Journal of Experimental Botany, Vol. 62, No. 9, pp. 3135–3153, 2011doi:10.1093/jxb/err105 Advance Access publication 21 April, 2011

REVIEW PAPER

Enhancing drought tolerance in C4 crops

Marta S. Lopes1, Jose Luis Araus1,2, Philippus D. R. van Heerden3 and Christine H. Foyer4,*

1 International Maize and Wheat Improvement (CIMMYT), Km. 45, Carretera Mexico-Veracruz, El Batan, Texcoco, CP 56130 Mexico2 Unitat de Fisiologia Vegetal, Universitat de Barcelona, 08028, Barcelona, Spain3 South African Sugarcane Research Institute, 170 Flanders Drive, Mount Edgecombe 4300, South Africa4 Centre for Plant Sciences, Faculty of Biology, University of Leeds, Leeds LS2 9JT, UK

* To whom correspondence should be addressed. E-mail: c.foyer@leeds.ac.uk

Received 20 December 2010; Revised 2 March 2011; Accepted 15 March 2011

Abstract

Adaptation to abiotic stresses is a quantitative trait controlled by many different genes. Enhancing the tolerance of

crop plants to abiotic stresses such as drought has therefore proved to be somewhat elusive in terms of plant

breeding. While many C4 species have significant agronomic importance, most of the research effort on improving

drought tolerance has focused on maize. Ideally, drought tolerance has to be achieved without penalties in yieldpotential. Possibilities for success in this regard are highlighted by studies on maize hybrids performed over the last

70 years that have demonstrated that yield potential and enhanced stress tolerance are associated traits. However,

while our understanding of the molecular mechanisms that enable plants to tolerate drought has increased

considerably in recent years, there have been relatively few applications of DNA marker technologies in practical C4

breeding programmes for improved stress tolerance. Moreover, until recently, targeted approaches to drought

tolerance have concentrated largely on shoot parameters, particularly those associated with photosynthesis and

stay green phenotypes, rather than on root traits such as soil moisture capture for transpiration, root architecture,

and improvement of effective use of water. These root traits are now increasingly considered as important targetsfor yield improvement in C4 plants under drought stress. Similarly, the molecular mechanisms underpinning

heterosis have considerable potential for exploitation in enhancing drought stress tolerance. While current evidence

points to the crucial importance of root traits in drought tolerance in C4 plants, shoot traits may also be important in

maintaining high yields during drought.

Key words: Effective use of water, heterosis, maize, photosynthesis, root mass at depth, stay green, sugarcane.

Introduction

Water is already a scarce commodity in many parts of the

world, and predicted climate changes will aggravate the

situation in future. In addition to decreases in the amount and

frequency of precipitation, global increases in temperature are

predicted to enhance water losses further through evapo-

transpiration (Intergovernmental Panel on Climate Change,2007). The increased irrigation required to meet the needs of

crop growth is expected to lead to groundwater depletion

(Konikow and Kendy, 2005; Oki and Shinjiro, 2006).

The availability of water (in addition to atmospheric

CO2) is considered to have been a major driving force for

the evolution and the ecological success of C4 plants

(Edwards et al., 2010; Westhoff and Gowik, 2010). C4

plants are often considered to have mastered the art of

drought control particularly as they are able to maintain

leaf photosynthesis with closed stomata. Sorghum, for

example, is considered to be better adapted to water-

limiting environments than most other crops (Ludlow and

Muchow, 1990; Sanchez et al., 2002). Even though photo-synthesis can be decreased under drought conditions

(Carmo-Silva et al., 2008), C4 grasses such as Panicum are

often classified as drought tolerant as they are able to grow

through the dry seasons. Within the context of an in-

creasingly water-poor world, the exploitation of C4 photo-

synthesis has great potential for the enhancement of crop

production and food security. Moreover, although C4

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plants comprise <4% of global terrestrial plant species, they

are important from an ecological, agricultural, and atmo-

spheric perspective, and contribute ;20% to global primary

productivity (Ehleringer et al., 1997). Moreover, the C4

plant maize (Zea mays L.) is the most important cereal crop

in the world in terms of annual metric tonnes of production

(FAOSTAT, 2009).

C4 plants have high water use efficiencies (WUEs), and thepresence of the CO2-concentrating mechanisms makes C4

photosynthesis more competitive in conditions that promote

carbon loss through photorespiration, such as high temper-

atures, high light intensities, and decreased water availability

(Edwards et al., 2004). C4 photosynthesis is characterized by

the presence of a metabolic CO2 pump that concentrates CO2

in the vicinity of the main enzyme of carbon dioxide fixation,

ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco;Edwards et al., 2001, 2004). This confers a number of

important advantages in terms of WUE because it allows high

rates of photosynthesis to occur even when stomata are closed

while limiting flux through the photorespiratory pathway

(Edwards et al., 2001). Although only a limited number of

studies have been undertaken to date in C4 species, available

data suggest that the enzymes that comprise the metabolic

CO2 pump are more resistant to water deficits than theenzymes of C3 photosynthesis (Ghannoum, 2009).

The identification of trends in the responses of C4 plants to

drought is complicated by the occurrence of different

C4 subtypes [including NADP-malic enzyme (NADP-ME),

NAD-ME, and phosphoenolpyruvate carboxykinase

(PEPCK)] (Carmo-Silva et al., 2007) which can exhibit

different strategies for coping with water deficits. Literature

evidence suggests that there is considerable diversity in theresponses to drought of different C4 subtypes (Ripley et al.,

2010). For example, Paspalum dilatatum (NADP-ME) and

Zoysia japonica (PEPCK) respond to water deficit by early

stomatal closure in order to conserve leaf water status,

whereas this response was not apparent in Cynodon dactylon

(NAD-ME), which lost water rapidly when deprived of water

(Carmo-Silva et al., 2007). Of these three species, C. dactylon

was classed as the most resistant to the rapid imposition ofwater deficits while P. dilatatum was the least resistant

(Carmo-Silva et al., 2007). Panicoid grasses of the C4 NADP-

ME type were found to be metabolically more sensitive to

drought than C3 species and they recover more slowly (Ripley

et al., 2010). The following discussion will focus on the main

traits that are considered to be useful in the selection of C4

varieties with improved drought tolerance characteristics. The

genetic variability in the responses of C4 plants to drought isreadily demonstrated, as illustrated in Fig. 1. Moreover, the

models used for the integration of complex gene–phenotype

relationships in breeding C4 plants such as maize are

becoming increasingly effective (Messina et al., 2011).

Adaptation to drought

The ability to escape from drought by accelerating the life

cycle or, alternatively, the evolution of avoidance and

tolerance mechanisms have long been considered to be

important strategies in drought adaptation (Levitt, 1972;

Chaves et al., 2003). Escape strategies often rely on success-

ful reproduction before the onset of severe stress (Camposet al., 2004) using a short life cycle with high growth rates

together with the efficient storage and use of reserves for

seed production. Dehydration avoidance mechanisms in-

volve the maintenance of a high (favourable) plant water

status during stress. Such strategies include minimized water

loss (e.g. stomatal closure, reduced leaf area, and senescence

of older leaves) or maximal water loss afforded by increased

root proliferation at depths where the water is available.However, there is inherent conflict between biomass accu-

mulation and stress avoidance via a reduction in transpira-

tion rates because the acquisition of photoassimilates is

dependent on stomatal aperture and leaf area (Araus et al.,

2008; Blum, 2009, 2011). Tolerance to low water potentials

requires the maintenance of plant functions under condi-

tions of limited water availability and/or the rapid recovery

of plant water status and plant function after stress. Thisrequires precise osmotic adjustments and may also involve

the rigidity of cell walls and/or small cells (reviewed in

Chaves et al., 2003).

