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Environmental and Experimental Botany 63 (2008) 289–296

Physiological responses of the legume model Medicago truncatula cv.

Jemalong to water deficit
Cátia Nunes a,b,∗ , Susana de Sousa Araújo 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 Ciências, Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal
b Universidade de Lisboa, Faculdade de Ciências, Centro de Engenharia Biológica, Campo Grande,

1749-016 Lisboa, Portugal

c Instituto de Tecnologia Quı́mica e Biológica, Laboratório de Biotecnologia de Células 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

Abstract
In Medicago truncatula Gaertn. cv. Jemalong plants some mechanisms involved in drought resistance were analysed in response to a progressive
water deficit imposed by suppression of soil irrigation. Withholding water supply until the soil had reached one-half of its maximum water content
had no significant effect on leaf RWC, gas exchanges or chlorophyll fluorescence parameters. Under severe drought conditions, the plants resistance
to water shortage involved mainly drought avoidance mechanisms through a decrease in stomatal conductance. The consequent decrease in the
internal CO2 concentration (Ci ) should have limited the net CO2 fixation (A). Since A decreased slightly more than Ci under severe water deficit,
non-stomatal limitations of photosynthesis may have also occurred. Analysis of A/Ci curves showed reduced carboxylation efficiency due to
limitations in RuBP regeneration and Rubisco activity, confirming the presence of non-stomatal limitations of photosynthesis. Drought tolerance
mechanisms involving osmotic adjustment and an increase in cell membrane integrity were also present. Altogether, these mechanisms allowed M.
truncatula cv. Jemalong plants to still maintain a quite elevated level of net CO2 fixation rate under severe water deficit conditions. These results
may contribute to identify useful physiological traits for breeding programs concerning drought adaptation in legumes.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Drought stress; Medicago truncatula; Model legume; A/Ci curves; Water relations

1. Introduction semi-arid conditions, namely to alkaline clay soils (Lesins and

Legumes are the main single source of vegetable protein in
human diets and livestock feed, having major impacts on agricul-
ture, environment and health (Graham and Vance, 2003; Young
et al., 2003; Dita et al., 2006). Due to the legumes capacity
of symbiotic nitrogen fixation, these plants are often used to
improve soil organic fertility and nitrogen economy (Somers
et al., 2003; Young et al., 2003; Howieson and Ballard, 2004).
Medicago truncatula, commonly known as barrel medic, is a
forage legume plant of Mediterranean origin, well adapted to
∗ Corresponding author at: Universidade de Lisboa, Faculdade de Ciências,
Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal.
Tel.: +351 21 750 00 00x24337; fax: +351 21 750 00 48.
E-mail address: catiamaria@gmail.com (C. Nunes).
Lesins, 1979). This species is considered a legume model hav-
ing characteristics that enable its use in molecular and genetic
studies (Barker et al., 1990; May, 2004) and recently is emerging
as promising molecular farming (Abranches et al., 2005). The
Jemalong cultivar, the most used in pastures and agricultural
practices (Crawford et al., 1989), has been chosen for genome
sequencing (http://www.medicago.org).
It has been estimated that two-thirds of the potential yield
of major crops are usually lost due to adverse growing environ-
ments (Bajaj et al., 1999). Most climate change scenarios predict
a worldwide increase in arid areas, including the Mediterranean
basin (IPPC, 2001 and 2007). Water deficit is therefore the most
important abiotic stress and strategies to sustainable use of water
(Kang et al., 2003) and improve plant drought resistance are
urgent and should integrate conventional breeding and biotech-
nological approaches (Chaves et al., 2003). In the legumes, these

