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Environmental and Experimental Botany 69 (2010) 279–285

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

he role of organic and inorganic solutes in the osmotic adjustment ofrought-stressed Jatropha curcas plants

vandro Nascimento Silvaa, Sérgio Luiz Ferreira-Silvaa, Ricardo Almeida Viégasb,oaquim Albenísio Gomes Silveiraa,∗

Laboratório de Metabolismo de Plantas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6004, CEP 60451-970, Fortaleza, Ceará, BrazilUniversidade Federal de Campina Grande, Brazil

r t i c l e i n f o

rticle history:eceived 4 November 2009eceived in revised form 30 March 2010ccepted 1 May 2010

eywords:atropha curcassmotic adjustmentsmolytesolutesater stress

a b s t r a c t

This study aimed to assess the accumulation of organic and inorganic solutes and their relative contri-bution to osmotic adjustment in roots and leaves of Jatropha curcas subjected to different water deficitintensity. Plants were grown in vermiculite 50% (control), 40%, 30%, 20% and 10% expressed in gravi-metric water content. The water potential, osmotic potential and turgor potential of leaves decreasedprogressively in parallel to CO2 photosynthetic assimilation, transpiration and stomatal conductance, asthe water deficit increased. However, the relative water content, succulence and water content in theleaves did not show differences between the control and stressed plants, indicating osmotic adjustmentassociated with an efficient mechanisms to prevent water loss by transpiration through stomatal closure.The K+ ions had greater quantitative participation in the osmotic adjustment in both leaves and rootsfollowed by Na+ and Cl−, while the NO3

− ion only showed minor involvement. Of the organic solutesstudied, the total soluble sugars showed the highest relative contribution to the osmotic adjustment inboth organs and its concentration positively increased with more severe water deficit. The free amino

acids and glycinebetaine also effectively contributed to the osmotic potential reduction of both the rootand leaves. The role of proline was quantitatively insignificant in terms of osmotic adjustment, in boththe control and stressed roots and leaves. Our data reveal that roots and leaves of J. curcas young plantsdisplay osmotic adjustment in response to drought stress linked with mechanisms to prevent water lossby transpiration by means of the participation of inorganic and organic solutes and stomatal closure. Ofall the solutes studied, soluble sugars uniquely display a prominent drought-induced synthesis and/or

ts an

accumulation in both roo

. Introduction

Water deficit is one of the most important environmen-al stresses affecting agricultural productivity around the worldHessine et al., 2009). Therefore, the knowledge of physiologicalnd biochemical mechanisms involved with the whole plant levelesponses to water stress generate considerable interest (Slama

t al., 2007). Consequently, many breeding programs and intensetudies have been carried out in order to identify physiologicalarkers which can be used for the selection of plants resistant to

rought (Lizana et al., 2006).

Abbreviations: GB, glycinebetaine; OA, osmotic adjustment; RWC, relative waterontent; TSS, total soluble sugars; TFAA, total free amino acids; �w, water potential;s, osmotic potential; WC, water content; DW, dry weight; A, CO2 photosynthetic

ssimilation rate; E, transpiration; gs, stomatal conductance.∗ Corresponding author. Tel.: +55 8533669821; fax: +55 8533669821.

E-mail address: silveira@ufc.br (J.A.G. Silveira).

098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2010.05.001

d leaves.© 2010 Elsevier B.V. All rights reserved.

The physiological and developmental mechanisms which allowa species to tolerate prolonged periods of water deficit can involvenumerous attributes. One means of increasing drought tolerance isby accumulation of osmotically active solutes, so that turgor andturgor-dependent processes may be maintained during episodesof dry-down. The osmotic adjustment allows water uptake, cellenlargement and plant growth during water stress associated withpartial stomata opening allowing the CO2 assimilation at low waterpotentials that are otherwise inhibitory (Alves and Setter, 2004).

In this context, the osmotic adjustment (OA) has been consid-ered as an important physiological adaptation character associatedwith drought tolerance and it has drawn much attention during thelast years (Hessine et al., 2009). OA involves the net accumulationof solutes in plant cells in response to falls in water potential in

the root medium. As a consequence, the cell’s osmotic potential isdiminished which in turn attracts water into the cell by tendingto maintain turgor pressure (Pérez-Pérez et al., 2009). Accordingto Martinez et al. (2005), compatible soluble like sugars, glycerol,proline or glycinebetaine can also contribute to this process.

