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www.publish.csiro.au/journals/fpb Functional Plant Biology, 2004, 31, 1149–1159

Physiological and morphological responses to water stressin Aegilops biuncialis and Triticum aestivum genotypes

with differing tolerance to drought

Istvan MolnarA,E, Laszlo GasparB, Eva SarvariB, Sandor DulaiC,Borbala HoffmannD, Marta Molnar-LangA and Gabor GalibaA

AAgricultural Research Institute of the Hungarian Academy of Sciences, Brunszvik u. 2, H-2462,Martonvasar, Hungary.

BDepartment of Plant Physiology, Eotvos University, Pazmany P. setany 1 / C, H-1117, Budapest, Hungary.CDepartment of Plant Physiology and Botany, Eszterhazy College, H-3301, POB 43, Eger, Hungary.

DGeorgikon Faculty of Agriculture University of Veszprem, Keszthely, Hungary.ECorresponding author. Email: [email protected]

Abstract. The physiological and morphological responses to water stress induced by polyethylene glycol (PEG)or by withholding water were investigated in Aegilops biuncialis Vis. genotypes differing in the annual rainfallof their habitat (1050, 550 and 225 mm year−1) and in Triticum aestivum L. wheat genotypes differing in droughttolerance. A decrease in the osmotic pressure of the nutrient solution from –0.027 to –1.8 MPa resulted insignificant water loss, a low degree of stomatal closure and a decrease in the intercellular CO2 concentration (Ci) inAegilops genotypes originating from dry habitats, while in wheat genotypes high osmotic stress increased stomatalclosure, resulting in a low level of water loss and high Ci. Nevertheless, under saturating light at normal atmosphericCO2 levels, the rate of CO2 assimilation was higher for the Aegilops accessions, under high osmotic stress, than forthe wheat genotypes. Moreover, in the wheat genotypes, CO2 assimilation exhibited less or no O2 sensitivity. Thesephysiological responses were manifested in changes in the growth rate and biomass production, since Aegilops(Ae550, Ae225) genotypes retained a higher growth rate (especially in the roots), biomass production and yieldformation after drought stress than wheat. These results indicate that Aegilops genotypes originating from a dryhabitat have better drought tolerance than wheat, making them good candidates for improving the drought toleranceof wheat through intergeneric crossing.

Keywords: Aegilops biuncialis, CO2 fixation, drought tolerance, stomatal conductance, wheat.

Introduction

Aegilops species are closely related to Triticum species(Van Slageren 1994) and are widely used as genetic resourcesfor wheat improvement, especially against pests and diseases.For example, genes Sr32, Lr35 and Pm13 have beentransferred from Aegilops sp. to wheat, resulting in resistanceagainst stem rust (McIntosh 1991), leaf rust (Kerber andDyck 1990) and powdery mildew (Ceoloni et al. 1992),respectively. However, little information is available on theabiotic stress tolerance (such as drought, heat and light stress)of Aegilops sp. (Comeau et al. 1993; Rekika et al. 1997,Zaharieva et al. 2001) and there is no information on gene

Abbreviations used: A, net CO2 assimilation rate; Ci, intercellular CO2 concentration; DW, dry weight; FW, fresh weight; Fv / Fm, optimal quantumyield of PSII; �F / Fm

′, effective quantum yield of PSII; gm, mesophyll conductance; gs, stomatal conductance; NPQ, non-photochemical fluorescencequenching; RWC, relative water content; SW, water-saturated weight; QA, the first stable electron acceptor of PSII; �L, leaf water potential.

transfer from Aegilops sp. to wheat in order to improveabiotic stress tolerance.

Aegilops biuncialis Vis., an annual, allo-tetraploid species(2n = 4x = 28) with a UUMM genome, originated from across between Aegilops umbellulata (U genome), as thefemale parent and Aegilops comosa (M genome) as the maleparent. As demonstrated by Fernandez-Kalvin and Orellana(1992) the UM genome of Aegilops and the AD genome ofwheat show strong homoeologous pairing affinity in meiosis,resulting in the easy crossability of wheat and A. biuncialis(Logojan and Molnar-Lang 2000; Linc et al. 2003). It isknown that some accessions of A. biuncialis are highly

© CSIRO 2004 10.1071/FP03143 1445-4408/04/121149

1150 Functional Plant Biology I. Molnar et al.

resistant to barley yellow dwarf luteovirus (Makkouk et al.1994), while others have good frost resistance (Ekmekciand Terzioglu 2002). A. biuncialis grows in Mediterraneanand western Asiatic regions characterised by a drysummer season with high temperature and high irradiance(Van Slageren 1994). The annual rainfall in these habitatsrange from 225 to 1250 mm. Nevertheless, no data have beenpublished on the response of A. biuncialis to drought stress.The successful adaptation of some A. biuncialis accessionsto a dry habitat may make them useful for the geneticimprovement of wheat against drought stress.