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2007.11.004
290 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296
strategies are vital (Sharma and Lavanya, 2002; Dita et al., 2006)
and the utilization of a model such as the M. truncatula, becomes
an advantage. Transgenic approaches to improve M. truncatula
drought resistance using stress-related genes were recently ini-
tiated (Araújo et al., 2004; Sheokand et al., 2005; Verdoy et
al., 2006). However, as far as we know, its systematic physio-
logical characterisation in response to water deficit has not yet
been performed, limiting the comparison between wild type and
transformed lines.
The aim of this work was to evaluate physiologically the
drought resistance of mature M. truncatula cv. Jemalong plants.
The two main components of drought resistance, drought avoid-
ance and drought tolerance (Levitt, 1980; Turner, 1986; Mundree
et al., 2002), were taken into account. The mechanism of drought
avoidance includes an efficient stomata regulation and those of
drought tolerance involve processes at the cellular level, par-
ticularly osmotic adjustment. These strategies were evaluated
through gas exchange and chlorophyll a fluorescence mea-
surements as well as through water relations and membrane
integrity determinations. The decrease in CO2 fixation under
water deficit conditions was discussed in terms of stomatal
and/or non-stomatal limitations.
2. Materials and methods
2.1. Plant material and growth conditions
Medicago truncatula Gaertn. cv. Jemalong seeds were scar-
ified in concentrated anhydrous sulphuric acid according to
Araújo et al. (2004) and sterilised in 50% (v/v) sodium hypochlo-
ryde. Afterwards they were immersed in 70% (v/v) ethanol for
2 min, rinsed with distilled water and placed in Petri dishes
with soaked filter paper. After 3 days at 4 ◦ C in dark con-
ditions, Petri dishes were transferred to the growth chamber
(thermoperiod of 25/18 ◦ C, day/night, and relative humidity
of approximately 40%) where seeds germinated within 2–3
days still in dark conditions. Germinated seeds were main-
tained another week in Petri dishes but under light conditions
(photoperiod of 16/8 h, day/night, and a PPFD of around
500 ␮mol m−2 s−1 obtained from white fluorescent and incan-
descent lamps, TLD58W/77, Halo Star 64476, L30W/77 and
L58W/77). Afterwards, seedlings were individually transferred
to 0.5 L pots with standard “terra de Montemor” commercial
soil (Horto do Campo Grande, Lisboa, Portugal), enriched with
an organic fertiliser (Shacham Ginatada Lda, Israel). The final
concentration of N, P and K was 0.02 to 0.03% each. The plants
were maintained in the growth chamber, under the same condi-
tions (photoperiod, thermoperiod and relative humidity referred
above), through the entire experiment, even during water stress
imposition.
2.2. Experimental design
The plants were irrigated every other day with tap water until
the maximum soil water content was achieved. Eight-week-old
plants were divided in three groups, corresponding to the dif-
ferent levels of soil water availability they would be submitted
to. One group of plants was maintained fully irrigated through-
out the experiment (control). For all the other plants, the soil
water content (SWC) was decreased by withholding water for
4 and 7 days (mild drought and severe drought, respectively).
The youngest fully expanded leaf of the main branch of 8–9-
week-old plants was used in all the experiments, unless stated
otherwise.
2.3. Water status
The soil water status was evaluated through the SWC,
expressed as a weight fraction (Coombs et al., 1987). The follow-
ing equation was used: SWC(%) = 100*(FW − DW)/DW, where
FW was the fresh weight of a soil portion of the internal area
of each pot and DW was the dry weight of the same soil por-
tion after had been oven-dried at 105 ◦ C for 4 days. Plant water
status was evaluated by the leaf relative water content (RWC),
determined according to Catsky (1960).
2.4. Gas exchange analysis
Photosynthetic gas exchange measurements were performed
in non-detached young and fully expanded leaf using a portable
infra-red gas analyser (IRGA, LCpro+ ADC BioScientific
Ltd., Herfordshire, UK) with controlled atmosphere (approx-
imately 370 ␮mol mol−1 CO2 , relative humidity of 60–70%
and 25 ± 2 ◦ C) and a saturating external light source of
800 ␮mol m−2 s−1 . The CO2 response curves were obtained by
increasing atmospheric CO2 concentration stepwise from 100
to 1000 ␮mol mol−1 CO2 according to Long and Bernacchi
(2003) and the leaf internal CO2 concentration (Ci ) and net CO2
assimilation rate (A) were recorded. The maximum Rubisco
carboxylation rate (Vcmax ) and the maximum rate of electron
transport driving regeneration of RuBP (Jmax ) were calculated
from the A/Ci curves according to von Caemmerer and Farquahar
(1981).
2.5. Chlorophyll a fluorescence
Chlorophyll a fluorescence was determined using a modu-
lated fluorometer PAM 101 (Heinz Walz, Effeltrich, Germany).
An optical fibre passing through the radiation shield of the leaf
IRGA chamber, at an angle of 60◦ , allowed the simultaneous
measurement of fluorescence parameters and photosynthesis
under atmospheric CO2 concentrations. The maximal PS II
photochemical efficiency (Fv /Fm ) was determined in leaves
pre-adapted to dark for 10 min according to Kitajima and
Butler (1975). When the measuring light was turned on, the
minimum level of fluorescence (F0 ) was attained. After a
saturating pulse of 5120 ␮mol m−2 s−1 , the maximal fluores-
cence (Fm ) emission was recorded. After 20 min under an
actinic light of 800 ␮mol m−2 s−1 the steady-state of photo-
synthesis was achieved and the leaf electron transport rate
(ETR) and the photochemical (qP) and non-photochemical
(qN) quenching coefficients were measured and calculated
according to Genty et al. (1989) and Schreiber et al. (1986),
respectively.
 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296 291

