Breeding cereals for Mediterranean conditions: ecophysiological clues for biotechnology application

129Ann. appl. Biol. (2003), 142:129-141Printed in UK

*Corresponding Author E-mail: [email protected]

© 2003 Association of Applied Biologists

Breeding cereals for Mediterranean conditions: ecophysiological clues forbiotechnology application

By J L ARAUS1*, J BORT1, P STEDUTO2, D VILLEGAS3 and C ROYO3

1Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028Barcelona, Spain

2CIHEAM-IAMB, Mediterranean Agronomic Institute of Bari, via Ceglie 9, 70010 Valenzano - Bari, Italy3Area de Conreus Extensius, Centre UdL-IRTA, Alcalde Rovira Roure 177, 25198 Lleida, Spain

(Accepted 3 March 2003; Received 26 November 2002)

Summary

Water stress is the main environmental factor limiting cereal yield in Mediterranean environments.For particular regions, such as the Mediterranean basin, the agroecological conditions are expected toget worse. In response to this challenge attempts are being made to improve crop yield through farm-management practices and plant breeding efforts. Here we examine traits that may be used as selectioncriteria for breeding C3 cereal crops with improved yield and stability in Mediterranean conditions.Emphasis is made on the potential implications of defining proper selection traits and targetenvironments when adopting biotechnology approaches in breeding programmes.

Key words: Water use efficiency, transpiration efficiency, harvest index, photosynthesis, carbon isotopediscrimination, marker assisted selection, transformation

Introduction

Water stress is the main environmental factorlimiting cereal yield in Mediterranean environments,in which barley (Hordeum vulgare L.), durum wheat(Triticum turgidum var. durum L.) and bread wheat(T. aestivum L.) are the main crops. Moreover, forregions such as the Mediterranean basin the agro-ecological conditions are expected to deteriorate dueto the difficulty of ensuring supplies of fresh water(in competition with demographic anddevelopmental priorities), and the foreseenenvironmental shift to greater aridity (Araus et al.,1997). The way to meet this challenge is to improvethe yield of rainfed and irrigated crops by farm-management practices and plant breeding (Hatfieldet al., 2001; Richards et al., 2002), the secondapproach being the most promising in the long term(Miflin 2000; Araus et al., 2002 and referencestherein). Several reviews on the contribution ofphysiology to breeding have been published recently(Araus et al., 2002; Condon et al., 2002; Richardset al., 2002) and a monographic website has beenposted (Blum, 2000, http://www.plantstress.com).

The last decade has witnessed an increase in theadoption of biotechnological methods inconventional breeding programmes. However, theapplication of biotechnology to the production ofnew cultivars with either higher yield potential orbetter performance under drought or other majorabiotic stresses has not been accomplished. Here weexamine morpho-physiological traits that may be

used as selection criteria for breeding C3 cereal cropswith improved yield and stability underMediterranean conditions. We also discuss, from aneco-physiological perspective, how such knowledgemay be successfully exploited by biotechnology.

Potential and Limitations of Biotechnology

Biotechnology offers (in addition to approachessuch as tissue culture and double haploid) two newways for improving cereals and other crop plants:one through the development and application ofmolecular markers and the other through geneticengineering.

The development of comprehensive genetic mapsbased on molecular markers has improved the powerof genetic analysis. Thus molecular markers linkedto traits (mostly grain quality traits) of economicimportance have been identified in wheat (seeexamples in Langridge et al., 2001 and a broadcatalogue in http://wheat.pw.usda.gov), and barley(Barr et al., 2000). Moreover, traits that in the pastwere recalcitrant to analysis, such as abiotic stressresponses, are now more amenable. Thusidentification of individual major genes andquantitative trait loci (QTLs) mediating the variationto stresses has been reported (see Snape et al., 2001;McCouch, 2001; Cattivelli et al., 2002; Tuberosa etal., 2002).

However, the impact of marker-based QTLanalysis on the development of new varieties withenhanced quantitative traits has been less than hoped

130 J L ARAUS ET AL.

(Tanksley & Nelson, 1996; Thomas 2003). In factthe few validation studies reported to date aredisappointing (see for example Romagosa et al.,1999 for QTLs on barley yield). Quantitative traitsare genetically complex and poorly understood atthe physiological and biochemical level (Langridgeet al., 2001). Identification of QTL is much morecomplex than for simple traits, and the genotype ×environment (G × E) interactions on the expressionof these QTLs are frequently large (e.g. Kjaer &Jensen, 1996; Romagosa et al., 1999). This isparticularly true for Mediterranean rainfedconditions, where genetic advance is hindered bylarge G × E (either seasonal or locational)interactions, mainly arising from unpredictablerainfall (Richards et al., 2002). Moreover,identification of QTL depends on the geneticbackground of the mapping population (Slafer &Araus, 1998).