Drought is one of the most important biomass-limitingstress factors in agriculture (Araus et al. 2002), causingthe retardation of stem and root growth (Blum et al. 1997;Frensch 1997; Munns 2002), a decrease in the assimilatingleaf area (Passioura 1988) and the number of tillers (Coneet al. 1995) resulting in substantial yield losses. Duringdrought stress, the relative water content (RWC) and waterpotential (�) of the plants decrease (Bajji et al. 2001), leadingto a significant decline in photosynthetic CO2 fixation. Thereduction in the net CO2 assimilation rate (A) is mainlydue to stomatal closure, which is the most efficient wayto reduce water loss (Cornic 2000). When the stomata areclosed, so that the conductance of water vapour through thestomata is low (gs), the diffusion of CO2 into the leaves islimited, resulting in a decrease in the leaf intercellular CO2concentration (Ci, Cornic 2000). However, during droughtstress, the diffusion of CO2 from the leaf intercellularspaces to the chloroplast of the mesophyll cells may also bereduced (Delfine et al. 1999; Loreto et al. 2003) dependingon the structure of the mesophyll cell (Bongi and Loreto1989) and the rearrangement of the intercellular spaces(Delfine et al. 1998); These processes are manifested as adecrease in mesophyll conductance (gm) and an increasein Ci. Besides the limitation of CO2 diffusion from the airto the chloroplasts, metabolic factors such as the reducedactivity or degradation of Rubisco, the disturbed regenerationof ribulose-1,5-bisphosphate (RuBP) through the inhibitionof the photosynthetic electron transport and / or insufficientATP may also play an important role in the reduction ofphotosynthetic CO2 assimilation (A) during drought stress(Centritto et al. 2003; Chaves et al. 2003). Moreover, dueto stomatal closure, the CO2 / O2 ratio may be modified,resulting in a shift from the carboxylase to the oxygenasefunction of Rubisco. In this case there is an increase inphotorespiration (Lawlor and Cornic 2002; Tezara et al.1999).

In addition to physiological modifications, plants are ableto adapt to water deficiency by shortening their growth cycle,but this reduces the total biomass production and therefore theyield. Another way of avoiding drought stress is to intensifyroot growth with the retention of normal shoot growth,resulting in a relative increase in water uptake comparedwith water loss (Serraj and Sinclair 2002). Physiological

and morphological acclimatisation traits tend to dependon the climate in the native habitat of the plant species(Zaharieva et al. 2001; Bultynck et al. 2003). Since theAegilops species are indigenous to the Mediterranean region,which is characterised by a dry summer season, these plantswere obliged to develop various acclimatisation strategies inorder to survive a shorter or longer period of drought.

The aim of the present study was to compare the responsesto drought stress in three Aegilops biuncialis accessions,adapted to different rainfall conditions, with several wheatgenotypes possessing different levels of drought tolerance.The drought stress was induced either by PEG treatment ina hydroculture system or by withholding water in soil potexperiments. The experiments were performed in order tofind A. biuncialis accessions suitable for improving wheatdrought tolerance through intergeneric crossing.

Materials and methodsPlant materials

A comparison was made of the responses to drought stress, inducedeither by PEG treatment in a hydroculture system or by withholdingwater in soil pot experiments, of three Aegilops biuncialis Vis.accessions to the responses of several wheat (Triticum aestivum L.)genotypes with various levels of drought tolerance. The threeA. biuncialis accessions (Ae1050, Ae550, Ae225), originating fromdifferent habitats (with annual rainfall of 1050, 550 and 225 mm) wereprovided by the ICARDA gene bank in Syria. The winter wheat Mv9kr1,a genotype containing a crossability gene (Molnar-Lang et al. 1996),has moderate drought tolerance. The wheat cultivar Sakha is drought-tolerant (Triverdi et al. 1991), while Cappelle Desprez is drought-sensitive (Galiba et al. 1992). Three wheat cultivars that are currentlygrown but have unknown drought tolerance (Mv Fatima, Mv Magmaand Renan) were also used in the soil pot experiments.

Experimental conditions

The water deficit occurring during drought stress was induced eitherby increasing the osmotic pressure of the hydroculture media throughthe addition of non-penetrating polymers of PEG as published byRanjbarfordoei et al. (2000) or by decreasing the water supply in the soil,as reported by Biehler and Fock (1996). The osmotic stress generatedby PEG and the water deficit stress in soil resulted in similar symptomsin many plant species, including a decrease in the relative water content(RWC), leaf water potential, photosynthetic CO2 fixation, biomassproduction and yield formation (Zhang and Kirkham, 1995), confirmingthe stimulation of drought stress by PEG-induced osmotic treatments. Inthese experiments, the drought stress lasted for 4–5 weeks, simulatingthe situation generally occurring in central Europe.