2.6. Pigment determination

WUE (␮molCO2 mmol−1 H2 O)

Effect of withholding water on soil water content (SWC) and its consequence on leaf M. truncatula relative water content (RWC), net photosynthetic rate (A), transpiration rate (E), stomatal CO2 conductance (gsca ),
Segments from the leaves used in the gas exchange and
fluorescence measurements were immersed for pigment extrac-
tion in 5 mL of methanol and left in the dark at 4 ◦ C for at
least 2 days. After spectrophotometric measurements at 470,

5.1 ± 0.3 b
4.2 ± 0.4 a
4.3 ± 0.5 a
652.4 and 665.2 nm (Unicam 8700 Series, UV/vis Spectrome-
ter Unicam 2 td., Cambridge, UK), chlorophyll a, chlorophyll
b and carotenoids concentrations were calculated according to
Lichtenthaler (1987).

Ci (␮mol mol−1 )
2.7. Electrolyte leakage measurements

192 ± 11 b
235 ± 11 a
219 ± 6 a
Two sets of five leaf discs (0.5 cm diameter) per sample
were rinsed three times with deionised water, to remove solutes
from damaged cells and veins, and floated for 15 h in the dark,
at room temperature, either in deionised water (control) or in

gsca (mmolCO2 m−2 s−1 )

40% (w/v) polyethylene glycol 8000. After PEG-induced dehy-
dration, discs were carefully washed and floated in 3 mL of
deionised water for 4 h, in order to allow re-hydration of tis-
sues. The conductivity of the solutions was monitored using

150 ± 26 b
310 ± 61 a
284 ± 36 a
a conductimeter (Con 5—EcoScan, Eutech Instruments, Sin-
gapore). Samples were heated at 90 ◦ C for 90 min and after

Each value represents the mean ± S.D. (n = 5–20). Different letters indicate significant differences (P ≤ 0.05) among treatments.
cooling to ambient temperature, a second measurement took
place. The relative membrane injury index (I%) was calculated
using electrolyte leakage according to Bajji et al. (2002).

