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Available online at www.sciencedirect.com

Environmental and Experimental Botany 63 (2008) 289–296

Physiological responses of the legume model Medicago truncatula cv.Jemalong to water deficit

Catia Nunes a,b,∗, Susana de Sousa Araujo c, Jorge Marques da Silva a,b,Manuel Pedro Salema Fevereiro a,c, Anabela Bernardes da Silva a,b

a Universidade de Lisboa, Faculdade de Ciencias, Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugalb Universidade de Lisboa, Faculdade de Ciencias, Centro de Engenharia Biologica, Campo Grande,

1749-016 Lisboa, Portugalc Instituto de Tecnologia Quımica e Biologica, Laboratorio de Biotecnologia de Celulas Vegetais,

Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal

Received 13 April 2007; received in revised form 4 September 2007; accepted 5 November 2007

bstract

In Medicago truncatula Gaertn. cv. Jemalong plants some mechanisms involved in drought resistance were analysed in response to a progressiveater deficit imposed by suppression of soil irrigation. Withholding water supply until the soil had reached one-half of its maximum water contentad no significant effect on leaf RWC, gas exchanges or chlorophyll fluorescence parameters. Under severe drought conditions, the plants resistanceo water shortage involved mainly drought avoidance mechanisms through a decrease in stomatal conductance. The consequent decrease in thenternal CO2 concentration (Ci) should have limited the net CO2 fixation (A). Since A decreased slightly more than Ci under severe water deficit,on-stomatal limitations of photosynthesis may have also occurred. Analysis of A/Ci curves showed reduced carboxylation efficiency due toimitations in RuBP regeneration and Rubisco activity, confirming the presence of non-stomatal limitations of photosynthesis. Drought tolerance

echanisms involving osmotic adjustment and an increase in cell membrane integrity were also present. Altogether, these mechanisms allowed M.runcatula cv. Jemalong plants to still maintain a quite elevated level of net CO2 fixation rate under severe water deficit conditions. These results

ay contribute to identify useful physiological traits for breeding programs concerning drought adaptation in legumes.2007 Elsevier B.V. All rights reserved.

ater

sLisaJps

eywords: Drought stress; Medicago truncatula; Model legume; A/Ci curves; W

. Introduction

Legumes are the main single source of vegetable protein inuman diets and livestock feed, having major impacts on agricul-ure, environment and health (Graham and Vance, 2003; Youngt al., 2003; Dita et al., 2006). Due to the legumes capacityf symbiotic nitrogen fixation, these plants are often used tomprove soil organic fertility and nitrogen economy (Somers

t al., 2003; Young et al., 2003; Howieson and Ballard, 2004).edicago truncatula, commonly known as barrel medic, is a

orage legume plant of Mediterranean origin, well adapted to

∗ Corresponding author at: Universidade de Lisboa, Faculdade de Ciencias,epartamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal.el.: +351 21 750 00 00x24337; fax: +351 21 750 00 48.

E-mail address: catiamaria@gmail.com (C. Nunes).

omabi(un

098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2007.11.004

relations

emi-arid conditions, namely to alkaline clay soils (Lesins andesins, 1979). This species is considered a legume model hav-

ng characteristics that enable its use in molecular and genetictudies (Barker et al., 1990; May, 2004) and recently is emergings promising molecular farming (Abranches et al., 2005). Theemalong cultivar, the most used in pastures and agriculturalractices (Crawford et al., 1989), has been chosen for genomeequencing (http://www.medicago.org).

It has been estimated that two-thirds of the potential yieldf major crops are usually lost due to adverse growing environ-ents (Bajaj et al., 1999). Most climate change scenarios predictworldwide increase in arid areas, including the Mediterraneanasin (IPPC, 2001 and 2007). Water deficit is therefore the most

mportant abiotic stress and strategies to sustainable use of waterKang et al., 2003) and improve plant drought resistance arergent and should integrate conventional breeding and biotech-ological approaches (Chaves et al., 2003). In the legumes, these

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90 C. Nunes et al. / Environmental and

trategies are vital (Sharma and Lavanya, 2002; Dita et al., 2006)nd the utilization of a model such as the M. truncatula, becomesn advantage. Transgenic approaches to improve M. truncatularought resistance using stress-related genes were recently ini-iated (Araujo et al., 2004; Sheokand et al., 2005; Verdoy etl., 2006). However, as far as we know, its systematic physio-ogical characterisation in response to water deficit has not yeteen performed, limiting the comparison between wild type andransformed lines.