Hydroculture system

Germinated seedlings of wheat and A. biuncialis were grown in half-strength modified Hoagland nutrient solution (Nagy and Galiba 1995)in a plant growth chamber (Conviron, Manitoba, Canada) with 12 / 12 hday / night cycles of 18 / 13◦C for 7 d followed by 20 / 18◦C for 7 d with70% relative humidity and 200 µmol m−2 s−1 light intensity. Osmoticstress was then imposed by applying PEG 6000 (Sigma, St Louis, MO)in 7-d cycles at increasing concentrations of 12, 15, 18 and 21% (w / v),resulting in osmotic potentials of –0.45, –0.72, –1.14 and –1.8 MPa,respectively (Fig. 1). For each genotype, 20 plants (five plants / 1.5-Lpot) were grown in a hydroculture system. The solution was renewedtwice a week. Two independent, identical experiments were performed.

Responses to water stress in wheat and Aegilops biuncialis Functional Plant Biology 1151

–2.5

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Time of treatment (d)

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a)

Measurement

0%PEG

12%PEG 15%

PEG 18%PEG

21%PEG

0%PEG

Fig. 1. Changes in the osmotic potential and PEG concentration ofHoaglands solution during the experiment. Measurements were madeat the points indicated by arrows.

Samples were taken before PEG application (control), on the seventhday after the application of various PEG concentrations and after 2 and7 d of regeneration without PEG. Only the youngest, fully expanded (i.e.exposed auricle), non-senescing leaves were used for the measurements.

Pot experiment

The pot experiments, arranged in a randomised complete block designwith four replicates, were conducted at the Georgikon Faculty ofAgriculture, University of Veszprem, Keszthely, Hungary. The plantswere grown in an unheated greenhouse with natural sunlight. Seedsof seven genotypes were sown in plastic pots filled with 6.5 kg ofsoil composed of 750 g eutric cambisol kg−1 and 250 g sand kg−1

(waterholding capacity: 340 g kg−1). After sowing, each pot was broughtto waterholding capacity. In the control experiment (well watered) thepots were irrigated daily until full ripening (Zadoks’ scale: 91; Zadokset al. 1974) The drought stress (dry treatment) was induced by addingonly 50% of the water given to the control pots. Stress treatment wasstarted at the mid-flowering stage of plant growth (Zadoks’ scale: 65)and continued till maturity, because in Hungary drought generallyoccurs during the grain-filling period. This experiment focused primarilyon growth (shoot and root weight, biomass production) and yieldparameters.

Determination of leaf water potential, water content and growthparameters

The leaf water potential values (�L) were determined in a pressurechamber (PMS Instrument Co., Corvallis, OR) with N2 gas. The watercontent of the leaves was expressed as relative water content (RWC)according to the following equation:

RWC = (FW − DW) × 100 / (SW − DW) (1)

where FW is the fresh weight, SW the water-saturated weight and DWthe dry weight after drying for 12 h at 105◦C.

The shoot and root weight and the relative biomass content of wholeplants were determined at the end of the growing period in terms of drymatter in the pot experiment. In the hydroculture system, the growingparameters were determined at the end of the –1.8 MPa osmotic stresstreatment.

Gas exchange measurements

The CO2 assimilation of intact leaves was measured in a standard gasmixture of 340 µmol mol−1 CO2 and 21% O2 in N2 using an infrared gas

analyser (LCA-2, Analytical Development Co., Ltd, Hoddesdon, UK).The light response curve of CO2 fixation was determined in the range100–1300 µmol m−2 s−1 as described by Darko et al. (1996). The ratesof net CO2 fixation (A), stomatal conductance (gs) and intercellularCO2 concentration (Ci) were calculated in the light-saturated state ofphotosynthesis using the equations of von Caemmerer and Farquhar(1981). In another experiment, A was determined at 21% O2 and then at1.5% O2 from a standard gas mixture (340 µmol mol−1 CO2 and 1.5%O2 in N2) at saturating light intensity.

Fluorescence induction and quenching analysis

The in vivo chlorophyll a fluorescence of intact leaves was measuredafter 30 min dark adaptation using a frequency and amplitude-modulatedchlorophyll fluorometer (PAM101–103, H. Walz, Effeltrich, Germany)as described by Dulai et al. (1998). Photosynthesis was induced for15 min by continuous actinic light of 200 µmol m−2 s−1 produced by aSchott KL-1500 light source (Schott, Essex, UK). The nomenclatureof van Kooten and Snel (1990) was used to determine the optimalquantum yield of PSII (Fv / Fm) measured after 30 min dark adaptation.This parameter describes the maximal quantum efficiency of chargeseparation in PSII, when all the QA (the first stable electron acceptorof PSII) is oxidised (Krause and Weis 1991). The quantum efficiencyof PSII photochemistry, reflecting the photochemical utilisation ofabsorbed light energy at a given light intensity (in the present case200 µmol m−2 s−1) was calculated as �F / Fm

′, as described by Gentyet al. (1989). The Stern-Volmer coefficient (NPQ = Fm / Fm

′ – 1) wasused to determine the non-photochemical fluorescence quenchingparameter, reflecting the alternative (heat) dissipation processes ofexcess excitation energy (Govindjee 1995). Both the effective quantumyield of PSII photochemistry (�F / Fm

′) and the non-photochemicalfluorescence quenching (NPQ) parameters were determined at thesteady-state level of photosynthesis.