E (mmolH2 O m−2 s−1 )

2.8. Leaf water relations

2.9 ± 0.5 b
4.9 ± 0.7 a
4.9 ± 0.5 a
Water relations parameters, osmotic potential at full tur-
gor (Π 100 ), osmotic potential and RWC both at turgor loss
point (Π 0 and RWC0 , respectively) and the elastic modulus
value (ε), were calculated from pressure–volume curves deter-
mined using a thermocouple psychrometer (HR 33T, Wescor,
A (␮molCO2 m−2 s−1 )

Logan, USA) equipped with a C-52 leaf chamber. Three leaflets

from three young fully expanded leaves were detached and
floated overnight (12 h) in distilled water, in the dark at ambi-
14.8 ± 2.3 b
20.9 ± 2.7 a
21.4 ± 1.7 a

ent temperature, to achieve full turgor. After the excess water

was removed, the leaflets were exposed to the laboratory envi-
intercellular CO2 concentration (Ci ) and water use efficiency (WUE)

ronment. Psycrometric measurements were made in leaf discs

(0.5 cm diameter), made from progressively dryer leaflets, after
equilibrium was reached inside the leaf chamber (20 min after
63.9 ± 3.7 b
72.6 ± 3.2 a
74.3 ± 0.8 a

leaf disc enclosure). A parallel leaflet was used for RWC deter-
RWC (%)

mination. The water relation parameters were calculated from

the pressure–volume data according to Schulte and Hinckley
(1985) and Bannister (1986).
81.6 ± 10.2 a

2.9. Statistical analysis

30.9 ± 4.8 b
17.6 ± 4.1 c
SWC (%)

Data obtained from the three groups of plants was analysed

by mean comparisons after evaluation of the normality. Either
a t-test or Duncan test was applied, to parametric and non-
parametric samples, respectively. Significant differences were
Severe drought

accepted at P < 0.05 and represented by different letters. The

Mild drought

relationship between different parameters was tested by regres-

Control
Table 1

sion analysis. The regression model that best fitted the data,
evaluated by an F-test, was chosen. All the analyses were per-
292 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296

Fig. 2. Mean values of net CO2 assimilation rate in the control (white
squares) and severe drought (black diamonds) M. truncatula plants at dif-
ferent internal CO2 concentrations. Measuring conditions were an irradiance
of 800 ␮mol m−2 s−1 , a relative humidity of 60–70% and a temperature of
25 ◦ C. Each value is the mean of 21 (control) and 8 (severe drought) mea-
surements ± S.D. The arrow indicates measurements taken at atmospheric CO2
concentration (370 ␮mol mol−1 ).

significantly affected (P < 0.01). Although A and E had simi-

Fig. 1. Influence of stomatal conductance to CO2 on (A) net CO2 assimilation
rate and on (B) internal CO2 concentration. Each data point corresponds to one
leaf of different RWC. The solid line is the linear regression fit to all points and
is significant for P ≤ 0.05.
formed using the programme “Statistical Package for Social
Sciences” (SPSS, 11.5, 2002).
3. Results
3.1. High photosynthetic resistance to water deficit
Despite a significant decrease of the soil water content
(SWC) (Table 1), no significant differences in leaf relative
water content (RWC), net CO2 assimilation rate (A), tran-
spiration rate (E), stomatal conductance to CO2 (gsca ) and
internal CO2 concentration (Ci ) were found between plants
under control and mild drought conditions. Only in severe
drought conditions, when the SWC reached values lower than
25%, the leaf RWC and the gas exchange parameters were
lar responses under severe drought, the relative decrease of E
was greater (28 and 42%, respectively), resulting in a signif-
icant increase (P < 0.01) of instantaneous water use efficiency
(WUE) (Table 1). The relationship between A and gsca and Ci
and gsca were linear (Fig. 1) but the stomatal closure induced
a higher decrease on A than on Ci . In fact, for a reduction of
100 mmol m−2 s−1 in gsca , A decreased 15% while Ci decreased
only 9.4%.
The maximal photochemical efficiency of PSII of dark-
adapted leaves (Fv /Fm ), the electron transport rate (ETR),
the photochemical quenching (qP), the non-photochemical
quenching (qN) and the leaf pigments content did not change
significantly between treatments (Table 2). Total chlorophyll
content was about 3.5 times higher than total carotenoids inde-
pendently of the SWC and leaf RWC.
3.2. Non-stomatal limitations of photosynthesis occurs
under severe water deficit
The A/Ci curves obtained for control and severe drought
plants were significantly different (Fig. 2). From A/Ci curves, the
in vivo maximum Rubisco activity (Vcmax ) and the maximum
rate of electron transport used in the regeneration of RuBP (Jmax )
were determined and a highly significant (P < 0.001) decrease
of both parameters was observed in the severe drought plants

