower than the high and the no added K

rate (P=0.01 - 0.03, respectively) (Figure 3.7). During the second drying no significant

differences between water and potassium treatments were measured.

-7.0

-3.5

0.0

3.5

7.0

10.5

4 5 10 11 13 17 19 21 22 23 24

Diff

eren

ce b

etw

een

Leaf

and

Am

bien

t Te

mpe

ratu

re (°

C)


No K Low K High K

No K Control Low K Control High K Control

2nd drying cycle

Watered

Watered

1st drying cycle

Watered* Watered*

No K (D) (D) (D)

No K (C)

Low K (D)

Low K (C)

High K (D)

High K (C)


P a g e | 50

Figure 3.7: Difference between leaf and ambient temperature of the drought-stressed

(D) and well-watered (C) canola plants treated with three different potassium rates (no

K, low K, high K) on days 10 and 13 of the experiment. Error bars show ±SE (n=3).

Different letters above the bars indicate significance at ≤0.05 between different

potassium rates within the same water treatment.

3.3.2.4 Water Use

Figure 3.8 shows the water use of control and drought-stressed samples treated with

three different potassium rates during two consecutive drying cycles by means of the

pot weights. The control samples were watered daily to maintain pot weights between

6250 g and 6150 g. The drought-stressed samples showed a rapid decrease in pot

weight down to approximately 6000 g during both drying cycles. Overall the no added

K treatment had the lowest average water use per pot, whereas the low K treatment

had the highest average water use. However, these differences were not significant a

P≤0.05.

-7.9

-5.3

-2.6

0.0

2.6

5.3


Dif

fere

nc

e b

etw

ee

n L

ea

f a

nd

Am

bie

nt

Te

mp

era

ture

(°C

)

Potassium Rate and Water Treatment Day 10 Day 13

ab

c d cd A B A β γ

a

b α


P a g e | 51

Figure 3.8: Pot weights of drought stressed (D) and control (C) samples treated with three

different potassium rates (no K, low K, high K) during two consecutive drying cycles.

‘Watered*’ indicates watering back to 64.5% available soil water to prevent premature dying

of plants.

3.3.2.5 Transpiration per Leaf Area

Contrasting trends in transpiration rate were observed between the well watered

control and drought-stressed treatments over the course of the experiment. The control

samples showed an increasing trend in transpiration per leaf area over the

experimental period. The drought-stressed samples showed a decrease in

transpiration per leaf area during each of the two drying cycles and a rapid increase

during the recovery phases (Figure 3.9). The transpiration per leaf area recovered for

all potassium treatments back to the values measured at the start of the experiment.

Towards the end of each drying cycle all potassium treatments reached similar

minimum transpiration values of about 0.04 g H2O cm-2.

In general the transpiration per leaf area of the control treatments was higher than for

the drought stressed samples, except on day 7 and days 12 and 13, at the time when

the second drying cycle was initiated, where the transpiration was very similar. All

drought stressed samples showed an increase in transpiration per leaf area from day 5

5800

5850

5900

5950

6000

6050

6100

6150

6200

6250

6300

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Pot W

eigh

t (g)



Watered 2nd drying cycle

1st drying cycle

Watered

Watered* Watered*


P a g e | 52

until day 7. The low K treatment of the control samples showed a significantly higher

transpiration than the no added and the high K treatments (P=0.008 and P=0.03,

respectively), except for day 21 when the second recovery period was initiated. The

high and no added K treatments showed no significant differences with the no added K

treatment having a slightly higher transpiration during the first drying cycle and the

recovery phases, whereas the high K treatment showed a slightly higher water loss

during the second drying cycle. The only exception was again day 21.

For the water stressed samples, it was again the low K treatment which had the

highest transpiration per leaf area, except for days 10 to 12 where it showed a very

similar water loss to the other treatments and days 15 to 17, where the no added K

treatment showed a slightly larger water loss per leaf area. For the other two

treatments, the high K treatment had the second largest water loss during the first

drying cycle and the lowest during the second drought period. However, no significant

differences were observed between the drought-stressed samples (Figure 3.9).

Figure 3.9: Transpiration per leaf area of drought stressed (D) and control (C) samples

treated with three different potassium rates (no K, low K, high K) during two consecutive

drying cycles measured by weight. ‘Watered*’ indicates watering back to 64.5% available

soil water to prevent premature dying of plants.

0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Tran

spira

tion

per L

eaf A

rea

(g H

2O c

m-2

)



Watered

2nd drying cycle

1st drying cycle

Watered

Watered*

Watered*


P a g e | 53

3.3.2.6 Transpiration Rate

In general the well-watered and the drought-stressed samples showed similar

transpiration trends; nevertheless, a significant difference could be measured between

the two water treatments (P=0.02) as the drought-stressed plants showed a lower

transpiration rate than the control treatments towards the ends of both drying cycles

(days 4 - 10 and 15 - 21). During the first drying cycle the transpiration declined for all

samples, but rapidly increased again during the two-day recovery phase, peaking on

the first day of the second drying cycle. During the second drying cycle the

transpiration of all samples decreased again before rising again after watering. On

average the no added K treatment of the stressed plants had a higher transpiration rate

than the other two droughted treatments; however, the differences between the no

added and high K treatments were only significant between days 2 and 8 during the

first drying cycle (P=0.03) (Figure 3.10). No further significant differences could be

observed between any of the treatments. The Licor malfunctioned between days 18 to

21. Consequently those days are not shown for the transpiration rate, stomatal

conductance and photosynthesis.

Figure 3.10: Transpiration rate of drought-stressed (D) and control (C) samples treated

with three different potassium rates (no K, low K, high K) during two consecutive drying

cycles. ‘Watered*’ indicates watering back to 64.5% available soil water to prevent

premature dying of plants.

0

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Tran

spira

tion

(mm

ol m

-2 s

-1)


No K Low K High K


Watered

2nd drying cycle 1st drying

cycle

Watered

Watered*

Watered*


P a g e | 54

3.3.2.7 Stomatal Conductance

In general the stomatal conductance followed a similar trend as the transpiration rate,

with an overall significant difference between the drought-stressed and well watered

control (P=0.01) (Figure 3.11). Furthermore, as observed for the transpiration rate, the

no added K treatment showed a significantly larger stomatal conductance than the low

K and high K treatment for the drought stressed plants between days 4 and 8 of the

first drying cycle with p-values of 0.02 and 0.002, respectively for the two days. There

were no other significant differences.

Figure 3.11: Stomatal conductance of drought-stressed (D) and control (C) samples

treated with three different potassium rates (no K, low K, high K) during two

consecutive drying cycles. ‘Watered*’ indicates watering back to 64.5% available soil

water to prevent premature dying of plants.

3.3.2.8 Rate of Photosynthesis during Two Consecutive Drying Cycles

Photosynthesis followed a similar trend in all drought-stressed treatments; it increased

for the first two days, then declined rapidly. After watering on day 11, the

photosynthetic activity increased again, reaching almost their initial values on days 14

and 15. During the second drying cycle a similar drop in photosynthesis was observed.

During the second recovery phase the photosynthetic rate of all samples increased

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Con

duct

ance

(mol

m-2

s-1

)



Watered

2nd drying cycle

1st drying cycle

Watered

Watered* Watered*


P a g e | 55

again. Interestingly, the drought-stressed no added K treatment showed the highest

photosynthetic activity during the two drying cycles followed by the low and high K

samples. However, differences between the high K and the no added and low K

treatments were only significant during days 2 to 8 (P=0.04). After the samples had

been watered again, the high K treatment recovered faster than both of the other

treatments (Figure 3.12).

The control samples showed similar trends to the drought-stressed samples but on

average maintained a higher photosynthetic rate throughout the experiment. Significant

differences were observed between the low K treatment and the no added and high K

treatments for the well watered control, particularly between days 13 to 17, where the

low K treatment showed a significantly lower photosynthetic rate than the other two

treatments with P-values of 0.009 and 0.01, respectively (Figure 3.12).

Figure 3.12: Photosynthetic rate of drought-stressed (D) and control (C) samples

treated with three different potassium rates (no K, low K, high K) during two

consecutive drying cycles. ‘Watered*’ indicates watering back to 64.5% available soil

water to prevent premature dying of plants.

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Phot

osyn

thes

is (μ

mol

m-2

s-1

)

Days after 30 July

No K Low K High K


2nd drying cycle 1st drying cycle

Watered

Watered Watered*

Watered*


P a g e | 56

3.3.2.9 Relative Turgor Pressure during Drought Stress

The leaf patch clamp pressure probes malfunctioned on some days at the start of the

first drying cycle during Experiment 1. Therefore, only the later data after day 13 was

presented. Both the monitored potassium treatments (no added and high K) showed

expected diurnal changes with high Pp-values (indicating low turgor pressure) during

the day and lower Pp-values at night. With increasing drought stress the Pp-values

showed an increase during the day with the no added K treatment showing close to

250 kPa and the high K treatment values up to 160 kPa. There was an unexpected

drop of the Pp-values of the no added K stress treatment during the night one day prior

to re-irrigation whereas the high K stress plants show a strong decrease of the Pp-

values two days prior to re-irrigation (Figure 3.13).

Figure 3.13: Examples of Pp measurements on canola subjected to different potassium

treatments. Figure A: no added K – drought = dotted line with filled circles; control = solid

line with open circles. Figure B: high K - drought = dotted line with filled squares, dotted

2nd drying cycle

Watering

13 15 17 19 21


Watering*


P a g e | 57

line; control = solid line with open squares. Figure C: corresponding ambient temperature

(T – black line and squares) and relative humidity (R.H. – grey line and squares). Black

bars top panel indicate irrigation events of control plants. Grey bar indicates irrigation event

of all plants. ‘Watered*’ indicates watering back to 64.5% available soil water to prevent

premature dying of plants.

3.3.3 Experiment 2

This second part of the results section first presents the leaf area and dry-mass data at

harvest then the leaf water potential of the three potassium treatments during the

drying cycle. This is followed by the diurnal changes of the control samples of the

different potassium levels and, finally, the transpiration rate, stomatal conductance,

photosynthesis and relative turgor pressure findings during the short-term recovery

phase after drought-stress are presented.

3.3.2.1 Leaf Area

The high K control treatment had the largest leaf area of all samples, with 759 cm2,

closely followed by the low K control treatment. The low K treatment showed the

largest leaf area of the drought-stressed samples, with 602 cm2, followed by the high K

and the no added K treatment. Nevertheless, no significant differences could be

observed (Figure 3.14).

Figure 3.14: Leaf area of drought-stressed (D) and well-watered (C) plants treated with

three different potassium rates (no K, low K, high K). Error bars show ± SE (n = 4).

-125

100

325

550

775

1,000


Le

af

are

a (

cm

2)


a

a a

a a

a


P a g e | 58

3.3.3.2 Root and Shoot Dry Weight

The low K treatment showed the highest shoot dry weight among both the well-watered

and the drought-stressed samples. The no added K treatment had a significantly lower

shoot dry weight than the low and high K treatments among both the well-watered and

the drought-stressed samples (P=0.001-0.009). The root dry weight was lowest for the

high K treatment for both controls and drought-stressed samples, showing a significant

difference to the no added K drought-stressed samples and to the low K control

samples (P=0.05). The largest root dry weight for the drought-stressed samples with

12.3 g was observed for the low K treatment (Figure 3.15).

Figure 3.15: Root and shoot dry weight of drought-stressed (D) and well-watered (C)

plants treated with three different potassium rates (no K, low K, high K). Error bars show ±

SE (n = 4). Different letters above the bars indicate significance at ≤0.05 between

different potassium rates within the same water treatment (potassium main effect: shoot

P<0.001, root: P=0.04; water main effect: shoot P<0.001, root P=0.62; interaction: shoot

P=0.98, root: P=0.65).

The high K treatment showed for both the control and drought-stressed treatments the

highest shoot to root ratio with 2.16 and 1.59, respectively, being significantly larger

(P=0.04) than the shoot to root ratio for the no added K treatment, which had the

smallest shoot to root ratio of all treatments (Figure 3.16).

0

4

8

12

16

20


Dry

We

igh

t (g

)


Shoot Root

a

b b

AB

A

CD C

A

d

c

B b


P a g e | 59

Figure 3.16: Shoot:root ratio of drought-stressed (D) and well-watered (C) plants treated

with three different potassium rates (no K, low K, high K). Error bars show ± SE (n = 4).

Different letters above the bars indicate significance at ≤0.05 between different potassium

rates within the same water treatment (potassium main effect P=0.009, water main effect

P=0.03, interaction P=0.93).

3.3.3.3 Pod Number

There were no significant differences between the different treatments, although the no

added K had the lowest pod number for both the control and drought-stressed

treatments (Figure 3.17).

Figure 3.17: Pod number of drought-stressed (D) and well-watered (C) plants treated with

three different potassium rates (no K, low K, high K). Error bars show ± SE (n = 4).

0.00

0.65

1.30

1.95

2.60


Sh

oo

t:R

oo

t R

ati

o


ab

b

c

cd

d

0

65

130

195

260

325


Po

d n

um

be

r


a a

a

a a

a

a


P a g e | 60

3.3.3.4 Leaf Water Potential

During the drought stress, the leaf water potentials of the no added and low K

treatment were significantly lower than the leaf water potential of the high K treatment

(P=0.003 and P=0.001, respectively). All of the well-watered samples showed similar

values of between 0.2 to 0.25 MPa (Figure 3.18).

Figure 3.18: Midday leaf water potential of drought-stressed (D) and well-watered (C)

plants treated with three different potassium rates (no K, low K, high K) measured on day 1

of the experiment. Error bars show ± SE (n = 3). Different letters above the bars indicate

significance at ≤0.05 (potassium main effect P<0.001, water main effect P<0.001,

interaction P<0.001).

3.3.3.5 Diurnal Changes of Photosynthesis, Transpiration and Stomatal Conductance

The temperature on the day of the diurnal measurements was 18°C in the morning,

gradually increasing to 35°C at 12:15 hrs, before steadily decreasing again to 21.5°C in

the evening. The relative humidity followed an opposite trend starting at 76% in the

morning, dropping down to 29% at lunch time before rising again to 60% at 6:00 hrs

(Figure 3.19).

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0 No K Low K High K No K Control Low K Control High K Control

Wat

er P

oten

tial (

MPa

)


a

b

c c c

a


P a g e | 61

Figure 3.19: Temperature and relative humidity on the day of the diurnal

measurements (18 September 2014).

The photosynthetic rate of the no added K treatment remained around 8 μmol m-2 s-1

until it increased to 17 μmol m-2 s-1 at 09:30 hrs. It then decreased to 8 μmolm-2s-1 again

at approximately 12:00 hrs and then further to 7.5 μmolm-2s-1 at around 13:00 hrs,

before rising again to 16.7 μmolm-2s-1. After a small decrease the no added K treatment

showed another increase in photosynthesis, leveling at 21.8 μmol m-2 s-1 before

declining again at 16:45 hrs (Figure 3.20 C).

The photosynthetic rate of the low K control plants gradually increased throughout the

day starting at 3.30 μmolm-2s-1 at around 09:40 hrs and reaching 21.4 μmolm-2s-1 at

17:19 hrs. The increase was slightly steeper during the morning hours and became

more moderate during the midday and afternoon hours. Small decreases in

photosynthesis were observed at around 10:00 hrs and 13:00 hrs (Figure 3.20 C).

During the morning of day 1 the high K control treatment had a photosynthetic rate of

around 15 μmol m-2 s-1. The photosynthetic rate increased to 25 μmol m-2 s-1 at 12:00

hrs before dropping back to 15 μmol m-2 s-1 at 13:30 hrs and further to 11 μmol m-2 s-1 at

around 15:00 hrs. Thereafter, it slowly increased again reaching 14 μmol m-2 s-1 by

18:00 hrs (Figure 3.20 C).

0

10

20

30

40

50

60

70

80

90

0

5

10

15

20

25

30

35

40

8:00

8:

20

8:40

9:

00

9:20

9:

40

10:0

0 10

:20

10:4

0 11

:00

11:2

0 11

:40

12:0

0 12

:20

12:4

0 13

:00

13:2

0 13

:40

14:0

0 14

:20

14:4

0 15

:00

15:2

0 15

:40

16:0

0 16

:20

16:4

0 17

:00

17:2

0 17

:40

18:0

0

Rel

ativ

e H

umid

ity (%

)

Tem

pera

ture

(°C

)

Time (hrs)

Temperature Relative Humidity


P a g e | 62

The stomatal conductance of the no added K treatment stayed around 0.10 mol m-2 s-1

until approximately 9:30 hrs. At this point it rapidly increased to 0.35 mol m-2 s-1 before

gradually decreasing again. The stomatal conductance showed another increase at

around 13:00 hrs and another rapid rise at 14:30 hrs, before falling back to 0.50 mol m-

2 s-1 at around 16:30 hrs. The transpiration rate of the no added K treatment followed

the same pattern (Figure 3.20 A and B).

In general the conductance and transpiration of the low K treatment followed a similar

trend as observed for the photosynthesis. Towards the late afternoon the stomatal

conductance of the low K treatment showed a very rapid increase similar to the no K

treatment, whereas the transpiration and photosynthesis of the low K treatment

remained relatively level (Figure 3.20 A and B).

As observed for the low K treatment the transpiration and conductance of the high K

treatment showed a similar trend to photosynthesis. The only difference was a more

rapid initial decline of the stomatal conductance after reaching its peak at 11:50 hrs

(Figure 3.20 A and B).


P a g e | 63

Figure 3.20: Average diurnal transpiration (A), stomatal conductance (B) and

photosynthesis (C) of well-watered plants (n = 5) treated with three different potassium

rates (no K, low K, high K) measured on day 1 of the experiment.


P a g e | 64

3.3.3.6 Short Term Recovery

The control samples showed a significantly higher photosynthesis rate than the

drought-stressed samples (P=0.001) (Figure 3.21). No significant differences could be

established between any of the three different potassium treatments during the

recovery period.

As for the photosynthesis rate, the control samples had a significantly higher

transpiration rate than the drought-stressed samples (P<0.001). The no added K

control and drought-stressed samples showed a decrease and then an increase in

transpiration as described for the photosynthesis. A similar trend was this time also

noticeable for the high K treatment. The low K control treatment showed a slight but

constant decrease over the three measurements. The low and the high K drought-

stressed samples both increased and then decreased in transpiration, whereby the low

K treatment showed a much stronger increase between the first and the second

measurement than the high K treatment. Nevertheless, no significant differences were

measured. The stomatal conductance followed the same trends as observed for the

transpiration (Figure 3.21).


P a g e | 65

Figure 3.21: Short term recovery of transpiration, stomatal conductance and

photosynthesis of drought-stressed (D) and well-watered (C) plants treated with three

different potassium levels (no K, low K, high K) measured 0-1 h, 1-2 h and 2-3 h after

watering. Error bars show ± SE (n=3).

0.000

0.002

0.003

0.005

0.006

0.008


Tra

ns

pir

ati

on

(m

mo

l m

-2 s

-1)

α

A

a a

a

b b b

A

A

A

B B B

α

α β β

β

0.0

0.2

0.4

0.5

0.7


Co

nd

uc

tan

ce

(m

ol m

-2 s

-1)

B

a a a

b b

b

A

A B

B

α

β

β

-9.38

0.00

9.38

18.75

28.13

37.50


Ph

oto

syn

the

sis

(μ

mo

l m

-2 s

-1)


Measurement 1 Measurement 2 Measurement 3

C

a a a

b b

b A

A B B

B

α α

α β

β

β


P a g e | 66

During the drying cycle slight diurnal changes were visible for the low and high

potassium treatments similar but not as pronounced as for the control samples.

However, with increasing drought stress the diurnal changes disappeared and the Pp

curve showed constantly high values with the low K treatment being significantly higher

compared to the high K treatment. Immediately after re-watering both potassium

treatments showed a rapid decrease of the Pp-values and started to depict diurnal

changes again. The low K treatment, here also, had slightly higher Pp-values than the

high K treatment (Figure 3.22).