The aim of this work was to evaluate physiologically therought resistance of mature M. truncatula cv. Jemalong plants.he two main components of drought resistance, drought avoid-nce and drought tolerance (Levitt, 1980; Turner, 1986; Mundreet al., 2002), were taken into account. The mechanism of droughtvoidance includes an efficient stomata regulation and those ofrought tolerance involve processes at the cellular level, par-icularly osmotic adjustment. These strategies were evaluatedhrough gas exchange and chlorophyll a fluorescence mea-urements as well as through water relations and membranentegrity determinations. The decrease in CO2 fixation underater deficit conditions was discussed in terms of stomatal

nd/or non-stomatal limitations.

. Materials and methods

.1. Plant material and growth conditions

Medicago truncatula Gaertn. cv. Jemalong seeds were scar-fied in concentrated anhydrous sulphuric acid according toraujo et al. (2004) and sterilised in 50% (v/v) sodium hypochlo-

yde. Afterwards they were immersed in 70% (v/v) ethanol formin, rinsed with distilled water and placed in Petri dishesith soaked filter paper. After 3 days at 4 ◦C in dark con-itions, Petri dishes were transferred to the growth chamberthermoperiod of 25/18 ◦C, day/night, and relative humidityf approximately 40%) where seeds germinated within 2–3ays still in dark conditions. Germinated seeds were main-ained another week in Petri dishes but under light conditionsphotoperiod of 16/8 h, day/night, and a PPFD of around00 �mol m−2 s−1 obtained from white fluorescent and incan-escent lamps, TLD58W/77, Halo Star 64476, L30W/77 and58W/77). Afterwards, seedlings were individually transferred

o 0.5 L pots with standard “terra de Montemor” commercialoil (Horto do Campo Grande, Lisboa, Portugal), enriched withn organic fertiliser (Shacham Ginatada Lda, Israel). The finaloncentration of N, P and K was 0.02 to 0.03% each. The plantsere maintained in the growth chamber, under the same condi-

ions (photoperiod, thermoperiod and relative humidity referredbove), through the entire experiment, even during water stressmposition.

.2. Experimental design

The plants were irrigated every other day with tap water untilhe maximum soil water content was achieved. Eight-week-oldlants were divided in three groups, corresponding to the dif-erent levels of soil water availability they would be submitted

((ar

imental Botany 63 (2008) 289–296

o. One group of plants was maintained fully irrigated through-ut the experiment (control). For all the other plants, the soilater content (SWC) was decreased by withholding water forand 7 days (mild drought and severe drought, respectively).he youngest fully expanded leaf of the main branch of 8–9-eek-old plants was used in all the experiments, unless statedtherwise.

.3. Water status

The soil water status was evaluated through the SWC,xpressed as a weight fraction (Coombs et al., 1987). The follow-ng equation was used: SWC(%) = 100*(FW − DW)/DW, whereW was the fresh weight of a soil portion of the internal areaf each pot and DW was the dry weight of the same soil por-ion after had been oven-dried at 105 ◦C for 4 days. Plant watertatus was evaluated by the leaf relative water content (RWC),etermined according to Catsky (1960).

.4. Gas exchange analysis

Photosynthetic gas exchange measurements were performedn non-detached young and fully expanded leaf using a portablenfra-red gas analyser (IRGA, LCpro+ ADC BioScientifictd., Herfordshire, UK) with controlled atmosphere (approx-

mately 370 �mol mol−1 CO2, relative humidity of 60–70%nd 25 ± 2 ◦C) and a saturating external light source of00 �mol m−2 s−1. The CO2 response curves were obtained byncreasing atmospheric CO2 concentration stepwise from 100o 1000 �mol mol−1 CO2 according to Long and Bernacchi2003) and the leaf internal CO2 concentration (Ci) and net CO2ssimilation rate (A) were recorded. The maximum Rubiscoarboxylation rate (Vcmax) and the maximum rate of electronransport driving regeneration of RuBP (Jmax) were calculatedrom the A/Ci curves according to von Caemmerer and Farquahar1981).