Table 2
Effect of water deficit on the maximal photochemical efficiency of PSII (Fv /Fm ), the electron transport rate (ETR), the photochemical and non-photochemical
quenching (qP and qN respectively) and pigment concentration values (chlorophyll a, total chlorophylls and carotenoids) of M. truncatula leaves
Fv /Fm ETR (␮mol m−2 s−1 ) qP qN Chlorophyll a Total chlorophylls Carotenoids
(mg g−1 DW) (mg g−1 DW) (mg g−1 DW)

Control 0.79 ± 0.04 744 ± 132 0.56 ± 0.10 0.76 ± 0.07 13.1 ± 3.0 14.3 ± 3.3 4.2 ± 0.9
Mild drought 0.77 ± 0.05 700 ± 53 0.55 ± 0.04 0.74 ± 0.13 14.5 ± 4.3 16.06 ± 4.8 4.4 ± 1.2
Severe drought 0.79 ± 0.02 821 ± 204 0.64 ± 0.14 0.74 ± 0.06 13.1 ± 1.4 14.48 ± 1.5 4.3 ± 0.5

Each value represents the mean ± S.D. (n = 5–20). There were no significant differences 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 3
Water deficit effect on maximum Rubisco carboxylation rate (Vcmax ) and elec-
tron transport driving regeneration of RuBP (Jmax ) of M. truncatula plants
estimated from A/Ci response curves (Fig. 2)
Vcmax (␮mol m−2 s−1 ) Jmax

Control 67.5 ± 15.7 a 109.9 ± 24.7 a

Severe stress 34.9 ± 8.4 b 71.9 ± 11.7 b

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

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

than in Jmax (36%).

3.3. Cell osmotic adjustment and membrane stability
contributed to drought tolerance
The water relation parameters (Table 4) calculated from the
pressure–volume curves (Fig. 3) show that severe drought plants,
with lower leaf RWC, had lower osmotic potential at full tur-
Fig. 4. Drought effect on membrane injury index. Vertical bars correspond to
mean values ± S.D. (n = 8). The membrane injury index value is significantly
different (P ≤ 0.05) for control and severe drought plants.
gor (Π 100 ) and lower osmotic potential at turgor loss point
(Π 0 ) than control plants. The RWC value at which cells lost
turgidity rose in severe drought plants, which is consistent with
a decrease in cell wall elasticity and, accordingly, with a rise
in the elastic modulus value (ε). An osmotic adjustment of
cells to the imposed water stress of −0.63 MPa, was obtained
from the difference between Π 100 in control and severe drought
conditions.
The injury index (I%) calculated from electrolyte leakage
measurements was significantly lower in severe drought plants
(Fig. 4). The cell membranes of control plants suffered more
injury with the water stress imposed by PEG solution than the
membranes of already stressed plants.

4. Discussion

The suppression of irrigation considerably reduced the soil

Fig. 3. Pressure–volume curves of (A) control and (B) severe drought M. trun-
catula plants. Each curve corresponds to a different leaf subjected to a gradual
dehydration induced under laboratory atmospheric conditions. Arrows indicate
the medium inflexion point that corresponds to the interception of the linear
portions of the curves.
water content but only under severe drought conditions the soil
water availability induced a decrease in leaf RWC of M. trun-
catula cv. Jemalong plants. In fact no difference in leaf RWC
was found when water supply withholding decreased maximum
soil water content to one-half. The utilization of leaf RWC as an
indicator of the plant water status is usual (Chaves, 1991; Lawlor
and Cornic, 2002). In several legumes cultivars of Phaselus vul-
garis (Cruz de Carvalho et al., 1998; Costa França et al., 2000),
Vigna unguiculata (Cruz de Carvalho et al., 1998; Scotti Campos
et al., 1999), Vigna glabrescens (Scotti Campos et al., 1999)
and Lupinus albus (Pinheiro et al., 2001) the close relationship
between RWC and predawn water potential found during pro-
gressive water deficit supports its utilization as an indicator of
the plant water status. The maintenance of leaf RWC in mild