Figure 3.22: Examples of Pp measurements on canola subjected to different K

treatments and the corresponding ambient temperature and relative humidity. Figure A)

Pp measurements on drought-stressed canola with low K (dotted line with black filled

triangles) and high K (dotted line with filled squares). B) Pp measurements on control

canola plants with high K (solid line with open squares). Black bars top panel and

middle panel indicate irrigation events.

1 2 3 4 Days after 8 September 2013


P a g e | 67

3.4 Discussion

The obtained findings of the two experiments have shown that canola plants fertilised

with a high rate of potassium are able to adapt their physiological drought-stress

responds more effectively than plants that have received a low or no added rate of

potassium. The following section will first discuss the plant morphological changes

produced by different levels of potassium supply, before the physiological mechanisms

are interpreted. The applied potassium levels during the two conducted experiments

represented a mild potassium deficiency for the no added K treatment and marginal/

optimal and above optimal potassium rate for the low K and high K treatments,

respectively, based on typical values found in the Western Australian cropping region

(Brennan et al., 2013). This is different to most other conducted studies that often focus

on more extreme differences in nutrient availability, not considering that a mild

deficiency might occur much faster in the field (Barranco and Benlloch, 2006; Brennan

et al., 2013).

Both experiments have highlighted significant differences between the well watered

control samples and the water-stressed samples for a range of different measurements

e.g. leaf area, shoot and root dry weight, stomatal conductance, transpirational water

loss and leaf temperature. This indicated that the applied water stress was severe

enough and highlighted the many physiological changes induced by water stress.

These findings are in agreement with the literature where drought stress is described

as one of the most severe environmental impacts on plant growth and productivity

(Mittler, 2006; Fanaeia et al., 2009). Furthermore, the osmotic potential, relative leaf

water content and water use efficiency for both experiments was determined. However,

no meaningful differences were established as all samples had been watered back to

field capacity at the time of the measurement, which would have impacted the findings

and the results are not further discussed here. Thus, further experiments assessing the

changes of those parameters in canola during drought stress are needed in order to

understand any osmotic adjustment strategies as described for other plants (Dhindsa

et al., 1975; Mengel and Arneke, 1982).

3.4.1 Plant Morphological Changes to Potassium and Drought Stress

The low K treatment (56 mg kg-1 soil) was found to provide enough potassium for

maximum growth in canola. A higher than adequate potassium rate did not increase

dry weight, leaf area and subsequent pod number in canola. The shoot dry weight of


P a g e | 68

the no added K treatment (41 mg kg-1 soil) of Experiment 2, on the other hand, was

significantly lower than the low K treatment, for both the well watered and drought

stressed samples. The significant response in Experiment 2 may have been due to the

extended overall growing period, which was two weeks longer than for Experiment 1,

so a prolonged potassium deficiency may have caused a significant reduction in dry

weight as seen in the second experiment (Rose et al., 2007). Furthermore, it could

indicate that a critical value determining plant growth in canola is lies in the range of 41

to 56 mg kg-1 soil, which is in agreement with Brennan et al. (2013).

Usually, a reduction in the plant stature is one of the more obvious consequences of

plant growth under potassium limited environments, which results directly from a

reduction of photosynthesis per unit leaf area and solar radiation interception

(Cassman et al., 1989; Heckman and Kamprath, 1992; Pettigrew and Meredith, 1997;

Ebelhar and Varsa, 2000). These two experiments suggest that there is no benefit of

applying a higher than adequate potassium rate to canola grown in pots as no

significant increase in dry weight, leaf area or pod number was observed with

increasing potassium rate among the two watering regimes. This is in contrast to the

well described effect of additional nitrogen application in canola, which would result in a

significant higher growth rate until the next limiting factor, usually water supply, has

been reached (Duff et al., 2006). Consequently, the effect of potassium on the dry

weight of canola will be monitored during later chapters under more severe potassium

levels in order to understand whether a higher than optimal rate of potassium produces

a significant increase in growth.

These experiments showed that a restricted availability of potassium increased root

growth in canola, relative to shoot growth. In Experiment 2 the shoot:root ratio was

significantly larger for the high K treatment compared to the no added K treatment. This

trend was also observed in Experiment 1 but the differences were not significant. A

reduced amount of available potassium has been shown to stimulate root growth in

plants, in particular under water limiting conditions (De Dorlodot et al., 2007). These

authors contend that plants manipulate root system architecture (RSA) towards a

better distribution of root systems to optimise their nutrient and water uptake in the soil

medium, to minimise the adverse effect of abiotic environmental impacts on yield.

Furthermore, Damon et al. (2007) mentioned a similar observation in their research,

explaining that root distribution and potassium efficiency in canola can be influenced by


P a g e | 69

different genotypes. Those observations are supported by several other studies, which

have also confirmed that root architecture and distribution might vary with the genotype

for grain crops (Siddique et al., 1990; Liao et al., 2006; Manschadi et al., 2006; Palta et

al., 2011).

On the other hand, the study by Ma et al. (2013) has found that wheat (Triticum

aestivum L) developed a smaller root dry weight under a reduced potassium availability

whereas the high K treatment showed a larger root weight, which would be in contrast

to the measurements obtained for Trigold Canola plants for this study. As those

studies, however, were conducted on different species and were based on a more

severe potassium deficiency, these findings (Ma et al., 2013) need to be further

investigated. Hence, in the following chapter more extreme potassium levels will be

applied to confirm the current findings and to identify whether different crops have

potentially different strategies to cope with a more limited potassium and water supply.

3.4.2 Plant Physiological Changes to Potassium and Drought Stress

A high potassium fertiliser rate reduced transpiration, stomatal conductance while

maintaining a higher relative turgor pressure under drought stress in canola. However,

this water saving strategy did not lead to a higher photosynthetic rate. Interestingly, the

no added K treatment showed a higher photosynthetic rate throughout Experiment 1

with significant differences obtained during the first drying cycle. Those differences can

be explained with the higher transpiration rate as well as stomatal conductance that

were measured for the no added K treatment (Sofo et al., 2008;

Oosterhuis et al.,

2014). Since potassium is substantially involved in stomatal adjustment, it is expected

that potassium starved plants would close their stomata in particular under water stress

(Thiel and Wolf, 1997; Oosterhuis et al., 2014).

However, the studies conducted by Benlloch-Gonzalez et al. (2008) and Arquero et al.

(2006), which investigated the role of potassium in plant stomatal regulation, are in

agreement with the findings made for Experiment 1, concluding that potassium starved

plants would also have relatively higher stomatal conductivity under drought stress.

Founier et al. (2005) also reported an increased water uptake and decreased water

use efficiency under potassium deprivation, although a specific level of the potassium

deficiency was not identified. Findings of this study suggest that a rate between 41 and

56 mg kg-1 potassium in sandy soil may lead to significant physiological changes in


P a g e | 70

canola, which is in agreement with a study by Brennan et al. (2013) who established

morphological changes in canola at the same potassium rates. Another study by Basile

et al (2003) concluded, on the other hand, that potassium would have no significant

effect on the stomatal regulation of almond and consequently it is unlikely to impact the

rate of photosynthesis. Nevertheless, a number of studies support the current findings

reporting a higher transpiration under potassium starved conditions Bednarz et al.,

1998; Sudama et al., 1998; Cabanero and Carvajal, 2007).

One way to explain this unexpected response of potassium deficient plants having a

higher transpiration is based on an interaction effect of the plants hormones ABA and

ethylene on stomatal adjustments (Tanaka et al., 2005; Benlloch-Gonzalez et al., 2010;

Oosterhuis et al., 2014). Because the closure of stomata is known to be regulated by

ABA and the synthesis of ethylene increases under low levels of potassium, the closing

of the stomates in ABA active plants could be inhibited through the interference of

ethylene causing a higher transpiration rate as observed for the no added K treatment.

This conclusion was as well supported by another experiment observing a decrease of

transpiration after the application of an ethylene synthesis inhibitor (Tanaka et al.,

2005; Benlloch-González et al., 2010). The described increase in stomatal

conductance and consequently transpiration rate of potassium deficient plants might,

thereby, be an adaption strategy developed to avoid potassium deprivation by an up-

regulation of the stomatal conductance in order to increase xylem sap movement in the

plant, thereby, increasing the uptake of potassium (Benlloch-Gonzalez et al., 2010;

Oosterhuis et al., 2014). However, further experiments are needed that focus on

measuring changes in ABA and ethylene under different potassium levels and drought

stress in canola to consolidate the observations and conclusions made for this study.

These observational studies should be supported by studies manipulating ABA and

ethylene, for instance inhibitor or mutant line studies.

Canola is not able to adjust to multiple drying cycles with different potassium rates, as

all drought stressed samples reached similar minima and maxima values during both of

the drying and recovery phases indicating no specific physiological adaption strategy to

the reoccurring water stress. A number of different studies in the literature have also

described only a partial recovery after water stress, not reaching the same level of

photosynthetic activity (Xu and Zhou, 2008; Pou et al., 2008). However, as the canola

samples were eventually able to recover their stomatal conductance, rate of


P a g e | 71

transpiration and photosynthesis at the start of the second drying cycle, the

implemented recovery period must have been too short for canola to fully recover

suggesting that individual plant species obviously adapt and respond differently to

drought stress. (Marschner, 1995; Xu and Zhou, 2008; Pou et al., 2008; Flexas, 2009;

Oosterhuis et al., 2014). Further studies assessing the response of different canola

cultivars to repetitive water stressed canola plants under different potassium levels

should be undertaken. This is in order to consolidate the observation that plants can

exercise some kind of stress memory (Lee et al., 2010; Ding et al., 2012).

Across all the potassium treatments, the drought stressed samples showed an

expected decrease in transpiration per leaf area during each of the two consecutive

drying cycles of Experiment 1, compared with the well watered. A rapid increase was

measured during each of the recovery phases peaking for the first recovery phase at

the start of the second drying cycle, which could indicate an over-compensation or

regulation of the plant’s stomata after the imposed drought stress (Gallé et al., 2007;

Xu et al., 2010). This was as well noticeable for Experiment 2, where the stomatal

conductance, transpiration and photosynthetic rate of the low and high K treatment

increased upon re-watering, but decreased again about 2 to 3 hours after irrigation

towards midday and this is commonly described as a midday depression (Raschke and

Resemann, 1986).

The no added K treatment of the drought stressed samples in Experiment 2, on the

other hand, recovered slower than the two potassium treatments showing first a further

decrease in photosynthesis, transpiration rate and stomatal conductance during the

first two hours after re-watering before increasing after three hours implying that a mild

potassium deficiency reduces the ability of plants to recover quickly from drought

stress (Oosterhuis et al., 2014). In general, a range of studies have reported that plants

are able to fully recover their photosynthesis after the elimination of drought stress

(Izanloo et al., 2008; Xu et al., 2009; Fortunati et al., 2008). Similar findings were for

example described for kidney beans highlighting a rapid recovery of transpiration,

stomatal conductance and photosynthesis within two days after a period of drought

stress recognising a close correlation between the leaf water potential and recovery

level and speed of those mechanisms (Miyashita et al., 2004). Ben-Gala et al. (2010)

reported that transpiration and photosynthesis continued for olive trees at reduced

rates during drought stress and with diurnal patterns shifting to the morning hours.


P a g e | 72

The obtained diurnal changes of the well watered potassium treatments have indicated

that a mild deficiency or low rate of potassium produces a higher stomatal conductance

and transpiration. While the high K treatment had the highest maximum value in

photosynthesis, both other potassium treatments showed a higher rate in the

afternoon. Those findings further confirm the observed higher transpiration rate,

stomatal conductance and photosynthesis that was measured for the drought stressed

samples in Experiment 1 for the no added and low K treatment.

In canola a high potassium level leads to a higher relative turgor pressure and a

significant lower xylem pressure under drought stress. Ehrenberger et al. (2011)

outlined that the described increase of Pp values can be utilised as a clear indication for

severe drought stress. A range of other studies on several fruit trees and crops

suffering under water limiting conditions have also described a characteristic change of

the profile of the diurnal curves which were derived from the output signals of the

LPCP-probe, indicating that plants have increasing difficulty to compensate losses of

turgor pressure by water uptake (Zimmermann et al., 2008; Ben-Gala et al., 2010;

Rueger et al., 2010; Fernandez et al., 2011; Ehrenberger et al., 2011). The slight

decrease of the Pp curve observed for the High and No Additional K treatment on the

second last day and last day of the second drying cycle, respectively, coincides with

the maintenance water that was applied to prevent the water stressed samples from

drying too quickly. Thus during that time a slight increase of the relative turgor pressure

of the two treatments occurred, which at the same time highlights the rapid response of

turgor pressure to a slight increase in soil moisture (Ben-Gala et al. (2010). Hsiao

(1973) also described that changes in turgor pressure of plants subjected to drought

were highly responsive compared to other parameters, such as photosynthesis,

substantiating that turgor is particularly senstive to changes in soil water potential.

Comparing those observations to the leaf water potential measurements conducted

during the drying cycle of Experiment 2, a significantly higher water potential was

obtained for the high K treatment compared to the low and no added K levels. This

confirms the measurements derived from the LPCP-probes and indicates the ability of

plants, sufficiently supplied with potassium, to better adjust to water limiting

environments (McRobbie, 1977; (Hsiao and Lauchi, 1986; Marschner, 1995; Reddya et

al., 2004). During the drought stress period of Experiment 2, the high K treatment was


P a g e | 73

able to maintain a constantly higher relative turgor pressure compared to the low K

treatment, which confirms the findings made for Experiment 1. The recovery of the

relative turgor pressure was instantaneous and similar for both potassium treatments,

as the Pp values decreased rapidly indicating an increase of Pc. The high K treatment

had a slightly higher relative turgor pressure than the low K treatment. A fast recovery

of the relative turgor pressure was also reported by Ben-Gala et al. (2010) in olive

trees. These authors found that a full recovery of the diurnal Pp curve was obtained

after 2 days. The canola plants in both conducted experiments of the underlying study

already started to show a diurnal trend of the Pp curve after one day and are, thus, in

agreement to the findings of other studies (Rueger et al., 2010; Fernandez et al.,

2011). However, more research on the changes in relative turgor pressure of drought

stressed canola is needed to further investigate the recovery speed after re-watering

by for instance monitoring smaller time intervals in order to better capture the rapid

responses and, thereby, identify differences between different potassium levels.

3.5. Conclusion

Overall, canola plants are able to better adjust to water stress under a sufficient

potassium level than plants with marginal or a mild deficient potassium supply. A high

rate of potassium causes a reduced transpiration and stomatal conductance under

drought stress consequently leading to an increase in relative turgor pressure and

xylem potential in canola. Furthermore, a high potassium supply in well watered canola

plants led to a decrease in the diurnal photosynthesis rate, stomatal conductance and

transpiration during the afternoon; whereas the low and no added K treatments

increased their photosynthetic activity over the course of the day.

Interestingly, a mild potassium deficiency was found to cause a significantly higher rate

of photosynthesis, stomatal conductance and transpiration under water limiting

conditions implying that the canola plants are compensating for the deficiency by

increasing the uptake of potassium. An interaction with the plant hormones ABA and

ethylene could be a major factor contributing to the measured up-regulation in stomatal

conductance for the no added K treatment. However, more research on this is needed.

The high K treatment was also able to recover its relative turgor pressure faster than

the no added K treatment. All drought stressed samples showed an over-compensation

in stomatal conductance, transpiration and photosynthesis during the recovery periods,

but could only reach their initial values at the beginning of the second drying cycle,


P a g e | 74

implying that the applied recovery phase was too short for a full recovery and that

canola plants cannot be trained to repetitive drought as they did not decrease their

stomatal conductance to reduce water loss during the second drying cycle.

Canola develops a significantly larger root system relative to the shoots under a mild

potassium deficiency and drought stress. Furthermore, a higher than optimal rate of

potassium was found to have no significant effect on the dry weight, leaf area or

number of pods as an indication for a larger yield. Moreover, a mild potassium

deprivation only causes significant differences in growth if the duration of the deficiency

is long enough, which was not the case in these experiments. Those conclusions will

be further investigated during the next chapters by focusing on more extreme

potassium rates to identify and confirm the physiological response of canola under

drought-stress and the effect on plant size and shoot:root ratio.

Chapter 4: Short Term Plant Water Relations of Potassium Exposed Canola under Water Stress

P a g e | 75

Chapter 4 Short Term Plant Water Relations of Canola Grown in Nutrient Solution and Exposed to Rapid Potassium and Water Stress

4.1 Introduction

Chapter 3 concluded that a mild potassium deficiency in canola produced a

significantly higher stomatal conductance, transpiration and rate of photosynthesis in

comparison to well potassium supplied plants and also results in a smaller shoot:root

dry weight ratio. However, further research is needed to understand the impact of more

severe potassium deficiency on the physiological mechanisms influencing plant water

dynamics of canola under water-stressed conditions, such as the regulation of stomata

and relative turgor pressure.

Potassium is the main osmotic solute in plants (Mengel and Arneke, 1982).

Accumulation of potassium in the cell generates the cell turgor required for growth,

which subsequently causes osmotic water uptake (De la Guardia and Benlloch, 1980;

Mengel and Arneke, 1982) as well as stomatal opening (Fischer and Hsiao, 1968).

Potassium is also involved in the control of leaf transpiration and in osmotic water

absorption by the root (Hsiao and Lauchli, 1986). Although some aspects of a plant's

response to reduced potassium availability have been described in the literature,

understanding the response to different potassium levels in relation to the regulation of

stomatal conductance, transpiration rate and relative turgor pressure has not yet been

characterised (Lez et al., 2009). In particular, ion transport from the root to shoot may

be dependent on transpiration, osmotic water uptake or a combination of both (Fischer,

1958; Epstein, 1972; Kramer, 1983; Lez et al., 2009; Oosterhuis et al., 2014).

However, most research on whole plants has failed to identify a direct relationship

between either the transpiration and the long-distance transport of solutes, including

potassium (Muenscher, 1922; Schulze and Bloom, 1984), or root uptake of ions

present in low concentrations in the external medium (Russell and Shorrocks, 1959;

Blom-Zandstra and Jupijn, 1987; Pitman, 1988). Thus, further investigation to identify

possible uptake mechanisms in canola in response to different potassium levels is

needed.


P a g e | 76

A more severe and rapid potassium deficiency in canola could, thereby, reveal whether

the process of stomatal regulation would be impacted immediately either inhibiting

stomatal opening (Fischer and Hsiao, 1968; Oosterhuis et al., 2014), or whether it may

enhance stomatal opening and transpiration through an interaction effect with the plant

hormones ABA and ethylene as concluded in Chapter 3 (Section 3.5) (Brag, 1972;

Sudama et al., 1998; Bednarz et al., 1998; Cabanero and Carvajal, 2007; Benlloch-

Gonzalez et al., 2008). The increase of stomatal conductance of potassium -starved

plants may be an evolved mechanism in order to increase xylem sap movement in the

plant avoiding potassium deficiency observable through an increased water uptake and

decreased water use efficiency under potassium deprivation (Founier et al., 2005;

Benlloch-Gonzale et al., 2010; Oosterhuis et al., 2014). Nevertheless, further work is

needed to verify those conclusions under more controlled conditions for canola.

Growing plants in a nutrient solution in order to mimic rapid changes in nutrient supply

could, thereby, be a suitable method and is often utilised in studies investigating the

effect of a specific plant nutrient (Burnett et al., 2005).

Therefore, the main research questions were:

I. How do very low and high levels of potassium influence stomatal aperture

under water stress?

II. How quickly does transpiration rate recover at the end of a drying cycle and is

this recovery related to different potassium rates?