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80 E.N. Silva et al. / Environmental and

Species and varieties of crop plants differ greatly in respect tohe types of solutes accumulated and their relative contribution inowering the osmotic potential and the primary osmolyte involvedn OA is species-dependent (Rhodes et al., 2002). In order to achievesmotic balance at the cellular level, these substances have to bellocated between the cytoplasm and the vacuole, and also betweenhe cytoplasm and the apoplast. Cellular osmotic homeostasis canesult from sequestration of a major fraction of the toxic substancesn the vacuolar compartment, while the non-toxic solutes shoulde preferentially located in the cytoplasm where they can act asompatible osmo-solutes (Gagneul et al., 2007).

In several studies, it has been reported that OA occurs in somepecies in response to water stress in both field and controlled envi-onmental conditions (Chimenti et al., 2006). However, substantialifferences also have been reported between species, cultivars or

andraces in terms of OA capacity and with respect to the naturef the major solutes contributing to osmotic potential (Bajji et al.,001). The degree of OA also could be affected by the rate of stress

ntensity and most particularly by organ type and plant age (Alvesnd Setter, 2004). The osmotic adjustment in the root axis of plantsultivated in dry soils is crucial to growth and drought resistanceSerraj and Sinclair, 2002).

It has been evidenced that the accumulation of proline (Pérez-érez et al., 2009) and glycinebetaine (Bajji et al., 2001) areommonly observed metabolic response of higher plants to watereficit. Similarly, changes in the potassium content may contributeubstantially to osmo-regulation (Shabala and Cuin, 2007) and mayccur in concert with changes in sugars and amino acids (Pérez-érez et al., 2009). In some cases, however, changes in sugars, aminocids, or organic acids were not accompanied by changes in potas-ium concentrations (Slama et al., 2007).

Jatropha curcas is distributed over the arid and semi-arid areas ofouth America and in all tropical regions and in the last years it hasecently received tremendous attention because its high seed oilontent which can be converted to biodiesel. This manner, it is beingonsidered as a universally accepted energy source crop (Kumar etl., 2008). This species grows in areas with extreme climates and soilonditions that could not be habited by most of the agriculturallymportant plant species (Francis et al., 2005).

Recently, we reported that J. curcas displayed an effectivesmotic adjustment in response to salinity (Silva et al., 2009a)nd, interestingly, it exhibited some mechanisms similar to thosemployed by the halophyte Atriplex nummularia (Silveira et al.,009). However, the involvement of the osmotic adjustment in theater stress resistance of J. curcas is still unknown. In addition, the

omprehension of the role that process in the drought tolerance inultivated plants is also poorly understood.

This study was carried out to test the hypothesis that J. cur-as roots and leaves display an effective osmotic adjustment andater deficit resistance by the use of a drought-induced net accu-ulation of K+ ions and organic solutes especially, glycinebetaine.lthough the J. curcas young plants have exhibited indicators offfective osmotic adjustment in response to a wide range of watereficit, of all the solutes studied, soluble sugars uniquely display arominent drought-induced synthesis and/or accumulation in bothoots and leaves.

. Materials and methods

.1. Plant material and growth conditions

J. curcas L. seeds, kindly provided by the Instituto Fazendaamandua, Brazil, and previously selected for size and weight,ere surface sterilized for 1 min with a 5% sodium hypochlorite

olution and germinated in sand. Eight days after germination, a

imental Botany 69 (2010) 279–285

homogeneous group of seedlings in height and having the samemorphological aspects was transplanted into pots (2 L) filled withvermiculite of medium texture, 70% of water holding capacity(m/m) and density of 0.12 g/cm3. Every 2 days, the pots werewatered with half strength Hoagland and Arnon solution (1950) inquantities enough to bring the substrate holding capacity to 70%.This initial phase of the experiment was carried out in a green-house under natural conditions, where the mean air temperaturevaried between 24 ◦C (minimum) and 36 ◦C (maximum) with amean temperature of 29 ◦C, a mean air relative humidity of 65%,a mean maximum photosynthetic photon flux density (PPFD) of700 �mol m−2 s−1 and a photoperiod of around 12 h. The physicnut plants were kept in the greenhouse until reach 23-day-old.