Table 4
Water stress effect on osmotic potential at full turgor (Π 100 ), osmotic potential and RWC both at turgor loss point (Π 0 and RWC0 respectively) and the elastic
modulus value (ε) of M. truncatula plants estimated from pressure–volume curves (Fig. 3)
Π 100 (MPa) Π 0 (MPa) RWC0 (%) ε (MPa)

Control −1.15 ± 0.22 a −1.96 ± 0.20 a 57.0 ± 2.4 a 1.65 ± 0.32 a

Severe drought −1.78 ± 0.10 b −2.44 ± 0.10 b 65.2 ± 3.6 b 2.51 ± 0.52 b

Each value represents the mean ± S.D. (n = 4). Different letters indicate significant differences (P ≤ 0.05) among treatments.
294 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296
drought M. truncatula plants show that these plants were able
to avoid tissue dehydration.
Drought avoidance includes frequently the minimisation of
leaf water loss through an efficient stomatal control and/or
decrease of light absorbance (Levitt, 1980; Chaves et al., 2003).
However, in this study mild drought conditions had no effect
on gsca , Ci and A, or chlorophyll a fluorescence and pigments
content. Also in potted alfalfa plants (Medicago sativa L. cv.
Aragón) no significant differences were observed in leaf RWC,
A, Ci , and chlorophyll a fluorescence when a similar drought
stress was applied (Aranjuelo et al., 2005). Avoiding tissue
dehydration could also involve an adjustment of plant growth
to water availability, either minimising water loss by reducing
shoots and/or maximising water uptake by increasing roots. We
cannot ignore that M. truncatula plants may have adopted this
strategy to better explore the drought soil maintaining the leaf
water status.
Plants under severe drought conditions had a small but sig-
nificant decrease in leaves RWC and a higher decrease in gas
exchange parameters. As said before, stomatal closure is a
drought avoidance response that allows leaf water content main-
tenance (Ludlow, 1980) but the consequent decrease of internal
CO2 concentration limits photosynthesis (Chaves, 1991; Cornic,
2000; Flexas and Medrano, 2002; Lawlor, 2002; Lawlor and
Cornic, 2002). The concomitant decrease in gsca , Ci and A,
and the linear relationship between A and gsca and Ci and gsca ,
observed in M. truncatula cv. Jemalong plants under severe
drought conditions indicate that the significant lower value of A
in the severe dehydrated plants was mostly induced by stomatal
closure. However, the decrease in A induced by stomatal closure
was more evident than the decrease observed in Ci suggest-
ing that non-stomatal limitations of photosynthesis also occur.
These could involve a lower efficiency of the photochemical
reactions and/or lower carboxylation efficiency. In the first case,
stomatal closure can decrease CO2 concentration at the chloro-
plast level and induce photodamage of PSII, because the light
energy reaching the reaction centres became excessive. This
injury leads to changes in chlorophyll a fluorescence param-
eters. In the present work, however, there were no differences in
those parameters between well-watered plants and plants under
severe drought conditions. The absence of differences in pig-
ment content, especially carotenoids, and in total chlorophyll to
total carotenoids ratio indicate that there was no triggering of
photoprotective mechanisms at the achieved leaf RWC values.
This supports the idea that leaf photochemistry in M. truncat-
ula plants is rather resistant to water shortage and the electron
transport chain efficiency was maintained at the imposed water
deficit. On the other hand, the A/Ci curves showed that RuBP
regeneration and Rubisco carboxylation efficiency were both
decreased in dehydrated plants. A decrease in RuBP regenera-
tion under drought conditions was referred in several studies and
related to a decrease in ATP availability (Tezara et al., 1999).
According to Lawlor (2002), RuBP synthesis is limited by an
inadequate ATP content, probably caused by progressive inacti-
vation or loss of coupling factor resulting from increasing ionic
(Mg2+ ) concentration and not to reduced capacity for electron
or proton transport, or inadequate trans-thylakoid proton gradi-