The main hypothesis was that a high potassium rate reduces or delays rapid loss of

turgor pressure under drought stress in comparison to plants fertilised with a low rate

or no potassium; and that this delay in loss of turgor is due to more effective reduction

of stomatal conductance and transpiration per leaf area. The sub-hypotheses were:

I. The high K-treated plants will close their stomata under drought, leading to a

higher relative turgor pressure and, consequently, more dry weight, because

plants are able to maintain their vital metabolic functions better under those

stress conditions compared to K-deficient plants.

II. Potassium starvation increases transpiration rate and in turn increases

potassium uptake by the plant.

To address these hypotheses, canola was grown in a nutrient solution and treated with

polyethylene glycol (PEG) to induce drought stress and to manipulate the potassium


P a g e | 77

supply (Burnett et al., 2005). Stomatal conductance, transpiration rate per unit leaf area

and turgor pressure were measured to quantify the effect of potassium supply on the

response of canola to drought stress. The findings will also be used to verify the

conclusions of the previous Chapter 3.

4.2 Methodology

The experiment was conducted in a heated glasshouse during the winter months at the

Institute of Applied Plant Nutrition (IAPN) of the George August University of

Goettingen, Germany (51.32°N, 9.56°E, 158 m above sea level). The experiment

commenced on 22 January 2013 and was harvested 51 days later on 13 March 2013.

The glasshouse temperatures ranged from 11.9°C at night to 31.6°C during the day,

which reflected comparable conditions to average temperatures during the growing

season in the Western Australian wheatbelt (Farré et al., 2007; Brennan and Bollard,

2007). Plants were positioned under growth lights with an average light intensity of 400

μE, providing additional light to the ambient light level. Temperature and humidity levels

in the glasshouse were monitored at 5-minute intervals by sensors positioned on the

benches where the plants were grown, as well as in 30-minute intervals by a sensor

located in the centre of the glasshouse. The experiment was conducted under

hydroponic conditions by growing the canola plants in nutrient solution in pots. Foam

lids were used to hold the plants in place. To ensure similar growth conditions, all pots

were regularly rearranged in the glasshouse.

4.2.1 Seed and Pot Preparation

Trigold canola seeds were pre-germinated using a moist paper roll. The paper roll was

made by wetting multiple layers of tissue paper and placing 10 to 12 seeds

approximately 0.5 cm from the top edge. The paper was rolled and placed in a glass

beaker with tap water (Stadtwerke Goettingen AG, 2014) and then covered by a plastic

bag to keep the seeds moist and to prevent evaporation. In order to expose the seeds

and emerging seedlings to the same growing conditions as the experiment, the glass

with the paper roll was located in the glasshouse. The germination process lasted for

10 to 14 days; the tap water was renewed every second day until the canola seedlings

had reached the 2–3 leaf stage and were big enough to transplant into the nutrient

solution. This method enabled the cotyledon to easily emerge out of the paper allowing

a good germination rate and uniform plant sizes. Twenty-four 5-litre pots were

prepared by lining them with a black plastic bag to keep the inside of the pot dark and


P a g e | 78

to prevent algae development. Foam lids were fitted to the pots and three holes were

drilled into the lids; 2 × 10 mm for the plants and 1 × 5 mm for the air tubes.

4.2.2 Transplantation of Seedlings

Germinated seedlings were transplanted after 11 days from the paper roll into the pots

containing nutrient solution. The soft foam material was cut appropriately to fit around a

single seedling, which was then inserted into one of the two ‘plant’ holes in the lid in

order to gently hold the plant in place. The additional seedling per pot served as a

backup in case one seedling died. The second plant was removed after 18 days. The

plastic tubes were connected to an air compressor and one tube per pot was inserted

through the lid into the nutrient solution. The holes around the air tubes were sealed

with adhesive tape to minimise evaporation (Figure 4.1).

Figure 4.1: Overview of experimental set up showing canola plants in nutrient solution

on automated balance system.

4.2.3 Nutrient Composition and Change of Nutrient Solution

The required plant nutrients were provided to the canola plants through the hydroponic

solution. All plants were initially supplied with a nutrient solution containing the nutrient

amounts listed in Table 4.1.


P a g e | 79

The nutrient solution in each pot was renewed every seven days once plants started

growing and every three days towards the end of the experiment. When changing the

nutrient solution, the lid with the canola plant was placed onto an empty pot in order to

minimise evaporation from the root system. The old solution was discarded and half

the pot was filled again with fresh deionised water. The plant nutrients were then added

to the pot before re-filling to 5 L to ensure even mixing of all nutrients. The lid with the

plant was again placed on the pot with the renewed nutrient solution ensuring that the

roots could easily reach the solution and that the air supply was working properly.

Table 4.1: Nutrient composition of applied fertiliser stock solution.

Nutrient Concentration of nutrient (mM) In form of

Ca 1.5000 Ca(NO3)2.4H2O

S 0.7500 MgSO4.7H2O

Mg 0.7500 MgSO4.7H2O

N 0.5000 NH4NO3

P 0.5000 NaH2PO4.2H2O

Fe 0.0500 Fe EDTA

B 0.0300 H3BO3

Mn 0.0025 MnSO4.7H2O

Zn 0.0010 ZnSO4.7H2O

Cu 0.0010 CuSO4.5H2O

Mo 0.0003 (NH4)Mo7O24.4H2O

4.2.3.1 Potassium Treatments

Prior to the treatments commencing, all plants received the same amount of potassium

(2.0 mM) in order to ensure even growth and similar plant sizes of all canola plants at

the start of the drought experiment. Four days before the start of the first PEG/drought

treatment, three potassium levels were implemented. One treatment received no

potassium (no K), the second received 0.2 mM (low K) and the third received the same

concentration as the stock solution of 2.0 mM (high K).

4.2.4 Water Stress

Polyethylene glycol (PEG) 6000 (Roth, Karlsruhe, Germany) was used to simulate

water stress and this is a commonly applied technique in studies investigating the


P a g e | 80

effects of plant nutrients and water stress (Burnett et al., 2005). PEG 6000 is used as

an osmoticum to simulate a drying cycle by forming hydrogen bonds with water,

thereby lowering the osmotic potential of the solution to mimic changes in matric

potential in a soil medium (Kjellander and Florin, 1981; Steuter, 1981). It is not known

whether PEG also forms bonds with nutrient ions restricting their uptake into plants.

However, Burnett (2004) pointed out that the electrical conductivity, used to estimate

the nutrient concentration of the substrate, decreases under an increasing level of

PEG. A second study confirmed these findings suggesting that PEG might bind some

of the nutrients in the substrate and that a morphological effect of PEG on treated

plants lead to a reduced dry weight (Burnett et al., 2005; Hamayun et al., 2010). These

negative effects on plant size only occurred after 14 days. Since the plants in the

current experiments only experienced short-term PEG application (three days) the

outlined effects were likely to be minimal and would impact all potassium treatments in

a similar way (Burnett, 2004).

All drought-stressed plants received 180 g PEG/L nutrient solution on the last day of

the drying cycle as per Steuter et al. (1980). The PEG consisted of a dry granule that

was dissolved in each pot by stirring the solution while constantly adding PEG. The

conducted drying cycle of the current experiment lasted three days. In order to allow

the canola plants to slowly adjust to the drought stress, rather than a shock treatment,

the rate of PEG during the simulated drying cycle was gradually increased over three

days until the last day of the water stress treatment i.e. 60 g PEG/L nutrient solution

was applied daily for three days. There were two water treatments (PEG-

stressed/drought (n=4) and control (n=4). The first drying cycle started on day 1 of the

experiment (4 March 2013) and lasted for three days under water stress; it was

followed by a 6-day recovery period which was terminated on day 9 (13 March 2013)

with the plants being harvested in the morning of day 10 (Table 4.2).

To confirm the results of the first drying cycle, a second drying cycle was carried out

using the control plants from 7 March 2013 to 10 March 2013 (Table 4.2). However,

during this drying cycle plants were only treated with 60 g PEG/L nutrient solution/day

during the first two days and 30 g PEG/L during the third day in order to observe the

effects at a lower stress level. Moreover, the plants were kept at the same PEG stress

level for a further day. The drying cycle was followed by a 2-day recovery period with

the plants being harvested on day 10 (Table 4.2).

Tabl

e 4.

2: T

imel

ine

of c

ondu

cted

PEG

indu

ced

dryi

ng c

ycle

s

Dat

e 4/

3/20

13

5/3/

2013

6/

3/20

13

7/3/

2013

8/

3/20

13

9/3/

2013

10

/3/2

013

11/3

/201

3 12

/3/2

013

13/3

/201

3

Day

of t

he

expe

rimen

t 1

2 3

4 5

6 7

8 9

10

1st P

EG in

duce

d dr

ying

cyc

le w

ith

3 K

- and

2 P

EG-

trea

tmen

ts w

ith

thre

e re

plic

ates

60 g

PE

G/L

nu

trien

t so

lutio

n

120

g PE

G/L

nu

trien

t so

lutio

n

180

g PE

G/L

nu

trien

t so

lutio

n

Star

t of

reco

very

fo

r wat

er

stre

ssed

sa

mpl

es

reco

very

re

cove

ry

reco

very

re

cove

ry

reco

very

ha

rves

t

2nd P

EG in

duce

d dr

ying

cyc

le w

ith

3 K

- tre

atm

ents

us

ing

the

cont

rol

plan

ts o

f 1st

run

with

thre

e re

plic

ates

wel

l w

ater

ed

wel

l w

ater

ed

wel

l w

ater

ed

60 g

PE

G/L

nu

trien

t so

lutio

n

120

g PE

G/L

nu

trien

t so

lutio

n

150

g PE

G/L

nu

trien

t so

lutio

n

150

g PE

G/L

nu

trien

t so

lutio

n

Star

t of

reco

very

fo

r wat

er

stre

ssed

sa

mpl

es

reco

very

ha

rves

t

P a g e | 81



P a g e | 82


The relative turgor pressure was monitored using leaf patch clamp pressure probes

(LPCP) (Yara ZIM plant technology, Hennigsdorf, Germany) as described by

Zimmermann (2008). For the conducted experiment, 18 canola plants were monitored

using the LPCP-probes, with one probe clamped onto the fourth fully-expanded leaf of

each plant using the 2.5 mm magnet in order to prevent any damage to the fragile

canola leaves. Six plants of each of the three K treatments were clamped: three

stressed plants and three control plants. This approach ensured comparable probe

readings as the leaves were fully developed but not too old to start senescing. The

clamping occurred in the morning when the plants had high turgor pressure, four days

before the start of the first PEG-induced drying cycle to allow time for the plants to

adjust to the probe and to get regular output readings (Zimmermann et al., 2008). The

applied clamping pressure ranged from 20 to 40 kPa. For the experiment, the relative

turgor pressure was logged every five minutes throughout the experiment. To allow

comparison of LPCP-probe pressure output (Pp) measurements, the Pp data was

normalized, as described in Chapter 3. Note that the Pp output pressure reflects a drop

in relative turgor pressure (Pc) i.e. an increase in Pp equals a decrease in Pc. The

baseline Pp is the linear regression of all minima Pp readings illustrating the trend of

relative turgor pressure (Rueger et al., 2010).

4.2.6 Pot Weight and Transpiration per Leaf Area

Three replicates per potassium and water stress treatment were placed on an

automated balance system (Model TQ30, ATP Messtechnik GmbH, Ettenheim,

Germany) which logged pot weight at 30-minute intervals throughout the experiment.

From this, the transpiration per leaf area of the canola plants was determined, based

on the final leaf area obtained after harvest.

4.2.7 Stomatal Conductance, Transpiration rate and Photosynthesis

Stomatal conductance of the canola plants was measured using a leaf porometer 7.0

from Decagon Devices (Washington, USA). Each sample was measured daily on the

last fully-expanded leaf between 11:00 and 12:00 h during the drying cycle and the

subsequent recovery phase.


P a g e | 83

The photosynthetic and transpiration rate was derived from measurements conducted

with a Portable Gas Exchange Fluorescence System GF-3000 (Heinz Walz GmbH,

Effeltrich, Germany). Due to the long recording time of the device for each plant,

measurements commenced one hour after the growth lights went on at 08:00 h and

finished at 14:00 h, and only the no K and high K treatments in control and PEG-

stressed canola plants could be measured. The light intensity for these measurements

was set to 500 μE m–2 s–1 and the rate of CO2 in the cuvette was maintained at 380

nmol mol–1.

4.2.8 Harvest and Isotope Analysis

Plants were harvested on day 10 (13 March 2013), by which time 70% of all plants had

reached the flowering stage. At harvest, all leaves were removed from each plant and

the leaf area was determined using a leaf area scanner (Lens System, Epson

Perfection V700 Photo scanner, Seiko Epson Corporation, Nagano, Japan). Shoots

were separated from the roots, and the entire shoot (leaves and stem) and root

material of each plant were dried in an oven for 48 h at 70°C to determine the dry

weight of each plant.

The root and shoot material was subsequently ground to 2 mm using a Culatti, mill type

DFH 48 (No. 177792, rpm 6000, 220 Volt, Culatti AG, Zuerich, Switzerland). The

potassium was extracted from the dried plant material using 65% nitric acid under

pressure and its concentration was determined using flame photometry. The shoot

material was then crushed using a ball mill from Retsch, MM 400, frequency 1 s–1

(Retsch International, Haan, Germany) in order to prepare the samples for isotope

analysis, which was conducted at the J. Dyckmans Centre for Stable Isotope Research

and Analysis of the University of Goettingen using the standard procedures outlined in

their manuscript ‘Stable Isotopes in Terrestrial Ecology’ (J. Dyckmans Centre for

Stable Isotope Research and Analysis, 2013).

4.2.9 Experimental Design and Statistical Analysis

The experiment was arranged in a randomised complete block design with four

replicates for each of the three potassium treatments (no K, low K and high K) and two

water treatments (PEG-stressed and well watered (control)). Data were analysed with

Microsoft Office Excel 2008 (Microsoft, Redmond, USA) and Origin V from OriginLab

(Northampton, MA) to calculate mean values and standard errors and, where


P a g e | 84

appropriate, an ANOVA with a Tukey-test as a posthoc test. Since the control plants

(n=4) were used to repeat the main drying cycle (Table 4.2), the leaf area, shoot and

root dry weights and potassium content of both drying cycles was combined for the

statistical analysis and presented together (n=8). In addition, the photosynthetic and

transpiration rate data collected over the duration of the drying cycle was combined

and presented as an overall average of the two water treatments for each potassium

rate in order to ensure sufficient replicates for each treatment.

4.3 Results

4.3.1 Environmental Conditions

During the drought experiments the temperature in the glasshouse ranged from 11.9°C

during the night to 31.6°C during the day, with an average temperature of 20.4°C for

the first three days. Slightly higher temperatures (28–31°C) were observed for the first

three days, before decreasing to a more constant maximum of 24°C during the day

(Figure 4.2A). Relative humidity averaged 28.8%; it was 31.7% on day 1 and steadily

increased to a maximum of 49.3% on day 5 before decreasing to a minimum 17.9% on

day 10 (Figure 4.2B).


P a g e | 85

Figure 4.2: Temperature and relative humidity during the experiment starting on 4

March 2013 and ending on 13 March 2013.

4.3.2 Leaf Area

The high K treatment had the largest leaf area with 2697 cm2, which was significantly

larger than the no K (2380 cm2) and low K treatments (P=0.03 and 0.01, respectively)

(Figure 4.3). Interestingly, the leaf area of the low K treatment was also significantly

smaller than that of the no K treatment (P=0.03).

0

5

10

15

20

25

30

35

0:0

0:0

0

12

:00

:00

0:0

0:0

0

12

:00

:00

0:0

0:0

0

12

:00

:00

0:0

0:0

0

12

:00

:00

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0:0

0

12

:00

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0

12

:00

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0

12

:00

:00

0:0

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12

:00

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0:0

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0

12

:00

:00

0:0

0:0

0

12

:00

:00

1 2 3 4 5 6 7 8 9 10

Tem

pera

ture

(°C

) A

0

10

20

30

40

50

60

0:00

:00

12:0

0:00

0:00

:00

12:0

0:00

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:00

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:00

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:00

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:00

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:00

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0:00

:00

12:0

0:00

1 2 3 4 5 6 7 8 9 10

Rel

ativ

e H

umid

ity (%

)

Days after 3 March 2013

B


P a g e | 86

Figure 4.3: Average leaf areas of the three different drought-stressed potassium

treatments (no K, low K, high K). Error bars show ± SE (n=8). Different letters above

the bars indicate significance at ≤0.05.

4.3.3 Shoot and Root Dry Weights

For both shoots and roots, the high K treatment had significantly more dry weight than

the no K treatment (P=0.01) (Figure 4.4). It also had a higher root dry weight than the

low K treatment (P=0.03). The no K and low K treatments resulted in similar root and

shoot dry weights. The shoot:root ratios were similar across all three potassium and

PEG treatments with no significant differences observed.

0

500

1000

1500

2000

2500

3000

No K Low K High K

Leaf

Are

a (c

m2 )

Potassium Rate

b

c

a


P a g e | 87

Figure 4.4: Mean shoot and root dry weights and shoot:root ratio for the three drought-

stressed potassium treatments (no K, low K, high K). Error bars show ± SE (n = 8).

Different letters above the bars indicate significance at ≤0.05.

4.3.4 Potassium Concentration

The potassium concentration of the water-stressed shoots and roots reflected the three

potassium treatments, with the high K treatment having significantly higher shoot and

root potassium concentrations than the other two treatments (P=0.01) and the no K

treatment had significantly smaller shoot and root potassium concentrations than the

low K treatment (P=0.02). The shoot:root ratio of the high K treatment was 1.13, which

was significantly lower than the no K and low K treatments, which averaged 1.25 and

1.32, respectively (P=0.0001 and P=0.004, respectively). However, no significant

differences were observed between the no K and low K treatments (Figure 4.5).

0

1

2

3

4

5

6

7

0

2

4

6

8

10

12

14

16

18

20

No K Low K High K

Shoo

t:Roo

t

Wei

ght (

g)

Potassium Rate

Shoot Root Shoot:Root

b

A A B

α α α a ab


P a g e | 88

Figure 4.5: Average potassium concentration in shoot and root of drought-stressed

canola plants treated with three different potassium rates (no K, low K, high K) and

shoot to root ratio of potassium concentration. Different letters above the bars indicate

significance at ≤0.05.

4.3.5 Isotope Analysis of Shoot Material

The observed trend for the 13C isotope analysis showed that the low K and high K

potassium drought-stressed treatments had significantly more 13C accumulated in their

tissues than the no K treatment (P=0.03 and P=0.01, respectively) (Figure 4.6A).

However, no significant differences were obtained for the 18O/16O stable isotope ratios

(Figure 4.6B).

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0

1

2

3

4

5

6

7

No K Low K High K

Shoo

t:Roo

t

% P

otas

sium

Potassium Rate

Shoot Root Shoot:Root

b

c

A B

C

α

β

a

α


P a g e | 89

Figure 4.6: Average 13C/12C (A) and 18O/16O (B) stable isotope ratios of the drought-

stressed canola plants treated with three different potassium rates. Error bars show ±

SE (n=8). Different letters above the bars indicate significance at ≤0.05.