Fig. 1. Inbred lines of tropical maize exhibiting contrasting

performance in relation to water deficit under field conditions. The

maize lines were grown in the field over winter (i.e. the dry season)

at the CIMMYT station in Tlaltizapan (Mexico).

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Responses to drought are species specific and often

genotype specific (Hall et al., 1982; Campos et al., 2004).

Moreover, the nature of drought responses of plants is

influenced by the duration and severity of water loss (Yang

et al., 1993; Pinheiro and Chaves, 2011), the age and stage

of development at the point of drought exposure (Chimenti

et al., 2006), as well as the organ and cell type experiencing

water deficits (Pastori and Foyer, 2002). Plants subjected tosoil water deficits typically exhibit characteristic losses in

leaf water content leading to decreased growth and leaf

elongation. This is accompanied by decreases in leaf

osmotic potential (Westgate and Boyer, 1985), stomatal

conductance, photosynthesis, and transpiration rates

(Ripley et al., 2007, 2010). The abundance of reactive

oxygen species (ROS) tends to increase in the tissues of

plants exposed to drought, as do the activities of antiox-idant enzymes (Jiang and Zhang, 2002). Hormone signalling

pathways are highly responsive to drought, particularly

those associated with increased synthesis and cycling of the

stress hormone abscisic acid (ABA; Bray, 2002; Pinheiro

and Chaves, 2011).

The responses of plants to drought observed under field

conditions are generally much more complex than those

measured under controlled environment conditions becauseof other factors accompanying water deficits which in-

fluence the nature of the stress response. Water deficits lead

not only to low tissue water status and cell dehydration but

also to nutrient deprivation and osmotic stress, and often

additionally heat stress linked to decreased transpiration.

This complicates the definition of target stress for plant

breeders as well as the environmental conditions required

for evaluation and selection of improved varieties. More-over, periods of soil water deficits can occur at any time

during the growing season, and not all stages of plant

development are equally sensitive in terms of impact on

ultimate crop yield. For example, crop species such as maize

are particularly sensitive to drought at the heading stage,

which is just before tassel flowering. In sugarcane, drought

stress occurring when the leaf canopy is already well

established has more serious impacts on final yield in termsof total biomass, stalk biomass, and stalk sucrose than

when drought occurs earlier during the season (Robertson

et al., 1999a). For simplicity, much of the following

discussion will focus on the traits and mechanisms that

have the potential to confer increased performance in maize

under mild or moderate water deficits (Araus et al., 2011).

Moreover, because maize is a cross-pollinating species that

is prone to failure of grain set under stressful conditions,traits that serve to prevent seed abortion will be considerd.

Strategies for enhancing yield potential andstress adaptation

The breeding value of any trait is intrinsically dependent on

the prevailing growing conditions (such as soil type and

climate) in the target regions in which genotypes will be

grown (Tardieu and Tuberosa, 2010; Araus et al., 2011).

The classical approach to breeding varieties with improved

drought tolerance traits has involved multilocation progeny

testing, in which variations in water availability across the

different sites are sufficient to facilitate random selection on

the basis of high and stable yield. While breeding for high

yield in drought-prone environments using multilocation

testing is inherently complicated by the year to year

variability in the amount and temporal distribution of

available soil water and the low heritability of drought

resistance traits under these conditions, selection for drought

tolerance in routine breeding programmes with adequate

weighting has yielded significant increases in maize yields

(Banziger et al., 2004). Higher shoot biomass can be achieved

through different, and in many cases conflicting, strategies

(Collins et al., 2008). If this is accompanied by higher root

biomass and improved root architecture, it should facilitate

better water extraction capabilities under field conditions.

However, while improved yield potential can translate into

better performance under stress, it also places a greater

demand on water and other resources. Thus in conditions of

limited soil water availability, a higher capacity for water

uptake by the plant can sometimes result in an increased

frequency of stress experiences. Thus, it is preferable that

a higher yield potential is accompanied by enhanced stress

tolerance.For moderate stresses that cause up to a 70% reduction in

potential yield, there are complementary strategies which

can be employed to optimize total yield, although this

sometimes occurs at the expense of yield stability. In the

absence of stress, the main yield optimization strategies are

as follows.

(i) Increasing yield potential through shoot mechanisms

such as increased transpiration rates or ‘stay green’

characteristic (i.e. the ability to retain chlorophyll pigments

in photosynthetic tissues and maintain a green leaf area at

the end of the crop cycle). Increases in yield may translate

to a higher biomass accumulation and grain yield undermild to moderate water deficit.

(ii) Maintaining growth and thus biomass accumulation

under decreasing water status. This strategy is not without

risk as crop failure may occur as a consequence of soil

dehydration, as discussed in detail below.

For more severe water deficit scenarios, breeding strate-gies tend to focus on reducing the risk of total yield loss.

This can be achieved by decreasing cumulative transpira-

tion, which often incurs a penalty in terms of yield potential

and performance under mild or moderate water deficit

conditions. Such strategies therefore often increase yield

stability at the expense of yield potential. These strategies

include, for example, the following.

(iii) Shortening the duration of the crop cycle (i.e. pheno-

logical adjustment) to escape from the drought period. This

strategy has proved to be most successful for C3 cereals

such as wheat or barley in environments, such as those of

the Mediterranean region, which experience drought at the

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end of the growing season, here referred to as ‘terminal

drought’ (Araus et al., 2002).

(iv) Reducing leaf area or stomatal conductance, traits

which frequently increase the WUE (i.e. the amount of

harvested biomass per unit transpired water).

Any trait can thus have different (positive, negative, or

neutral) impacts on yield, depending on the drought

scenario. Water conservation traits have beneficial effects in

the most severe drought conditions or in terminal drought

scenarios, in which it is useful to save water for the end of

the crop cycle and to decrease the numbers of sinks for

assimilates in order to obtain larger seeds. Traits that serve

to conserve water (conservative traits) include low stomatalconductance, low leaf growth rate, high WUE, or deep but

sparse root systems. Conversely, growth ‘maintenance’

traits have beneficial effects under milder drought condi-

tions, in which the soil profile is periodically re-watered.

Biomass production is tightly linked to transpiration,

WUE, and nitrogen accumulation. Blum (2011) has argued

that breeding for high WUE under drought conditions will

ultimately result in low-yielding genotypes with reduceddrought tolerance because WUE is increased by reduced

transpiration and water use. Therefore, biomass production

under most drought conditions (i.e. except for marginal

drought-prone areas) can only be enhanced by maximizing

soil moisture capture for transpiration, which also involves

minimized water loss by soil evaporation. Collectively, this

property has been called effective use of water (EUW;

Blum, 2011).Biomass production is tightly linked to transpiration, and

hence breeding for maximal soil moisture capture for

transpiration is perhaps the most important target for yield

improvement under drought stress conditions. In addition,

breeding for EUW in order to improve plant water status

helps to sustain assimilate partitioning and reproductive

success, resulting in increased harvest indices. This is

particularly important in maize, because the reproductivestage is the most susceptible to drought.