Statistical analysis

The results were obtained in two independent series of experimentsand are the means ± LSD (5%) of six measurements per treatmentfor CO2 gas exchange and fluorescence quenching analysis and eightmeasurements per treatment for RWC, �L, biomass production andgrowth parameters. The measurements were performed on differentplants. Differences between the treatments and genotypes weredetermined by means of two-factor analysis of variance (ANOVA) atthe P<0.01 or P<0.05 level and three-factor ANOVA in the case ofCO2 gas-exchange measurements at low O2 concentration.

Results

Effects of PEG-induced osmotic stress on the watercontent and water potential of the leaves

In order to determine the physiological responses of Aegilopsand wheat species to drought stress, drought stress wasinduced by gradually decreasing the osmotic pressure ofthe nutrient solution from –0.025 to –1.8 MPa by addingPEG in increasing concentrations every 7 d (Fig. 1). Mildosmotic stress (to –0.7 MPa osmotic potential) only resultedin a slight reduction in the RWC of the leaves (Fig. 2A)and in �L (Fig. 2B) in all the genotypes compared with thecontrols. A decrease in the water potential of the solutioncaused a more rapid reduction in RWC and �L in theA. biuncialis accessions than in the wheat genotypes. Thegreatest decline in �L (–2.3 MPa) was recorded for Ae225,not for Ae550, which exhibited the greatest water loss. A


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Osmotic treatment Recovery

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a)

2 d 7 d

B

Fig. 2. Effect of increasing osmotic stress followed by 7 d regeneration on (A) the relativeleaf water content and (B) the leaf water potential of three wheat varieties (dotted lines)and three A. biuncialis genotypes (continuous lines). (�) Mv9kr1, (•) cv. Cappelle Desprez,(�) cv. Sakha, (�) Ae1050, (◦) Ae550, (�) Ae225. The results are means ± LSD (5%) of dataof 16 plants per treatment.

comparison of RWC and �L revealed a linear relationshipfor all the genotypes but, the steepness of the straight linediffered from one genotype to the other (data not presented).During regeneration in Hoagland’s solution containing noPEG, the three A. biuncialis accessions and the wheat lineMv9kr1 regained their normal water contents by the endof the second day, while Cappelle Desprez and Sakha stillexhibited substantial water deficiency on the seventh day.

Changes in gas-exchange parameters duringPEG-induced osmotic stress

As the stomata play an important role in the regulationof transpirational water loss, gas-exchange measurementswere made during the osmotic stress treatment in orderto determine the changes in the stomatal conductance(gs), which is proportional to the stomatal aperture, in

the intercellular CO2 level (Ci) and the CO2 assimilationcapacity.

Parallel with the decrease in water content, gs decreasedin all the genotypes (Fig. 3A). The most intensive stomatalclosure was observed for wheats, especially for Mv9kr1and Cappelle Desprez, which exhibited the least water loss.The stomata of the Aegilops accessions (especially Ae225and Ae550) remained more widely open even in the caseof severe (–1.8 MPa) osmotic stress (Fig. 3A) than those ofwheat. Parallel to stomatal closure, there was a large increasein the intercellular CO2 level (Ci) in the drought-sensitiveCappelle Desprez and even in the relatively drought-tolerantSakha. In contrast, in accessions Ae225 and Ae550, mildosmotic stress induced a slight but significant (P<0.05)decrease in Ci, which remained below the control level evenunder severe stress (Fig. 3B). In Mv9kr1 and Ae1050, which


gs

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Fig. 3. Effect of increasing osmotic stress followed by 7 d regenerationon (A) stomatal conductance (gs), (B) intercellular CO2 concentration(Ci) of the leaves and (C) net CO2 assimilation rate (A) in three wheatvarieties (dotted lines) and three A. biuncialis genotypes (continuouslines). (�) Mv9kr1, (•) cv. Cappelle Desprez, (�) cv. Sakha, (�)Ae1050, (◦) Ae550, (�) Ae225. The results are means ± LSD(5%) ofdata of six plants per treatment.

was adapted to the wettest habitat, although there was aninitial decrease in Ci, severe osmotic stress led to a slightincrease in Ci compared with the control level, as in the otherwheats.

Besides the modification of gs and Ci, the net CO2assimilation (A) also decreased during PEG-induced osmoticstress. Before osmotic treatment, no significant differencecould be detected between the different genotypes in the netCO2 assimilation rates. In the case of mild osmotic stress,although the net CO2 assimilation rate declined in all thegenotypes, a substantial decrease could only be observed

in the drought-sensitive variety Cappelle Desprez (Fig. 3C).When the osmotic stress was severe, the CO2 fixationwas strongly inhibited. However, the net CO2 assimilationremained significantly higher in the A. biuncialis accessions,especially in Ae225 and Ae550, than in the wheat genotypes,despite the smaller Ci and more rapid water loss.