2.2. Water-deficit treatments and harvesting

The pots were transferred from the greenhouse into a growthchamber with controlled environmental conditions of: PFFD of400 �mol m−2 s−1, air temperature of 27 ◦C, air humidity of 70% andphotoperiod of 12 h. In order to have a wide range of water avail-ability to 23-day-old seedlings (± eight leaves stage), a regime fromwell-watered to severely water-stressed conditions was imposed.The water content utilized in the control was equal to 70% of thevermiculite holding capacity (approx. 50% in a gravimetric basis)and the water deficit was progressively applied by restricting thelevel of irrigation by 75%, 50%, 25% and 0% (water withdrawal) incomparison with the control. During the experiment all of the potswere daily weighed and corrected for water loss with full-strengthHoagland and Arnon (1950) solution when needed. The valuesof vermiculite gravimetric water content measured at end of theexperimental period were approximately 50%, 40%, 30%, 20% and10% as defined by following expression: � = (WW − DW)/WW × 100(where WW is the wet weight and DW is dry weight of the ver-miculite sample). The plants were kept in the treatments for 10days. At the end of the experiment, the plants were harvested, sep-arated into leaves and roots, then frozen and stored at −80 ◦C forlyophilization and further chemical and biochemical analyses.

2.3. Water status and osmotic adjustment measurements

The leaf water potential (�w) was evaluated immediately aftersampling using the pressure chamber method (Scholander et al.,1965) at pre-dawn (�w, at 6:00 h) in leaves similar to thoseused for leaf gas exchange and chlorophyll fluorescence mea-surements. The leaf relative water content (RWC) and leaf watercontent (water weight/dry weight) were determined as previ-ously described (Silveira et al., 2009). Thirty leaf discs (diameter1.0 cm) were sampled and immediately weighed (FW). Then, theywere immersed in distilled water in Petri dishes for 6 h at 25 ◦Cunder a photon flux density of 40 �mol m−2 s−1, blotted on filterpaper, and the turgid weight (TW) was determined. The discs weredried in an oven at 75 ◦C for 48 h and the dry weight (DW) wasobtained. The RWC was calculated using the following equation:RWC = (FW − DW)/(TW − DW) × 100. The leaf succulence (LS) wascalculated by the equation (FW)/A, where A is the area of thirty leafdiscs (diameter 1.0 cm), as described by Silveira et al. (2009).

For determining the osmolality, small segments from fullyexpanded leaves and 5 cm-segments of terminal roots were macer-ated in a mortar. After extract filtration in a miracloth membrane,the sap was centrifuged at 10,000 × g for 10 min at 4 ◦C. Theresultant supernatant was used to determine the osmolality (c)

with a vapor pressure osmometer (Vapro 5520, Wescor, USA).The osmotic potential was determined using the formula: �s(MPa) = −c (mosmol kg−1) × 2.58 × 10−3, according to the Van’tHoff equation. For the measurement of osmotic potential at full tur-gor (�s100), intact leaves and root segments of stressed and control

E.N. Silva et al. / Environmental and Experimental Botany 69 (2010) 279–285 281

Table 1Leaf water potential, relative water content, leaf and root osmotic potential, leaf succulence and leaf and root osmotic adjustment in J. curcas plants subjected to different %of vermiculite gravimetric water content for 10 days. Data refer to mean values (n = 4). The same letters are not significantly different to 0.05 by Tukey’s test.

Vermiculite gravimetric watercontent (%)

�w (−MPa) RWC (%) �s (−MPa) Leaf succulence (mg H2O cm−2) OA

Leaf Root Leaf Root

50 −0.55a 64.04a −0.85a −0.78a 40.07a.96b.97b.05b.10c

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40 −0.71b 64.77a −030 −0.89c 65.81a −020 −0.98c 66.13a −110 −1.05d 72.49b −1

lants were fully hydrated on moistened filter paper in Petri dishesor 24 h at 4 ◦C in the dark. The total OA was calculated as the differ-nce of osmotic potential at full turgor between the control (�sc100)nd salt stress (�ss100) conditions (Martinez-Ballesta et al., 2004).