.5. Chlorophyll a fluorescence

Chlorophyll a fluorescence was determined using a modu-ated fluorometer PAM 101 (Heinz Walz, Effeltrich, Germany).n optical fibre passing through the radiation shield of the leaf

RGA chamber, at an angle of 60◦, allowed the simultaneouseasurement of fluorescence parameters and photosynthesis

nder atmospheric CO2 concentrations. The maximal PS IIhotochemical efficiency (Fv/Fm) was determined in leavesre-adapted to dark for 10 min according to Kitajima andutler (1975). When the measuring light was turned on, theinimum level of fluorescence (F0) was attained. After a

aturating pulse of 5120 �mol m−2 s−1, the maximal fluores-ence (Fm) emission was recorded. After 20 min under anctinic light of 800 �mol m−2 s−1 the steady-state of photo-ynthesis was achieved and the leaf electron transport rate

ETR) and the photochemical (qP) and non-photochemicalqN) quenching coefficients were measured and calculatedccording to Genty et al. (1989) and Schreiber et al. (1986),espectively.

Experimental Botany 63 (2008) 289–296 291

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.6. Pigment determination

Segments from the leaves used in the gas exchange anduorescence measurements were immersed for pigment extrac-

ion in 5 mL of methanol and left in the dark at 4 ◦C for ateast 2 days. After spectrophotometric measurements at 470,52.4 and 665.2 nm (Unicam 8700 Series, UV/vis Spectrome-er Unicam 2 td., Cambridge, UK), chlorophyll a, chlorophyll

and carotenoids concentrations were calculated according toichtenthaler (1987).

.7. Electrolyte leakage measurements

Two sets of five leaf discs (0.5 cm diameter) per sampleere rinsed three times with deionised water, to remove solutes

rom damaged cells and veins, and floated for 15 h in the dark,t room temperature, either in deionised water (control) or in0% (w/v) polyethylene glycol 8000. After PEG-induced dehy-ration, discs were carefully washed and floated in 3 mL ofeionised water for 4 h, in order to allow re-hydration of tis-ues. The conductivity of the solutions was monitored usingconductimeter (Con 5—EcoScan, Eutech Instruments, Sin-

apore). Samples were heated at 90 ◦C for 90 min and afterooling to ambient temperature, a second measurement tooklace. The relative membrane injury index (I%) was calculatedsing electrolyte leakage according to Bajji et al. (2002).

.8. Leaf water relations

Water relations parameters, osmotic potential at full tur-or (Π100), osmotic potential and RWC both at turgor lossoint (Π0 and RWC0, respectively) and the elastic modulusalue (ε), were calculated from pressure–volume curves deter-ined using a thermocouple psychrometer (HR 33T, Wescor,ogan, USA) equipped with a C-52 leaf chamber. Three leaflets

rom three young fully expanded leaves were detached andoated overnight (12 h) in distilled water, in the dark at ambi-nt temperature, to achieve full turgor. After the excess wateras removed, the leaflets were exposed to the laboratory envi-

onment. Psycrometric measurements were made in leaf discs0.5 cm diameter), made from progressively dryer leaflets, afterquilibrium was reached inside the leaf chamber (20 min aftereaf disc enclosure). A parallel leaflet was used for RWC deter-

ination. The water relation parameters were calculated fromhe pressure–volume data according to Schulte and Hinckley1985) and Bannister (1986).

.9. Statistical analysis

Data obtained from the three groups of plants was analysedy mean comparisons after evaluation of the normality. Eithert-test or Duncan test was applied, to parametric and non-

arametric samples, respectively. Significant differences were

ccepted at P < 0.05 and represented by different letters. Theelationship between different parameters was tested by regres-ion analysis. The regression model that best fitted the data,valuated by an F-test, was chosen. All the analyses were per- Ta

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292 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296

Fig. 1. Influence of stomatal conductance to CO2 on (A) net CO2 assimilationrli

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Fig. 2. Mean values of net CO2 assimilation rate in the control (whitesquares) and severe drought (black diamonds) M. truncatula plants at dif-ferent internal CO2 concentrations. Measuring conditions were an irradianceof 800 �mol m−2 s−1, a relative humidity of 60–70% and a temperature of2sc

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ate and on (B) internal CO2 concentration. Each data point corresponds to oneeaf of different RWC. The solid line is the linear regression fit to all points ands significant for P ≤ 0.05.

ormed using the programme “Statistical Package for Socialciences” (SPSS, 11.5, 2002).