Use of stable isotopes to screen for wateruse efficiency

WUE, defined as the ratio of biomass accumulated to watertranspired, has been traditionally considered to be the key

determinant of drought tolerance (Tambussi et al., 2007). In

C3 cereals, carbon isotope discrimination (D13C) has been

used to screen for genotypes with higher WUE (Araus et al.,

2002). In contrast, few reports are available concerning the

use of D13C in C4 crops such as maize (Monneveux et al.,

2008; Cabrera-Bosquet et al., 2009c). Moreover, the results

obtained to date are not encouraging, mainly because of thenature of C4 photosynthesis.

Much attention has focused on improving WUE when

breeding for drought adaptation. However, it would now

appear that EUW is a more important adaptive trait than

WUE except in severe drought conditions (Araus et al.,

2008; Blum, 2009). EUW is related to the genotypic

capacity to use available water and, therefore, to sustain

transpiration under unfavourable environmental conditions.

Oxygen isotope enrichment (D18O) can also be useful in

selecting for drought adaptation in both C3 and C4 grasses.

Such measurements are independent of the photosynthetic

process and have therefore been proposed as a ‘time-

integrative’ indicator of transpiration and EUW in differentplant species (Barbour, 2007; Farquhar et al., 2007;

Cabrera-Bosquet et al., 2011a) including maize (Cabrera-

Bosquet et al., 2009b; Araus et al., 2010). Moreover,

associated genotypic differences in D18O and grain yield

have been reported in cereals such as wheat (Cabrera-

Bosquet et al., 2009a) and maize (Cabrera-Bosquet et al.,

2009b). Negative relationships between D18O and grain yield

have been observed under both well-watered and moderatewater deficit conditions (Cabrera-Bosquet et al., 2009b).

These findings would suggest that genotypes with higher

transpiration traits were the most productive. Heterosis in

maize, as discussed in more detail later, appears to confer

higher transpiration. Maize hybrids have lower D18O values

and higher ash contents compared with the inbred lines

regardless the growing conditions (Araus et al., 2010).

Given the cost, technical skills, and facilities involved inoxygen isotope analysis, its large-scale application to

breeding programnes may not be feasible for small/medium

breeding programmes and therefore other alternatives for

assessing plant transpiration are often required. Thus, the

accumulation of mineral or ash content in vegetative tissues

may be an inexpensive and easy way to predict yield and

genotypic adaptation to drought in different cereals (Araus

et al., 1998). Mineral accumulation in vegetative tissues canbe explained through the passive transport of minerals via

the xylem driven by transpiration (Araus et al., 2002). Leaf

ash has been correlated with yield in barley (Voltas et al.,

1998) and wheat (Araus et al., 1998) plants grown under

different water regimes. While this approach can be used

successfully in both C3 and C4 crops, to our knowledge

there is only one report of the application of leaf ash

measurements in maize and that is a recent study from theCIMMYT where a close relationship between leaf ash

content and isotope enrichment was observed (Cabrera-

Bosquet et al., 2009c). Based on these results, however, it is

possible to conclude that leaf ash content is a viable

alternative criterion to D18O for assessing yield performance

in maize grown under drought conditions. Moreover, the

analysis of ash (or total mineral) content is not costly and it

does not require extensive facilities or highly qualifiedtechnical staff. Hence, this trait is ideal for breeding

programmes in developing countries.

Studies on maize conducted at the CIMMYT (Cabrera-

Bosquet et al., 2009c; Araus et al., 2010) and other

unpublished results using CIMMYT germplasm support

the potential usefulness of both D18O measurements (partic-

ularly in kernels) and ash content in leaves as secondary

traits for direct selection of maize genotypes with improvedperformance under drought through higher EUW. While

further studies are needed to match both traits to usefulness

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criteria, specifically with respect to the extent of genotypic

variance, heritability, and genetic correlation of both D18O

and ash content with grain yield, the findings to date clearly

indicate the potential value of these techniques. In addition

these two traits may be estimated in a fast and low-cost

manner by near-infrared reflectance spectroscopy (Cabrera-

Bosquet et al., 2011b). Moreover, it will be equally

important to characterize what root traits, as discussed indetail below, might be responsible for the observed geno-

typic variation in D18O and mineral contents.

An additional tool for the assessment of water uptake

capacity by the root system is the isotope composition

of oxygen (d18O) and hydrogen (d2H) in stem water

(Ehleringer and Dawson, 1992). This technique, which has

largely been developed in trees to date, is based on the

existence of evaporative gradients in the isotope composi-tion of soil water that tends to be more enriched in the

heavier isotope at the soil surface than in deeper layers

because of evaporation. A comparison of the isotopic

composition of stem water (i.e. the water taken up by the

plant) with that of soil water at different depths has shown

that it is possible to determine which parts of the root

system contribute most to water uptake (see, for example,

Ferrio et al., 2005; Patz, 2009; Holst et al., 2010). Inaddition, genetic differences in water uptake parameters are

significant and may be partly responsible for the observed

genetic variability in d18O data (Retzlaff et al., 2001; Ferrio

et al., 2007; Voltas et al., 2008). Traditionally, stable

isotopes have been determined by isotope ratio mass

spectrometry (IRMS), which is an expensive technique

requiring costly equipment and a complex laboratory

infrastructure. Moreover, standard IRMS devices cannotbe used to analyse d18O and d2H simultaneously. This

means that the time and cost of the analyses are effectively

doubled. However, commercial alternatives based on high

precision infrared laser spectroscopy have recently emerged,

providing cheap, fast, and accurate determinations of both

d18O and d2H in a single measurement (Aggarwal et al.,

2006; Lis et al., 2008).

Root system architecture and root mass atdepth

Plants use different strategies, ranging from avoidance todesiccation tolerance, to cope with dry soils. Desiccation

avoidance is associated with the minimization of water loss

and the maximization of water uptake (Ludlow and

Muchow, 1990). Evidence suggests that the adverse effects

of drought can be successfully avoided by changing carbon

allocation patterns to allow the formation of a deep root

system before the onset of a growth-limiting water shortage

(Jordan et al., 1983; Jones and Zur, 1984; Blum, 1985;O’Toole and Bland, 1987; Sponchiado et al., 1989; Sinclair

and Muchow, 2001; Campos et al., 2004; Manschadi et al.,

2006 2008; Reynolds et al., 2007; Lopes and Reynolds,

2010, 2011). Deep root systems have a proven advantage

when water is available deep in the soil profile but not in

shallow soils (Sponchiado et al., 1989). Positive relation-

ships between root ramification and yield under drought

have not always been found and, moreover, a significant

association between a lower root mass and increased ear

growth was observed under drought in a tropical maize

population (Bolanos and Edmeades; 1993). However, the

latter results are possibly a rather exceptional response to

specific environmental conditions (Bolanos and Edmeades;1993).

Root system architecture, which is defined as the spatial

configuration of a root system in the soil, is used to describe

the shape and structure of root systems (Dorlodot et al.,

2007). This parameter is important because major soil

resources are heterogeneously distributed in the soil. The

spatial deployment of roots determines the ability of a plant

to secure edaphic resources (Dorlodot et al., 2007). There isoverlap between quantitative trait loci (QTLs) for root

features and plant productivity in terms of yield, water use,

or nutrient capture (Tuberosa et al., 2002; Steele et al.,

2007). In maize, for example, QTLs for the elongation rate

of axile roots, the number of axile roots, and yield were co-

localized (Trachsel et al., 2009). Such evidence indicates that

root architecture is an important determinant of yield.