In this context, there is a need to examine the G ×E interaction and to define the selection traits andthe target environment (Fig. 1). For example, themost effective way to improve the productivity ofcereal crops grown in less-favoured Mediterraneanareas (e.g. barley yields well below 1 t ha-1) is to uselocally adapted germplasm and select in the targetenvironment (Ceccarelli et al., 1998). Molecularmarkers have been focused frequently on traitsconferring stress tolerance, therefore improving cropsurvival in low-yielding environments (Cattivelli et

al., 2002; Tuberosa et al., 2002). However, selectionfor yield potential under favourable conditionsfrequently leads to higher yields in mild tomoderately drought stressed Mediterraneanenvironments (Richards, 2000; Richards et al., 2002;Araus et al., 2002).

A further problem highlighted by QTL analysis isthe association of abiotic stress traits with geneticloci of agronomic importance (Forster et al., 2000b).For instance, genetic linkage between salt toleranceat germination and ABA (abscisic acid) response hasbeen found from QTL mapping in barley (Mano &Takeda, 1997). Important genes for adaptation totarget environments such as genes responsive tovernalisation and photoperiod and semi dwarf genesfrequently show pleiotropic effects on stresstolerance. In addition, QTLs for stress responses cancoincide with yield and quality QTLs or otherimportant genomic regions (Forster et al., 2000a).

Moreover, there is concern that much of the geneticvariability for improving abiotic stress tolerance insmall grain cereals has been lost duringdomestication, selection and modern breeding(leaving pleitropic effects of the selected genes fordevelopment and adaptation). In barley, as manyother crops, greater variation in the response toabiotic stress is found in gene pools of primitivelandraces and related wild species (Ellis et al., 2000;Forster et al., 2000a). The polyploidy of bread wheatand durum wheat makes the use of molecularmarkers for breeding and selection more complexthan in barley. Moreover, bread and durum wheatapparently have lower polymorphism than barleyand other cereals (Chao et al., 1989; Devos et al.,1995; but see Harker et al., 2001), and the level ofpolymorphism is not consistent across genomes orcrosses (Langridge et al., 2001).

The ability to use genetic transformationtechnology (and in general functional genomics)opens up new opportunities. Progress in cerealtransformation has been faster in rice and maize (e.g.Lazzeri & Shewry, 1993; Tanksley & McCouch,1997; Sheehy et al., 2000; Dunwell, 2000) than inwheat or barley (see Barceló et al., 1998 for wheat),but attention is now turning to the modification ofcereals for agronomic characteristics. However, thedevelopment of efficient genetic transformation(which depends among other factors on the type ofpromoter or the genetic background used) is notenough; there still the need to recognise alteredphenotypes and to rapidly associate phenotypicchanges with specific sequence alterations(McCouch, 2001).

The identification of the right trait is basic whenattempting to transform cereals for improvedperformance under stress. From an eco-physiologicalpoint of view, a shortcoming in efforts to achieveplant transformation to improve performance under

Gra

in y

ield

(t h

a-1)

Environment index (t ha-1)

Cultivarsselected understress are moreproductive andsteady in verypoorenvironment, inexchange to alower yieldpotential

Selection foryield potentialreleases verylow-yieldinggenotypesunder severestress

Cultivar

�1�

(selec

ted in

stres

s-free

envir

onmen

ts)

Cultivar �2�

(selected in stressful environments)

Fig. 1. Theoretical relationships between grain yieldof a given cultivar and the environmental index,understood as the average yield of all participatinggenotypes assayed within a given environment.Environment is here equivalent to a trial (combinationof a site and a year). The schematic diagram shows acommon negative G × E interaction, implying acrossover at relatively low levels of environmentalindex (arrow), when cultivars were selected for yieldper se in near optimum conditions (cultivar �1�, stress-free) or under severe stress (cultivar �2�, stressful).

131Ecophysiological clues for cereal breeding

Increasing water use is feasible when there is stillsoil water available at maturity or when a significantpart of the soil water is lost through evaporation (Fig.2). Soil evaporation indirectly affects dry matteraccumulation by limiting the water available to roots,and by modification of temperature and humidity inthe canopy (Jamieson et al., 1995). Therefore

drought is that they have mainly focused onprocesses at the cellular scale. In contrast, the mainprocesses involved in crop responses to watershortage occur at higher scales, and thus are usuallylargely polygenic (Richards, 1996). Anotherdrawback is that plant transformation has focusedon traits conferring cell survival (e.g. desiccation ordehydration protectants, antioxidants) and thus ontolerance to fast developing water stress (Dunwell,2000 and references therein), which may be relevantonly for very marginal drought-prone areas.However, increasing yield under water limitedconditions in the field is related more frequently tostress avoidance and higher yield potential (Richardset al., 2002; Araus et al., 2003a).