4.3.6 Transpiration per Leaf Area

As the well-watered control pots were kept at the same water level throughout the

drying cycle and the main focus was monitoring differences between potassium

treatments under water stress, only drought-stressed plants are presented. Figure 4.7

shows the transpiration per unit leaf area of canola plants treated with three potassium

rates undergoing a 3- or 4-day simulated drying cycle. Both sets of plants (3- and 4-

day) showed similar trends in the low K treatment with a significantly higher

-32.4

-32.2

-32.0

-31.8

-31.6

-31.4

-31.2

-31.0 No K Low K High K

13C

/12C

V-P

DB

(°/°°

)

Potassium Rate

b

A

29.2

29.4

29.6

29.8

30.0

30.2

30.4

No K Low K High K

18O

/16O

(°/°

°)

Potassium Rate

B

a

a

a

a

b


P a g e | 90

transpiration per leaf area than the no K and high K treatments during the first two days

of drying (P=0.02 and P=0.03, respectively). Interestingly this trend reversed on the

last day of both drying cycles and the first day of each recovery phase, with the low K

treatment showing a lower transpiration per leaf area than the no K and high K

treatments. However, this difference at the end of the drying cycle/early recovery was

only significant for the first set of plants (3-day) with P-values of 0.02 and 0.01,

respectively. In the following days of recovery, all three treatments approached similar

transpiration values and no further significant differences were observed.


P a g e | 91

Figure 4.7: Transpiration per leaf area of canola plants treated with three different

potassium treatments (no K, low K, high K) undergoing a 3- (A) and 4-day (B) drying

cycle, respectively.

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0:18

:00

4:48

:00

9:18

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8:00

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00

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8:00

3:

18:0

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48:0

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8:00

15

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00

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8:00

0:

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48:0

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06:0

9 13

:36:

09

16:4

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20

:51:

11

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:11

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:11

10:2

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11

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11

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13:2

1:11

17

:51:

11

22:2

1:11

2:

51:1

1 7:

21:1

1 11

:50:

27

1 2 3 4 5 6 7

Tran

spira

tion

per L

eaf A

rea

(g H

2O c

m-2

)


No K Low K High K

Drying Cycle Recovery

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0:18

:00

4:48

:00

9:06

:09

13:3

6:09

16

:47:

43

20:5

1:11

1:

21:1

1 5:

51:1

1 10

:21:

11

14:5

1:11

19

:21:

11

23:5

1:11

4:

21:1

1 8:

51:1

1 13

:21:

11

17:5

1:11

22

:21:

11

2:51

:11

7:21

:11

11:5

0:27

16

:20:

27

20:5

0:27

1:

20:2

7 5:

50:2

7 10

:20:

27

14:0

3:53

18

:33:

53

23:0

3:53

3:

33:5

3 8:

03:5

3 12

:33:

53

17:3

2:19

22

:02:

19

2:32

:19

7:02

:19

1 2 3 4 5 6 7

Tran

spira

tion

per L

eaf A

rea

(g H

2O c

m-2

)


No K Low K High K

Drying Cycle Recovery B

A


P a g e | 92

4.3.7 Transpiration Rate

Overall the drought-stressed plants had lower transpiration rates than the controls; a

significant difference was only measured for the no K treatment (P=0.03) (Figure 4.8).

No significant differences were observed between the two potassium treatments during

the drought and recovery phases. Interestingly, the no K control treatment had a

significantly higher transpiration rate than the high K control treatment (P=0.03).

Figure 4.8: Average transpiration rate of PEG- and non-PEG treated no and high K

fertilized canola plants. Error bars show ± SE (n=8). Different letters above the bars

indicate significance at ≤0.05 (potassium main effect P=0.88, water main effect


4.3.8 Stomatal Conductance

All potassium treatments showed a downward trend during each of the PEG-induced

drying cycles and a subsequent increase in stomatal conductance during the recovery

periods (Figure 4.9). Significant differences were only observed on days 4 to 5 for the

first set (3-day drying) of plants (Figure 4.9A), with the low K treatment being

significantly lower than the no K and high K treatments (P=0.03 and P=0.04,

respectively).

0

1

2

3

4

5

6

7

Drying Cycle Recovery Control

Tran

spira

tion

(mm

ol m

-2 s

-1)

Water Treatment

No K High K

b

c a a a

a


P a g e | 93

Figure 4.9: Average daily stomatal conductance of drought-stressed canola plants

treated with three different potassium rates (no K, low K, high K) undergoing a (A) 3-

and (B) 4-day drying cycle, respectively.

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4 5 6 7

Con

duct

ance

(mol

m-2

s-1

)


No K Low K High K

Drying Cycle Recovery A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1 2 3 4 5 6 7

Con

duct

ance

(mol

m-2

s-1

)


No K Low K High K

Drying Cycle Recovery B


P a g e | 94

4.3.9 Photosynthesis

The average rate of photosynthesis during the PEG simulated drying cycle was

significantly smaller for the no K treatment than the high K treatment (P=0.02) (Figure

4.10). Interestingly, no significant differences were observed between the two

potassium treatments during the recovery phase. Only the no K PEG-treated plants

had significantly lower rates of photosynthesis than the no K control plants (P=0.03).

There were no other significant differences between the PEG-treated and control

plants (Figure 4.10).

Figure 4.10: Average photosynthetic rate of PEG- and non-PEG treated no and high K

fertilized canola plants. Error bars show ± SE (n=8). Different letters above the bars

indicate significance at ≤0.05 (potassium main effect P=0.18, water main effect



During the PEG-induced drought stress of the first drying cycle, the relative turgor

pressure of the low K treatment did not recover fully at night (i.e. the relative turgor did

not decrease to the same level as the previous night), as indicated by an increasing

trend in the night-time baseline. For the no K treatment this was only visible on the last

day of the drying cycle. Some of the patch pressure probes malfunctioned for the high

treatment over the duration of the drying cycle, therefore only the no K and low K

treatments are displayed over the full three days (Figure 4.11).

0

5

10

15

20

25

30

Drying Cycle Recovery Control

Phot

osyn

thes

is (μ

mol

m-2

s-1

)

Water Treatment

No K High K

b c bc bc

bc

a


P a g e | 95

Figure 4.11: Representative examples of Pp measurements on canola subjected to

different potassium treatments and the corresponding ambient temperature and

relative humidity. A) Pp measurements on drought-stressed no K sample (dotted line

with black filled circles) and corresponding control (solid line with open circles); B) Pp

measurements on drought-stressed low K samples (dottled line with black filled

triangles) and corresponding control control (solid line with open triangles). Black bars

top panel and middle panel indicate change of nutrient solution.

Figure 4.12 shows the relative turgor pressure of drought-stressed plants treated with

three potassium rates on the last day of PEG-induced drying. After the final dose of

A

B



P a g e | 96

PEG, there was a rapid response in the no K treatment, such that the no K treatment

significantly increased its output patch pressure i.e. a rapid decrease in relative turgor

pressure more than the high K treatment. In contrast, the low K treatment gradually

increased in output patch pressure i.e. decreased its relative turgor pressure after the

final addition of PEG.

Figure 4.12: Representative examples of Pp measurements on drought-stressed

canola subjected to different potassium treatments and the corresponding ambient

temperature and relative humidity on Day 3 (6 March 2014) of the experiment. A) Pp

measurements on drought-stressed no K sample (dotted line with black filled circles);

B) Pp measurements on drought-stressed low K samples (dottled line with black filled

triangles); C) Pp measurements on drought-stressed high K samples (dottled line with

black filled squares).

A

B

C

Time (hrs)


P a g e | 97

4.4 Discussion

This section highlights the effect of potassium on the water dynamics of canola under

water stress. Firstly the changes in relative turgor pressure and transpiration per leaf

area are discussed, followed by the morphological response in plant size and leaf area,

and the distribution and effect of potassium on the rate of stomatal conductance and

photosynthesis.

4.4.1 Changes in Relative Turgor Pressure and Transpiration per Leaf Area under Water Stress and Different Potassium Levels

A high K rate led to a higher relative turgor pressure during drought stress but did not

prevent rapid water loss at the end of the drying cycle. The baseline for relative turgor

pressure in all potassium treatments increased during the first two PEG applications

indicating increased stress levels and lower leaf water contents, which is consistent

with the work by Zimmermann (2008). The initial observations at the start of the drying

cycle were expected, as increasing the amount of PEG increases water stress

(Kjellander and Florin, 1981; Steuter, 1981). On day 3 of the drying cycle, the baseline

of the no K and high K treatments increased rapidly after the third PEG application

while the baseline of the low K treatment increased gradually during the day.

The rapid loss of turgor pressure for the high K and no K treatments may have been

caused by xylem cavitation due to the fast change in matric potential after the third

PEG application (Zimmermann, 1983). Tyerman (2007) describes xylem embolism as a

well-known phenomenon in plants, thereby inhibiting water flow. It can occur as a

consequence of abiotic factors such as water stress, freezing and mechanical stress in

plants. The PEG treatment would have significantly lowered the water tension of the

nutrient solution almost immediately after being added, causing the water column to

cavitate particularly as the plants were transpiring. This is in line with the observations

of Holbrook et al (2001) who showed, with the help of magnetic resonance imaging

(MRI), cavitation in grapevine stems while the plants were transpiring. Drought-induced

xylem embolism in plants usually occurs due to xylem rapidly filling with water vapour

caused by air entry through conduit pit membranes, as explained by Zimmermann

(1983) in his ‘air seeding’ hypothesis. This theory is supported by many other studies

that also report the occurrence of xylem cavitation as a result of drought stress, for

instance in grapevines (Milburn and Johnson, 1966; Sperry et al., 1987; Sperry and

Tyree, 1988).


P a g e | 98

After removal of the PEG solution, all three treatments started to recover, as indicated

by an increase in transpiration per leaf area over a short period even while their xylem

vessels were under negative tension, as the change of nutrient solution occurred in the

morning and the plants continued to transpire. This interpretation agrees with much of

the evidence presented in the literature, which also describes the reversal of cavitation

in plants (McCully et al., 1998; McCully, 1999; reviewed in Meinzer et al., 2001;

Clearwater et al., 2004; reviewed in Clearwater and Goldstein, 2005). According to a

study conducted by Holbrook et al. (2001), the presence of membrane-bound water

channels (aquaporins) in the surrounding xylem parenchyma cells could play a major

role for the mechanism controlling the movement of water back into the embolised

vessels. Alternatively, re-filling cavitated vessels in plants occurs from the generation of

positive root pressure (Sperry et al., 1987). On the other hand, embolism repair at night

can also occur when xylem tension is close to zero (Holbrook et al., 2001). Holbrook

and Zwieniecki (1999) further introduced a mechanism that includes the anatomy of

xylem vessels and their surrounding living xylem parenchyma cells to repair embolised

xylem vessels. However, additional experiments with different PEG treatments are

needed to assess a precise threshold level where canola plants start to cavitate and to

investigate their recovery particularly in regard to relative turgor pressure. Furthermore,

it would be interesting to assess whether cavitation can be avoided by increasing the

amount of PEG in smaller steps.

4.4.2 Morphological Impacts of Potassium on Canola under Drought

The measured differences in plant size between the three potassium treatments

indicated that a higher potassium availability will significantly increase plant size in

canola; the high K treatment had the greatest shoot and root dry weight as well as leaf

area as previously described in Chapter 3 (Section 3.3), which also agrees with the

findings for other species (Kimbrough et al., 1971; Wolf et al., 1976; Longstreth and

Nobel, 1980; Huber, 1985; Pier and Berkowitz, 1987; Pettigrew and Meredith, 1997;

Bednarz et al., 1998; Jordan-Meille and Pellerin, 2004; Oosterhuis et al., 2014).

Hossain et al. (2010), for instance, reported for Hibiscus cannabinus grown in a

nutrient solution that higher rates of potassium led to significant increases in plant size.

As a consequence and as expected, the no K treatment in this research had the

smallest shoot and root dry weights with the low K treatment having similar values.


P a g e | 99

There were, however, no significant differences between the dry weight shoot:root

ratios of the three potassium treatments. This could be due to the significant effect on

plant growth of the nutrient solution as a growth medium i.e. nutrients are easily

accessible until they are depleted and, consequently, deficiencies occur only once all

nutrients are below a critical value (Steingrobe and Claassen, 2000; Van Iersel, 2004;

Römheld and Kirkby, 2010; Hossain et al., 2010). Van Iersel (2004) described an

increasing shoot:root ratio for several horticultural crops permanently grown under an

increasing supply of nutrients in a hydroponic solution. As those plants were

continuously grown under the same conditions, the larger effect of a long-term nutrient

supply would have been more significant than for the canola plants of the underlying

experiment, which were only exposed to different potassium rates for the last two

weeks before harvest. The study by Ma et al. (2013) also reported that root growth in

wheat is more depressed than shoot growth under potassium deficiency. Thus, the

long-term effect of different potassium rates on the shoot:root ratio in canola should be

further investigated in order to identify whether there is a different morphological

response to potassium supply between different plant species as previously observed

in Chapter 3 (Section 3.3).

4.4.3 Potassium Distribution in Water Stressed Canola and the Effect on Stomatal Conductance and Photosynthesis

Potassium starvation in canola leads to a significantly higher relative accumulation of

potassium in the shoots than in the roots compared with those supplied with a high rate

of potassium (Ashley et al., 2005). These significantly higher shoot:root ratios for

potassium concentration in the no K and low K treatments suggest that plants try to

compensate for the potassium deprivation by accumulating potassium in shoots, which

has been discussed in Chapter 3 (Section 3.4) and by others (Benlloch-Gonzalez et al.,

2010; Oosterhuis et al., 2014). Those interpretations also agree with the significantly

higher transpiration rate of the no K control treatment in comparison to the high K

control. The absolute amount of potassium that was taken up by canola plants,

however, reflected the different potassium treatments, with the high K treatment

showing values of around 5.5% (considered high) compared to values of only around

1.25% for the no K treatment. This indicates that higher rates of potassium applied

results in more potassium accumulation in the plant (Steingrobe and Claassen, 2000;

Oosterhuis et al., 2014). This also agrees with Oddo et al. (2013) who reported an 80%

increase in potassium xylem sap content in Laurus nobilis L in the high-K treatment


P a g e | 100

after 24 hours compared with the no-K treatment. Further experiments to assess the

long-term effect of potassium on the shoot and root concentration in canola grown in a

soil medium are investigated in Chapter 5.

In this study, a high or low rate of potassium led to an overall reduction in stomatal

conductance under water stress, whereas potassium deficient (no K) plants kept their

stomata open longer. This is indicated by the significantly higher accumulation of ∆13C

in the high K and low K treatments compared with the no K treatment (Ferrio and

Voltas, 2003). Interestingly, the low-K treatment had significantly higher transpiration

per leaf area during the first two days of the drying cycle, which is in contrast to the

findings of the isotope analysis. However, during the third day of the drought-stress

period and the first day of the recovery phase, the observed trend reversed with the

low K treatment having significantly lower transpiration per leaf area than the no K

treatment and even the high K treatment. Those results are supported by a significantly

smaller stomatal conductance in the low K treatment for the same days. This could

also explain the smaller increase of the Pp output curve for relative turgor pressure on

the third day of the drying cycle (Arquero et al., 2006; Benlloch-Gonzalez et al., 2008;

Benlloch-Gonzalez et al., 2010).

The deprivation of potassium under well-watered conditions triggers potassium uptake

in canola, as indicated by a significantly larger transpiration rate for the no K treatment

in the control plants, which significantly differed from the no K drought-stressed plants.

This was confirmed by a significantly lower ∆13C content compared to the two other

potassium treatments and has been reported by Kochian and Lucas (1982), Siddiqi

and Glass (1987) and Benlloch et al. (1989). These results agree with the findings in

Chapter 3 of higher transpiration with no K and where an interaction effect with the

plant hormones ABA and ethylene on stomatal adjustment was proposed (Section 3.4)

(Bednnarz et al., 1998; Sudama et al., 1998; Tanaka et al., 2005; Cabanero and

Carvajal, 2007; Benlloch-Gonzalez et al., 2010; Oosterhuis et al., 2014; Saradadadevi

et al., 2014). All drought-stressed canola plants fully recovered their transpiration per

leaf area during the recovery phase or even slightly over-compensated after the

imposed water stress as was observed in other studies (Gallé et al., 2007; Xu et al.,

2010).


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Interestingly, water-limiting conditions had a greater impact on the rate of

photosynthesis than potassium deprivation. Nevertheless, a high rate of potassium did

counteract the impact of water stress on the rate of photosynthesis i.e. water stress in

the absence of potassium significantly reduced photosynthesis, whereas water stress

with high K had no effect. This may be explained with the monitored reduction of

stomatal conductance and transpiration rate of the high K treatment under drought

stress, which was also observed in Chapter 3 (Section 3.3). This in turn leads to a

higher relative turgor pressure i.e. a better water supply of the leaf may have a greater

significant impact on maintaining photosynthesis than a reduced availability of CO2

because of partially closed stomata (Oosterhuis et al., 2014). Besides that, potassium

primarily influences the turgor pressure of the leaf through osmoregulation, which

subsequently controls the stomata mechanism and, consequently, the CO2 influx,

which then impacts the rate of photosynthesis (Marschner, 1995).

The potassium concentration in the no K treatment was, as expected, significantly

smaller than the two other drought-stressed potassium treatments, which was also

shown in a study on rubber plants by Samarappuli et al. (1994). The differences

represent different uptake kinetics between the three potassium treatments and

highlight the role of potassium as a primary osmoticum in maintaining high water

potential of plant tissues especially under drought stress (Fanaeia et al., 2009). Other

studies have confirmed these conclusions for other Brassica species where osmotic

adjustments were closely associated with stomatal conductance and canopy

temperature (Kumar and Singh, 1998; Singh et al., 1985). Nevertheless, more

experiments linking osmotic potential to a specific potassium rate and related stomatal

conductance and photosynthesis are needed to further understand the regulatory

mechanisms of canola plants to reduce water loss under drought stress.

4.5 Conclusion

Canola plants that are well supplied with potassium can maintain higher relative turgor

pressure under water-limiting conditions. However, a sudden increase in the water

stress may lead to cavitation in the xylem, which was reversible during the recovery

phase and could not be prevented by the high K treatment. This was as well confirmed

with the improved regulation of stomata adjustment in canola with sufficient potassium,

shown as earlier closing of the stomata, with significantly higher accumulation of ∆13C

in the low K and high K treatments under water stress. Consequently, the high K


P a g e | 102

treatment could maintain a higher rate of photosynthesis compared to the no K

treatment under drought. The high K treatment also produced significantly more dry

weight than the other two K treatments. Nevertheless, there was no significant

difference observed for the shoot:root ratio between the three potassium rates.

A higher transpiration rate for potassium-starved canola plants may be a strategy to

increase the uptake of potassium into the plant. This interpretation is supported by

relatively higher accumulation of potassium in the shoots than the roots when

compared with high-K plants. An interaction effect of the plant hormones ABA and

ethylene may explain those observations, which have been described in Chapter 3.

However, the absolute amount of potassium taken up by canola still depends on the

overall availability. More research is needed to investigate the distribution strategies of

potassium starved canola plants and the effect of different potassium levels on

transpiration per leaf area and plant size in a soil medium to consolidate the

conclusions drawn from this chapter. In addition, the interaction between the amount of

potassium available to canola plants grown continually at different soil moisture levels

should be further investigated in order to monitor any effects on plant size, shoot:root

ratio, relative turgor pressure and transpiration per leaf area.

Chapter 5: The Longer Term Effects of Potassium and Water Stress

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Chapter 5 The Longer Term Effects of Potassium and Water Stress on

Canola Growth and Transpiration Rate

5.1 Introduction

The previous two chapters focused on the physiological effects of potassium on

drought stress in canola plants. This experimental chapter has greater emphasis on the

effect of different long term potassium rates and soil moisture levels on the

morphological response of Trigold canola plants and only some physiological

parameters such as relative turgor pressure and transpiration rate per leaf area were

monitored.

Previous research findings on potassium physiology have indicated a reduced

transpirational water loss for crops with high potassium concentrations, which was as

well observed for canola suffering under water stress during the two previous chapters

(Sections 3.3 and 4.3) (Lösch et al., 1992; Brag, 1972). Furthermore, in the context of

morphological drought adaptation, a deeper root system has been considered as a

significant beneficial trait in acquiring moisture from sub-soil layers. Rain-fed crops,

especially under Mediterranean environments, usually develop a vigorous root-system

for enhanced growth and yield. However, this strategy can be at risk if plants deplete

the available soil moisture well before the crop has completed the grain filling stage

(Palta et al., 2011). There is some knowledge about the potassium requirements and

yield expectations of canola, however, morphological and physiological adaptations of

plants suffering under drought stress still require further investigation (Brennan and

Bolland, 2009; Brennan, Mason and Walton, 2000; Farré et al., 2007).