. Results

.1. High photosynthetic resistance to water deficit

Despite a significant decrease of the soil water contentSWC) (Table 1), no significant differences in leaf relativeater content (RWC), net CO2 assimilation rate (A), tran-

piration rate (E), stomatal conductance to CO2 (gsca) and

nternal CO2 concentration (Ci) were found between plantsnder control and mild drought conditions. Only in severerought conditions, when the SWC reached values lower than5%, the leaf RWC and the gas exchange parameters were

irwo

able 2ffect of water deficit on the maximal photochemical efficiency of PSII (Fv/Fm),uenching (qP and qN respectively) and pigment concentration values (chlorophyll a

Fv/Fm ETR (�mol m−2 s−1) qP q

ontrol 0.79 ± 0.04 744 ± 132 0.56 ± 0.10 0ild drought 0.77 ± 0.05 700 ± 53 0.55 ± 0.04 0

evere drought 0.79 ± 0.02 821 ± 204 0.64 ± 0.14 0

ach value represents the mean ± S.D. (n = 5–20). There were no significant differen

5 ◦C. Each value is the mean of 21 (control) and 8 (severe drought) mea-urements ± S.D. The arrow indicates measurements taken at atmospheric CO2

oncentration (370 �mol mol−1).

ignificantly affected (P < 0.01). Although A and E had simi-ar responses under severe drought, the relative decrease of Eas greater (28 and 42%, respectively), resulting in a signif-

cant increase (P < 0.01) of instantaneous water use efficiencyWUE) (Table 1). The relationship between A and gsca and Cind gsca were linear (Fig. 1) but the stomatal closure inducedhigher decrease on A than on Ci. In fact, for a reduction of

00 mmol m−2 s−1 in gsca, A decreased 15% while Ci decreasednly 9.4%.

The maximal photochemical efficiency of PSII of dark-dapted leaves (Fv/Fm), the electron transport rate (ETR),he photochemical quenching (qP), the non-photochemicaluenching (qN) and the leaf pigments content did not changeignificantly between treatments (Table 2). Total chlorophyllontent was about 3.5 times higher than total carotenoids inde-endently of the SWC and leaf RWC.

.2. Non-stomatal limitations of photosynthesis occursnder severe water deficit

The A/Ci curves obtained for control and severe droughtlants were significantly different (Fig. 2). From A/Ci curves, the

n vivo maximum Rubisco activity (Vcmax) and the maximumate of electron transport used in the regeneration of RuBP (Jmax)ere determined and a highly significant (P < 0.001) decreasef both parameters was observed in the severe drought plants

the electron transport rate (ETR), the photochemical and non-photochemical, total chlorophylls and carotenoids) of M. truncatula leaves

N Chlorophyll a(mg g−1 DW)

Total chlorophylls(mg g−1 DW)

Carotenoids(mg g−1 DW)

.76 ± 0.07 13.1 ± 3.0 14.3 ± 3.3 4.2 ± 0.9

.74 ± 0.13 14.5 ± 4.3 16.06 ± 4.8 4.4 ± 1.2

.74 ± 0.06 13.1 ± 1.4 14.48 ± 1.5 4.3 ± 0.5

ces between treatments for any of the measured parameters at the 5% level.

C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296 293

Table 3Water deficit effect on maximum Rubisco carboxylation rate (Vcmax) and elec-tron transport driving regeneration of RuBP (Jmax) of M. truncatula plantsestimated from A/Ci response curves (Fig. 2)

Vcmax (�mol m−2 s−1) Jmax

Control 67.5 ± 15.7 a 109.9 ± 24.7 aS

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evere stress 34.9 ± 8.4 b 71.9 ± 11.7 b

ach value represents the mean ± S.D. (n = 8–21). Different letters indicateignificant differences (P ≤ 0.001) among treatments.

Table 3). However, the decrease in Vcmax was higher (48%)han in Jmax (36%).

.3. Cell osmotic adjustment and membrane stabilityontributed to drought tolerance

The water relation parameters (Table 4) calculated from theressure–volume curves (Fig. 3) show that severe drought plants,ith lower leaf RWC, had lower osmotic potential at full tur-

ig. 3. Pressure–volume curves of (A) control and (B) severe drought M. trun-atula plants. Each curve corresponds to a different leaf subjected to a gradualehydration induced under laboratory atmospheric conditions. Arrows indicatehe medium inflexion point that corresponds to the interception of the linearortions of the curves.