Which Traits to Select for? � An Eco-Physiological Perspective

Accordingly to Passioura (1977), crop yield indry conditions depends on (i) water use (i.e. theamount of water used by the crop), (ii) the water useefficiency (WUE: the efficiency for producingabove-ground dry biomass per unit of water used),and (iii) the Harvest Index (HI: the proportion ofthe above-ground biomass allocated to grain). Thesethree variables are not fully independent and (asdiscussed below) modifying a given trait may affectmore than one variable. However, the modelproposed by Passioura (1977) remains a simple, butuseful, approach when looking for breeding traits toimprove yield in Mediterranean conditions (Arauset al., 2002). As pointed out by Richards et al. (2002)this framework is based on yield alone, with no focuson protection or survival under drought (i.e. droughttolerance), which have been largely unsuccessfulapproaches.

Barley is the best adapted cereal for marginalMediterranean areas (characterised by drought-proneconditions and poor soil fertility), whereas durumwheat, and even more so bread wheat, need betterwater and nutrient conditions. WUE increases inbarley under water stress, more so than in wheat,triticale and oat (Good & Bell, 1980; Loboda, 1993;Rekika et al., 1995; Araus, 2002; Cattivelli et al.,2002), WUE being positively correlated with yield(Dakheel et al., 1994; Simpson & Siddique, 1994).Therefore, a priori, it makes more sense to selectfor drought tolerance in barley than in other cereals.The underlying mechanisms conferring tolerance tostress have been extensively reviewed elsewhere(e.g. Araus, 2002 for barley) and will be onlymarginally addressed in this paper.

Water useWater used (mainly transpired) by plants is directly

associated with dry matter accumulation, as watervapour and carbon dioxide pass through the stomata.

ET

A

C

B

D

ET1 ET2

M4

M3

M2

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ET

A

C

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D

ET1 ET2

M4

M3

M2

M1E

ET3

M5

Abo

ve-g

roun

d bi

omas

(M)

ET

Fig. 2. Variable impacts of different crop characteristicson the relationships between above-ground biomass(M) and crop season evapotranspiration (ET).Case A: initial conceptual relationship for a given crop,where for a given cumulated evapotranspiration (ET1)a corresponding biomass production (M1) is obtained.Case B: the crop expresses a higher water use efficiency(WUE: above-ground biomass per unit of watertranspired) than in Case A, so that the slope of therelationship is increased. Therefore, for the samecumulated evapotranspiration (ET1) a higher amountof biomass production (M2) is obtained.Case C: the crop has the same WUE of the Case A,and the same sowing date, but expresses a faster �earlygrowth�, so that more water of the cumulatedevapotranspiration (ET1) is allocated in transpiration(productive water) than in evaporation (unproductivewater). Therefore, for the same cumulatedevapotranspiration (ET1) a higher amount of biomassproduction (M3) is obtained.Case D: the crop has the same WUE and the sameinitial growth of the Case A, but it is able (e.g. by havingdeeper root growth) to capture more water from thesoil. Therefore, a higher cumulated evapotranspirationis obtained (ET2) with a consequent higher biomassproduction (M4).Case E: the crop (e.g. a long-cycle variety) is sownearlier, and therefore both higher allocation ofevapotranspiration into transpiration and morecumulated evapotranspiration (ET3) are foreseen.Case E is represented with the same WUE (i.e. sameslope) as Case A, but frequently a higher WUE mayalso be expected due to a lower vapour pressure deficitduring grown. This is apparently the best option interms of biomass production (M5) but frequently it isnot feasible for Mediterranean environments, eitherbecause soil water is exhausted well before the end ofthe crop cycle or due to late freezing episodes.


allocating soil evaporation into plant transpirationby increasing early vigour (seedling establishment,i.e. the capacity of fast leaf-ground cover) oradjusting phenology (matching crop developmentand seasonal rainfall pattern) or capturing more waterfor transpiration, through for example more rootgrowth, are sound options (Araus, 2002; Richardset al., 2002). Long cycle (early sowing and or lateflowering) genotypes may have advantages in termsof improving root depth and distribution (Richards,2000), but frequently these are options not feasiblefor non-irrigated Mediterranean environments.

Diminishing soil evaporationEmbryo and leaf structural traits of seedlings

conferring early seedling vigour have already beenproposed (Richards et al., 2002 and referencesherein). However interaction with low temperature(Araus et al., 2000), or the adoption of gibberellicacid insensitive semi-dwarf cultivars (Richards etal., 2002) may limit the feasibility of these traits.Recent reports on transgenic cereals with HVA1gene, over-expressing late-embryogenesis-abundant(LEA) proteins, and showing early vigour may openup new alternatives (Sivamani et al., 2000).

The single most important attribute (other than theyield itself), tackled by breeding programmes inMediterranean conditions has been phenologicaladjustment; specifically crop earliness, even whenthis approach may face the problem of late-frost,coinciding with the reproductive stage of the crop(Araus et al., 2002). Thus yield potential QTLanalysis has been carried out for heading time(Sourdille et al., 2000). Marker assisted selectiontechnology has allowed the development ofstrategies for stress amelioration based on changingthe phenology of genotypes or introducing genes thatenable the plant to tolerate cold stress. For instance,genes for vernalisation response and cold tolerancehave been located close together, on chromosomesof homeologous Group 5 of wheat (Snape et al.,2001). Selection in this region is therefore likely totailor wheat varieties with particular life cycle timesand thus, changed yield potential.