When the osmotic stress was relieved, both the stomatalconductance and the values of Ci and CO2 fixation returnedto the initial level within 2 d in the A. biuncialis accessions,indicating the good regeneration capacity of these plants(Fig. 3A–C). In Mv9kr1, however, the restoration of gs andCi could be observed after 2 d, but the CO2 assimilation ratedid not return to the original (control) level until the seventhday. In the case of Sakha and Cappelle Desprez regenerationwas not complete even after a week.

The decrease in CO2 assimilation induced by droughtstress was influenced by diffusional and metabolic factors(Centritto et al. 2003). To determine the existence of thesemetabolic factors, the maximal CO2 fixation at atmosphericCO2 level was measured at low [O2] in the absenceof photorespiration. According to Lawlor (2002), whenmetabolic limitation occurs, the maximal CO2 fixationcapacity fails to return to the control values. No significantdifferences were found between the genotypes for the Avalues measured either at 21% O2 or at low O2 before PEGtreatment (Fig. 4). In the case of accessions Ae225 and Ae550,the CO2 assimilation at low O2 was able to return to thecontrol values after exposure to mild stress (–0.7 MPa). Atsevere stress (–1.8 MPa), although A measured at low O2 wasstill higher than that measured at 21% O2, it did not reach

0

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A (

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Control 1.5% O2

Mild stress 21.0% O2

Mild stress 1.5% O2

Severe stress 21% O2

Severe stress 1.5% O2

Fig. 4. Effect of low O2 concentration (1.5%) on the rate of net CO2

assimilation during mild (–0.7 MPa) and severe (–1.8 MPa) droughtstress in A. biuncialis accessions and wheat genotypes. The rate of netCO2 assimilation was measured under saturating light intensity at 340µmol mol−1 CO2. The results are means ± LSD (5%) of data of sixplants per treatment. CD, Cappelle Desprez.


the control values. In the case of wheat and Ae1050, onlya slight increase in A could be observed after mild stress. Inthe wheat genotypes, the highly reduced rates of CO2 fixationwere not modified significantly as the result of low O2 undersevere stress conditions (Fig. 4), indicating the complete O2insensitivity of CO2 assimilation.

Photosynthetic electron transport processes duringosmotic stress

One of the metabolic factors affecting CO2 assimilation isthe inhibition of photosynthetic electron transport processes.Among these processes, the organisation and maximal chargeseparation capacity of PSII, the photochemical quantumefficiency and the alternative light-energy dissipation(known as non-photochemical fluorescence quenching) wereinvestigated by chlorophyll a fluorescence measurements.

During PEG-induced osmotic stress, no significantdifference was found in the optimal quantum yield (Fv / Fm)of PSII, which reflects the maximal charge separationcapacity and the relative number of active PSII reactioncenters and only small decrease was observed in theeffective quantum yield (�F / Fm

′) of PSII photochemistryat 200 µmol m−2 s−1 light intensity in genotypes Ae550,Ae225 and Cappelle Desprez (Table 1). These results indicatethat the decrease in CO2 assimilation as the result ofPEG-induced osmotic stress is not due to either thelimitation of PSII charge separation or the modification ofphotosynthetic electron transport processes in any of thegenotypes. A substantial change was only detected in theNPQ parameter during osmotic stress. The NPQ calculatedfrom the quenching parameters recorded in leaves at a lightintensity of 200 µmol m−2 s−1 increased considerably as theresult of severe drought stress in genotypes Ae550, Ae225 andCappelle Desprez (Table 1), but no increase was observed forAe1050 or Mv9kr1, while in the genotype Sakha there was a

Table 1. Optimum quantum yield of PSII (Fv / Fm), steady-state effective quantum yield ofPSII (�F / Fm

′) and non-photochemical fluorescence quenching (NPQ) parameters at the endof a period of osmotic stress at –1.8 MPa (Stress) and in control plants of similar age grown in

nutrient solution without PEG (Control)Significant differences are indicated: ∗, P<0.05; ∗∗, P<0.01; ns, not significant. n = 6.

CD, Cappelle Desprez

Fv / Fm �F / Fm′ NPQ

Control Stress Control Stress Control Stress

Ae1050 0.80 0.80 0.56 0.55 0.56 0.66Ae550 0.81 0.80 0.585 0.43∗∗ 0.50 1.19∗∗

Ae225 0.80 0.81 0.62 0.52∗ 0.46 0.95∗∗

Mv9kr1 0.81 0.78 0.59 0.58 0.48 0.57CD 0.81 0.81 0.58 0.49∗ 0.57 1.01∗∗

Sakha 0.81 0.79 0.60 0.61 0.50 0.30

Significance levelsGenotype (A) ns ∗∗ ∗∗

Treatment (B) ∗∗ ∗∗ ∗∗

A × B ns ∗∗ ∗∗

slight decrease in NPQ. The increase in NPQ in the above-mentioned genotypes indicates that the dissipation of excessexcitation energy to heat may help to avoid the overreductionof photosynthetic electron transport compounds and thus,oxidative damage to PSII.