.4. Determination of CO2 photosynthetic assimilation,ranspiration, stomatal conductance and leaf and root dry weight

Leaf gas exchange was measured with an infrared gas analyzerIRGA (LCi, ADC, Hoddesdonm, UK) operating in an open system

nd with an air flow of 200 mL min−1. Measurements of leaf CO2hotosynthetic assimilation rate (A), transpiration (E) and stom-tal conductance (gs) were performed in a mature expanded leaf.oots were carefully separated from vermiculite by washing with0.1 mM CaSO4 solution and the leaf and root dry weight was

erformed after complete drying by lyophilization.

.5. Determination of organic and inorganic solutes

Lyophilized leaf and root samples were transferred into her-etically closed tubes containing deionized water and placed in100 ◦C water bath for 1 h. The extracts were then filtered and

tored at −20 ◦C for later analyses. The Na+ and K+ contents wereetermined by flame photometry and the Cl− content throughitration with AgNO3. The NO3

− concentration was determined byhe method described by Cataldo et al. (1975). The total solubleugar content was determined using the phenol-sulfuric methodDubois et al., 1956) and the total free amino acids were mea-ured by reaction with ninhydrin (Yemm and Cocking, 1955). Theroline concentration was determined according to the method ofates et al. (1973). Quaternary ammonium compounds (QACs) werextracted and measured as glycinebetaine equivalents according torieve and Grattan (1983). All of the results of solute concentrationre expressed as mmol solute kg−1 tissue water after correction forhe humidity in the leaf and root samples. The relative contributionRC) of each solute to the osmotic potential was estimated as % of thesmolality calculated by following ratio: RC = solute concentrationmmol kg−1 water tissue)/osmolality (mmol kg−1 solvent) accord-ng to Silveira et al. (2009).

.6. Statistical analysis

The experiment had a completely randomized design, with fivereatments and four independent replicates, each one consistingf an individual plant per pot. Data were analyzed by ANOVA andeans were compared by Tukey’s test at the 0.05 confidence level.

he standard deviation is plotted in all of the graphics.

. Results

In this study, well-watered and water-stressed J. curcas younglants were studied to assess the effects of water deficit over 10ays on the osmotic adjustment and water relations mechanisms

−0.85a 40.18a 0.21d 0.14c−0.85a 40.39a 0.43c 0.24b−0.84a 40.54a 0.55b 0.33a−0.91b 40.68a 0.65a 0.38a

in both the roots and leaves. As expected, the leaf water poten-tial (�w) declined gradually with decreasing water supply andit varied between −0.55 and −1.05 MPa (Table 1) over the rangeof water availability used during the experiment. The same stan-dard was observed for the osmotic potential (�s), which variedbetween −0.85 and −1.10 MPa in the leaves and between −0.78and −0.91 MPa in the roots (Table 1). The leaf turgor potential alsodecreased progressively as the water deficit increased, varying from+0.30 to +0.05 MPa (Table 1). Moreover, reinforcing that J. curcasyoung plants were able to display osmotic adjustment in responseto drought stress, no effect of water deficit levels on the relativewater content (RWC) and succulence was observed (Table 1).

The water deficit induced a prominent and progressive decreasein the CO2 photosynthetic assimilation rate, transpiration andstomatal conductance. These changes in the gas exchange of J. cur-cas leaves were positively correlated with the leaf and root dryweight (Table 2). These data evidence that the maintaining of agood hydration status and relatively high water potential (approx.−1.0 MPa in the most stressed leaves) were associated with anefficient mechanism of stomatal control and restriction in the pho-tosynthesis. Interestingly, the leaf water content (WC) was notchanged by drought stress (Table 2).

The Na+ concentration in the leaves of the drought-stressedtreated plants was slightly higher than in plants grown with anadequate water supply (Fig. 1A) whereas the leaf Cl− ions accu-mulated more intensively than Na+ in response to drought stress.Conversely, the concentrations those ions in the roots remainedpractically unaffected in all treatments (Fig. 1B). The importance ofthese inorganic solutes in carrying out osmotic adjustment in the J.curcas plants exposed to water deficit is highlighted by their relativecontribution to the total osmolality. The relative contribution of theNa+ + Cl− sum to the total osmotic potential reached approximately30% in the leaves and 26% in the roots (Table 3).