In Chapter 3 potassium deficient canola plants were shown to develop a larger root

system relative to the shoots under water stress i.e. smaller shoot:root ratio in

comparison to high fertilised plants. Moreover, canola plants suffering under a mild

potassium deprivation may be able to compensate plant growth if the deficiency is not

too long. Furthermore, Chapter 4 has shown that a short term potassium deprivation

may lead to higher potassium concentration in the shoot relative to the root i.e. greater

shoot:root ratios, which could be due to the higher rates of transpiration that was also


P a g e | 104

described. Thus, those observations will be further investigated in this chapter under a

longer term experiments.

Two experiments were conducted in order to quantify the effect of potassium nutrition

on canola growth. The trials were conducted in sequence and are separately described

in the methodology and results sections. The main hypothesis for both experiments

was that canola plants that are well fertilised with potassium develop a larger plant size

while maintaining a higher relative turgor pressure due to reduced water loss under

drought stress conditions in comparison to long term potassium starved plants, which

will have a significantly smaller plant size. It is also expected that the potassium

deficient plants will adapt to the moisture and nutrient stress by decreasing their

shoot:root ratio to aid the uptake of water and potassium into the plant in particular

under long term water deficiency during the growing period.

The sub-hypotheses for the two conducted experiments were:

I. Under drought stress and potassium starved conditions canola plants accumulate

relatively more potassium in their shoots than in their roots in comparison to well

supplied plants due to an increased rate of transpiration, to compensate for the

deficiency.

II. A higher than optimal rate of potassium does not lead to any significant

advantage in plant size e.g. shoot and root dry weight.

III. A severe long term potassium deprivation in canola leads to a significant

reduction in dry weight, whereby, plants under a mild potassium deficiency are

able to compensate their growth.

5.2 Methodology

The methodology described applies to both conducted experiments, unless indicated

otherwise. The experiments were grown under controlled environmental conditions in a

climate chamber at the University of Goettingen (Germany, in 2012) with temperatures

ranging from 5°C, during the night to 20°C, during the day. The humidity was set to

60% and light intensity to 300 μE with a day length of 14 hours.

For the first experiment, 40 canola plants of the cultivar Trigold were grown for a period

of 46 days until 70% of the plants reached the flowering stage. Five different potassium

treatments (no added K, low K, medium K, recommended K and high K) were


P a g e | 105

implemented at the start of the growing season in order to be able to monitor longer

term effects of potassium on the plant as outlined in Table 5.2. When the canola plants

reached bud development, they were subjected to a five day drying cycle by

completely withholding all irrigation water until they showed significant wilting

symptoms. After this, all samples were watered back to field capacity based on the pot

weight for each plant as described below.

For the second experiment, 24 Trigold canola plants were grown for a duration of 69

days only using two potassium treatments, one with a mild potassium starvation (low

K) and one with adequate potassium (recommended K) (Table 5.2). In this experiment

three different moisture treatments were used, which were also applied from the start

of the experiment in order to monitor any different morphological adaptation strategies

by canola under different soil moisture levels. The three long term water treatments

were:

1. 1000 ml deionised water per 3.5 kg dry soil approximately 28% gravimetric soil

moisture content (equivalent to field capacity).

2. 600 ml deionised water per 3.5 kg dry soil approximately 17% gravimetric soil

moisture content (equivalent to 60% of field capacity).

3. 300 ml deionised water per 3.5 kg dry soil approximately 8.5% gravimetric soil

moisture content (equivalent to 30% field capacity)

The watering of the three (water) treatments was then stopped at the early bud stage

and all plants were dried down until they showed severe wilting symptoms before

eventually all plants of all water regimes were as well watered back to field capacity as

outlined for the previous experiment, ranging from 9 to 13 days depending on the

individual plants and different water treatments.

5.2.1 Pot and Soil Preparation

For both experiments, the canola plants were grown in 4-litre round plastic pots (18 cm

high and 12 cm wide). The bottom of each pot was sealed with a filter paper only

allowing water to pass through while keeping the soil inside the pot. In order to aid

irrigation through capillary rise and to minimise water and nutrient lost through

leaching, the pots were placed in shallow trays in which the water was added every

second day after seedling emergence making sure that the pots were not constantly in


P a g e | 106

water in order to prevent potassium loss via diffusion. The maximum amount of water

taken up into the soil through this method was used as field capacity for both

experiments. Pots were filled with a synthetic soil mixture that was created by

thoroughly mixing 92% sand (from a local quartz sand supply in Goettingen), 5% silt

(farm soil near Goettingen) and 3% organic turf material, which was fertilised

sufficiently to ensure that only potassium was limiting growth (Table 5.1). Prior to

mixing, each of the three soil components were analysed for potassium concentration

using the method described by Schüller (1969) and the sum of the data utilised to

establish the implemented potassium treatments, whereby, the measured potassium

concentration in the unfertilised soil equated to the no added K treatment (Table 5.2).

All soil components, except for the turf, were passed through a 2 mm sieve before

being air dried in a glasshouse for 72 hours. After that, the components were

thoroughly mixed to a homogeneous substrate and then divided into portions of 3.5 kg

for each pot. Every pot, therefore, received the same amount of fertiliser. Only the rate

of potassium was altered into five different treatments for the first experiment and two

treatments for the second experiment (Table 5.2).

Table 5.1: Fertiliser composition

Table 5.2: Potassium fertilser rates

Fertiliser rate Amount of K (mg kg-1 soil) In form of

No K+ 18

KCl

Low K+ 72

Medium K+ 127

Recommended K+ 182

High K+ 236

Fertiliser Amount of nutrient (mg kg-1 soil) In form of

P 53 Ca(H2PO4)2

S 81 MgSO4

Mg 81 MgSO4

N 142 Ca(NO3)2


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5.2.2 Planting

For both experiments, one canola seed was planted in each pot in the centre of the pot

about 1 cm deep. During the first few days, 10 to 20 ml of deionised water was added

to the centre area of each pot in the vicinity of the seeds to improve germination. This

was in addition to the water applied into the tray of each treatment. The plants of both

experiments were grown until they reached the bud to early flowering development

stage. At that stage all samples were subjected to the drying cycle by stopping the

irrigation. All plants were then watered back to field capacity through capillary rise by

adding the lost amount of deionised water into the tray underneath each pot. During

each drying cycle the pot weight and relative leaf turgor pressure were monitored.

5.2.3 Pot Weight and Transpiration per Leaf Area

The pots of both experiments were weighed at regular intervals four times a day during

the drying cycles at 7:00, 10:00, 13:00 and 16:00 hrs. From this the transpiration per

leaf area of the canola plants was determined utilising the final leaf area obtained after

harvesting at the end of each experiment. The average transpiration per leaf area of

well-watered canola plants was obtained by averaging the transpiration per leaf area of

three consecutive days immediately prior to the drying cycle before the same samples

were subjected to the water stress cycles, to reduce plant to plant variability. Since the

main focus was on monitoring differences between the potassium treatments under

water stress and not the effect of potassium on water stress, only the trend of the

drought stressed samples of both conducted experiments are presented in the result

section.


The relative turgor pressure was monitored using leaf patch clamp pressure probes

(LPCP) (Yara ZIM plant technology, Hennigsdorf, Germany) as described by

Zimmermann (2008). For both experiments, twelve canola plants were monitored using

the LPCP-probes, with one LPCP-probe clamped onto the fourth fully expanded leaf of

each plant using the smaller of the two available magnets in order to prevent any

damage to the fragile canola leaves. For the first experiment, four of the plants from the

no added K treatment, low K and high K treatment were clamped. The four plants of

each potassium treatment were initially used as control samples during the growing

period in order to monitor the Pp trend of well-watered canola plants and subsequently


P a g e | 108

subjected to a drying cycle. This approach ensured a direct comparison of individual

plants and allowed comparable probe readings as the leaves were fully developed but

not too old to be the next senesced leaf by the plant. During the second experiment ,

two plants of the low K and recommended K treatments for each water level were

clamped in the same way as outlined above. For both experiments, the clamping

occurred in the morning when the plants had a high turgor pressure. The applied

clamping pressure ranged from 20 to 40 kPa.

For the two experiments, the relative turgor pressure was logged every five minutes

throughout the experiments. To allow comparison of the diurnal LPCP-probe output

pressure readings (Pp), were normalised as described previously. Note that an increase

Pp output pressure reflects a drop in relative turgor pressure (Pc) Furthermore, the

baseline is referred to as the regression line between all minima Pp readings illustrating

the general trend of relative turgor pressure (Rueger et al., 2010).

5.2.5 Harvest

The plant shoots and roots of both experiments were harvested at the completion of

each drying cycle. Roots were separated from soil by washing them over a sieve. A

collective soil sample out of every pot was taken. The diameter of the taproot was

measured at the point where the stem was cut off the shoot. All leaves were cut off

each plant and the leaf area determined using a Lens System, Epson Perfection V700

Photo scanner (Seiko Epson corporation, Nagano, Japan) before the shoots were

separated from the roots. The complete shoot material (leaves and stems) as well as

the roots of each sample were then dried in an oven for 48 hours at 70°C to determine

the dry weight of each plant. The potassium was extracted from the dried root and

shoot material material using 65% nitric acid under pressure and its

concentration was determined using flame photometry. The potassium content of

the collected soil samples, on the other hand, were analysed as described by Schüller

(1969).

5.2.6 Experimental Design and Statistical Analysis Both experiments were set up in a complete randomised block with eight replicates per

potassium treatment for the first experiment and four replicates per potassium and soil

moisture treatment for the second experiment. All plants were randomly moved every

second day within the different treatments in the climate chamber to ensure similar


P a g e | 109

growth conditions for every plant. The statistical analysis was conducted using Origin

(OriginLab, USA, Northampton, MA) version 8.6 as well as Microsoft Office Excel 2008

(Microsoft, Redmond, USA), to calculate mean values, standard error and where

appropriate an ANOVA with a posthoc Tukey-test.

5.3 Results

5.3.1 Experiment 1

5.3.1.1 Leaf Area

The mean leaf area of the five potassium treatments at final harvest showed an

increase with increasing potassium rate. The mean leaf area of the no added K

treatment was with 1194 cm2 less than half the size of the highest potassium treatment

(2585 cm2). This was significantly smaller than all other potassium treatments with P-

values between 0.0001 and 0.016 for comparing the no added K treatment with the

other treatments. No significant differences were observed between the other

potassium rates (Figure 5.1).

Figure 5.1: Average leaf area for canola plants treated with five different potassium

rates. Error bars show ± SE (n = 4). Different letters above the bars indicate

significance at ≤0.05.

0

500

1000

1500

2000

2500

3000

No K Low K Medium K Recommended K High K

Leaf

Are

a (c

m2 )

Potassium Rate

b b b

b

a


P a g e | 110

5.3.1.2 Shoot and Root Dry Weight, and Shoot:Root Ratio

The shoot dry mass increased with increasing potassium fertilisation with the no added

K treatment showing a significantly lower shoot dry mass than all the other potassium

rates (P-values: 0.0008 - 0.019). The differences between the other potassium

treatments were, however, not significant, although there was a trend for higher shoot

weight with increasing potassium rate (Figure 5.2 A).

As observed for the shoot dry weight, the root dry weight of the no added K treatment

was lower with 1.18 g and this was significantly smaller than for all the other treatments

(P-values: 0.004 - 0.04). For the other four treatments the root dry weight followed a

trend inverse to the one observed for the shoot dry weight with the root dry weights

decreasing with increasing potassium fertilisation. Yet these differences were again not

significant (Figure 5.2 B).

A significant difference in the shoot:root ratio was observed between the low and the

high K treatment (P=0.03) with the low K treatment showing the lowest shoot:root ratio

of 4.34 and the high K treatment showing the highest shoot:root ratio of 7.9. The other

three potassium treatments showed no significant differences with shoot:root ratios

around 5.5 (Figure 5.2 C).


P a g e | 111

Figure 5.2: Average shoot (A) and root dry weights (B), as well as shoot:root ratio (C) for

canola plants treated with five different potassium rates. Error bars show ± SE (n = 4).

Different letters above the bars indicate significance at ≤0.05.

0

5

10

15

20

25

No K+ Low K+ Medium K+ Recommended K+ High K+

Shoo

t Dry

Wei

ght (

g)

A

b b

b

0.0

0.5

1.0

1.5

2.0

2.5

3.0

No K+ Low K+ Medium K+ Recommended K+ High K+

Roo

t Dry

Wei

ght (

g)

B

a

b b b

b

0

1

2

3

4

5

6

7

8

9

10


Shoo

t:Roo

t Rat

io

Potassium Rate

C

ab ab

a

b

a

b

ab


P a g e | 112

5.3.1.3 Root Diameter

The no added K treatment had a significantly lower root diameter, with 7 mm, than the

four other potassium treatments (P-value: 0.002 – 0.01), which all showed a root

diameter around 12.9 mm. Thus, no further significant differences could be observed

(Figure 5.3).

Figure 5.3: Average root diameter of canola plants treated with five different potassium

rates. Error bars show ± SE (n = 4). Different letters above the bars indicate significance at

≤0.05.

5.3.1.4 Potassium Concentration in Shoot and Root, as well as Shoot:Root Ratio

The shoot potassium concentration for the no added and the low K treatments were

significantly smaller than for all other potassium rates with P-values ranging from 0.004

to 0.01 (Figure 5.4 A). The root potassium concentration of the no added and low K

treatments were only significantly smaller than the root K content of the high K

treatment with P-values of 0.01 and 0.03, respectively. Moreover, a significant

difference could be observed between the low K and the medium K treatment (P=0.02)

(Figure 5.4 B). There were no further significant differences between the shoot

potassium concentrations of the other treatments.

The shoot:root ratios for potassium concentration of the no added, medium and high K

treatments were very similar ranging from 1.8 to 2.4. With 4.4 and 4.7, respectively, the

low and recommended K treatments had the highest shoot:root potassium ratios.

0

2

4

6

8

10

12

14

16


Roo

t Dia

met

er (m

m)

Potassium Rate

a

b b b

b


P a g e | 113

However, no significant differences were observed due to the large standard errors

present (Figure 5.4 C).

Figure 5.4: Average shoot (A) and root (B) potassium content, as well as shoot:root ratio

(C) for canola plants treated with five different potassium rates. Error bars show ± SE (n =

4). Different letters above the bars indicate significance at ≤0.05.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5


Shoo

t K (%

) A

a

b

b

b

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8


Roo

t K (%

)

B

a

b

ab

bc

0 1 2 3 4 5 6 7 8 9

10


Shoo

t:Roo

t Rat

io

Potassium Rate

C a

a

a a

a

a

b

ab


P a g e | 114

5.3.1.5 Pot Weights and Transpiration per Leaf Area

The average pot weights of all five potassium treatments declined during the drying

period. All treatments started with a pot weight of 4.97 kg (Figure 5.5). Whereas the pot

weights of the medium, recommended and high K treatments all declined at a very

similar rate showing pot weights of around 4.25 kg on day 4 of the drying cycle, the no

added and low K treatments showed a slightly slower decrease with pot weights of

4.42 and 4.33 kg on day 4, respectively. However, a significant difference in total water

loss was only observed between the no added and the recommended K treatments

with a P-value of 0.03.

Figure 5.5: Average pot weights of canola plants treated with five different potassium rates

during drying cycle.

The no added K treatment had on average a significantly higher transpiration per leaf

area than all other treatments (P-values: 0.0004 – 0.01) (Figure 5.6). The high K

treatment showed the lowest transpiration per leaf area over the drought period,

however, it was only significantly different to the no added K and medium K treatment

with a P-value of 0.03. Overall, all treatments showed a decreasing trend in

transpiration per leaf area over the drying period.

4,100

4,200

4,300

4,400

4,500

4,600

4,700

4,800

4,900

5,000

5,100

16:00 7:00 10:00 13:00 16:00 7:00 10:00 13:00 16:00 7:00

1 2 3 4

Pot W

eigh

t (g)

Day of Drying Cycle



P a g e | 115

Figure 5.6: Average transpiration per leaf area of canola plants treated with five different

potassium rates during drying cycle.

Under well-watered conditions the average transpiration per unit leaf area ranged from

0.115 g cm-2 to 0.148 g cm-2 with the low K treatment showing a slightly higher

transpiration per unit leaf area than all the other treatments. However, there were no

significant differences. (Figure 5.7).

Figure 5.7: Average transpiration per leaf area of well-watered canola plants treated with

five different potassium rates. Error bars show ± SE (n = 8).

0

0.01

0.02

0.03

0.04

0.05

0.06

16:00 7:00 10:00 13:00 16:00 7:00 10:00 13:00 16:00 7:00

1 2 3 4

Tran

spira

tion

per L

eaf A

rea

(g c

m-2

)

Days of Drying Cycle


0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20


Tran

spira

tion

per L

eaf A

rea

(g c

m-2

)

Potassium Rate

a

a a a

a


P a g e | 116

5.3.1.6 Relative Turgor Pressure

Figure 5.8 shows the relative turgor pressure of plants treated with five different

potassium rates during the drying cycle. All samples, besides the low K treatment,

showed an increase in the Pp baseline with increasing drought stress. Interestingly, the

recommended and high K treatments needed to be re-watered first and their Pp

baseline was higher than for all other treatments. At the same time their baseline

returned to lower values than for all other treatment after re-watering. However, the low

K treatment peaked to the highest output pressures followed by the no added K

treatment. These two treatments also sustained the drying cycle the longest.


P a g e | 117

Figure 5.8: Examples of Pp measurements on canola subjected to five different potassium

treatments and the corresponding ambient light and relative humidity. A) Pp measurements

on drought-stressed canola treated with high K (dotted line with black filled squares); B) Pp

measurements on drought-stressed canola treated with recommended K (dotted line with

filled hexagon); C) Pp measurements on drought-stressed canola treated with medium K

(dotted line with filled diamond); D) Pp measurements on drought-stressed canola treated

with low K (dotted line with filled triangles; E) Pp measurements on drought-stressed canola

plants treated with no added K (dotted line with filled circles). Black bars indicate irrigation

events. Grey background indicates night time and light background indicates day time.

A

B

C

D

E

Days of Drying Cycle


P a g e | 118

It was observed that the Pp increased rapidly from the nighttime baseline when the light

in the growth rooms were switched on in the growth room, prior to the drying cycle. The

Pp then dropped very soon after and stabilized at a level above the baseline, following

which it slowly increased during the day. This ‘overshoot’ (Figure 5.9) (i.e. initial rapid

increase then drop of Pp/low relative turgor), was only occasionally visible during the

drying cycle (Figure 5.8). The high K treatment, thereby, peaked higher on day one in

comparison to the no added K treatment.

Figure 5.9: Overshoot examples of no added and high K treatment prior to the drying

cycle.

4 3 2 1 Days prior to Drying Cycle

4 3 2 1

Days prior to Drying Cycle


P a g e | 119

5.3.2 Experiment 2

5.3.2.1 Leaf Area

The mean leaf area of both potassium treatments increased significantly with

increasing soil moisture level (P<0.01), ranging from 22 cm2 for the recommended K at

8.5% soil water treatment to 1181 cm2 for the recommended K 28% soil water

treatment. There were no significant differences observed between the two potassium

treatments within each water treatment (Figure 5.10).