Effect of PEG-induced osmotic stress on rootand shoot growth

At the end of the PEG-induced osmotic stress treatments,the root and shoot growth parameters were determined andthe data were compared with the values obtained for controlplants of similar age grown in Hoagland’s solution withoutPEG (Table 2). In the wheat genotypes, a significant reductionwas found in both the shoot and root growth compared withthe untreated samples. This reduction was no more than 10%in the A. biuncialis accessions. Osmotic stress induced greaterroot growth in the case of Ae225 and Ae550, leading to anincrease in the root / shoot ratio in these plants. In the caseof Sakha and Mv9kr1 the increase in the root / shoot ratiowas due to the more intensive inhibition of shoot growththan of root growth. The root and shoot growth inhibitionduring osmotic stress was manifested in a decrease in biomassproduction, determined from the dry weight of the shootsand roots. During osmotic treatment the biomass productionwas 70% and 78% of the control in A. biuncialis accessionsoriginating from drier habitats (Ae225 and Ae550), whileit was approx. 50% in Ae1050 and in the wheat genotypes(Table 2). It could also be observed that the development ofSakha accelerated during osmotic stress, resulting in earlyflowering and spike formation.

Drought-induced changes in growth, biomassproduction and yield formation in pot experiments

From the agronomic point of view, the most important effectof drought stress is the decrease in yield formation. Since it


Table 2. Root length, shoot length and biomass production expressed in terms of dry matter at the end of a period of osmotic stressat –1.8 MPa induced by PEG treatment (Stress) and in control plants of similar age grown in nutrient solution without PEG (Control)

Significant differences are indicated: ∗, P<0.05; ∗∗, P<0.01; ns, non-significant. n = 16. CD, Cappelle Desprez

Shoot length (cm) Root length (cm) Biomass (g)Control Stress Control Stress Control Stress

Ae1050 42.6 40.8 (95.8%) 19.2 18.9 (98.4%) 0.855 0.459∗ (53.6%)Ae550 34.9 31.6∗ (89.7%) 13.0 15.66∗∗ (120.4%) 1.171 0.919 (78.4%)Ae225 39.3 36.3 (92.2%) 18.0 21.0∗ (116.6%) 1.043 0.735 (70.4%)Mv9kr1 46.1 34.7∗∗ (75.3%) 20.1 17.1∗ (85.1%) 1.094 0.567∗∗ (54.0%)CD 52.7 42.7∗∗ (81.0%) 20.0 14.8∗∗ (74.0%) 1.416 0.796∗∗ (56.2%)Sakha 59.0 33.8∗∗ (57.2%) 22.8 19.7∗ (86.4%) 1.400 0.795∗∗ (56.7%)

Significance levelsGenotype (A) ∗∗ ∗∗ ∗∗

Treatment (B) ∗∗ ∗∗ ∗∗

A × B ∗∗ ∗∗ ns

is very difficult to study yield formation in a hydroculturesystem, pot investigations were also performed. Droughtstress was induced by withholding water from the plants,starting at the mid-flowering stage of development. In theseexperiments, the RWC of the leaves, the shoot and rootgrowth, the total biomass production calculated as the totalmass of below- and aboveground parts (data not shown) andthe yield formation were determined. Besides the Aegilopsaccessions and wheat genotypes used in the PEG treatments,additional, widely cultivated wheat genotypes with highyields were also investigated. As in the case of PEG-inducedosmotic stress, the RWC of the leaves decreased in all thegenotypes after withholding 50% water for 2 weeks. Thisdecrease was more pronounced in the Aegilops accessions(Ae1050 and Ae550) than in wheat.

Despite the fact that the shoot and root growthparameters were determined at the end of the growingperiod, when the plants were approximately 3 months old, and

Table 3. Effect of drought stress (50% less water compared with the control) on relative water content (RWC), root dry weight, shoot dryweight and yield in two A. biuncialis accessions and five wheat genotypes

RWC was measured after withholding 50% water for 2 weeks; the growth parameters, biomass production and yield formation were measured atmaturity. Significant differences are indicated: ∗, P<0.05; ∗∗, P<0.01. n = 8. CD, Cappelle Desprez

RWC Shoot (g) Root (g) Yield (g)Control Stress Control Stress Control Stress Control Stress