The K+ content was not significantly altered in the leaves or inroots by any water-deficit treatment (Fig. 1C). In spite of this, theK+ ion contributed most to the osmotic adjustment in both organsin all treatments (Table 3). In general, the relative contribution ofK+ to the osmotic potential in stressed plants was approximately25% in the leaves and 29% in the roots. By contrast, increased NO3

−

concentrations were present in leaves with more severe water-deficit treatments (Fig. 1D). However, our data for the analyzedinorganic solutes showed that NO3

− did not contribute as much tothe osmotic potential in the stressed plants (∼5% in leaves and 4%in roots) (Table 3).

The total free amino acid (AA) content in the leaves increasedsignificantly (p < 0.05) at and below of the treatment of 20% of ver-miculite water content, but was not significantly different amongin the roots with reduced water availability (Fig. 2A). The AA frac-tion accounted for 9% and 8.5% of the overall osmotic adjustment

in the leaves and roots of the water-stressed plants, respectively(Table 4). Conversely, the total soluble sugar content (TSS) of thesetwo organs increased with reduced water availability to be 3-foldhigher in the leaves of the most stressed plants, compared to control(Fig. 2B). The maximum contribution of AA and TSS to the osmotic

282 E.N. Silva et al. / Environmental and Experimental Botany 69 (2010) 279–285

Table 2Leaf and root dry weight, leaf water content, CO2 photosynthetic assimilation rate, transpiration and stomatal conductance in J. curcas plants subjected to different % ofvermiculite gravimetric water content for 10 days. Data refer to mean values (n = 4). The same letters are not significantly different to 0.05 by Tukey’s test.

Vermiculite gravimetricwater content (%)

DW (g plant−1) WC (g H2O g−1 DW) A (�mol m−2 s−1) E (mmol m−2 s−1) gs (mol m−2 s−1)

Leaf Root

50 1.50a 1.03a 3.71a 9.98a 3.22a 0.30a40 1.31b 0.92a 3.82a 7.34b 2.88a 0.18b30 1.22b 0.81b 4.07a 4.38c 1.58b 0.06c20 1.08c 0.70c 4.01a 3.82d 1.54b 0.06c10 0.95c 0.62c 3.79a 1.40e 0.96c 0.02d

Table 3Relative contribution of inorganic solutes in the osmotic adjustment of leaves and roots of J. curcas plants subjected to different % of vermiculite gravimetric water contentfor 10 days. Data refer to mean values (n = 4). The same letters are not significantly different to 0.05 by Tukey’s test.

Vermiculite gravimetricwater content (%)

Na+ Cl− K+ NO3−

% of osmolality

Leaf Root Leaf Root Leaf Root Leaf Root

50 15.7a 17.2a 13.7b 9.5b 32.7a 30.1a 5.1a 5.0a40 17.9a 16.0a 13.2b 12.0a 30.4a 32.9a 5.0a 4.3b30 16.4a 18.7a 15.4a 9.9b 23.9b 29.0b 4.6a 3.8b20 16.4a 16.3a 13.9b 8.3c 23.3b 28.0b 4.8a 3.9b10 13.3b 16.7a 16.4a 6.6d 21.3b 25.6c 5.3a 4.1b

Fig. 1. (A) Na+ content (B) Cl− content (C) K+ content and (D) NO3− content in the leaves (�) and roots (©) of Jatropha curcas plants subjected to different % of vermiculite

gravimetric water content. Data are means of four replicates ± SD.

Table 4Relative contribution of organic solutes in the osmotic adjustment of leaves and roots of J. curcas plants subjected to different % of vermiculite gravimetric water content for10 days. Data refer to mean values (n = 4). The same letters are not significantly different to 0.05 by Tukey’s test.

Vermiculite gravimetricwater content (%)

TFAA TSS GB Proline

% of osmolality

Leaf Root Leaf Root Leaf Root Leaf Root

50 8.7b 10.1a 7.7d 10.6c 7.4b 8.6a 0.1b 0.1b40 7.7b 7.7b 9.8c 9.7c 7.1b 9.2a 0.1b 0.1b30 7.9b 9.7a 16.2b 12.4b 6.6b 8.7a 0.1b 0.1b20 10.2a 8.1b 18.2a 19.6a 8.4a 8.3b 0.1b 0.1b10 9.5a 9.1a 20.7a 21.4a 8.8a 10.0a 0.2a 0.2a

E.N. Silva et al. / Environmental and Experimental Botany 69 (2010) 279–285 283

F lycined tes ± S

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ig. 2. Concentrations of amino acids (A), total soluble sugar (B), proline (C) and gifferent % of vermiculite gravimetric water content. Data are means of four replica

otential of leaves and roots of stressed J. curcas plants was higherhan the other analyzed organic solutes and made up approx. 30%f the total osmotic adjustment in both organs (Table 4).