Root traits are notoriously difficult to measure under fieldconditions. Of the root traits that can be easily measured in

such circumstances, few are positively related to yield. The

‘root mass at depth’ parameter but not total root mass has

been shown to correlate with yield under drought condi-

tions, with no yield penalty under full irrigation (Lopes and

Reynolds, 2011). Genetic variation in root mass at depth has

been shown in C3 plants such as wheat (Lopes and

Reynolds, 2010) and C4 plants such as maize (Hund et al.,2009) and sorghum (Ludlow et al., 1990; Santamaria et al.,

1990). Moreover, remote sensing techniques might be useful

indirect indicators of the root mass at depth parameter. For

example, canopy temperature has been suggested as a possi-

ble marker, cooler canopy temperatures indicating geno-

types that have the ability to maintain transpiration because

the roots can access water deep in the soil profile. Negative

correlations between canopy temperatures at grain fillingand root dry weight at depth have been observed (Lopes

and Reynolds, 2010). Similar associations under drought

have been found in four bean cultivars, where genotypes

with deeper roots showed better seed yield, crop growth,

lower canopy temperature, and lower soil moisture extrac-

tion, in all but poor soils where root variations were no

longer observed (Sponchiado et al., 1989).

Taken together, such findings suggest that the spatialdistribution of the roots in the soil, and not total root mass

per se, defines the ability of a root system to take up water.

An ‘ideal’ root system, with an even distribution of ;1 cm

root m�3 of soil (root length density or RLD) is considered

to be the most suitable under field conditions (Tardieu

et al., 1992). Higher RLDs are adequate when evaporative

demand increases and soil water reserve decreases. However

high RLD values might ultimately prove to be wasteful ofphotoassimilates and other resources without giving appre-

ciable increases in water uptake. The conductivity

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properties and water retention capacity of the soil have to

be taken into account when defining the required hydraulic

properties of the roots, and target types for root architec-

ture and efficiency (Javaux et al., 2008). Root angles and

branching are considered to play a basic role in improving

water acquisition by plants. Novel approaches have been

applied to improve our current understanding of the

complexities of maize root architecture, together with theuse of fractals and associate derivatives (Bohn et al., 2006;

Zhong et al., 2009). However, it is important to note that

many of the root architecture traits described above have

only been assessed in controlled environment studies.

Unfortunately, conditions in controlled environment cham-

bers or cabinets are not always representative of the stresses

experienced by plants in the field. For instance, root mass at

depth, which is expected to improve yields under post-anthesis drought (Lopes and Reynolds 2010), cannot be

determined in gel chambers or growth columns without root

growth restrictions. Thus, fine mapping of genes and

markers related to root traits has to be assessed in the field,

and only then can the data obtained be effectively in-

corporated in breeding programmes.

Exploitation of heterosis to enhance droughttolerance in maize

Heterosis in maize is associated with higher yield potential

and it confers adaptation to a wide range of growingconditions that might be successfully exploited to enhance

drought tolerance. Traits associated with heterosis have

potential to reveal new mechanisms that contribute to

drought tolerance. A recent study undertaken at the

CIMMYT revealed that maize hybrids have better water

use than inbred lines, regardless of the water regime during

cultivation (Araus et al., 2010). Thus, the effects of heterosis

on growth and yield parameters in maize may, at least inpart, be explained in terms of variations in water use and

status, factors that are also present under well-watered

conditions. Therefore, the study of root characteristics

associated with heterosis is promising in terms of determin-

ing traits conferring improved EUW. Heterosis in root

architecture can already be seen at the very early stages of

root development, a few days after germination. Thus

lateral root density shows the highest degree of heterosis(Hoecker et al., 2006), and maize hybrid seedlings develop

a greater number of fine roots than their parents (Li et al.,

2008). Similarly, heterosis in the total length of lateral roots,

together with a higher ratio of lateral root length to that of

the axial root, was already found in 20-day-old maize

seedlings grown under different N conditions (Chun et al.,

2005). Together with the post-embryonic shoot-borne root

system, lateral roots, which become dominant later in rootdevelopment, are responsible for most of the water and

nutrient uptake by the root system (Hochholdinger et al.,

2004). Moreover, heterosis exists for water uptake ability at

the root cell level under well-watered conditions (Liu et al.,

2009). However, to date, the above studies on roots have

been performed only on seedlings under controlled growth

conditions. The difficulties associated with performing

systematic studies of root traits in heterosis on adult plants

in the field should not be underestimated.

Shoot architecture and stay greenphenotypes

Using a set of historical yield trends in maize, Hammer et al.

(2009) demonstrated that changes in root architecture and

water capture are associated with biomass accumulation.

While changes in canopy architecture are likely to have littledirect effect on water capture, they may have important

indirect effects via leaf area retention and partitioning of

carbohydrate to the ear (Hammer et al., 2009). Stay green

phenotypes in C4 crops such as sorghum are often

a consequence of other earlier traits, such as a slow leaf

growth rate. Such genotypes tend to save water for the end

of the crop cycle and have a deep root system (Harris et al.,

2007). Strong evidence in support of the value of stay greencharacteristics in the adaptation to abiotic stresses comes

from retrospective comparisons of the performance of

temperate maize hybrids produced over the last 70 years in

the USA (Tollenaar and Lee, 2006). A high level of

association between yield stability and general stress toler-

ance is observed in new temperate maize hybrids, where

yield stability does not appear to have declined with

increasing yield potential (Tollenaar and Lee, 2002; Duvicket al., 2004). Higher dry matter accumulation during grain

filling in newer compared with the older hybrids can be

attributed, at least in part, to a longer duration of the grain-

filling period in the newer hybrids which are adapted to

higher planting densities (Tollenaar and Lee, 2006). There-

fore, the higher yield potential of the newest hybrids is

related to a higher expression of stay green characteristics,

together with the ability to grow well under higher plantingdensities. These findings suggest that as well as having

a positive role in increasing the amount of light used

effectively in photosynthesis for the production of assim-

ilates to drive growth and biomass production, stay green is

a consequence of the requirement of the newest hybrids to

cope with growth conditions where nutrient and water

resources are limiting (Blum, 2011). Water deficits will tend

to be higher when plants are subjected to high densitypropagation, and thus tolerance of high plant densities must

inherently involve a high resistance to drought stress

(Tollenaar and Wu, 1999).

The benefits of stay green phenotypes have been de-

scribed in sorghum (Borrel et al., 2000), where the rate of

leaf senescence was shown to be negatively correlated with

yield under conditions of late (pre- and post-flowering)

water deficits (Borrel et al., 2000). It has been claimed thatstay green is a constitutive trait in sorghum, with important

contributions to yield that are expressed under well-watered

conditions. However, the expression effects of stay green

relative to the faster senescence of genotypes with wild-type

patterns of senescence become more prominent under severe

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drought stress (Blum, 2011). Moreover, the utilization of

stay green expression as a secondary trait can incur

problems related to interactions with other variables. For

example, stay green can result from late flowering or low

reproductive sink strength, and stay green phenotypes are

highly prone to variation in response to environmental

factors (Rosenow and Clark, 1995). Taken together, these

findings indicate that while stay green shows potential forroutine use in breeding programmes seeking to enhance

abiotic stress tolerance, any interactions of stay green

expression with other complicating factors that might

adversely affect yield should first be eliminated.