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able 4ater stress effect on osmotic potential at full turgor (Π100), osmotic potential andodulus value (ε) of M. truncatula plants estimated from pressure–volume curves (F

Π100 (MPa) Π0 (MPa

ontrol −1.15 ± 0.22 a −1.96 ±evere drought −1.78 ± 0.10 b −2.44 ±ach value represents the mean ± S.D. (n = 4). Different letters indicate significant di

ig. 4. Drought effect on membrane injury index. Vertical bars correspond toean values ± S.D. (n = 8). The membrane injury index value is significantly

ifferent (P ≤ 0.05) for control and severe drought plants.

or (Π100) and lower osmotic potential at turgor loss pointΠ0) than control plants. The RWC value at which cells losturgidity rose in severe drought plants, which is consistent with

decrease in cell wall elasticity and, accordingly, with a risen the elastic modulus value (ε). An osmotic adjustment ofells to the imposed water stress of −0.63 MPa, was obtainedrom the difference between Π100 in control and severe droughtonditions.

The injury index (I%) calculated from electrolyte leakageeasurements was significantly lower in severe drought plants

Fig. 4). The cell membranes of control plants suffered morenjury with the water stress imposed by PEG solution than the

embranes of already stressed plants.

. Discussion

The suppression of irrigation considerably reduced the soilater content but only under severe drought conditions the soilater availability induced a decrease in leaf RWC of M. trun-

atula cv. Jemalong plants. In fact no difference in leaf RWCas found when water supply withholding decreased maximum

oil water content to one-half. The utilization of leaf RWC as anndicator of the plant water status is usual (Chaves, 1991; Lawlornd Cornic, 2002). In several legumes cultivars of Phaselus vul-aris (Cruz de Carvalho et al., 1998; Costa Franca et al., 2000),igna unguiculata (Cruz de Carvalho et al., 1998; Scotti Campost al., 1999), Vigna glabrescens (Scotti Campos et al., 1999)

nd Lupinus albus (Pinheiro et al., 2001) the close relationshipetween RWC and predawn water potential found during pro-ressive water deficit supports its utilization as an indicator ofhe plant water status. The maintenance of leaf RWC in mild

RWC both at turgor loss point (Π0 and RWC0 respectively) and the elasticig. 3)

) RWC0 (%) ε (MPa)

0.20 a 57.0 ± 2.4 a 1.65 ± 0.32 a0.10 b 65.2 ± 3.6 b 2.51 ± 0.52 b

fferences (P ≤ 0.05) among treatments.

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94 C. Nunes et al. / Environmental and

rought M. truncatula plants show that these plants were ableo avoid tissue dehydration.

Drought avoidance includes frequently the minimisation ofeaf water loss through an efficient stomatal control and/orecrease of light absorbance (Levitt, 1980; Chaves et al., 2003).owever, in this study mild drought conditions had no effectn gsca, Ci and A, or chlorophyll a fluorescence and pigmentsontent. Also in potted alfalfa plants (Medicago sativa L. cv.ragon) no significant differences were observed in leaf RWC,, Ci, and chlorophyll a fluorescence when a similar droughttress was applied (Aranjuelo et al., 2005). Avoiding tissueehydration could also involve an adjustment of plant growtho water availability, either minimising water loss by reducinghoots and/or maximising water uptake by increasing roots. Weannot ignore that M. truncatula plants may have adopted thistrategy to better explore the drought soil maintaining the leafater status.Plants under severe drought conditions had a small but sig-

ificant decrease in leaves RWC and a higher decrease in gasxchange parameters. As said before, stomatal closure is arought avoidance response that allows leaf water content main-enance (Ludlow, 1980) but the consequent decrease of internalO2 concentration limits photosynthesis (Chaves, 1991; Cornic,000; Flexas and Medrano, 2002; Lawlor, 2002; Lawlor andornic, 2002). The concomitant decrease in gsca, Ci and A,nd the linear relationship between A and gsca and Ci and gsca,bserved in M. truncatula cv. Jemalong plants under severerought conditions indicate that the significant lower value of An the severe dehydrated plants was mostly induced by stomatallosure. However, the decrease in A induced by stomatal closureas more evident than the decrease observed in Ci suggest-

ng that non-stomatal limitations of photosynthesis also occur.hese could involve a lower efficiency of the photochemical

eactions and/or lower carboxylation efficiency. In the first case,tomatal closure can decrease CO2 concentration at the chloro-last level and induce photodamage of PSII, because the lightnergy reaching the reaction centres became excessive. Thisnjury leads to changes in chlorophyll a fluorescence param-ters. In the present work, however, there were no differences inhose parameters between well-watered plants and plants underevere drought conditions. The absence of differences in pig-ent content, especially carotenoids, and in total chlorophyll to