It is important to emphasize the significant role of the solubleugars in the osmotic adjustment of stressed plants. The concen-rations of these solutes markedly increased in both the leaf andoot with increase of water deficit. As a consequence, their rela-ive contribution to total osmotic adjustment was increased frompproximately 8% to 22% in leaves and from 11% to 20% in rootsTable 4). As the leaf water content was not changed by droughttress, the increase in the soluble sugar concentration representednet accumulation of those organic solutes possibly triggered by

ncrease in their synthesis from starch hydrolysis, reduction in theirtilization due to restriction in translocation or decrease as carbonkeleton source for biosynthesis and plant growth.

The proline content in the leaves was greater in the stressedlants than in the well watered control; however, a significanthange was not observed in the roots with different treatmentsFig. 2C). Although stressed plants accumulated significantly moreroline in the leaves, the amount recorded (approximately 0.1% ofsmolality) was at a level very low to osmotic adjustment of J. cur-as plants would be expected (Table 4). Conversely, leaf and rootlycinebetaine concentrations (GB) were higher at and below a sub-trate moisture of 20% of vermiculite water content (Fig. 2D). Unlikeroline, GB played a role in reducing the osmotic potential of J. cur-as plants in both the drought-stressed and well-watered plantsith a relative contribution of approximately 8% in the leaves and

% in the roots (Table 4).The above-mentioned results indicate that K+ relatively con-

ributed (% of total osmolality) most to the osmotic adjustment inhe leaves and much more in the roots in the drought stressed andot stressed J. curcas plants. Interestingly, Na+ and Cl− also had aignificant contribution to the osmotic potential in both organs forll of the treatments, while nitrate presented a minor contribution.

he average contribution of these inorganic solutes to leaf and rootsmotic adjustment of water-stressed plants was of approximately5%. Except for proline, all of the other organic solutes analyzed inhe current study contributed significantly to the osmotic adjust-

ent as in well watered as in drought-stressed plants. Altogether

betaine (D) in the leaves (�) and roots (©) of Jatropha curcas plants subjected toD.

TSS, TFAA and GB had a relative contribution to the osmotic poten-tial of approximately 40% in both leaves and roots of stressed plants.

4. Discussion

Osmotic stress is a physiological event often associated withexcessive water deficit that can reduce plant growth through mech-anisms not yet fully known. Osmotic adjustment is a cellularadaptive mechanism vital for water-stress-tolerant plants, allow-ing for plants to continue growing in the case of drought. Osmoticadjustment is usually defined as a decrease in the cell sap osmoticpotential resulting from a net increase (discounted the concentra-tion effect due to drought-induced reduction in cell volume) ofintracellular solutes rather than from a loss of cell water (Kusaka etal., 2005). That phenomenon in plants is very difficult to measureas well as evaluate its importance for beneficial effects in droughttolerance (Serraj and Sinclair, 2002).

The results of this study evidence that J. curcas plants havean efficient adaptive mechanism to avoid a severe drought stressby maintaining a good leaf water status and an effective osmoticadjustment. This strategy is also associated with a rapid growthrestriction and impairment of photosynthesis by means of an effec-tive stomatal closure (Table 2). Under these stressing conditions,the leaf water potential was slightly reduced whereas the leaf rela-tive water content (RWC), water content and leaf succulence weremaintained at level of well-watered plants. This strategy is commonin some semi-arid adapted species like cowpea when subjectedto drought stress (Souza et al., 2004) and salinity (Cavalcanti etal., 2004). Similar results were found for lemon plants exposed todrought for 15 days (Pérez-Pérez et al., 2009) and for annual cloversunder water stress imposed by withholding water (Iannucci et al.,2002).