The three separate systems of genes that controlflowering time in wheat and barley (those controllingvernalisation, those controlling photoperiod responseand those controlling developmental rate, the socalled �earliness per se� genes) have pleiotropiceffects on other aspects of plant growth anddevelopment. Pleiotropic and/or the co-location ofgenes is a problem, the effect of different alleles offlowering time genes needs further study (Snape etal., 2001). It has been demonstrated that theCheyenne alleles for vernalisation requirement andfrost tolerance on chromosome 5A of wheatsignificantly increased sugar concentrations,particularly sucrose and fructan, and hence improve

osmotic potential and frost tolerance (Galiba et al.,1997).

Drought during grain filling (terminal drought)hastens leaf senescence of cereals in Mediterraneanconditions, leading to premature death. Assuming ahigh correlation between net carbon output andchlorophyll content (an assumption by no meansjustified in practice; see Thomas & Howarth, 2000),stay-green genotypes, with a delayed leafsenescence, might be an alternative option fordiminishing evaporation, while eventually increasingwater use and water use efficiency. Thus geneticvariation to delay leaf senescence and extend theduration of grain filling has been reported fordifferent C4 cereals (Russell, 1991; Borrell et al.,2000). In contrast, transgenic approaches to producestay-green plants may include enhanced endogenouscytokinins and/or reduced ethylene production orperception, (see references in Thomas & Howarth,2000; Dunwell, 2000). Alternatively, QTL analysisand marker-assisted selection mapping and marker-assisted selection could be applied to develop lineswith delayed senescence, even when the expressionof the character is strongly influence byenvironmental conditions (Mahalakshmi & Bidinger,2002). However, cereals studies with stay-greengenotypes have been performed more in C4 than inC3 species (Thomas & Smart 1993; Thomas &Howarth, 2000). Moreover the stay-green seemsmore relevant for intermittent drought than forterminal drought (Ludlow & Muchow, 1990; but seeMahalakshmi & Bidinger, 2002). In fact, this traitmay still be useful for environments where there isa high probability of rainfall during grain filling(Richards et al., 2002).

Enhancing water extractionRoot system development may be enhanced by

osmolyte accumulation (Serraj & Sinclair, 2002).This trait has been extensively studied and genesencoding osmolyte biosynthesis have beenintroduced in cereals. Thus, Snape et al. (2001)concluded that the Group 5 chromosomes of wheatcarry genes controlling a range of stress responsesrelated to osmolyte accumulation such as toleranceto freezing, drought, osmotic stress and hightemperatures. Sugar synthesis seems to play a rolein drought tolerance in providing compatible solutesfor osmotic adjustment (Bohnert et al., 1995) orthrough various protective roles including theprotection of membranes (Crowe et al., 1992).

QTL analyses have been carried out for differentdrought-related traits including leaf ABA content inwheat (Quarrie et al., 1994, 1995), and water status,water-soluble carbohydrate, osmotic adjustment,plant architecture, growth habit and chlorophyllcontent in barley (Mano & Takeda 1997; Teulat etal., 1997, 1998, 2001; Teulat-Merah et al., 2000). It


has been postulated that relatively simplebiochemical processes involved in drought that maylend themselves to genetic transformation includeosmotic adjustment, repair and degradation ofproteins, and structural adjustment, for example, ofthe cell wall (Ingram & Bartels, 1996).

However, there is little evidence that crop yieldsbenefit by increased osmolyte accumulation (Serraj& Sinclair, 2002). For traits such as aquaporin(Tyerman et al., 2002), ABA (Passioura, 2002; Qin& Zeevaart, 2002) and cell wall-enzymes (Wu &Cosgrove, 2000) there is evidence for their basic rolein plant water relations, but their integration at thewhole plant level is still not resolved, which makestheir inclusion in breeding difficult. Whereas theinhibitory signals driven by soil conditions mayaffect stomatal conductance, cell expansion, celldivision and the rate of leaf appearance, their natureis still under debate. This debate is becomingincreasingly complex, which probably signifies thata network of hormonal and other responses isinvolved in attuning the growth and developmentof a plant to its environment (Passioura, 2002).

Indicators of crop transpirationA major function of transpiration in plants is leaf

cooling. When plant water status is reduced andstomata close leaf temperature rises due to the lackof transpirational cooling. Hence, canopy

temperature can serve as an indirect measure of planttranspiration and plant water status. This surrogatehas been used in plant breeding with the developmentof the infrared thermometer that can sense canopytemperature remotely and speedily. Thus, cerealgenotypes having lower canopy temperature atmidday have relatively better water status and areassumed to be better drought avoiders (Blum et al.,1982, 1990; Garrity & O�Toole, 1995). Canopytemperature has also been used to select for highyield potential wheats (Reynolds et al., 1994, 1998,1999; Amani et al., 1996).