Ae1050 77.01 54.01∗∗ (70.1%) 10.45 9.76 (93.4%) 1.73 2.83∗ (163.5%) 9.82 7.77∗ (79.1%)Ae550 74.00 48.98∗∗ (66.2%) 8.64 8.9 (103%) 1.56 2.71∗ (173.7%) 8.00 7.90 (98.7%)Mv9kr1 88.01 72.99∗∗ (83.0%) 15.98 15.11 (94.5%) 2.85 4.00∗ (140.3%) 13.50 9.97∗∗ (73.8%)CD 83.00 65.99∗∗ (79.5%) 24.94 21.1∗∗ (84.6%) 4.73 4.55 (96.2%) 16.70 11.19∗∗ (67.0%)Fatima 84.37 74.64∗ (88.5%) 18.18 14.53∗∗ (79.9%) 3.46 3.27 (94.5%) 15.77 11.16∗∗ (70.7%)Magma 85.00 78.00 (91.7%) 18.31 15.22∗∗ (83.1%) 5.37 5.13 (95.5%) 14.54 11.44∗∗ (78.6%)Renan 88.29 65.60∗∗ (74.3%) 21.21 17.93∗∗ (84.5%) 4.48 5.47 (122.0%) 17.22 11.53∗∗ (66.9%)

Significance levelGenotype (A) ∗∗ ∗∗ ∗∗ ∗∗

Treatment (B) ∗∗ ∗∗ ∗∗ ∗∗

A × B ∗∗ ∗∗ ∗∗ ∗∗

the measurements were based not on length, as in the PEGtreatments, but on weight, little or no inhibition of shootgrowth and an increase in root growth could again beobserved in the Aegilops accessions during drought stress. Inwheat, the inhibition of shoot growth was more pronouncedthan in the Aegilops accessions, while the root growthwas reduced, contributing to lower biomass production, asalso found after PEG treatment. These results indicate thatdrought stress had a similar effect on the growth parameterswhether it was induced by PEG treatment or by withholdingwater.

This experiment was focused mainly on yield formation.On the basis of the results, withholding 50% water did notresult in a yield decrease in Ae550. Significant differencesin yield were found between Ae550 and all the investigatedwheats, and between Ae1050, which originated from thewettest habitat, and the most drought-sensitive wheats,Cappelle Desprez and Renan (Table 3). These results indicate


that there was greater relative yield formation during droughtstress in the Aegilops accessions, especially that originatingfrom a dry habitat (Ae550), than in wheat.

Discussion

This is the first report of experiments conducted to investigatethe effects of drought stress on A. biuncialis accessionsadapted to different rainfall conditions and to compare theseresults with those achieved for wheat genotypes with differentdrought sensitivities. The aim was to determine whethersuitable A. biuncialis accessions could be found for use asgene sources to improve the drought tolerance of cultivatedwheat.

The A. biuncialis accessions, especially those adaptedto low-rainfall conditions, responded to increasing osmoticstress with a more rapid reduction in RWC and leaf waterpotential (Fig. 2A, B), and less intensive stomatal closure(Fig. 3A) than the wheats. However, despite this considerablewater loss, A. biuncialis genotypes, were capable ofmaintaining a relatively high rate of net photosyntheticactivity compared with wheat (Fig. 3C).

Stomatal closure has been described as the most efficientway to reduce transpirational water loss (Cornic 2000).However, it may restrict CO2 diffusion into the leaves (Cornic2000), leading to a reduction in Ci and to a decrease in A(Flexas and Medrano 2002). When mesophyll conductanceand metabolic factors do not limit the CO2 carboxylationprocess, the decrease in A is caused primarily by a decreasein the Ci level. This appears to be the case in A. biuncialisaccessions originating from dry habitats (Ae550 and Ae225),where gs and A decreased, while Ci remained below thecontrol level throughout the stress period (Fig. 3). A similarinterpretation was given by Escalona et al. (1999) and Earl(2002) in studies on drought stress in wine grapes andsoybean. In contrast, the wheat varieties Cappelle Desprez(drought- sensitive) and Sakha (relatively drought-tolerant)showed a significant increase in Ci during increasing osmoticstress, while more intensive stomatal closure and a greaterreduction in A could be observed, indicating that besidesstomatal limitation, other metabolic factors may also play animportant role in the limitation of CO2 assimilation. This isalso supported by the slower regeneration of A after osmotictreatment (Fig. 3C). The behaviour of Mv9kr1 and Ae1050was intermediate, since there was a slight decrease in Ciduring mild osmotic stress (as in the Aegilops accessionsoriginating from dry habitats) and a slight increaseduring severe stress (as found in Sakha and CappelleDesprez).

Downton et al. (1988) reported that conclusions basedon Ci values are very uncertain due to non-homogeneousstomatal closure occurring during sudden drought stress,resulting in the overestimation of Ci values. Although inthe present work the effects of slowly increasing drought

stress were investigated and heterogeneous stomatal closureis not a general phenomenon (Gunasekera and Berkowitz1992), further investigations were performed to determinethe contribution of metabolic factors to the limitation of netCO2 assimilation.