The strategy of water maintenance in leaf and root tissues, byavoiding water loss by transpiration, employed by J. curcas plants,

was accomplished with an effective osmotic adjustment mecha-nism. The simultaneous occurrence of these two mechanisms isvery efficient to cope with drought stress conditions (Martinez-Ballesta et al., 2004). The reduction in �w, in response to waterstress, is influenced by leaf age and this effect seems to be due to the

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84 E.N. Silva et al. / Environmental and

eaf capacity to adjust osmotically and to maintain a good hydra-ion status (Bajji et al., 2001). The tolerance to drought is associatedith ability to combine effectively osmotic adjustment with stom-

tal control mechanisms in order to allow a continued growth. Theccurrence of active osmotic adjustment (OA) can be establishednly if a net increase of solute concentration occurs (Silveira et al.,009).

Our data reveal that inorganic solutes are involved in the OA ofoth stressed and not stressed J. curcas plants, especially K+, Na+ andl−, in both leaves and roots. However, increased concentrations inesponse to drought stress occurred only for Cl− and NO3

− in leaves,n spite of this latter anion have shown a minor quantitative con-ribution to osmotic adjustment. Increased concentrations of ionsriggered by water deficits have not been commonly found in theissue of higher plants (Iannucci et al., 2002). The K+ concentrationn these two organs was high and its relative contribution to thesmotic potential of J. curcas plants under water stress was higherhan other inorganic ions. The K+ ion is known to be quite solublend to play a key osmo-regulatory role in guard cells and similarlyn turgor maintenance (Shabala and Cuin, 2007). Our results are inine with those obtained by Patakas et al. (2002), who reported onhe importance of K+ in the osmotic adjustment of grapevine plantsnder water stress.

In this study, it was observed an increase of leaf Cl− concentra-ion in the stressed plants whereas in roots it decreased slightly,oth in comparison with the control. The Na+ concentrations didot change significantly by effect of drought stress in both leaf andoot. Interestingly, the Cl− and Na+ ions showed a significant con-ribution to osmotic adjustment in leaves and roots of non-stressednd stressed plants. These results are unusual for plants underater stress, but not for plants under salt stress. J. curcas plants

re characterized by as a salt includer species, especially for Na+

nd Cl−, even when cultivated with low concentrations of thoseons (Silva et al., 2009b). This involvement of Na+ and Cl− ions isemonstrated by the high relative contribution of these ions in thesmotic potential of J. curcas in both organs in all of the tested con-itions. However, this plant species has a high affinity for these

ons, even under low availability in the root substrate, as previouslyemonstrated (Silva et al., 2009b).

As already mentioned, our current results are in agreementith those obtained by Patakas et al. (2002), who observed an

ncrease in the Na+ and Cl− concentrations in grapevine leavesnder water stress. Conversely, Pérez-Pérez et al. (2009) demon-trated a negligible participation of Na+ and Cl− in the osmoticdjustment in lemon plants subjected to drought. An increase inhe NO3

− content probably comes from the inhibition of reduc-ase activity, which has been observed in many species, even under

ild stress (Kameli and Losel, 1995). Although the NO3− contribu-

ion to the osmotic adjustment is not quantitatively comparableo other solutes, its relative participation to the osmotic poten-ial can be important in both organs in the untreated and treatedlants.

The organic solutes similarly participated in the osmotic adjust-ent in the leaves and roots of J. curcas plants, especially TSS, TFAA

nd glycinebetaine. The increase in the leaf TFAA contents in theore severe drought treatments indicates major protein degra-

ation as viewed by the reduction in protein content (data nothown). On the other hand, the leaf and root TSS contents increasedn all of the water-stress treatments provably due to a drought-nduced starch hydrolysis and restriction in sucrose translocationrom leaves and diminished use of assimilates by leaf and root

rowth induced by water stress (Sturm and Tang, 1999). Our dataeinforce the hypothesis that soluble sugars are the organic soluteshat most contribute to the osmotic adjustment in the leaves andoots of higher plants under water-stressed conditions (Iannucci etl., 2002).

imental Botany 69 (2010) 279–285

Although the proline content is not changed in roots, prolineaccumulation is higher in leaves under the more severe droughttreatment. Nevertheless, the leaf proline content has not significanteffect on the osmotic potential in J. curcas plants. Proline may havedifferent roles in drought mechanisms, such as in the scavengingof free radicals and thereby protecting cellular structures againstoxidative damage and denaturation (Girija et al., 2002), and canalso serve as a carbon and nitrogen reserve for growth after stressrelief (Silveira et al., 2003). However, our data showed that pro-line not accumulates in J. curcas tissues in sufficient quantities tofunction either as an osmolyte or a protein protectant. The accu-mulation of proline in this study is likely due to the response of thetissue to stress-induced damage than to acclimation or adaptationfor drought tolerance.