Growth regulation and stress tolerance

A decrease in leaf growth occurs rapidly in plants exposed

to water deficits, occurring even before stomatal closure

(Saab and Sharp, 1989). There is considerable genetic

variability in the sensitivity of leaf expansion to water

deficits in temperate and tropical maize varieties (Welckeret al., 2007). The high sensitivity of leaf growth to water

deficits is considered to be a ‘stress avoidance’ mechanism,

which enables plants to tolerate severe drought scenarios by

saving soil water. Leaf rolling or epinasty is a short-term,

reversible mechanism that fulfils essentially similar func-

tions by decreasing the active leaf area. However, high

sensitivity of growth and leaf movements to water availabil-

ity has several potential drawbacks. First, the geneticdeterminism of leaf growth is partially linked to reproduc-

tive growth (Welcker et al., 2007). Secondly, loss of leaf

area may limit photosynthesis and thus drought-induced

decreases in leaf area may lead to a reduced rate of biomass

accumulation. However, the differential regulation of pho-

tosynthesis in the two sides (adaxial and abaxial) of the

leaves of maize and other C4 monocotyledonous species

may avoid limitations on photosynthesis caused by leafcurling. C4 monocotyledonous leaves are essentially divided

into two compartments, the upper adaxial side and the

lower abaxial sides. The upper and lower sides of the leaves

operate independently in terms of the regulation of

photosynthesis and stomatal conductance, features that are

very useful for the optimization of whole-leaf photosynthe-

sis in relation to prevailing environmental conditions

(Soares-Cordeiro et al., 2009, 2011). The lower abaxialsurface, which becomes exposed to light when the C4 leaves

curl, has much higher rates of photosynthesis when directly

exposed to incident light than it does in its usual orienta-

tion. High photosynthesis rates that are similar to whole-

leaf values under adaxial illumination under well-watered

conditions can thus be maintained solely by the abaxial side

of the leaves when the leaves curl. Moreover, leaf water loss

is decreased because the stomata on the adaxial surface arecompletely closed under these conditions and those on the

abaxial surface are also partially closed (Soares-Cordeiro

et al., 2009).

Many mechanisms, such as the cell cycle, hormonal

regulation, hydraulics, and cell wall mechanical properties,

contribute to the control of growth. All of these are

adversely affected when plants are subjected to stress, and

there is little compelling evidence for one individual process

being more affected than others. Candidate water stress-

responsive mechanisms that control growth include plant

hydraulic properties and their control by aquaporins.

A functional analysis of the aquaporins, which are proteins

which form channels to facilitate water transport throughmembranes, thereby increasing the hydraulic conductivity

of tissues, has been conducted in several species including

maize (Hachez et al., 2008). The amount of aquaporin

proteins and the regulation of their gating properties

contribute to the control of hydraulic processes when ABA

levels increase as a result of water deficit or anoxia

(Tournaire-Roux et al., 2003; Boursiac et al., 2008).

Similarly, osmotic adjustments maintain turgor underdrought or osmotic stress, thereby maintaining growth

(Zhang et al., 1999). However, osmotic adjustment mecha-

nisms may not be particularly relevant to growth regulation

in species such as maize under conditions of water

limitation (Tardieu, 2006).

Drought-triggered seed abortion and earlyseed growth

Seed number, which is a major component of yield

potential, is mainly determined at flowering and slightly

afterwards. While the number of ovules greatly exceeds thenumber of seeds that are finally produced in most species,

exposure to water deficits decreases the seed/ovule ratio

even more because of flower abortion (Barnabas et al.,

2008). Flower abortion is considered to be an adaptive

mechanism, which allows the remaining seeds to be filled

appropriately in relation to the availability of fixed carbon

provided by photosynthesis. Thus, flower abortion can have

either a positive or a negative effect on yield, depending onthe drought scenario. However, from a breeding perspec-

tive, flower abortion is considered as a negative trait for

both tropical and temperate maize.

Sucrose feeding experiments and metabolite measure-

ments have shown that carbon metabolism and transport

make a significant contribution to early seed growth and

that the absence of an appropriate carbon supply leads to

flower abortion (Zinselmeier et al., 1995; McLaughlin andBoyer, 2004). However, the relationships between sugar

content and flower viability are complex and so flower

abortion does not result solely from limitations in sugar

supply, because sugar levels are usually maintained in

ovules even in plants experiencing water deficits. Therefore,

flower abortion may result from a combination of drought-

mediated disruption of metabolic pathways and changes in

ear development (Carcova and Otegui, 2007). Other pro-cesses, such as a delay in silk growth (anthesis–silking

interval; ASI) also have a major impact on seed number and

the susceptibly of maize to stress-induced flower abortion

(Bolanos and Edmeades, 1996). Over the last 50 years the

ASI has been successfully modified by genetic improvement

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(Duvick, 2005; Duvick et al., 2004). However, further

substantial improvements in the ASI trait are unlikely in

relation to drought adaption in maize (Monneveux et al.,

2008). Moreover, it is not easy to determine whether a larger

ASI is the cause of decreased grain numbers and yield,

because exposure to drought may simply diminish assimi-

late availability for growing silks (as discussed above) as

well as affecting plant water status and growth. The geneticcontrol of the ASI under water deficit is therefore consid-

ered to be shared with the sensitivity of leaf elongation rates

to drought, thereby contributing to the interest in alleles

maintaining leaf growth under water deficit (Welcker et al.,

2007).

Drought-induced limitations onphotosynthesis

The reduction in plant growth resulting from the imposition

of drought is in part related to changes in whole-plant

carbon status, which in turn depends on changes in thepartitioning of assimilate between different organs and also

the balance between photosynthesis and respiration (Flexas

et al., 2006a). While drought-induced inhibition of photo-

synthesis has been extensively studied (e.g. Cornic, 2000;

Lawlor, 2002; Chaves et al., 2003; Flexas et al., 2004;

Pinheiro and Chaves, 2011) and there has been an extensive

characterization of drought-induced changes in plant

physiology, metabolism, and gene expression patterns(Chaves et al., 2003; Huang et al., 2008; Ghannoum, 2009;

Hayano-Kanashiro et al., 2009), a recent literature review

concluded that relatively few general trends were apparent

in the metabolic and molecular responses of photosynthesis

to water stress (Pinheiro and Chaves, 2011). The photosyn-

thetic systems of C4 plants are as sensitive to drought-

induced inhibition as they are in C3 plants (Ripley et al.,

2010). Both stomatal and metabolic limitations on photo-synthesis occur during drought stress (Tezara et al., 1999;

Lawlor and Cornic, 2002; Medrano et al., 2002; Flexas

et al., 2006a; Lawlor and Tezara, 2009). While there is

ongoing debate as to which of these processes plays the

more dominant role, there is a general consensus that

reduced movement of CO2 from the atmosphere to the site

of carboxylation within the chloroplast is a major cause for

decreases in CO2 assimilation capacity under most droughtstress situations (Chaves and Oliveira, 2004; Flexas et al.,