Rubisco has both carboxylase and oxygenase functions,depending mainly on the CO2 / O2 ratio in the chloroplast(von Caemmerer 2000). This ratio may be substantiallymodified during drought stress, since stomatal closure,mesophyll conductance and metabolic factors may all alterit. Dutilleul et al. (2003) and Novitskaya et al. (2002)demonstrated that if metabolic factors play an important rolein the decrease in CO2 assimilation during drought stress,the A measured at low O2 level is lower than the maximalCO2 assimilation capacity measured under non-stressedconditions. Therefore, in order to determine the maximal CO2assimilation capacity, A was also measured at atmosphericCO2 level with low O2 (1.5%) concentration and saturatinglight intensity during increasing osmotic stress in the Aegilopsand wheat genotypes, when photorespiration (the oxygenasefunction of Rubisco) is eliminated. In the case of accessionsAe225 and Ae550, the metabolic limitation of photosyntheticCO2 assimilation was only observed to a slight extent undersevere osmotic stress, since in the absence of photorespiration(at low O2) A reached the control values under mildosmotic stress, and increased, though without reaching thecontrol level, under severe osmotic stress. These resultssuggest that the decrease in A during mild osmotic stressin Ae225 and Ae550 was mainly due to the limitation of CO2diffusion into the chloroplast, while there was little metaboliclimitation even under severe osmotic stress. In Ae1050, whichoriginated from a relatively rainy area, however, the metaboliclimitation of CO2 assimilation was observed even under mildosmotic stress, as in the wheat genotypes. In the case ofwheat, not only did the reduced CO2 assimilation underphotorespiratory conditions (21% O2; Fig. 4 column 2) fail toregain the maximal CO2 fixation capacity (Fig. 4 column 1),but little difference was observed between the A valuesmeasured at high and low O2 during mild and especiallysevere osmotic stress (Fig. 4 columns 3 and 4). These resultssupport the hypothesis suggested by the Ci values and theslow recovery of photosynthesis, that besides intense stomatalclosure, the inhibition of the metabolic processes involved inCO2 fixation also plays an important role in the limitationof CO2 fixation during osmotic stress in wheats. However,further investigations and analytical evidence will be requiredfor the exact determination of the ratio of diffusional andmetabolic limitation of CO2 assimilation (Lawlor 2002) inwheat and A. biuncialis genotypes.

Several studies have provided evidence that the inhibitionof photosynthetic electron transport activity during waterdeficiency could be one possible reason for the metaboliclimitation of CO2 assimilation (Keck and Boyer 1974;


Giardi et al. 1996). However, in the present study, no damageto the structure and function of PSII was observed in theA. biuncialis and wheat genotypes, when the drought stresswas induced by osmotic treatments as indicated by theunchanged Fv / Fm (Table 1). Nevertheless, at the most severeosmotic stress level the photosynthetic electron transportprocesses were slightly down-regulated in genotypes Ae550,Ae225 and Cappelle Desprez resulting in an increase in thedissipation of excess excitation energy by heat, as reflectedin the decrease in �F / Fm

′ parameters and in the increasein NPQ parameters. These processes could take part in theprotection of the photosynthetic apparatus (Lawlor 2001;Medrano et al. 2002).

The decrease in photosynthetic CO2 assimilation as theresult of drought stress was manifested in a greater reductionin both total biomass production and shoot growth in thewheat genotypes and in Ae1050, which originated from thewettest habitat, while in genotypes Ae550 and Ae225, whichare adapted to a drier climate, the dry matter productionand shoot growth exhibited less inhibition, and an increasein root growth was even observed during PEG-induceddrought stress. Similar results for shoot and root growth andbiomass production were found when water deficiency stresswas induced in soil by withholding irrigation. Differencescould also be observed between the Aegilops accessions andthe wheat, including cultivated wheat. From the agronomicpoint of view high yield stability (a relatively smalldifference in yield) is the most important trait indicative ofdrought tolerance in plants (Guttieri et al. 2001). As wellas maintaining considerable photosynthetic activity underwater stress and having only a slight reduction in biomass,the decrease in yield in Ae550 was not so great as thatfound in wheat, indicating the greater drought tolerance ofAe550.

Therefore, on the basis of the results it seems thatA. biuncialis genotypes, Ae225 and Ae550, originating fromdrier habitats were able to maintain a considerable level ofCO2 fixation, biomass production and yield formation inspite of the substantially more rapid water loss. The fact thatthere is little metabolic limitation of CO2 fixation in theseAegilops accessions, combined with intensive root growth,may contribute to their rapid regeneration after a short period(4–5 weeks) of drought stress. These properties make Ae225and Ae550 good candidates for chromosome-mediated genetransfer aimed at improving the drought tolerance of wheatin central Europe.

Acknowledgments

This work was supported by Wheat Consortium 4 / 38 / 2001and by OTKA grants T043 120 and T043 502. We alsowish to thank Laszlo Stehli for his excellent technicalassistance and Geza Kovacs for his help in the statisticalanalysis.

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Manuscript received 23 July 2003, received in revised form 28 April2004, accepted 8 October 2004

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