In spite of the J. curcas plants not undergoing prominent changesin the leaf and root GB concentrations in stressed plants underwater stress (it was significantly increased only at the lowestmoisture level), the GB contribution to osmotic adjustment of well-watered and drought stresses is significant. There is circumstantialevidence that water stress-induced GB synthesis is an adaptiveresponse since it may function as a non-toxic osmolyte or anosmoprotectant primarily in the cytoplasm and organelles such aschloroplasts (Bajji et al., 2001). The subcellular location of GB in thecell cytoplasm is important for drought tolerance, and improvedGB synthesis is triggered by both water and salt stress in mostChenopodiaceae species (Martinez et al., 2005; Hessine et al., 2009).

If the GB accumulation is exclusively confined into the cytosol,which usually makes up 10% of the total cell volume, then its effec-tive concentration can be 10-fold higher (Silveira et al., 2009) in allof the studied treatments. In this case, GB jointly with soluble sug-ars and K+ would contribute mostly to OA in the cytoplasm of leafand root cells. Besides being considered as an efficient osmolyte,GB is thought to improve tolerance to dehydration (Sakamoto andMurata, 2002), to stabilize the protein structure of the PSII complexand to prevent oxidative damages and protection of cell mem-branes of drought-stressed plants (Yang et al., 2007; Hassine et al.,2008).

In this context, our data reinforces the suggestion that GB mayhave a central role in both cellular protection and cytosol OA in leafand root cells of J. curcas exposed to water stress. Previously, wedemonstrated that GB is an important solute for osmotic adjust-ment of J. curcas leaves subjected to salt stress (Silva et al., 2009a).Apparently the high level of GB in leaf and root tissues of J. curcasplants is constitutive, i.e. its synthesis is not triggered by drought(Fig. 2) or under salinity conditions (Silva et al., 2009a.). Conversely,the accumulation and/or synthesis of soluble sugars are stronglystimulated by drought stress and this process is dependent of waterdeficit level. Sucrose represented approximately 25% of total solu-ble sugars in roots and leaves of drought-stressed plants (data notshown).

Although the more severe drought levels have decreased signif-icantly the photosynthesis (CO2 assimilation rate) and dry matteryield in J. curcas, our data suggest that this species was able toexhibit an effective acclimation to drought, especially in terms ofosmotic adjustment and maintenance of a good water status inthe leaves even under severe conditions of stress. In addition, theplants did not exhibit any visual symptoms of drought stress suchas chlorosis or drying in the stressed leaves. These data suggest thatthose physiological mechanisms employed by physic nut plantscould be able to confer drought resistance under field conditions.

In conclusion, roots and leaves of J. curcas young plants display

an effective osmotic adjustment mechanism in response to a widerange of drought stresses involving the participation of some inor-ganic (K+, Na+, Cl−, and NO3

−) and organic solutes (TSS, TFAA, GB).However, of all of the studied solutes, soluble sugars uniquely dis-play a prominent drought-induced synthesis and/or accumulation

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development, growth and carbon partitioning. Trends Plant Sci. 4, 401–407.Yang, X., Wen, X., Gong, H., Lu, Q., Yang, Z., Tang, Y., Liang, Z., Lu, C., 2007. Genetic

E.N. Silva et al. / Environmental and

n both roots and leaves. The osmotic adjustment in J. curcas isssociated with mechanisms linked to restriction of water loss andaintaining a good water status in leaves.

cknowledgements

We thank Conselho Nacional de Desenvolvimento Científico eecnológico (CNPq) and Fundacão Cearense de Apoio ao Desen-olvimento Científico e Tecnológico (FUNCAP) for financial support..A.G.S. is a CNPq research scientist and E.N.S. holds a CNPq fel-owship. The authors gratefully acknowledge the Tamanduá Farmnstitute, Santa Terezinha-PB (Brazil) for supplying the physic nuteeds.

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