2006b; Warren, 2008). The reduced movement of CO2

involves two important components, namely stomatal

closure, which restricts CO2 uptake from the atmosphere

into the leaf, and decreased internal conductance (gi), which

restricts CO2 movement from the intercellular air spaces

within the leaf to the site of carboxylation. Although

decreased stomatal conductance (gs) during drought stressis a very well known phenomenon, it has only been

suggested fairly recently that decreased gi also represents an

important limitation to CO2 movement (Flexas and

Medrano, 2002; Warren et al., 2004; Warren, 2008). As

a consequence the actual chloroplastic CO2 concentration

(Cc) could be substantially lower than the measured in-

tercellular CO2 concentration (Ci) even under conditions of

mild drought stress. A noteworthy implication of reduced giis that it may, at least in part, explain reduced CO2

assimilation rates at common Ci, or reduced apparent

carboxylation efficiencies (slope of the A–Ci curve), symp-

toms of drought stress that are normally regarded as

indicative of metabolic limitation.The relative effects of stomatal and metabolic limita-

tions are species dependent and that they are also

influenced by growth and experimental conditions (Lawlor

and Tezara, 2009). A conceptual model was therefore

proposed in which mild drought stress leads to decreased

gs, with CO2 assimilation rates falling in the presence of

only minor reductions in Ci and Cc (Lawlor and Tezara,

2009). Lawlor and Tezara (2009) suggested that themagnitude of changes in Cc might often be overestimated

in the literature because of assumptions and potential

errors in the estimation of gi.

While the responses of C4 photosynthesis to drought have

been much less studied than those of C3 photosynthesis,

literature evidence suggests that drought-treated C4 plants

also show stomatal and metabolic limitations on photosyn-

thesis (Ghannoum, 2009). Photosynthesis is decreased in theleaves of C4 plants subjected to drought through stomatal

and non-stomatal limitations (Foyer et al., 1998; Ghan-

noum et al., 2003; Carmo-Silva et al., 2007; Ripley et al.,

2007). Recent evidence suggests that C4 photosynthesis is

prone to metabolic limitations on photosynthesis and has

poor rates of recovery following drought (Ripley et al.,

2010). The available evidence suggests that there is prefer-

ential inhibition of the activity of C3 cycle enzymes duringdrought (Ghannoum, 2009). The differential inhibition of

the C3 and C4 cycles could lead to a rise in the CO2

concentration inside the bundle sheath cells and a build-up

of a CO2 gradient across the bundle sheath membrane

that would increase CO2 leakage (Bowman et al., 1989;

Saliendra et al., 1996).

The pathway of photorespiration is much reduced as

a result of the C4 pathway of photosynthesis. However,photorespiration is still required in C4 plants as it plays

important roles in cellular redox homeostasis and nitrogen

metabolism (Foyer et al., 2009). Photorespiration rates do

not increase markedly in C4 plants subjected to drought

stress (Carmo-Silva et al., 2008). The mesophyll chloro-

plasts of C4 plants have a high capacity for the Mehler

reaction, which generates hydrogen peroxide (Siebke et al.,

2003), and this pathway may provide an alternative sink forelectrons during drought stress, particularly if it becomes

partially uncoupled from ATP synthesis. The C4 pathway

enzymes located in the mesophyll chloroplasts of plants

could be inactivated by enhanced oxidation during drought

stress, and this might also explain why recovery of

photosynthesis after drought is more delayed in C4 than in

C3 plants (Ibrahim et al., 2008). Impaired metabolism is

a particularly important limitation on photosynthesis in C4

crops such as sorghum and sugarcane (Countour-Ansel

et al., 1996; Du et al., 1996).

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Drought and the production of sugarcane

Sugarcane is important in tropical and subtropical

agriculture in >80 countries (Molinari et al., 2007), but

unfortunately our understanding of sugarcane physiology

has lagged behind that of other major food and fibre

crops (Inmam-Bamber et al., 2005). Drought stress is

common in dryland sugarcane production (Inmam-Bam-

ber and Smith, 2005; Smit and Singels, 2006). Drying-off

prior to harvest through the withholding of irrigation is

an important strategy in irrigated sugarcane production,

as it serves to enhance stalk sucrose content and to

facilitate infield operations during harvesting (Robertson

et al., 1999b; Inmam-Bamber, 2004). With the gradual

development of water deficit during drying-off, stalk

elongation declines rapidly along with lower rates of leaf

development. Reduced stalk and leaf growth lowers the

demand for photoassimilate and, because photosynthesis

is less sensitive to water deficit than growth, more sucrose

thus becomes available for storage in millable stalks

(Robertson et al., 1999c). However, there are certain risks

associated with drying-off because if the water deficit

becomes too severe, photosynthesis is inhibited with an

associated lowering of cane and sucrose yields (Robertson

et al., 1999b). Increases in stalk sucrose content during

periods of low rainfall under dryland production of

sugarcane occur naturally during a process known as

natural ripening, but excessive drought stress, which

lowers photosynthetic capacity, frequently has large

negative impacts on cane and sucrose yields depending

on the timing of the stress (Robertson et al., 1999a).

The effects of drought stress on sugarcane growth and

photosynthesis were measured (Koonjah et al., 2006)

under rainshelter conditions as illustrated in Fig. 2. Plant

extension rates were the first process to be affected by

drought stress when the midday leaf water potential

decreased below –0.4 MPa. Photosynthetic rates werethen reduced when the midday leaf water potential

reached –0.7 MPa. Ultimately radiation use efficiency

was decreased by 50% when water potential reached

–1.5 MPa. A similar sequence of events relative to mid-

day leaf water potentials was observed in potted sugar-

cane plants subjected to drought stress, (Inmam-Bamber

and de Jager, 1986) and under field conditions (Koonjah

et al., 2006). At midday leaf water potentials of –1.0 MPato –1.7 MPa green leaf area was reduced, plant extension

ceased completely, and gs reached a minimum. The higher

drought sensitivity of growth processes compared with

photosynthesis was clearly demonstrated by Inmam-

Bamber et al. (2008), who found that only when stalk

extension rate was reduced by 33% did drought stress also

cause a reduction in photosynthetic rates. Besides slower

growth rates, shoot and leaf senescence is also acceleratedby severe drought stress in sugarcane (Fig. 3) and, with

the concomitant slower development of new leaves, the

reduction in radiation use efficiency is further aggravated

(Inman-Bamber and de Jager, 1986; Koonjah et al., 2006;

Smit and Singels, 2006). The combined effects of drought

stress on the extent of the green leaf canopy, and thus whole-

plant photosynthetic capacity, and reduced photosynthetic

rates on a leaf area basis per se explain why severe drought

stress can have substantial negative effects on sugarcane yield

components. It has been shown that drought stress that

occurs when the leaf canopy is already well established will

have more serious impacts on final yield in terms of totalbiomass, stalk biomass, and stalk sucrose than when drought

stress occurs earlier during the season in a younger crop

(Robertson et al., 1999a). The reduction in biomass pro-

duction was mainly due to lower photosynthetic rates of the

leaves, which decreased sharply because of rapid development

of drought stress in the crop with the larger leaf area index.

Fig. 2. A comparison of the effects of drought on the growth of

sugarcane (cultivar NCo 376) in experiments conducted in an

automatic rain shelter at the South African Sugarcane Research

Institute (SASRI), South Africa.