otal carotenoids ratio indicate that there was no triggering ofhotoprotective mechanisms at the achieved leaf RWC values.his supports the idea that leaf photochemistry in M. truncat-la plants is rather resistant to water shortage and the electronransport chain efficiency was maintained at the imposed watereficit. On the other hand, the A/Ci curves showed that RuBPegeneration and Rubisco carboxylation efficiency were bothecreased in dehydrated plants. A decrease in RuBP regenera-ion under drought conditions was referred in several studies andelated to a decrease in ATP availability (Tezara et al., 1999).ccording to Lawlor (2002), RuBP synthesis is limited by an

nadequate ATP content, probably caused by progressive inacti-ation or loss of coupling factor resulting from increasing ionicMg2+) concentration and not to reduced capacity for electronr proton transport, or inadequate trans-thylakoid proton gradi-

msia

imental Botany 63 (2008) 289–296

nt. When A is limited by ATP content, the excess excitationnergy could be dissipated via non-photochemical quenching,ut there were also no significant differences in this parameter.o prevent photodamage, alternative electron sinks such as pho-

orespiration, Mehler ascorbate peroxidase pathway and darkespiration may be involved (Lawlor and Cornic, 2002).

The decrease in maximum Rubisco carboxylation rateVcmax) in dehydrated leaves may be a consequence of a lowernzyme activation state, of depleted enzyme content or the pres-nce of tight-binding inhibitors (Parry et al., 2002). In M. sativalants grown in elevated CO2, the Vcmax decrease was suggestedo be related with a lower Rubisco to protein ratio (Aranjuelot al., 2005). Also in M. sativa plants under drought conditionshe A/Ci and A/Irradiance curves showed that the inhibition ofhotosynthesis was essentially caused by non-stomatal factorsAntolın and Sanchez-Dıaz, 1993). Our results with M. truncat-la suggest that stomatal closure may be the major limiting factorf photosynthesis under severe water deficit, but non-stomatalimitations also occur.

Besides drought avoidance through decrease in stomatal con-uctance, the severe drought M. truncatula plants also exhibitedrought tolerance mechanisms involving osmotic adjustment.he significant differences obtained for osmotic potential at fullaturation and at turgor loss point show that osmotically activeolutes were accumulated in the cells during the period of watertress induction allowing the maintenance of turgidity at lowWC. This evidence is also supported by the elastic modu-

us values that suffer a significant increase with the imposedater stress. The small decrease in leaf RWC suggests that theecrease in the osmotic potential was not a volume effect but aesult of an effective osmoregulation. Also, the electrolyte leak-ge test, used to assess cell integrity and stability during watertress periods (Costa Franca et al., 2000; Bajji et al., 2002),howed that cell membrane in severe drought plants acquiredome degree of resistance given by a lower injury index. Underevere water deficit, the drought tolerance of M. truncatulalants at the cellular level allows them to maintain the stom-ta open and concomitantly a significant net CO2 fixation rate.ll these mechanisms seem to be reversible after re-watering,

ince in preliminary studies the net CO2 fixation rate tended topproach control values in few days (results not shown).

. Concluding remarks

M. truncatula cv. Jemalong plants were resistant to mildrought conditions maintaining identical leaf RWC, gsca, pho-ochemical and biochemical photosynthetic processes when theoil water content decreased to one-half of its maximum, sug-esting that the plants are able to avoid leaf dehydration. Inevere drought conditions, the resistance to water shortagenvolves mainly drought avoidance mechanisms, through stom-ta closure, but drought tolerance strategies were also adopted.ltogether, these mechanisms allowed M. truncatula plants to

aintain the net CO2 fixation rate significantly high under severe

tress. This study should contribute to identify useful phys-ological traits for breeding programmes concerning droughtdaptation in legumes and the results shall be used as control

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C. Nunes et al. / Environmental and

or the behaviour of plants transformed for increased droughtesistance.

Further studies, particularly growth analyses, the nature of theccumulated osmolytes and the limitation in Rubisco carboxy-ation efficiency could improve the knowledge of M. truncatulaesponses to water deficit.

cknowledgments

This work was supported by research and equipment grantsrom Fundacao para a Ciencia e Tecnologia and FEDER throughOSC (POCTI/BIO/56659/2004).

We thank Ana Sofia Soares for her help in A/Ci curve analy-is, and Maria Celeste Arrabaca and Elisabete Carmo-Silva foromments on the manuscript.

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