Figure 5.10: Average leaf area for canola plants treated with two different potassium and

three different water rates. Error bars show ± SE (n = 4). Different letters above the bars

indicate significance at ≤0.05 (potassium main effect P=0.86, water main effect P<0.001,


5.3.2.2 Shoot and Root Dry Weight, and Shoot:Root Ratio

The shoot and root dry weights increased significantly with increasing soil moisture

levels (P<0.05). However, there were no significant differences between the two

potassium treatments within each water treatment (Figures 5.11 A and B). The

shoot:root ratio of both potassium treatments increased significantly (P<0.05) with

increasing soil moisture except for the low K treatment between the 17 and 28% soil

moisture treatment, which showed similar shoot:root ratios. Nevertheless, there were

no significant differences measured between the two potassium treatments (Figure

5.11 C).

0

200

400

600

800

1000

1200

1400

8.5 17 28

Leaf

Are

a (c

m2 )

Soil Water Concentration (%)

Low K Recommended K

a

b

c

a

b

c


P a g e | 120

Figure 5.11: Average shoot and root dry weights, as well as shoot:root ratio for canola

plants treated with two different potassium and three different water rates. Error bars show

± SE (n = 4). Different letters above the bars indicate significance at ≤0.05 (potassium

main effect A: P=0.10, B: P=0.79, C: P=0.21; water main effect A: P<0.001, B: P<0.001, C:

P=0.04; interaction A: P=0.99; B: P=0.74, C: P=0.90).

0

5

10

15

20

25

8.5 17 28

Shoo

t dry

wei

ght (

g)

A

a

b b

c

c

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ght (

g)

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c c

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io


Low K Recommended K

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c

a

a


P a g e | 121

5.3.2.3 Root Diameter

As observed for the shoot and root dry weight, the tap root diameter increased

significantly (P<0.05) with increasing soil moisture level. Interestingly, the tap root

diameter of the low K treatment was significantly larger than for the recommended K

treatment at the 8.5% soil moisture level (P=0.04). No further significant differences

could be established (Figure 5.12).

Figure 5.12: Average root diameter of canola plants treated with two different

potassium and three different water rates. Error bars show ± SE (n = 4). Different

letters above the bars indicate significance at ≤0.05 (potassium main effect P=0.43,

water main effect P<0.001, interaction P=0.53).

5.3.2.4 Potassium Concentration in Shoot and Root, as well as Shoot:Root Ratio

The shoot potassium concentration for the recommended K treatment was significantly

higher than the potassium concentration for the low K treatment across all soil moisture

levels (P<0.05) (Figure 3.13 A). The root potassium concentration of the recommended

K treatment, on the other hand, was only significantly larger than the low K treatment

for the 17% soil moisture level (P= 0.003) (Figure 3.13 B). There were no further

significant differences between the potassium treatments. Interestingly, there were

significant differences between the different soil moisture levels within the same

potassium treatment, with the low K shoot potassium concentration being significantly

0

2

4

6

8

10

12

14

16

18

20

8.5 17 28

Roo

t Dia

met

er (m

m)

Soil water concentration (%)

Low K Recommended K

b

c c

d d

a


P a g e | 122

larger when the soil moisture was 8.5% instead of 17 or 28% (P<0.0001) (Figures 3.13

A and B).

The recommended K treatment showed a significantly larger shoot:root ratio for

potassium concentration than the low K treatment when treated with 28% soil moisture

(P=0.04). The recommended K treatment also showed significant differences in the

shoot:root ratio between the three soil moisture treatments with the 8.5 and 28% rate

having a significantly larger ratio than the 17% moisture level (P=0.03). The low K

treatment also showed a significantly larger shoot:root ratio for the 8.5% soil moisture

level in comparison to the other two soil moisture levels (P=0.01 and P=0.001,

respectively (Figures 3.13 C).


P a g e | 123

Figure 5.13: Average shoot and root potassium content, as well as shoot:root ratio for

canola plants treated with two different potassium and three different soil moisture rates.

Error bars show ± SE (n = 4). Different letters above the bars indicate significance at

≤0.05 (potassium main effect A: P=0.001, B: P=0.74, C: P=0.18; water main effect A:

P<0.001, B: P=0.43, C: P=0.08; interaction A: P=0.14; B: P=0.29, C: P=0.48).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

8.5 17 28

Shoo

t K (%

)

A b

c

d

c

d

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

8.5 17 28

Roo

t K (%

)

B

a

a

ac

a a

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

8.5 17 28

Shoo

t:roo

t rat

io


Low K Recommended K

C

b b

c

a

b

a

a

ab


P a g e | 124

5.3.2.5 Pot Weights and Transpiration per Leaf Area

The average pot weights of all treatments declined during the drying period, whereby,

interestingly, the low and recommended K treatments treated with 17% soil moisture

sustained the drying cycle four days longer than the samples treated with 8.5 and 28%

soil moisture. The recommended K treatment showed significantly higher pot weights

for the 8.5% soil moisture level (P=0.003), but was significantly lower to the low K

treatment for the 28% water rate (P=0.03). When treated with the 17% water rate the

two treatments showed no significant differences (Figures 5.14 A - C).


P a g e | 125

Figure 5.14: Average pot weights of canola plants treated with two different potassium

and three different soil moisture rates (A: 8.5%, B: 17%, C: 28% soil moisture) during

drying cycle.

3,800

3,900

4,000

4,100

4,200

4,300

4,400

4,500

4,600

4,700

7:00

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Pot w

eigh

t (g)

A

3,800

3,900

4,000

4,100

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4,300

4,400

4,500

4,600

4,700

7:00

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Days after 11 April 2012

Recommended K Low K


P a g e | 126

The transpiration per leaf area during the conducted drying cycle decreased

significantly for both potassium treatments with increasing soil moisture level (P<0.05).

However, there were no significant differences between the 17 and 28% moisture

levels for the low K and the 8.5 to 28% levels for the recommended K treatment. Both

potassium treatments showed a decreasing trend in transpiration over the duration of

the drying cycle. However, there were no significant differences between the two

potassium treatments (Figure 5.15).


P a g e | 127

Figure 5.15: Average transpiration per leaf area of canola plants treated with two

different potassium and three different soil moisture rates (A: 8.5%, B: 17%, C: 28%

soil moisture) during drying cycle.

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

7:0

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rea

(g c

m-2

) A

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)


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C


P a g e | 128

5.3.2.6 Relative Turgor Pressure

The relative turgor pressure during the drying cycle decreased over the time of drying

indicated by an increase in Pp output pressure across all soil moisture treatments.

Overall, the recommended K treatment showed a lower Pp output pressure than the

low K treatment for most days and, thus, maintained a higher relative turgor pressure

throughout the drying cycle (Figures 5.16 - 5.18).

Figure 5.16: Pp output pressure of representative canola plants treated with low K (A)

and recommended K (B) grown at 8.5% soil moisture during the drying cycle. Bottom

panel shows ambient light level and relative humidity. Black bars indicate irrigation


Black arrow indicates irrigation event.


A

B


P a g e | 129


and recommended K (B) grown at 17% soil moisture during the drying cycle. Bottom




A

B



P a g e | 130


and recommended K (B) grown at 28% soil moisture during the drying cycle. Bottom




5.4 Discussion

First the impact of different potassium rates on the morphology of water stressed

canola will be discussed, followed by the physiological response in transpiration per

leaf area and relative turgor pressure.


A

B


P a g e | 131

5.4.1 Changes in Dry Weight of Longer Term Water and Potassium stressed Canola

A long term potassium deficiency has a significant impact on the plant size of water

stressed canola, however, there were no differences from the low through to the high

rates i.e. no further significant differences were observed for the shoot or root dry

weight or measured leaf area between any of the other potassium treatments for the

plants of both conducted experiments. This could indicate the ability of canola to

compensate its plant biomass independent of a large range of potassium levels as long

as the availability of potassium does not become too severe, i.e. smaller than 72 mg

kg-1. Interestingly, the difference between the no added K and other potassium

treatments was as well reflected in the taproot diameter of the five potassium

treatments. Those findings are in agreement to the observations made in Chapter 3

(Section 3.4) for soil grown canola plants where a mild potassium deprivation only

caused a significant difference in growth if the duration of the deficiency was long

enough. This was also supported by the findings of Brennan et al. (2013) who

described a 90% relative yield for canola if potassium was available at a critical value

ranging from 40 to 60 mg kg-1 soil depending on soil type and growing conditions. A

similar effect of potassium nutrition on tomato plant growth and fruit developments has

also been shown by other researchers supporting the underlying findings (Besford and

Mow, 1975).

In addition, Rasul (2012) could as well only report a significant growth advantage of

high fertilized (120 kg/ha) canola plants. This was as well confirmed by an earlier study

conducted by Khan et al. (2004). The authors also described no further growth or yield

benefits for a large range of potassium rates if the potassium deficiency does not

become too severe. These findings support the above statement. However, more

detailed research is needed, in particular to understand the effect of different

potassium levels on the taproot diameter in canola since the morphology of the roots

have been thought to significantly influence nutrient uptake into the plant and could,

consequently, be a first indicator for a potassium starvation, (Nye and Tinker, 1977;

Barber, 1995), as shown in this research.

However, a reduced amount of available potassium to the canola plant led to a

significantly decreased shoot:root ratio in dry weight with the low K treatment being

smaller in comparison to the high K treatment, as already described in Chapter 3


P a g e | 132

(Section 3.4). Interestingly, the no added K treatment was neither different to the low

nor the high K treatment. The severity of the potassium deficiency, therefore, must

influence the morphology of canola differently especially if the effect is enhanced or

impacted by other growth conditions such as additional drought stress (Khan et al.,

2004; De Dorlodot et al., 2007). Moreover, the decrease of the root dry weight with

increasing potassium rate (at rates above 18 mg kg-1 soil), could also have been

caused by an inhibiting effect of potassium chloride on plant growth with increasing

fertiliser rate similar to the work conducted by Lee and Van Iersel (2008), who have

observed a decrease of plant growth of Chrysanthemum morifolium under an

increasing sodium chloride application. However, as the applied rates of the high K

treatment was higher than adequate but not at a toxic level (Brennan et al., 2013), it is

more likely that canola shows a different morphological response depending on the

duration and the intensity of the stress as described by Bednarz et al. (1998).

In contrast, wheat (Triticum aestivum L.) developed a higher shoot:root ratio for dry

weight under a reduced potassium availability in comparison to the high K treatment

(Ma et al., 2013). Thus, there seems to be a fundamental difference in the

morphological response between the two species (Damon et al. (2007; Brennan et al.,

2013, Ma et al., 2013). One strategy of canola plants to optimise the uptake of plant

nutrients and water under drought stress conditions could be based on a larger taproot

diameter and a better distribution of the root system in the soil medium, which is

supported by the observations made for the underlying study (Siddique et al., 1990;

Liao et al., 2006; Manschadi et al., 2006; Palta et al., 2011). Those interpretations were

as well confirmed by the findings of the second experiment of this chapter, with the low

K treatment developing a significantly thicker taproot than the recommended K

treatment under the 8.5% soil moisture level suggesting some mechanism of the low K

canola treatment to modify its root morphology to optimise water and nutrient uptake

under a combination of long term potassium and water stress (Nye and Tinker, 1977;

Barber, 1995). Nevertheless, further experiments should be conducted to explain the

described differences between the two species and to establish possible mechanisms

behind each strategy.

Moreover, water significantly impacts the size of canola, as the measured leaf area as

well as the shoot and root dry weight in the second experiment significantly increased

with increasing soil moisture levels for both potassium treatments. A higher rate of


P a g e | 133

potassium did not reduce the negative effect of lower soil moisture level on the

morphology of canola, with no significant differences obtained for the leaf area and

shoot and root dry weight of the two potassium treatments. Those findings are in line

with a number of other studies that have investigated the effect of water on plant

growth and other parameters such as transpiration rate or photosynthetic activity

reporting smaller growth under restricted soil moisture (Zhi, 2011; Wang et al., 2012).

Additional experiments are, however, required assessing the impact of more extreme

potassium rates on the morphology of canola under long term moisture stress as the

low K treatment of the conducted experiment only represented a mild potassium

deficiency (Brennan et al., 2013).

5.4.2 Potassium Concentration

The relative concentration of potassium in the shoots of canola suffering under long

term potassium deficiency and drought stress is significantly lower than for well

fertilised plants. These potassium deficient plants also had a lower shoot:root ratio

compared with well fertilized plants, with the low K treatment showing a significantly

smaller shoot:root ratio than the recommended K treatment under drought stress in the

second experiment of this chapter. There were no further significant differences

obtained. This is in contrast to the findings made for Chapter 4 where a potassium

deficiency caused a relative larger accumulation of potassium in the shoot than in the

roots, which may have been caused by the significant higher transpiration per leaf area

for the same samples as a strategy to compensate the deficiency (Section 4.4) (Nye

and Tinker, 1977; Barber, 1995). Nevertheless, the canola plants for the experiment in

Chapter 4 were grown in a nutrient solution, which would have represented more

extreme growth conditions, with soil media having greater buffering capacity.

The findings of this chapter are in line with Rose et al. (2007), who examined the

change of potassium over the growing period of several canola and wheat genotypes.

They illustrated an initial increase and a later decrease of the potassium content in the

leaves during the vegetation process. The potassium content in the roots for Trigold

Canola was about 4 to 6 times lower than measured in the leaves, but did not decrease

as much as the leaf potassium content towards the end of the growing period resulting

in a similar amount of potassium in shoots and roots at harvest. The samples of the

two conducted experiments still showed slightly larger amounts of potassium in the

shoots in comparison to the roots at harvest. However, as the harvest of the


P a g e | 134

experiment already occurred at the flowering stage i.e. during the growing period of

canola before ripening and there was only one potassium analysis conducted at the

end of the drying cycle, the obtained results of this experiment cannot show a change

of potassium content in the plant parts and further research is required to investigate

the distribution of potassium in canola under water stress by more frequent biomass

sampling throughout the growing period.

5.4.3 Response of Transpiration per Leaf Area and Relative Turgor Pressure under Drought

Potassium deficiency may lead to a significantly higher transpiration per leaf area in

canola under drought stress. This might indicate a reduced ability to regulate stomatal

opening and closing, as described in Chapters 3 and 4. The influence of potassium on

the physiology and water use of plants was also described by several other studies for

a variety of different crops (Fischer and Hsiao, 1968; Brag, 1972; Lösch et al., 1992;

Brennan and Bolland, 2007).

Moreover, as expected the pot weight and transpiration per leaf area decreased

constantly over the time of drying for both experiments, indicating an increased water

stress level (Aroca et al., 2001; Jackson et al., 2003). Ritchie (1981), on the other

hand, observed that there was only little change in plant response to transpiration until

less than 30% of the extractable water remained in the soil, suggesting that

physiological responses of plants to drought stress only become visible after a certain

fraction of extractable soil moisture content has been reached. This was supported by

the findings of the second experiment, where canola plants of the 17% soil moisture

level (60% available soil water) were able to sustain the drying cycle significantly

longer than all other treatments. In addition to that, the significant differences in plant

size would have also influenced the speed of how quickly the samples use up the

available water in the pot. In order to further investigate the effect of different

potassium rates and soil moisture levels on the transpiration rate of canola, more

experiments are needed focusing on the lower fractions of available soil moisture to

identify at what threshold value the transpiration rate significantly declines.

Canola plants open and close their stomata at various times during the day under

drought stress, with an observed peak in transpiration per leaf area in the morning and

a second peak in the afternoon. As during the underlying experiment the transpiration


P a g e | 135

per leaf area was only recorded every three hours, it was not possible to measure

more frequent changes in transpiration. Nonetheless, previous studies have also

reported this phenomenon of autonomous, cyclic opening and closing of stomata cells

during the day (Barrs and Klepper, 1968; Barrs, 1971). Those authors described

periods less than 3 hours during which an opening and closing movement of stomata

cells occurred. And when they plotted the periodic variation in transpiration against

time a sine wave with a period of 10 - 90 min was obtained. Besides that, the described

oscillatory transpiration per leaf area could also have been caused by a range of other

factors. For example; the failure of stomatal control systems (Hopmans, 1971),

imbalances in water potential between subsidiary and guard cells (Gumowski, 1983),

internal fluctuations (CO, 1978), or imbalances in water potential between the plant

roots and canopy (Lang et al., 1969). Plants might, thereby, gain selective advantages

through oscillatory transpiration by regulating leaf temperature (Mahan and Upchurch,

1988), improving water-use efficiency (Cowan, 1977; Gumowski, 1981; Upadhyaya et

al., 1988) or modifying xylem water potential in order to transmit substances or

frequency signals between different regions of the plant (Due, 1989; Gumowski, 1981).

Consequently, further investigations are required to better understand the mechanisms

behind the observed oscillation in transpiration per leaf area in canola.

There was a peak in transpiration per leaf area in the morning which was also reflected

in an obtained peak in output pressure of the leaf patch pressure probes when the

lights were switch on prior to the drying cycle, indicating a drop in relative turgor

pressure, which almost disappeared during the drying cycle. One reason to explain the

observed overshoot could be that the underlying experiment has been conducted

under artificial, controlled environmental conditions in a climate chamber, thus, the

observed oscillatory transpiration might also have been caused by sudden and large

perturbations i.e. the on and off switching of the growth lights (Ehrler et al., 1965).

These sudden environmental shifts may have triggered responses of stomata cells

resulting in the monitored overshoot of transpiration and peak of output pressure

before the plants were adapting to the optimum stomata aperture (Barrs and Klepper,

1968; Ehrler et al., 1965). This overshoot was also outlined by Amer et al. (1993), who

could monitor a rapid opening of stomata cells in roses within 10 minutes after the

lights went on. Interestingly, canola plants were able to respond to the environmental

change as the observed overshoot in the morning disappeared as the drying cycle

progressed, which could indicate a response of the plants to the environmental


P a g e | 136

conditions by adjusting their transpiration rate and hydraulic conductivity. This way the

plants are also preventing damage in their tissue caused by a too low water potential

while at the same time optimising their water use efficiency (Wallach et al., 2010).

Canola plants that are well supplied with potassium are able to better maintain a high

relative turgor pressure under drought stress in comparison to plants with a low or

deficient rate of potassium. However, relative turgor pressure declined over the drying

period for all canola treatments; there were some differences in the diurnal pattern of

relative leaf turgor pressure between the potassium treatments. The observed increase

of output pressure values (Pp), thereby, indicates a decrease in relative turgor pressure

and, hence, a decline of water availability in the leaf tissue i.e. a drop of turgor

pressure (Pc), which was as well visible in the decrease of transpiration per leaf area

(Zimmermann et al., 2008; Westhoff et al., 2009; Zimmermann et al., 2010;

Ehrenberger et al., 2011). Interestingly, the recommended and high K treatments

recovered faster than the other treatments after re-watering as the baseline of the

output pressure (Pp) curve decreased further, i.e. the relative turgor pressure went up,

in comparison to the other treatments.

Those findings are in agreement with the observations made in Chapters 3 and 4

where the canola plants that received a high rate of potassium were able to maintain a

higher relative turgor pressure under drought stress due to reduced water loss,

because of the down regulation of stomata opening (Sinclair et al., 2005).

Nevertheless, as the leaf patch pressure probes in both conducted experiments

malfunctioned for some samples during parts of the measuring period and multiple re-

clamping of the probes were required in order to get regular Pp output readings, further

measurements are needed to verify the obtained findings of this study by for example

using a larger number of probes to minimise the chances of failure. Similar

observations have so far not been made for other plant species and would require

further investigation (Zimmermann et al., 2008; Westhoff et al., 2009; Rueger et al.,

2010).

5.5 Conclusion

Canola plants that were grown under a long term potassium deficiency and exposed to

water stress were significantly smaller than well fertilised plants. However, canola has

the ability to compensate plant dry weight and leaf area under a mild potassium


P a g e | 137

deficiency and water stress. Results suggest that a threshold value below 70 mg kg-1

significantly impacts the morphology of canola plants. Potassium starved canola plants

also develop a smaller shoot:root ratio than plants well fertilised with potassium. This

could be a morphological adaptation by canola to aid the uptake of potassium ions into

the plant. This was as well supported by potassium starved canola plants under long

term water stress, which produced a significantly thicker taproot diameter than well

supplied plants. Furthermore, the impact of long term water stress on plant size could

not be significantly counteracted by a high potassium supply in comparison to low

fertilised canola plants.