Fig. 3. The effects of drought on sugarcane growth under field

conditions. Sugarcane (cultivar N25) was grown at Komatipoort

(South Africa) under irrigation in treatments where three soil water

regimes were maintained, namely 75, 50, and 25% of total available

soil moisture (TAM).

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Based on an analysis of available evidence it was

proposed that with declining leaf water status, C4

photosynthesis goes through three successive stages

(Ghannoum, 2009). The initial stage is mainly character-

ized by a decline in stomatal conductance, which may or

may not be accompanied by reduced rates of CO2

assimilation. This is because the CO2-concentrating

mechanism in C4 plants is capable of saturating photo-synthesis even at relatively low Ci. As leaf water status

drops further, a combination of stomatal and non-

stomatal factors are introduced, while during the third

stage of photosynthetic decline at very low leaf water

status, mainly non-stomatal factors are involved in the

severe inhibition of photosynthesis. A survey of the

limited evidence available suggests that photosynthetic

decline in sugarcane during drought stress generallyfollows this pattern. At midday leaf water potentials

above –0.85 MPa the decline in CO2 assimilation rates in

sugarcane was mainly caused by stomatal closure, while

at leaf water potentials below –0.85 MPa, non-stomatal

components became increasingly important (Du et al.,

1996). In these experiments the CO2 assimilation rate and

gs were already reduced by 50% when midday leaf water

potential reached approximately –0.7 MPa, while theactivities of five key photosynthetic enzymes only reached

the 50% inhibition level at a leaf water potential of

approximately –1.5 MPa (Du et al., 1996).

When considering the non-stomatal factors involved,

the response in sugarcane during drought stress appears

to be as diverse as in C3 plants. Du et al. (1996)

demonstrated that severe drought stress in sugarcane

inhibited the activities of Rubisco, fructose bisphospha-tase (FBPase), phosphoenolpyruvate carboxylase (PEP-

case), NADP-ME, and pyruvate,orthophosphate dikinase

(PPDK). In particular, PPDK activity correlated very

closely with CO2 assimilation rates during the latter

stages of drought stress, and it was concluded that PPDK

was very likely be the limiting factor in non-stomatal

components in sugarcane during drought stress (Du et al.,

1996). However, Saliendra et al. (1996) noted a decline inthe Rubisco:PEPcase activity in sugarcane during

drought stress, mainly because Rubisco activity was not

inhibited while PEPcase activity actually increased. In

another study on sugarcane, drought stress caused in-

hibition of Rubisco activity but not PEPcase activity (Vu

and Allen, 2009), which fits the general idea of preferen-

tial inhibition of key C3 cycle enzymes by drought stress,

rather than key C4 cycle enzymes (Ghannoum, 2009).Differences in drought sensitivity between sugarcane

cultivars (e.g. Inmam-Bamber and de Jager, 1986; Smit

and Singels, 2006; Silva et al., 2007; Rodrigues et al.,

2009), the duration and intensity of the drought treat-

ments, and various other factors could perhaps explain

these differences in findings. Clearly, further research is

required to advance our understanding of the complex

and interacting mechanisms involved in the droughtresponse of photosynthesis in sugarcane and why culti-

vars differ in their sensitivity.

Looking to the future: increasedatmospheric CO2 and drought adaptation inC4 plants

Atmospheric CO2 concentrations have risen from ;270 ll l�1

in pre-industrial times to close to 390 ll l�1 at present, and

values could reach between 530 ll l�1 and 970 ll l�1 by the

end of this century (Intergovernmental Panel on Climate

Change, 2007). Growth of maize under CO2 enrichment hasan impact on the transpiration rates and water status of C4

plants even under well-watered conditions that is particu-

larly evident in the older leaf ranks (Prins et al., 2011).

Under well-watered conditions, elevated CO2 has little

effect on the photosynthesis or growth of C4 plants in either

controlled environments (Ziska and Bunce, 1999; Driscoll

et al., 2006; Soares et al., 2007; Prins et al., 2010) or in the

field free-air concentration enrichment experiments (FACE)studies (Leakey et al. 2009a, b). However, the negative

effects of drought on yield and biomass are attenuated at

high CO2 in both C3 and C4 plants because of stomatal

closure (Conley et al., 2001; Wullschleger et al., 2002; Wu

et al., 2004). The combined effects of high elevated CO2 and

drought were assessed by comparing the performance of

two CIMMYT tropical maize hybrids under conditions of

optimal water supply, moderate water stress, and severewater stress (Table 1 and Figs 4–7). Both maize genotypes

accumulated more shoot biomass under drought conditions

at elevated CO2 compared with ambient CO2 conditions

(Table 1). This finding is consistent with previous observa-

tions of higher biomass production in water-stressed maize

plants at elevated CO2 compared with air (Samarakoon and

Gifford, 1996; Ottman et al., 2001). Water loss was slower

when maize plants were grown at elevated CO2 comparedwith ambient CO2 conditions (Fig. 4). Drought-induced

decreases in CO2 assimilation rate and in stomatal conduc-

tance were more rapid in maize plants grown under ambient

CO2 conditions compared with elevated CO2 conditions

(Figs 5, 6). Moreover, transpiration efficiencies were signifi-

cantly higher at elevated CO2 (Fig. 7). These observations

clearly illustrate the better performance of maize leaf photo-

synthesis under conditions of drought when plants are grownat high CO2. This may be due to a combination of factors

including enhanced WUE, lower stomatal conductance rates,

and possibly enhanced root proliferation (Wullschleger et al.,

2002).

Drought stress may also be triggered in leaves by

increased temperatures. While growth under elevated CO2

can partially ameliorate the negative effects of drought in

both C3 and C4 species, it is unlikely that high CO2 willoffer any protection from heat stress (D. Wang et al., 2008).

Rather, high CO2 may exacerbate the negative effects of

high temperatures. Plants grown under drought stress and/

or elevated CO2 tend to exhibit higher leaf temperatures

because transpiration rates are decreased and thus less

latent heat is lost. Photosynthesis may therefore be more

sensitive to acute heat stress under conditions of drought

and/or elevated CO2. High temperatures often occur alongwith drought, and little is known about the interaction of

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the two stress factors—because most studies concentrate on

one of them at a time (due to the inherent complexity). So,

basically, more studies on this interaction are needed.

Final remarks and perspectives

In previous decades, the increases in the yield potential in

C3 crops such as wheat were largely achieved through

improvements in harvest index. While further large

increases in harvest index are considered to be unlikely, it

may be possible to increase productive biomass through

enhanced photosynthetic capacity and efficiency (Parry

et al., 2011). The yield potential of maize hybrids has

increased steadily over the past 70 years in the USA(Tollenaar and Lee, 2002). However, major advances, if not

breakthroughs, in breeding strategies are required for

further increases in the yield potential of C4 crops such as

maize (Duvick and Cassman, 1999). Moreover, enhanced

drought tolerance must be achieved without creating

penalties in yield potential (van Ginkel et al., 1998). While

genotypes with high yield potential generally perform well

under rain-fed and irrigated treatments (Tollenaar and Wu,1999; Tollenaar and Lee, 2002), the selection for high

yielding varieties under largely stress-free conditions has to

a certain extent improved yield under water-limiting con-

ditions (Cattivelli et al., 2008). In contrast, the selection for

drought adaptation has often resulted in lower genetic gains

(Campos et al., 2006). Despite the lower genetic gains

obtained by the slower conventional breeding approaches,

some literature examples bear testimony to the fact that

selection for drought tolerance in routine maize breeding

programmes with adequate weighting can result in signifi-cant increases in yield (Banziger et al., 2004).