Stable carbon isotope discrimination (∆, a wellestablished surrogate for transpiration efficiency: seebelow) may also be considered as an indirectindicator at the crop level of the amount of wateravailable for the plant in moderate and non waterstressed environments (Araus et al., 1997, 1999,2003b). Thus, whereas ∆ depends on the ratio ofintercellular to external CO2 concentration, stomatalconductance is by far the main factor affecting sucha ratio (Richards, 2000). Therefore, genotypes ableto keep the stomata more open are those moreproductive. This would explain the positivephenotypic relationships between ∆ (usually fromgrains, see Table 1) and yield in moderately waterstressed (with average yields as low as 2-3 Mg ha-1)to fully irrigated Mediterranean conditions (Voltaset al., 1999; Richards et al., 2002; Araus et al.,

Year 1997 1998 1999 AverageRainfed Irrigated Rainfed Irrigated Rainfed Irrigated Rainfed Irrigated

∆ in mature grains∆- Grain yield 0.37 0.50** 0.46* 0.54** 0.49* 0.44* 0.18 0.46*∆- TKW 0.03 0.07 0.14 0.67*** 0.10 0.50* 0.09 0.52**

∆- NES 0.04 0.17 -0.09 0.19 -0.21 -0.18 -0.26 -0.10

∆- HI 0.09 0.22 0.21 0.24 0.03 -0.14 0.10 0.02

∆- NGS 0.10 0.14 0.29 0.12 0.23 -0.04 0.09 0.15

∆- NGM2 0.14 0.28 0.23 0.29 0.02 -0.14 -0.10 0.07

∆- GWS 0.26 0.14 0.54** 0.58** 0.24 0.37 0.34 0.53**

∆ in seedlings∆- Grain yield -0.37 0.45* 0.10 -0.30 -0.28 0.14∆- TKW -0.23 -0.18 0.29 -0.03 0.19 -0.05∆- NES 0.14 0.29 -0.18 -0.06 -0.16 0.35

∆- HI -0.26 0.41* -0.32 -0.15 -0.40* 0.17

∆- NGS -0.16 0.03 -0.10 -0.10 -0.28 -0.10

∆- NGM2 -0.08 0.31 -0.20 -0.14 -0.35 0.17

∆- GWS -0.46* -0.07 0.13 -0.08 -0.28 -0.28TKW = Thousand kernel weight, NES = Number of spikes per m2, HI = Harvest index, NGS = Number of grains per spike, NGM2 =Number of grains per m2, GWS = Grain weight per spike. Empty cells indicate data not available* P < 0.05, ** P < 0.01,*** P < 0.001

Table 1. Pearson correlation coefficients between carbon isotope discrimination (∆) in mature grains and inseedlings at the end of tillering, and grain yield and yield components for each trial and on irrigated and rainfed

trials independently. Results correspond to durum wheat trials of NE-Spain (reported in part in Royo et al., 2002;Araus et al., 2003b)


2003b).Therefore it may be postulated that, given a similar

crop area and similar phenology, plants with a higher∆ and canopy temperature depression (CTD) willhave higher transpiration. Although ∆ and CTD donot provide information on the mechanisms by whichthe plants absorb water (e.g. larger root system, betterosmotic adjustment, etc.), they do identify crops withbetter performance potential.

Transpiration-driven CTD is measured within afew seconds on a plot basis, using an infraredthermometer, which is inexpensive and easy tohandle. CTD provides an instantaneous picture ofthe water status of the whole crop. By contrast, ∆analyses are more expensive and time consumingbut when performed in dry matter they give anintegrated assessment of the water status during theentire time of carbon fixation. Ideal conditions forcanopy temperature measurements are irrigatedcrops under high radiation and vapour-pressuredeficit. However for strict rainfed (i.e. withoutirrigation) Mediterranean conditions it may be lessuseful for predicting crop performance (Villegas etal., 2000; Royo et al., 2002). Guidelines for usingthe canopy temperature are given at http://w w w . p l a n t s t r e s s . c o m / a d m i n / f i l e s /IRT_protocol.htm. Performance of ∆ predicting yieldappears less affected by growing conditions (Royoet al., 2002; Araus et al., 2003a; but see below) evenwhen it depends on the organ analysed (Acevedo,1993; Araus et al., 1998; Table 1).

QTL mapping populations in maize (Sanguinetiet al., 1999) and sunflower (Herve et al., 2001) haveshown that leaf conductance is under genetic controlat multiple loci. By contrast, Clarke (1997) reportedsegregation at a single locus for leaf conductance ina durum wheat cross. Forster (2001) reported sevenQTLs affecting the response of carbon isotopecomposition (δ13C) to salt stress in barley (see alsoEllis et al., 2002 in barley). Other authors have alsoreported several major QTLs for ∆ (or δ13C) in otherplant species under drought (Masle et al., 1993;Thumma et al., 2001; Specht et al., 2001; Brendelet al., 2002). AFLP molecular markers have widegenome coverage and can account for much of thevariance in carbon isotope discrimination in breedingpopulations of bread wheat (Richards et al., 2002).