Potassium starved canola plants have similar to significantly higher transpiration per

leaf area under water stress and well-watered conditions than plants that have

adequate potassium available. This was as well reflected in the relative leaf turgor

pressure, which was higher in well fertilized plants. Moreover, canola oscillates its

stomatal opening and closing under drought stress throughout the day indicated by a

rise and drop of transpiration per leaf area and relative turgor pressure, which might be

a strategy to maintain a higher leaf water potential preventing damage to the tissue

and, at the same time, enhancing the water use efficiency of the plant. Assessing the

oscillation mechanism in greater detail would help to understand whether potassium

has an influence on this mechanism and identifying reasons why canola plants show

this behaviour under drought stress.

Further experiments are required to determine a threshold level of potassium at which

canola plants are able to produce similar sized plants to those well fertilised. Additional

work should also focus on canola strategies for uptake and distribution of potassium

under different potassium rates with water limiting conditions.

P a g e | 138

Chapter 6: The Role of Potassium in Water Relations and Yield of Field Grown Canola

P a g e | 139

Chapter 6 The Role of Potassium in Water Relations and Yield of Field

Grown Canola in the WA Grain Belt 6.1 Introduction

Previous chapters have discussed the effect of potassium and drought stress on

responses of Trigold canola under glasshouse conditions. The aim of this experiment is

to test the effect of potassium on drought response under field conditions in the WA

grain belt. Hence, the overall objective is to test if canola plants that are well supplied

with potassium are able to better regulate their water dynamics than plants suffering

under a mild potassium deficiency leading to a greater plant biomass and consequent

yield under field conditions.

The grain belt region of WA has been traditionally classified as having a

Mediterranean-type climate, characterized by hot, dry summers and cool, wet winters

(Farré et al., 2007; To et al., 2011). The production of canola in this region is

vulnerable to large variations in rainfall and frequent droughts ranging from early

seedling water stress to terminal drought at the end of a growing period (Farré et al.,

2007; To et al., 2011; Seymour et al., 2012). In this variable climate, soil moisture is the

most limiting factor for crop establishment and growth, in particular in regions of the

Wheatbelt with low to medium precipitation (Moir and Morris, 2011). Thus, greater

water use efficiency of canola through potassium fertilisation would contribute to

improved yields and crop reliability (Farré et al., 2007; Brennan and Bolland, 2007).

Previous field studies have indicated a significant yield benefit from increasing nitrogen

and potassium fertilization. There was a significant interaction between the two

nutrients, leading to greater plant sizes i.e. an increasing amount of N required an

increasing amount of K to produce greater yields and vice versa (Brennan and Bolland,

2009).

In light of the results from the previous chapters, it is hypothesised that the high

potassium treatments will produce higher yields in field conditions as the plants are

able to better regulate their stomata aperture leading to a higher turgor pressure, rate

of photosynthesis and transpiration. The effect of potassium will be enhanced in

particular during water limiting periods throughout the growing season, enabling canola


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to better counteract drought stress while maintaining a greater yield in comparison to

potassium starved plants.

The hypothesis will be addressed by monitoring stomatal conductance, transpiration,

leaf temperature, turgor pressure, osmotic potential and leaf water content at different

stages of the growing period. In addition to that, regular biomass cuts and a yield

determination at the end of the experiment will provide information on the yield and

water use efficiency of the different potassium treatments.

6.2 Methodology

The field experiment was conducted during the 2013 growing season at a field side on

Antonio Road near Meckering in the WA Wheatbelt (S31° 38’, E117° 00’). The

experimental site had been sown to wheat in the previous year and the soil type was a

deep pale sand. The mean bulk density at the site was 1.48 g cm-3. The weather data

used for the experiment was obtained approximately 5.2 km from the site at a nearby

weather station in Carterville, where daily maximum and minimum temperatures as well

as rainfall and evaporation were logged (Jeffrey et al., 2001).

6.2.1 Experimental Design

The field experiment included both potassium and nitrogen treatments. The nitrogen

treatments were included as this was part of a larger experiment conducted by the

Department of Agriculture and Food WA, whereby, the part of this research focused on

the water relations and yield of the different potassium treatments. The design was a

complete randomised block with four replicates, with a factorial arrangement of two

potassium (0 and 100 kg K ha-1) and nitrogen (30 and 50 kg N ha-1) with the following

designations: 1) 0 kg K ha-1 and 30 kg N ha-1 (no added K/ low N), 2) 100 kg K ha-1 and

30 kg N ha-1 (high K/ low N), 3) 0 kg K ha-1 and 50 kg N ha-1 N (no added K/medium N),

and 4) 100 kg K ha-1 and 50 kg N ha-1 N (high K/medium N). For comparison purposes

to the previous chapters, the applied potassium rates equate to approximately 30 mg

kg-1 for the no added K/ medium N treatment and 98 mg kg-1 of soil for the high K/

medium N treatment determined from bulk soil samples at a depth of 10 cm at the field

site. Each plot had a length of 20 m and a width of 1.45 m and plot centres 2 m apart.

All plots were sown on the 23 May 2013 using a seven-row plot seeder fitted with no-

tillage knife points. The canola variety ‘CV Crusher’ was sown at 4 kg ha-1 at 1 cm

depth and P, Mn, Cu and Zn were placed 3 cm below the seed at 20, 3, 1.25 and 1.15


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kg ha-1 respectively. Nitrogen treatments were applied as urea; 30 kg N ha-1 was

applied to the surface at seeding and an additional 20 kg N ha-1 was applied to the high

N treatments 4 weeks after seeding. Potassium was applied to the soil surface at

seeding as muriate of potash (KCl).

6.2.2 Soil analysis

Based on a pre-soil analysis of the trial site, all essential plant nutrients were applied in

the form of a basal fertiliser. This ensured that nitrogen and potassium were the only

growth limiting nutrients according to the critical values presented by Brennan et al.

(2013) for plant growth and development. Soil samples were collected from a depth of

10 cm and dried for 48 hours in a forced-draught oven set at 40°C. All samples were

passed through a 2 mm sieve. The soil potassium and nitrogen concentrations as well

as phosphorus, magnesium and calcium elements and the soil pH (using 0.01 M CaCl2)

were determined using the methods outlined by Colwell (1963), Searle (1984), and by

Rayment and Lyons (2011), respectively.

6.2.3 Measurements

The following measurements were conducted on two different dates during the growing

season of the canola field experiment, unless indicated otherwise. The first series of

measurements was carried out on the 25/08/2013 at the bud-development stage and

the second on the 21/09/2013 at flowering stage. The sampling of canola plants for

every plot occurred in three random locations.

6.2.3.1 Biomass Cuts

Three biomass cuts were conducted to monitor changes in plant size and nutrient

composition throughout the crop development. The three biomass cuts were carried

out on the 25/07/2013, 15/08/2013 and on the 16/09/2013. At each sampling date,

shoots were cut at ground level for 2 adjacent rows of 1 m length at 3 locations within

each plot. Shoot samples were dried in an oven at 70°C for 48 hours before weighing.

Potassium and nitrogen uptake (kg ha-1) was calculated as shoot concentration

multiplied by shoot biomass as outlined by McQuaker et al. (1979) and Rayment and

Lyons (2011), respectively. This way the uptake of those nutrients into the plant and

any interaction effects between potassium and nitrogen, which could affect plant

growth during the crop development of canola, could be identified.


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6.2.3.2 Photosynthesis, Stomatal Conductance and Transpiration Rate

Photosynthesis, stomatal conductance and transpiration measurements were

conducted at the bud development and flowering-stages using a LI-6400 portable

photosynthesis system from Licor (Lincoln, USA). The light intensity was set to 1000

µmol m-2 s-1 and the rate of CO2 in the cuvette was kept at 380 µmol mol-1. Three

canola plants per plot were measured between 09:00 hrs and 12:00 hrs, while being

sure to use the third youngest fully developed leaf on the plant.

6.2.3.3 Leaf Temperature

Leaf temperature was measured with a TiR32 infrared camera from Fluke (Everett,

USA). Three images were taken for each plot from a height of 1.5 m above ground

level and at an angle of 45°. The camera position on the field side was always chosen

in a way that the adjacent no added K and high K plots were in one image in order to

allow a better comparison of the two treatments. The images were taken on both

measuring days between 12:00 hrs and 13:00 hrs aiming to have similar light

conditions for each recording coinciding with high levels of solar radiation. Ambient

temperature was also recorded by a temperature sensor that was positioned within

each plot every time an image was taken as well as the temperature of a metal plate

probe that was always included in the image, which served as a reference point for

calibration. For the analysis of the images, the calibrated temperature of three leaves

per sample was determined and the mean leaf temperature per sample and per

treatment calculated using the software provided with the camera called ‘Smartview’

(Fluke, Everett, USA). 6.2.3.4 Leaf Water Potential

The midday leaf water potential of the high K/medium N and no added K/medium N

treatments was obtained using a PMS 1000 pressure bomb from PMS Instruments

(Albany, USA). One leaf was cut from each of six sample plants within each plot. Each

leaf was put in a plastic clip seal bag once cut off the canola plant to minimise any

evaporational water losses and immediately placed in the pressure chamber, which

was then pressurised until plant sap was visible at the cut end. At this point the

pressure was noted and used as the leaf water potential of the measured sample as

described by Scholander et al. (1964).


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6.2.3.5 Osmotic Potential

Leaf osmotic potential was measured using the method by Callister et al. (2006). A 2

cm sized leaf disc was taken from the same canola plant that had been tested for its

leaf water potential (Section 6.2.3.4) and placed in a vial and stored on dry ice. In the

laboratory the leaf disc of each sample was used to determine the osmotic potential of

the two potassium treatments using a Fiske 110 osmometer (Norwood, USA). To do

this, leaf sap was extracted from a single leaf of each tested plant using a plant sap

press and pipetted into the osmometer cell for analysis (Callister et al., 2006).

6.2.4 Harvest

Grain yield was determined on the whole plots using a plot harvester on 19/11/2013.

6.2.5 Statistical Analysis

The data was analysed with Microsoft Office Excel 2008 (Microsoft, Redmond, USA)

and GenStat 15th addition 2012 (VSN International Ltd, Hemel Hempstead, UK) to

calculate mean values, standard error and where appropriate an ANOVA with a Tukey-

test as a posthoc test with differences at p ≤ 0.05 being regarded as significant.

6.3 Results

6.3.1 Soil and Climate

The available potassium concentration at the trial site ranged from 18.5 to 36.5 mg

kg-1, whereas the available mineral nitrogen (nitrate + ammonium) ranged from 1 to 3.5

mg kg-1 (approx. 1.5 to 5.25 kg N per hectare, using a bulk density of 1.5 g cm-3). The

average maximum temperature remained around 20°C from May to September before

rapidly increasing to 26.3°C in October and 31.8°C in November (Figure 6.1). The

average minimum temperature, on the other hand, gradually decreased from 7.9°C in

May to 3.6°C in July before steadily increasing to 14.8°C in November. The total rainfall

over the entire growing period was 247.5 mm. After seeding on 23 May 2013 the site

received 20 mm of rainfall until the end of the month. This was followed by a dry period

in June with a total precipitation of 3.6 mm. The total monthly rainfall in July, August

and September was around 70 mm and then dropped down to 19 mm in October. After

that, there was no further rainfall recorded during the trial. The total monthly

evaporation showed a gradual increase throughout the growing period from 22 mm


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during the last week in May after seeding to 163 mm in October and a further 156 mm

until the harvest on 19 November 2013.

Figure 6.1: Average minimum and maximum temperature as well as evaporation and

rainfall over the growing period from 23 May 2013 and 19 November 2013.

6.3.2 Biomass Cuts and Grain Yield

Only nitrogen had a significant effect within the high K treatments 12 weeks and 16

weeks after seeding, with the high K/low N samples showing a significantly lower dry

weight than the high K/medium N samples (P=0.02 and P=0.04, respectively) (Figure

6.2). The dry weight of the three shoot biomass cuts increased over the experimental

growing period. However, there were no significant differences between the no added

and the high K treatments for each of the N rates.

0

5

10

15

20

25

30

35

0

20

40

60

80

100

120

140

160

180

May June July August September October November

Tem

pera

ture

(°C

)

Evap

orat

ion/

rain

fall

(mm

)

Time (month)

Evaporation Rainfall Maximum temperature Minimum temperature


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Figure 6.2: Average shoot biomass of field grown canola plants treated with two different

(no added and high K) and nitrogen (low and medium N) rates. Error bars show ± SE (n =

4). Different letters above the bars indicate significance at ≤0.05 (potassium main effect

P=0.24, nitrogen main effect P=0.001, interaction P=0.98).

The final grain yield after harvest ranged from 130 kg ha-1 for the no added K/low N

treatment and 455 kg ha-1 for the no added K treatment. However, there were no

significant differences obtained between any of the treatments (P>0.05) (Figure 6.3).

Figure 6.3: Average grain yield of field grown canola plants treated with two different (no added and high K) and nitrogen (low and medium N) rates. Error bars show ± SE (n = 4).

0

500

1000

1500

2000

2500

8 12 16

Bio

mas

s (k

g ha

-1)

Weeks after seeding (23/05/2013)

No added K/Low N High K/Low N No added K High K No added K/ Medium N

High K/ Medium N

B

β

AB

αβ β

a a a

0

50

100

150

200

250

300

350

400

No added K/Low N High K/Low N No added K High K

Gra

in Y

ield

(kg

ha-1

)

Potassium/Nitrogen Rate

No added K/ High K/ Medium N

a a

a

α

A AB

a

a


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6.3.3 Shoot Potassium and Nitrogen Concentration

Shoot potassium concentration was higher in the 100K compared to 0K treatments for

all three sampling times (Figure 6.4). The shoot potassium concentrations reflected the

potassium treatments with both the high K treatments showing significantly higher

potassium levels than the no added K treatments for both N rates throughout the

growing period (P<0.05). Overall the shoot potassium concentrations decreased over

the time of growing; only the shoot potassium concentrations of the high K/medium N

treatment showed a slight increase from week 8 to week 12 before decreasing again in

week 16 (Figure 6.4).

Interestingly, the no added K/low N and the high K/low N showed a higher

accumulation of potassium in the shoot throughout the growing period than their

respective high N treatments (no added K and high K), however significant differences

were only obtained 16 weeks after seeding (P=0.01 and P=0.001, respectively) (Figure

6.4).

Figure 6.4: Average shoot potassium concentration of field grown canola plants

treated with two different potassium (no added and high K) and nitrogen (low and

medium N) rates. Error bars show ± SE (n = 4). Different letters above the bars indicate

significance at ≤0.05 (Potassium main effect P<0.001, nitrogen main effect P=0.21,


0

1

2

3

4

5

6

8 12 16

Shoo

t K (m

g kg

-1)

Weeks after Seeding (23/05/2013)


High K/ Medium N

b

a

b

A

B

A

B

α

β

γ

α

a


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Differences in shoot nitrogen concentration occurred at 8 and 16 weeks after seeding

though the differences were greatest at 8 weeks after seeding. Eight weeks after

seeding the shoot N concentration followed the fertilisation rates with both the medium

N treatments showing significantly larger N accumulation in the shoot than the

respective low N treatments (P<0.05). No significant differences between the different

N rates could be observed 12 weeks after seeding (Figure 6.5). Sixteen weeks after

seeding the no added K/low N treatment again showed a significantly lower nitrogen

shoot content than the no added K/medium N treatment, whereas the high K/low N

treatment now had a significantly higher N shoot content than the high K/medium N

treatment (P<0.05).

Figure 6.5: Average shoot nitrogen concentration of field grown canola plants treated

with two different potassium (no added and high K) and nitrogen (low and medium N)

rates. Error bars show ± SE (n = 4). Different letters above the bars indicate

significance at ≤0.05 (potassium main effect P=0.27, nitrogen main effect P<0.001,


6.3.4 Canopy Temperature

The difference between leaf and ambient temperature across the two potassium and

nitrogen treatments increased slightly for both high K treatments from the bud

development to the flowering stage ranging from 0.6 to 2.9°C. The high K treatments

had lower leaf temperatures than the low K treatments, especially for medium N,

although the differences were not significant at P≤0.05 (Figure 6.6).

0

1

2

3

4

5

6

7

8

8 12 16

Shoo

t N (m

g kg

-1)

Weeks after Seeding (23/05/2013)


High K/ Medium N

d

a

c

β β α

A A

α

A

b

A


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Figure 6.6: Difference between leaf and ambient temperature of field grown canola plants

treated with two different potassium (no added and high K) and nitrogen (low and medium

N) rates during bud development and flowering stage. Error bars show ± SE (n = 4).

6.3.5 Transpiration Rate

The transpiration rate across all treatments increased significantly from the bud

development to the flowering stage (P≤0.05). For the no added K treatments the low N

was found to have a significantly higher transpiration rate than the medium N during

both development stages. During the flowering stage the high K/medium N treatment

showed a significantly larger transpiration than the high K/low N treatment (P=0.03). No

other significant differences could be established (Figure 6.7).

Figure 6.7: Average transpiration rate of field grown canola plants treated with two different

potassium (no added and high K) and nitrogen (low and medium N) rates during bud

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Bud development Flowering

Diff

eren

ce b

etw

een

Leaf

and

Am

bien

t Te

mpe

ratu

re (°

C)

Development Stage No added K/Low N High K/Low N No added K High K No added K/

Medium N High K/ Medium N

a

a

a

a a

a

a

0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045


Tran

spira

tion

(mm

ol m

-2 s

-1)

Development Stage


High K/ Medium N

ab

A A

B

A

a

b

ab

a


P a g e | 149

development and flowering stage. Error bars show ± SE (n = 4). Different letters above the

bars indicate significance at ≤0.05 (potassium main effect P=0.04, nitrogen main effect


6.3.6 Stomatal Conductance

The stomatal conductance also increased significantly from the bud development to the

flowering stage as observed for the transpiration rate. The no added K/medium N

treatment had a significantly lower stomatal conductance than the high K/medium N

treatment during both growth stages (P<0.02). Furthermore, during the flowering stage

the high K/medium N treatment showed a significantly higher stomatal conductance

than the high K/low N treatment (P=0.0007). There were no other significant

differences (Figure 6.8).

Figure 6.8: Average stomatal conductance of field grown canola plants treated with

two different potassium (no added and high K) and nitrogen (low and medium N) rates

during bud development and flowering stage. Error bars show ± SE (n = 4). Different

letters above the bars indicate significance at ≤0.05 (potassium main effect P<0.001,

nitrogen main effect P=0.01, interaction P=0.02).

6.3.7 Photosynthesis Rate

The rate of photosynthesis increased significantly for all treatments from the bud

development to the flowering stage (P≤0.05) except for the no added K treatment.

During the bud development stage the no added K/low N and the high K/medium N

0.0

0.2

0.4

0.6

0.8

1.0

1.2


Con

duct

ance

(mol

m-2

s-1

)

Development Stage

No added K/Low N High K/Low N No added K High K High K/ Medium N

No added K/ Medium N

a b

B B

A

C

ab ab


P a g e | 150

treatments had a significantly higher rate of photosynthesis than the no added

K/medium N and the high K/low N treatments (P=0.03). During the flowering stage both

high K treatments (low N and medium N) had significantly showed a significantly larger

photosynthesis rate than their no added K counterparts (P=0.03 and P=0.003,

respectively) (Figure 6.9).

Figure 6.9: Average photosynthesis of field grown canola plants treated with two

different potassium (no added and high K) and nitrogen (low and medium N) rates

during bud development and flowering stage. Error bars show ± SE (n = 4). Different

letters above the bars indicate significance at ≤0.05 (potassium main effect P<0.001,

nitrogen main effect P=0.70, interaction P<0.001).