Breeding for high yield in drought-prone environments

using multilocation testing remains inherently complicated

by the year to year variability in the amount and temporal

distribution of available soil water and the low heritability

of drought resistance traits under these conditions. An

increasing array of useful molecular tools that can be

applied in classical breeding approaches is now available toplant breeders, together with a greater knowledge of the

traits that sustain yield under drought. Transcript profiling

studies on maize leaves exposed to drought has revealed an

Table 1. A comparison of the effects of drought on the growth of

two CIMMYT maize genotypes (14 and 9) that were selected on the

basis of differing ASI (anthesis–silking interval) values, under ambient

CO2 levels (350 ll l– 1) or with CO2 enrichment (700 ll l�1).

Plants were grown for 5 weeks under an optimal watering regime(WW), and two additional water regimes (WR) were then applied for7 d, involving either a moderate drought stress (MS) where waterwas provided every other day or severe stress (SS) where water wascompletely withheld for the whole 7 d period of the experiment. Thetotal fresh weight (FW) and dry weight (DW) values of the shootswere determined on day 7 of the drought treatment experiment. Theanalysis of variance and probabilities were then calculated in relationto CO2, genotype, and WR effects.

Maize genotype GrowthCO2

WR Shoot biomass(g FW)

Shoot biomass(g DW)

Genotype 9 Air MS 136.9626.6 11.660.9

SS 96.968.4 8.961.4

Elevated MS 162.4616.0 13.362.3

SS 101.1610.7 9.862.2

Genotype 14 Air MS 125.9635.3 9.663.9

SS 92.1612.0 6.861.0

Elevated MS 151.2627.3 12.061.1

SS 109.4615.9 9.761.4

CO2 <0.05 <0.005

Genotype NS <0.05

CO23genotype NS NS

WR <0.0001 <0.0001

CO23WR NS NS

CO23genotype3WR NS NS

NS, not significant.

Fig. 4. A comparison of the effects of drought on soil water

content in the pots in two CIMMYT maize genotypes (14, top

panel, and 9, bottom panel) that were selected on the basis of

differing ASI (anthesis–silking interval) values, under ambient CO2

levels (350 ll l– 1) or with CO2 enrichment (700 ll l�1). Plants were

grown for 5 weeks under an optimal watering regime (WW), and

two additional water regimes (WR) were then applied for 7 d,

involving either a moderate drought stress (MS) where water was

provided every other day or severe stress (SS) where water was

completely withheld for the whole 7 d period of the experiment.

Dark grey triangles, severe water deficit at ambient CO2; light grey

circles, severe water deficit at elevated CO2; dark grey squares,

moderate water deficit at ambient CO2; open circles, moderate

water deficit at elevated CO2; dark grey diamonds, well watered at

ambient CO2; dark grey crosses, well watered at elevated CO2.

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Fig. 5. A comparison of the effects of drought on photosynthesis (A) in two CIMMYT maize genotypes [genotype 9 corresponds to (a) and (c)

and genotype 14 corresponds to (b) and (d)] that were selected on the basis of differing ASI (anthesis–silking interval) values, under ambient

CO2 levels (350 ll l– 1) in (a) and (b) or with CO2 enrichment (700 ll l�1) in (c) and (d). Plants were grown for 5 weeks under an optimal watering

regime (WW), and two additional water regimes (WR) were then applied for 7 days, involving either a moderate drought stress (MS) where

water was provided every other day or severe stress (SS) where water was completely withheld for the whole 7 d period of the experiment.

Photosynthetic gas exchange measurements were performed daily throughout the course of the drought treatment. Measurements were

performed at an irradiance of 1000 lmol m�2 s�1 and with either ambient (350 ll l�1) or high (700 ll l�1) CO2 levels.

Fig. 6. A comparison of the effects of drought on stomatal conductance (gs) in two CIMMYT maize genotypes [genotype 9 corresponds to (a)

and (c) and genotype 14 corresponds to (b) and (d)] that were selected on the basis of differing ASI (anthesis–silking interval) values, under

ambient CO2 levels (350 ll l– 1) in (a) and (b) or with CO2 enrichment (700 ll l�1) in (c) and (d). Plants were grown for 5 weeks under an optimal

watering regime (WW), and two additional water regimes (WR) were then applied for 7 d, involving either a moderate drought stress (MS)

where water was provided every other day or severe stress (SS) where water was completely withheld for the whole 7 d period of the

experiment. Photosynthetic gas exchange measurements were performed daily throughout the course of the drought treatment.

Measurements were performed at an irradiance of 1000 lmol m�2 s�1 and with either ambient (350 ll l�1) or high (700 ll l�1) CO2 levels.

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induction of stress-associated genes (Zheng et al., 2004; Jia

et al., 2006; Yue et al., 2008). Such molecular approaches

provide essential candidate gene sequences that allow

dissection of QTLs or transgenic approaches to drought

tolerance (Shou et al., 2004; Cattivelli et al., 2008; Wang

et al., 2008). The most effective QTLs and/or transgenescan be accumulated in elite genotypes, parental inbreds, and

hybrids without detrimental effects on yield potential.

Prediction models are becoming more accurate as genetic

analyses are effectively combined with high throughput

phenotyping in the field (Collins et al., 2008; Tardieu and

Tuberosa, 2010; Araus et al., 2011). Molecular genetic

approaches are increasingly used to characterize the com-

plex network of drought-related traits in C4 plants (Bolanosand Edmeades, 1993; Bruce et al., 2002), and traits such as

stay green are also proving to be useful in selection for

enhanced drought tolerance in maize (Haussman et al.,

2002). To date, however, relatively few applications for

DNA marker technologies have emerged in practical maize

breeding programmes, presumably because of the complex-

ities associated with genetic background, developmental

stage, and environment on QTL effects, as well as in-adequate phenotyping, gene-by-gene effects and the time

and cost considerations in developing these tools. Since

the costs of applying marker-assisted selection or genomics

for many QTLs/markers with small effects are often greater

than those of conventional cross-breeding, it may be

preferable to target only major QTLs/markers with a con-

siderable effect that is consistent across germplasm. Un-

fortunately, to date, studies have not identified a QTL/

marker in maize with sufficiently large effects to be used

effectively. Thus, an improved conceptual framework for

the molecular breeding of drought tolerance in C4 plants isrequired, together with its implementation in future QTL

and -omics studies.

Acknowledgements

JLA acknowledges the support of the DTMA project (Bill

and Melinda Gates Foundation) and the 08.7860.3-001.00

project (BMZ, Germany). We thank Dr M. A. Smit

(SASRI) for Figs 2 and 3. CHF acknowledges the support

of the European Union (Projects ITN: PITN-GA-2008-

215174 Chloroplast Signals and FP7- PIRSES-GA-2008-

230830 Legume Improvement). We thank J.P. Ferrio (UdL,Spain) and L. Cabrera-Bosquet (UB, Spain) for critical

reading of the manuscript.

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