Water use efficiencyWater use efficiency (WUE) here is defined as

above-ground dry biomass per unit of watertranspired, and it therefore depends primarily onphotosynthetic transpiration efficiency (TE: netphotosynthetic assimilation per unit watertranspired). Alternative definitions are frequentlyused for WUE (e.g. total dry biomass production toevapotranspiration) and TE (e.g. above ground drybiomass per unit water transpired) and therefore

caution is needed when reading literature on thisissue (Steduto, 1996).

Increasing WUEAs for water use, WUE may also be improved by

early vigour and phenological adjustment (namelycrop earliness), but this time through a reduction intranspiration rate driven by a lower vapour pressuredeficit during the period of maximum biomassaccumulation. Providing enough genotypicvariability is available, other alternatives forimproving WUE may consist in increasing surfacereflectance (Richards et al., 1986; Febrero et al.,1998) or decreasing residual transpiration (Rawson& Clarke 1988; Araus et al., 1991), or selecting forsmaller and more erect leaves (Richards et al., 2002).A longer term alternative to increasing WUE maybe by enlarging the photosynthetic capacity(Dunwell, 2000). While selection for higher rates ofleaf photosynthesis has not generally resulted inimproved yield under temperate conditions, greatersuccess might be expected under abiotic stress (seereferences in Araus et al., 2002), where molecularbiology techniques may play an important role. Analternative possibility is the transfer of C4photosynthetic metabolism into C3 plants. In thisregard the large amount of work done in transgenicrice shows the way forward for other cereals (seereferences in Sheehy et al., 2000; Dunwell, 2000).Moreover, recent findings on the lack of requirementof Kranz anatomy for a C4 photosynthesis interrestrial plants (Sage, 2002) may add new cluesfor redesigning C3 cereal photosynthesis bysimplifying the requirements for introducing C4metabolism.

QTLs have been reported for traits conferringperformance of cereals under Mediterraneanconditions such as WUE, and other complexquantitative yield-determining characteristics (Teulatet al., 1997, 1998; Slinkard, 1998; Yin et al., 1999).Transgenic wheat plants with higher WUE andimproved total biomass, root and shoot dry weightsunder water deficit were obtained by introducing theABA-responsive barley gene HVA1, a member ofGroup 3 LAE protein genes (Sivamani et al., 2000).However WUE is a complex trait, involving manyQTLs (usually with small effects). Reports on themultiplicity of QTLs for ∆ (or δ13C) for a given plantspecies (see above) also support this conclusion.Moreover the G × E interactions on the expressionof these QTLs are frequently large (e.g. Kjaer &Jensen, 1996).

Traits that improve stress tolerance may eventuallyprevent early senescence and help maintain high TE.Thus ABA may also have a role in tolerating celldehydration. Thus genes encoding for LEA proteinsproduced in developing seeds are similar to thoseexpressed in drought stressed vegetative tissue of


wheat (Curry et al., 1991), and ABA can induceexpression of these proteins. Antioxidants such assuperoxide dismutase and ascorbate peroxidaseincrease in response to drought stress (Mittler &Zilinskas, 1994) and may play a role in tolerancesince excess radiation and increased photorespirationassociated with stress can result in accumulation ofactive oxygen species. Xanthophylls also may playa role in tolerating excess energy during water stress.This can be assessed indirectly throughspectroradiometry indices (Tambussi et al., 2002).However, even though expectations are promising(see Dunwell, 2000) the performance of all thesetraits in selecting cereal genotypes with higher stresstolerance has not yet been established, and many ofthem may be short-term symptoms rather than long-term tolerance mechanisms.

Higher versus lower Transpiration EfficiencyTranspiration efficiency (TE) is difficult to

measure. However, it can be assessed in a faster andeasier way through the analysis of the carbon isotopecomposition of the plant material and its furthertransformation to carbon isotope discriminationvalues. The physiological basis of the negativerelationship between TE and ∆ is well establishedfor both wheat (Farquhar & Richards, 1984) andbarley (Hubick & Farquhar, 1989). Since then, theusefulness of ∆ as a breeding tool has beenextensively studied in bread wheat (e.g. Farquhar &Richards, 1984), durum wheat (Araus et al., 1998)and barley (Acevedo, 1993). The advantages of ∆in breeding are related to its integrative value (Table1), genetic variability, the relatively low G × Einteractions and high heritability values and the easeof sample preparation and the automation of analysis.Moreover, although the cost of ∆ analysis isrelatively high there are surrogates that allow a faster,cheaper and non-destructive assessment of grain ∆(Ferrio et al., 2001).