6.3.8 Leaf Water Potential

The midday leaf water potential across all potassium and nitrogen treatments ranged

from -4.4 to -6.8 MPa during the bud development and from -3.5 to -5.5 during the

flowering stage. However, due to large standard errors there were no significant

differences observed between any of the treatments (P>0.05) (Figure 6.10).

0

5

10

15

20

25


Phot

osyn

thes

is (μ

mol

m-2

s-1

)

Development Stage


High K/ Medium N

a a

B C

b b A

C


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Figure 6.10: Average midday leaf water potential of field grown canola plants treated

with two different potassium (no added and high K) and nitrogen (low and medium N)

rates during bud development and flowering stage. Error bars show ± SE (n = 6).

6.4 Discussion

First the climatic conditions in 2012 are discussed, followed by the biomass and yield

results, concluding with the plant physiological parameters.

6.4.1 Environmental Growth Conditions and Biomass Production of Field Grown Canola

The climatic conditions at the field site during the experimental period generally

reflected the expected trend of the Mediterranean type climate for that region i.e. cool

wet winters and increasing temperatures and less rainfall towards the end of the

growing season in November often causing terminal drought conditions (Farré et al.,

2007; Brennan and Bollard, 2007). However, water stress occurred at the start of the

season in June, when the only 3.6 mm of rainfall was recorded for the month. After this

sufficient moisture was available during the main growing and measurement phase of

the experiment, i.e. the bud and flowering stages, from July to September. Towards the

end of the growing season, the rainfall decreased significantly and no rain occurred in

November close to harvest while the evaporation rate increased indicating longer

sunshine hours and higher temperatures during the day (Jeffrey et al., 2001).

-9

-8

-7

-6

-5

-4

-3

-2

-1

0 Bud development Flowering

Wat

er P

oten

tial (

MPa

)

Development Stage


High K/ Medium N

a

a a

a a

a a

a


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Based on the initial soil samples at the site (average of 28 mg kg-1), the no added K

treatments were considered to have experienced a mild potassium deficiency (Brennan

et al., 2013). This experiment showed that a mild potassium deficiency of field grown

canola with early seedling drought stress does not impact biomass production and

subsequent yield over the growing season in comparison to well potassium supplied

canola plants under the same conditions as there was no significant difference in dry

matter production as well as grain yield established between the potassium treatments

during the experiment. Those observations are in line with the findings made for

Chapter 5, where water stressed canola plants suffering under a long term mild

potassium deficiency were also found to be able to produce similar dry weights to

plants with higher levels of potassium fertilizer. Thus, the combination of water stress

and relative low levels of applied potassium in the soil has little effect on canola

compared with high rates of potassium and water stress, leading to the observed

compensation of dry weight and yield which is consistent with work by other

researchers (Khan et al., 2004; De Dorlodot et al., 2007).

However, the early onset of drought during the establishment period of the crop could

have resulted in a similarly decreased availability of potassium for the high K/ medium

N treatment as observed for the no added K/medium N treatment due to water being

the dominant factor restricting potassium uptake into the plant (Duff et al., 2006; De

Dorlodot et al., 2007). Consequently, a higher rate of potassium can only cause a

beneficial effect to canola if the accumulation in the plant tissue is not limited by other

factors such as water, which was found to have a more significant effect on the

morphology of the plant than a mild deficiency of potassium (Section 5.3) (Barber,

1995). In addition to that, a mechanism of potassium deficient canola plants to increase

their growth rate under water stressed conditions i.e. thicker root diameters to aid the

uptake of nutrients and water into the plant as already observed in Chapter 5, could

also have been a reason why no significant difference in plant dry weight was

measured between the high and no added K treatments (Nye and Tinker, 1977;

Barber, 1995).

Nitrogen, on the other hand, had a significant impact on the biomass of field grown

canola that has experienced early seedling drought stress, if potassium is not a limiting

factor. This significant growth advantage due to a higher supply of nitrogen is well

known for canola (Duff et al., 2006). However, the conducted experiment has also


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shown that this effect may be diminished under low levels of potassium as the no

added K/medium N treatment did not have a significantly larger dry weight than the no

added K/low N treatment; this may highlight an important interaction between those

two nutrients on plant growth as observed in other studies (Rose et al., 2007;

Bengough et al., 2010). Nevertheless, further experiments should be conducted to

verify the observations made for this study in order to establish a threshold level of

potassium in conjunction with different rates of nitrogen at which canola is not able to

compensate its growth.

6.4.2 Shoot Potassium and Nitrogen Concentration

The shoot potassium and nitrogen concentration of field grown canola exposed to early

seedling drought stress decreases over the growing period for well-supplied and

deficient plants, which is generally known for annual crops (Marschner, 1995). The

concentration of potassium in the shoot was, thereby, higher in both of the nitrogen

reduced canola plots throughout the growing season with significant differences

measured 16 weeks after seeding, which may have been caused by a biomass dilution

effect in the higher fertilised plants (Marschner, 1995; Duff et al., 2006). This was as

well reflected in a significant decrease in osmotic potential from the bud-development

stage to the flowering-stage for all potassium and nitrogen treatments. A similar

observation was made for the nitrogen concentration of the two low applied potassium

treatments, 8 weeks and 16 weeks after seeding, showing significantly larger N

concentrations than the two well with potassium fertilised treatments, which may,

besides the dilution effect, also indicate an uptake preference of canola for nitrogen

over potassium under the observed growing conditions i.e. soil type and development

stage of the plant (Brady and Weil, 2002; Havlin et al., 2005).

As already observed in Chapters 4 and 5, the potassium and nitrogen shoot

concentrations reflected the fertiliser rates used. The uptake of potassium and nitrogen

into canola, therefore, depends on the availability of the nutrient in the surrounding

medium to the plant (Steingrobe and Claassen, 2000). Rose et al. (2007) examined the

change of potassium over the growing period of several canola and wheat genotypes,

describing an initial increase and a later decrease of the potassium concentration in the

leaves during the vegetation period. Those observations could be confirmed for the

shoot material of the high K treatment, which also first showed an increase in the

potassium concentration from 8 to 12 weeks after seeding before starting to decrease


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again 16 weeks after seeding during the current experiment. Additional research under

controlled conditions should be conducted to investigate whether there may be an

uptake preference for nitrogen over potassium.

6.4.3 The Effect of Potassium and Nitrogen on Transpiration Rate, Stomatal Conductance and Photosynthesis of Field Grown Canola

The transpiration rate across all fertiliser treatments of field grown canola increased

significantly from the bud-development stage to the flowering-stage, which was as well

observed by an increase in the difference between leaf and ambient temperature and a

significant increase in stomatal conductance across the two growth stages. An

increasing demand of photosynthates during that time to aid the growth and

development of canola may be an explanation for the observed findings, which was

also supported by a significant increase in photosynthesis for all treatments except the

no added K/ medium N treatment (Smith and Baxter, 2002; Duff et al., 2006; McCaffery

et al., 2009). Furthermore, a lower shoot potassium content lead to a larger difference

between leaf and ambient temperature compared to a high shoot potassium

concentration, indicating a larger transpiration for the high K treatment.

Nitrogen may have an impact on the rate of transpiration and stomatal conductance of

field grown canola. This is highlighted in the significantly higher transpiration rate of the

no added K/low N treatment in comparison to the no added K/medium N treatment and

the significantly lower transpiration rate and stomatal conductance of the high K/low N

treatment compared to the high K/medium N treatment during the two measurement

days. Those findings may indicate that nitrogen could be an additional factor to

potassium and plant hormones impacting the regulation of the stomata cells in canola

(Radin et al., 1981; Marschner, 1995; Founier et al., 2005; Benlloch-Gonzalez et al.,

2008; Oosterhuis et al., 2013). There were no further significant differences obtained

between the two potassium rates, which may have been caused by a sufficient level of

available soil moisture as the measurements of the current experiments were made

after the early season drought stress period. This was as well indicated by moderate

leaf water potentials around -4 MPa (McRobbie, 1977; Hsiao and Lauchi, 1986;

Marschner, 1995; Reddya et al., 2004) with no significant differences obtained between

any of the treatments.


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The rate of photosynthesis of field grown canola was significantly influenced by low

levels of potassium as the no added K/medium N treatment showed a lower rate of

photosynthesis during the bud-development and flowering-stages compared to all other

fertiliser treatments. In addition to that, the no added K/low N treatment was as well

significant lower than the high K/low N treatment. Interestingly, a low rate of nitrogen

under potassium deficient conditions (lower than 40 mg/kg extractable soil K), led to a

higher rate of photosynthesis compared to the no added K/medium N treatment

confirming a possible interaction between potassium and nitrogen as described before

for the transpiration rate, stomatal conductance as well as biomass production (Rose et

al., 2007; Brennan et al. 2013). Those findings, however, need to be further

consolidated focusing on the interaction of nitrogen and potassium on transpiration

rate, stomatal conductance and photosynthesis.

6.5 Conclusion

The biomass production and subsequent yield of field grown canola that experienced

water limiting conditions during early seedling development under a mild potassium

deprivation and similar nitrogen levels is not significantly impacted in comparison to

well potassium fertilised plants. However, if potassium is not a limiting factor, the

biomass production of canola is significantly increased through nitrogen applications.

Nevertheless, the conducted experiment has as well indicated that there may be an

interaction effect between nitrogen and potassium on the dry matter production of

canola.

Furthermore, the amount of potassium and nitrogen in the shoot of canola decreases

over the growing season after an initial increase of potassium until 12 weeks after

seeding. The uptake of potassium and nitrogen into the canola plant, thereby, depends

on the amount of those nutrients present in the surrounding medium. Interestingly,

canola may also have an uptake preference for nitrogen over potassium leading to

significantly higher nitrogen accumulations in the shoot of both, potassium deficient

and well supplied canola plants, which requires further investigation.

There may be an interaction between nitrogen and potassium in canola influencing the

rate of transpiration and stomatal conductance leading to an increase of transpiration

under low levels of potassium and nitrogen compared to plants under the same

potassium level but higher nitrogen treatment.


P a g e | 156

Additionally, field-grown canola increases its rate of photosynthesis from the bud-

development to the flowering-stage, which was as well indicated by an increase in

transpiration rate, stomatal conductance and difference between ambient and leaf

temperature. However, more research is needed to investigate the possible interaction

between nitrogen and potassium on the stomatal conductance, rate of transpiration

and photosynthesis, which will, consequently, impact the biomass production in canola

as observed during the conducted experiment.

Chapter 7: General Discussion and Conclusion

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Chapter 7 General Discussion and Conclusion

This section will highlight the main findings from the experiments described in

preceding chapters and discuss the main conclusions of this PhD research project. The

overall hypothesis for this study was that an adequate amount of potassium will

enhance the drought tolerance of canola by reducing water loss through closed

stomata leading to an increased relative turgor pressure, increased rate of

photosynthesis and a decreased transpiration rate and consequently to greater plant

growth in comparison to plants that do not have an adequate potassium supply. Six

experiments were conducted to investigate the different morphological and

physiological aspects of this hypothesis.

7.1 Impact of potassium on Growth in Canola Suffering Under Water Stress

Plant growth of canola is impacted by different potassium levels. Potassium deficiency

(less than 42 mg kg-1 soil K) significantly reduces the plant dry weight of canola

compared to plants with adequate potassium supply. Moreover, potassium cannot

counteract the impact of long term water stress on the dry weight of canola as drought

inhibits a range of physiological functions causing a reduction in overall plant growth

and development (Gimenez et al., 1992; Medrano et al., 1997; Tezara et al., 1999;

Lawlor and Cornic, 2002; Mittler, 2006; Fanaeia et al., 2009). However, a mild

potassium deficiency (35-42 mg kg-1 soil K) only decreases the dry weight significantly

if plants are exposed to a long term potassium deprivation as described in Chapters 3

and 5 (Ebelhar and Varsa, 2000; Rose et al., 2007; Brennan et al. 2013; Oosterhuis et

al., 2014). Furthermore, canola maintained a similar dry weight under a range of

different soil potassium rates (greater than 56 mg kg-1 soil K), as monitored across all

conducted experiments. This was confirmed in the biomass production and subsequent

yield of the conducted field experiment and is also in agreement with the study by

Brennan et. al. (2013) who described that a critical rate of 45-55 mg kg-1 soil K would

lead to 90% relative yield in canola depending on soil type and growing conditions

(Chapter 6). Nevertheless, those observations need to be further investigated under

field conditions in order to account for differences in potassium fertiliser application and

potassium availability in the soil.


P a g e | 158

The similar observed plant size over a range of applied potassium levels could be due

to a morphological adaptation strategy of canola to manipulate root growth towards a

smaller shoot:root ratio. A relatively larger root system of canola plants supplied with a

low level of potassium (41-72 mg kg-1 soil K) would optimise the plant’s nutrient and

water uptake in the soil medium, which was reflected in the significantly smaller

shoot:root ratio of the potassium deficient canola plants in comparison to well fertilised

plants (Sections 3.3 and 5.3). This would also minimise the adverse effect of abiotic

environmental impacts, e.g. drought stress, and result in larger yields, which was as

well consolidated through a significantly larger taproot diameter of the low potassium

and water supplied canola samples (Section 5.3). However, further experiments should

determine whether the larger root weights are due to greater root lengths or other root

parameters to support the findings.

Those interpretations are in line with conclusions drawn from other researchers

reporting that a reduced amount of available potassium stimulates root growth in plants

in particular under drought stress conditions (De Dorlodot et al., 2007). Furthermore,

Damon et al. (2007) reported a similar observation from their study highlighting that

different genotypes may influence root distribution and potassium efficiency in canola.

Additionally, a number of other studies have described that root architecture and

distribution might vary with the genotype for grain crops (Siddique et al., 1990; Liao et

al., 2006; Manschadi et al., 2006; Palta et al., 2011). Nevertheless, wheat (Triticum

aestivum L) has been reported to develop a smaller root dry weight under low soil

potassium levels i.e. a larger shoot:root ratio and is, thus, in contrast to the

observations made in this study for canola (Ma et al., 2013). Therefore, further

research is required to investigate the two different adaption strategies to potassium

and water stress of these species.

There may be an important interaction effect between nitrogen and potassium on the

biomass production of canola as described in Chapter 6. If potassium is not a limiting

factor, a higher nitrogen application was observed to cause a significant increase in

biomass production, which is well known for nitrogen (Section 6.3) (Duff et al., 2006).

This difference, however, was not visible under a low potassium level but similar

nitrogen rate indicating an interaction of nitrogen and potassium on plant growth

supporting the findings of other studies (Rose et al., 2007; Bengough et al., 2010). This


P a g e | 159

potential interaction effect needs to be further investigated focusing on whether higher

nitrogen applications require higher potassium rates to achieve a significantly larger

biomass production in canola.

7.2 Potassium Concentration and the Impact on Transpiration, Stomatal Conductance, Photosynthesis and Relative Turgor Pressure in Canola under Drought

In water-stressed canola, grown under controlled conditions in a nutrient solution using

polyethylene glycol, a short term potassium deficiency led to higher potassium

shoot:root ratios compared to sufficiently with potassium supplied plants. Canola

exposed to drought stress and under a long-term potassium deprivation in a soil

medium, on the other hand, had an overall reduced shoot:root ratio of potassium

compared with well fertilised plants. An explanation for this might be that potassium

deficient canola plants increase their transpiration per leaf area significantly allowing a

rapid uptake of potassium into the shoot if the deficiency is only short and plants did

not experience any previous potassium starvation as observed in Chapter 4, which was

conducted in a nutrient solution, enabling a fast alteration of the potassium

concentration (Burnett et al., 2005). This effect, however, is not visible in soil grown

canola plants that have been exposed to long term potassium deprivation and water

stress. Nevertheless, those plants still showed a higher transpiration rate, which is in

agreement with a number of studies that have reported an increase of osmotic

absorption by the roots and of potassium flux through the xylem of potassium deficient

plants (Kochian and Lucas, 1982; De la Guardia et al. 1985; Siddiqi and Glass, 1987;

Benlloch-Gonzalez et al., 1989; Quintero et al., 1998; Schraut et al., 2005). Further

studies are, hence, required to investigate the detailed interactions of drought and

other stresses on various physiological plant properties in order to understand the

different potassium distribution strategies in canola (Rose et al., 2007; Prasad et al.,

2008).

After an initial increase 12 weeks after seeding, the shoot potassium concentration

decreases in canola over the duration of the growing period agreeing with findings

made by Rose et al. (2007). This decrease may have been caused by a dilution effect

in the higher fertilised plants i.e. an increased overall biomass as described in previous

chapters and is commonly known for annual crops (Section 6.3) (Marschner, 1995;


P a g e | 160

Duff et al., 2006). Nevertheless, additional research to better understand the seasonal

accumulation patterns of canola under field conditions is required.

The low K treatments had increased transpiration and stomatal conductance and

reduced relative turgor pressure compared with high K, under drought stress. This was

due to the stomata remaining open in the low K and closed in the high K treatment as

described in Chapters 3 and 4 (Sofo et al., 2008;

Oosterhuis et al., 2014). Moreover, it

was reflected in larger accumulation of 13C isotopes of the high K treatment in Chapter

4. The higher supply of potassium also had a higher rate of photosynthesis under water

stress as previously reported for rubber plants by Samarappuli et al. (1994).

Furthermore, potassium levels around the critical rate identified for canola in this study

and presented by Brennan et al. (2013) (41-56 mg kg-1 soil K) also led to significantly

higher rates of photosynthesis, which could explain why there was no difference in

plant size for this treatment level (Section 3.4). The higher stomatal conductance and

associated transpiration rate of the low potassium rates may be caused by an

interaction effect of the plant hormones ABA and ethylene as outlined in previous

chapters (Section 3.4) (Brag, 1972; Sudama et al., 1998; Bednarz et al., 1998; Founier

et al., 2005; Cabanero and Carvajal, 2007; Benlloch-Gonzalez et al., 2008).

Interestingly, an interaction effect between nitrogen and potassium on the stomatal

conductance, transpiration rate and photosynthesis was again noticeable as

highlighted for the biomass production and is discussed in Chapter 6 (Radin et al.,

1981; Marschner, 1995; Founier et al., 2005). However, further studies are needed to

assess the different response mechanisms of canola suffering under different

combinations or severities of potassium and drought stress on the stomatal adjustment

and consequently on the rates of transpiration and photosynthesis (Sadras and Milroy,

1996; Sinclair et al., 1998). At what stress level are abscisic acid and ethylene

impacting those mechanisms and what impact has a possible interaction between

nitrogen and potassium?

Overall, this study has shown that an adequate amount of potassium (greater than 56

mg kg-1 soil K) will enhance the drought tolerance of canola by reducing water loss

through closed stomata leading to an increased relative turgor pressure and a

decreased transpiration rate and consequently to greater plant growth in comparison to

plants that are potassium deficient (less than 42 mg kg-1 soil K) generally confirming

the main hypothesis of this project. Nevertheless, future research should in particular


P a g e | 161

concentrate on the range between 40-60 mg kg-1 soil K, which may in sandy soils

represent a critical potassium level from which different plant physiological

mechanisms are triggered leading to the observed open stomata and an increase in

transpiration and photosynthesis and, consequently, to the monitored morphological

changes such as a reduced shoot:root ratio to aid the uptake of water and nutrients

into the plant. This could be because of the highlighted interaction effect that may exist

between the plant hormones abscisic acid and ethylene on stomatal conductance and

transpiration. Moreover, the interaction between nitrogen and potassium on stomatal

regulation, transpiration and photosynthesis resulting in better growth in canola and

experiments focusing on the distribution of potassium in the plant should as well be

further investigated under controlled environmental conditions.

P a g e | 162

P a g e | 163

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