ent. When A is limited by ATP content, the excess excitation
energy could be dissipated via non-photochemical quenching,
but there were also no significant differences in this parameter.
To prevent photodamage, alternative electron sinks such as pho-
torespiration, Mehler ascorbate peroxidase pathway and dark
respiration may be involved (Lawlor and Cornic, 2002).
The decrease in maximum Rubisco carboxylation rate
(Vcmax ) in dehydrated leaves may be a consequence of a lower
enzyme activation state, of depleted enzyme content or the pres-
ence of tight-binding inhibitors (Parry et al., 2002). In M. sativa
plants grown in elevated CO2 , the Vcmax decrease was suggested
to be related with a lower Rubisco to protein ratio (Aranjuelo
et al., 2005). Also in M. sativa plants under drought conditions
the A/Ci and A/Irradiance curves showed that the inhibition of
photosynthesis was essentially caused by non-stomatal factors
(Antolı́n and Sánchez-Dı́az, 1993). Our results with M. truncat-
ula suggest that stomatal closure may be the major limiting factor
of photosynthesis under severe water deficit, but non-stomatal
limitations also occur.
Besides drought avoidance through decrease in stomatal con-
ductance, the severe drought M. truncatula plants also exhibited
drought tolerance mechanisms involving osmotic adjustment.
The significant differences obtained for osmotic potential at full
saturation and at turgor loss point show that osmotically active
solutes were accumulated in the cells during the period of water
stress induction allowing the maintenance of turgidity at low
SWC. This evidence is also supported by the elastic modu-
lus values that suffer a significant increase with the imposed
water stress. The small decrease in leaf RWC suggests that the
decrease in the osmotic potential was not a volume effect but a
result of an effective osmoregulation. Also, the electrolyte leak-
age test, used to assess cell integrity and stability during water
stress periods (Costa França et al., 2000; Bajji et al., 2002),
showed that cell membrane in severe drought plants acquired
some degree of resistance given by a lower injury index. Under
severe water deficit, the drought tolerance of M. truncatula
plants at the cellular level allows them to maintain the stom-
ata open and concomitantly a significant net CO2 fixation rate.
All these mechanisms seem to be reversible after re-watering,
since in preliminary studies the net CO2 fixation rate tended to
approach control values in few days (results not shown).
5. Concluding remarks
M. truncatula cv. Jemalong plants were resistant to mild
drought conditions maintaining identical leaf RWC, gsca , pho-
tochemical and biochemical photosynthetic processes when the
soil water content decreased to one-half of its maximum, sug-
gesting that the plants are able to avoid leaf dehydration. In
severe drought conditions, the resistance to water shortage
involves mainly drought avoidance mechanisms, through stom-
ata closure, but drought tolerance strategies were also adopted.
Altogether, these mechanisms allowed M. truncatula plants to
maintain the net CO2 fixation rate significantly high under severe
stress. This study should contribute to identify useful phys-
iological traits for breeding programmes concerning drought
adaptation in legumes and the results shall be used as control
 C. Nunes et al. / Environmental and Experimental Botany 63 (2008) 289–296
for the behaviour of plants transformed for increased drought
resistance.
Further studies, particularly growth analyses, the nature of the
accumulated osmolytes and the limitation in Rubisco carboxy-
lation efficiency could improve the knowledge of M. truncatula
responses to water deficit.
Acknowledgments
This work was supported by research and equipment grants
from Fundação para a Ciência e Tecnologia and FEDER through
POSC (POCTI/BIO/56659/2004).
We thank Ana Sofia Soares for her help in A/Ci curve analy-
sis, and Maria Celeste Arrabaça and Elisabete Carmo-Silva for
comments on the manuscript.
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