In principle (pace Passioura�s) genotypes showinglower ∆ (i.e. higher TE) would be more productiveunder water-limited conditions. However, underMediterranean field conditions, except for very poorenvironments, this is not the case, and positiverelationships between ∆ and yield are usuallyreported. Thus Voltas et al. (1999) found for barleythat genotypes with lower ∆ performed better in low-yielding environments. On the contrary, a highgenotypic ∆ was an advantage in medium and high-yielding environments. The same trend has beenreported for durum wheat (Araus et al., 2003b). Evenif phenology is involved in this positive relationship(earlier genotypes having higher ∆ and yield thanlater genotypes in drought conditions) it is not theonly factor involved (Araus et al., 1998, 2003b;Voltas et al., 1999). Results on barley also supportedthe idea that a constitutively high ∆ may have been

driven by a large genotypic reproductive sink (Voltaset al., 1999).

Different studies dealing with genetic progress onbread wheat under irrigated conditions havedemonstrated strong, positive associations betweenleaf conductance (as well as with ∆ and CTD) andgrain yield (Fischer et al., 1998; Lu et al., 1998).Providing constancy in vapour pressure deficit theseresults mean that genotypes performing better underirrigation are those with a lower TE values. Highstomatal conductance in recent CIMMYT wheatcultivars was associated with cooler canopies andhigher photosynthetic rates (see references in Arauset al., 2002, 2003a). On the contrary, reduced leafconductance slows transpiration and water use toenhance crop water-use efficiency for bread wheatcrops with a limited water supply (Condon et al.,1990; Morgan & LeCain, 1991). For conditions otherthan those typical of the Mediterranean (whererainfalls occur mainly in winter and spring) such anapproach may be sound. Thus in summer-dominantrainfall environments (such as in the northernAustralian wheat belts) where cultivation relies onwater accumulated in the soil before sowing,selection for reduced ∆ increases aerial biomass andgrain yield of rainfed bread wheat; yield advantageincreasing in lower rainfall environment (Condonet al., 2002; Richards et al., 2002; Rebetzke et al.,2002).

These observations further support the assumptionthat drought tolerance and high yield potential undernon-limiting growing conditions may be antagonisticconcepts in cereals.

Harvest indexWhereas harvest index (HI) is reaching its ceiling

in favourable environments (Austin, 1993, 1999;Araus et al., 2003a), in Mediterranean conditionsthere is still room for improvement through changesin crop development. Thus drought stress at criticalstages, relative proportions of pre- to post-anthesisbiomass, mobilisation of pre-anthesis assimilates toreproductive organs and patterns of water supplyduring the cycle may all limit HI and thus final yield(Araus et al., 2002; Richards et al., 2002). Forexample an alternative to raising HI withoutpenalising response to drought would rely on theselection of cultivars with low tillering capacity (i.e.preventing the production of wasteful tillers), whichseems to result in substantially larger ears. Otherstrategies rely on the duration of the spike growthperiod to allow more assimilate supply to thegrowing florets, thus reducing floret abortion justbefore anthesis. This longer stem-elongation periodwould be compensated for by a reduced earlierperiod so that there would be no relevant differencesin the amount of water extracted (Slafer et al., 2001).Heading date has been a major trait studied in all


cereals. The genetic location of major genes andQTL for heading time is well documented and thepleiotropic effects on other traits studied (Sourdilleet al., 2000; Langridge et al., 2001).

In addition, selecting for a high HI under optimalconditions usually translates into higher yields inmoderate to mild drought Mediterranean conditions(Sayre et al., 1997; Araus et al., 2002; Richards etal., 2002 and references therein). Thus traits such asphotoperiod and vernalisation response and dwarfinggenes, which contributed significantly to increasedyields in the past, are often considered to be usefulfor future gains in yield potential (Law et al., 1991;Worland et al., 1998; Sears, 1998; Slafer et al., 2001),but interest should also be focused on producingcultivars with faster growth and greater biomassduring grain filling (Austin, 1999; Villegas et al.,2001), even though these are much more complextraits.

Increasing source capacity during grain fillingTraditionally when photosynthetic activity of

cereals has been studied, emphasis has been placedon leaves, neglecting other photosynthetic sources.However, the photosynthetic contribution of the earcould be more important than that of the flag leaf indetermining HI and yield, not only under wellwatered conditions (Fig. 3), but also under waterstress (Fig. 4). Several factors may be involved:higher total photosynthetic capacity of the ear thanthe flag leaf, photosynthetic tissues within the earclose to the grains, delayed senescence of the earcompared with flag leaf under Mediterraneandrought conditions as well as perhaps some specificcharacteristics conferring a higher TE (Araus et al.,1993; Bort et al., 1994, 1996) or a better osmoticadjustment (Blum, 1985; Teulat et al., 1998, 2001).Therefore genotypic variability in ear photosynthesisshould also be considered when attempting toimprove source capacity during grain filling.

Some Lessons for the Future

Currently basic research emphasises genomics andgenetic discovery. This is leading to a genocentricview of priorities in which the focus is on finding afunction for each of the genes in the genotypes(Miflin, 2000). In addition, marker-mediatedanalyses are revealing detailed information about thegenetic control of adaptation and stress responses,and the influence of specific genes on yieldperformance in different environments (Langridgeet al., 2001). However this is not sufficient for cropimprovement, because yield is dependent on cropgenotype, and the interaction between genotype andenvironment is crucial, particularly when stressesare present. Thus paraphrasing Miflin (2000) toimprove crops and to meet the challenges ahead, the

genotypic view and emphasis on genomics needs tobe balanced by a phenocentric approach. In thecontext of breeding for stress conditions thisapproach is crucial to screening for and measuringimportant phenotypic traits if full exploitation of theopportunities offered by marker technology are tobe realised.

Functional genomics may reveal the genetic basisof any given phenotypic response to the environmentand hence open up the possibility for genetictransformation. This has opened many expectationsfor producing progenitors of performing breeding

1800 20 40 60 80 100 120 140 160

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2

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4

5

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3

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7

1800 20 40 60 80 100 120 140 160

A (nmol CO2 organ-1 s-1)

FLAG LEAF

EAR

(a)

(b)

Gra

in y

ield

per

pla

nt (g

pla

nt-1)

Fig. 3. Correlations between the total netphotosynthesis of the ear (a) and the flag leaf blade(b) with the total grain yield per plant at anthesis fordurum wheat plants growing at different levels ofavailable water in the substrate (ASW). Each pointrepresents a single measurement (either of gasexchange or grain yield per plant) of a pot plant, withthree plants measured for each of the six genotypesused. Figure redrawn from Abbad et al. (2003).(a) ! 10% ASW, r2 = 0.774

! 20% ASW, r2 = 0.608" 40% ASW, r2 = 0.276" 60% ASW, r2 = 0.679# 100% ASW, r2 = 0.605

(b) ! 10% ASW, r2 = 0.551! 20% ASW, r2 = 0.459" 40% ASW, r2 = 0.542" 60% ASW, r2 = 0.145# 100% ASW, r2 = 0.051


(i.e. environmental condition, different plant tissue,phenological stage, time of day, dosage etc.). Thetechniques involved in genomics are now beingapplied to specific biological issues including abioticstress. At present there is a massive database resourceto explore (e.g. http://wheat.pw.usda.gov/genome).The challenge now is to sift through these databasesto identify genes associated with QTLs for droughtand other stress tolerances (Forster et al., 2000b).Considerable investment will be required beforegenetic engineering as a means of improving cerealcultivars for different target environments becomesroutine. Furthermore, as stressed above, much of theresearch at the molecular level considers processesrelated to dehydration tolerance and recovery(Cushman & Bohnert, 2000) rather than droughtavoidance strategies. Crop productivity depends onstrategies for avoiding stress through deep rooting,and the timing of developmental events such thatWUE is optimised with respect to grain formation(Richards et al., 2001).

Further advances are expected to benefit from theamalgamation of concepts and methodologies fromdifferent disciplines rather than applying themselectively. As concluded by McCouch (2001)�efforts to understand the functional significance ofa gene or suite of genes in virtually any biologicalcontext involve both keen observation of phenotypeand intensive computational comparisons of thestructural properties of DNA or protein�. For plantbreeding this means that physiological understandingof crop responses to environment is necessary if anefficient integration of biotechnologies is to berealised.

Acknowledgements

This study was supported in part by the researchproject AGL2002-04285-C03 from the SpanishMinistry of Science and Technology and theEuropean Union project WASAMED (ICA3-CT2002-10013).

Red

uctio

n (%

) in

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ry w

eigh

t by

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40

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10 15 20 25 30Ear gross photosynthesis (nmol CO2 s

-1 ear-1)

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earDarkenedflag leaf

Darkening treatment

Ker

nel d

ry w

eigh

t (m

g)To

tal g

rain

yie

ld p

er e

ar (g

)Fig. 4 (see left). (a) Relationship between ear grossphotosynthesis (Pn + Rd) 2 wk after anthesis and thereduction in kernel dry mass by darkening either theflag (blade + sheath) leaf or the ear from 1 wk afteranthesis to maturity. Data are from five irrigated durumwheat genotypes. Redrawn from Araus et al. (1993).(b) Effect of darkening the flag leaf or the ear on (i)grain yield per ear and (ii) kernel dry weight of awnlessand awned genotypes. Values are averages and SE ofthe mean of six to 10 replicates of irrigated barley.Redrawn from Bort et al. (1994).(a) ! r2 = 0.82* ear darkening

! r2 = 0.90* leaf darkening(b) Awnless geotype, empty bars; awned genotype,

grey bars.

lines in the forthcoming years (Dunwell, 2000).However, as outlined above, adaptation to stress atthe whole plant level involves the interaction ofmany genes which are expressed at multiple levels


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Breeding cereals for Mediterranean conditions: ecophysiological clues for biotechnology application