+++++Response of Nitrogen Fixation in Relation to Nodule Carbohydrate Metabolism in Medicago Ciliaris Lines Subjected to Salt Stress

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Journal of Plant Physiology 166 (2009) 477488

Response of nitrogen fixation in relation to nodule

carbohydrate metabolism in Medicago ciliaris linessubjected to salt stress

Imene Ben Salaha, Alfonso Albaceteb, Cristina Mart nez Andujarb,Rabiaa Haoualac, Nehla Labidia, Fethia Zribia, Vicente Martinezb,Francisco Perez-Alfoceab, Chedly Abdellya,

aLaboratoire dAdaptation des Plantes aux Stress Abiotiques, CBBC, BP 901, 2050 Hammam-Lif, TunisiabDepartment of Plant Nutrition, CEBAS-CSIC, Campus de Espinardo, 30100 Mucia, SpaincDepartement de Biologie, Institut Superieur de Biotechnologie de Monastir, BP 74, 5000 Monastir, Tunisia

Received 1 February 2008; received in revised form 2 June 2008; accepted 25 June 2008

KEYWORDS

Medicago ciliaris;

Nitrogen fixation;Nodule carbonmetabolism;Salt stress;Sucrose transport

SummaryThe effect of salt stress on nitrogen fixation, in relation to sucrose transport towards

nodules and other sink organs and the potential of sucrose breakdown by nodules,was investigated in two lines of Medicago ciliaris. Under salt stress conditions, thetwo lines showed a decrease of total biomass production, but TNC 1.8 was lessaffected by salt than TNC 11.9. The chlorophyll content was not changed in TNC 1.8,in contrast to TNC 11.9. Shoot, root, and nodule biomass were also affected in thetwo lines, but TNC 1.8 exhibited the higher potentialities of biomass production ofthese organs. Nitrogen fixation also decreased in the two lines, and was moresensitive to salt than growth parameters. TNC 1.8 consistently exhibited the highervalues of nitrogen fixation. Unlike nodules, leaves of both lines were well supplied innutrients with some exceptions. Specifically, the calcium content decreased in thesensitive line leaves, and the nodule magnesium content was not changed in eitherline. The tolerant line accumulated more sodium in its leaves. The two lines did notshow any differences in the nodule sodium content. Sucrose allocation towards

nodules was affected by salt in the two lines, but this constraint did not seem toaffect the repartition of sucrose between sink organs. Salt stress inducedperturbations in nodule sucrolytic activities in the two lines. It inhibited sucrosesynthase, but the inhibition was more marked in TNC 11.9; alkaline/neutral activitywas not altered in TNC 1.8, whereas it decreased more than half in TNC 11.9. Thus,the relative tolerance of TNC 1.8 to salt stress could be attributed to a better use ofthese photoassimilates by nodules and a better supply of bacteroids in malate.

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0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved.doi:10.1016/j.jplph.2008.06.016

Corresponding author. Tel.: +216 71 430 855; fax: +216 71 430 934.E-mail address: [email protected] (C. Abdelly).
http://www.elsevier.de/jplphhttp://dx.doi.org/10.1016/j.jplph.2008.06.016mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.jplph.2008.06.016http://www.elsevier.de/jplph


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The hypothesis of a competition for sucrose between nodules and other sink organsunder salt stress could not be verified.& 2008 Elsevier GmbH. All rights reserved.

Introduction

In plants, the major carbon transport from sourceleaves towards heterotrophic sink tissues is bysucrose. Sucrose is channeled into various pathwaysin different subcellular compartments. It may beused for the production of ATP and NADH and alsofor the biosynthesis of primary metabolites im-portant for tissue growth and development (Sturm,1999). In legumes, an additional synthetic processcan take place: N2 fixation, which occurs in nodule.It is the product of a complex interaction betweenlegume plant and soil bacteria collectively calledRhizobia. This process, like all synthetic processes,

imposes an energy burden on the plant (Minchinet al., 1981; Warembourg and Roumet, 1989), andthe carbon cost for this process is high. The priorityof nodules for sucrose is still a subject ofcontroversy. Some studies have argued thatthese organs represent strong sink for sucrose(Gordon et al., 1985; By Janez et al., 1997) andthat 4050% of each days photosynthetic productsare processed by these organs, with approximately50% of this lost as respired CO2 (Ryle et al., 1985,1986; Gordon et al., 1987). Other reports, however,have suggested the contrary (Boller and Heichel,1983; Cralle and Heichel, 1985; Kouchi et al.,

1985).Salt stress has been studied extensively, as soil

salinity represents an increasing problem foragriculture in many countries. Adaptation tosalinity is associated with osmoregulation adjust-ment, which leads to the accumulation of severalorganic solutes, such as proline, glycine betaineand also sugars. Under this constraint, the sucrosetransport towards sink organs is diminishedand competition for photoassimilates betweendifferent physiological processes (including toxicions exclusion, selective nutrient uptake and

osmotic adjustment) and between sink organscould occur and result in plant growth and yieldreduction (Munns, 1993; Balibrea et al., 1996,2000; Yeo, 1998). Nitrogen fixation by root nodulesis known to be inhibited by several mechanisms:a decrease in oxygen diffusion to the infected zone(Serraj et al., 1995), enhancement of reactiveoxygen species generation in the nodules (Mhadhbiet al., 2004; Jebara et al., 2005) and also alimitation in the energetic substrates shortageto bacteroids following photosynthetic activityand nodule sucrose breakdown reduction (Soussi

et al., 1998, 1999; Galvez et al., 2005; Lopez et al.,2007).

Annual medics (Medicago spp.) are winter annuallegumes. These species are capable of forming rootnodules in symbiosis with soil bacteria that can fixatmospheric nitrogen (N2). These plants are there-fore good candidates for the improvement ofmarginal or degraded lands with low fertility and/or high salinity such as Sebkha edges. In such areasin Tunisia, several medic species live in associationwith halophytes and can produce, in rainy years,40% of the vegetative cover (Abdelly et al., 1999).These medics promote halophyte growth throughimproving the soil quality, resulting from its

aptitude to enrich the soil in nitrogenous com-pounds. Among these medics, Medicago ciliaris isthe most salt-tolerant species. Under controlledconditions and in the presence of mineral nitrogenand 100 mM NaCl, this species maintained itsbiomass production in comparison with Medicago

polymorpha, Medicago truncatula and Medicagominima (Abdelly et al., 1995).

In this work, the physiological responses and thepotential of sucrose breakdown by nodules werecompared in two Medicago ciliaris lines cultivatedin symbiosis with Sinorhizobium medicae. Further,

particular attention was paid to

14

C-sucrose trans-port to young leaves, root tips and nodules to verifyif whether there is eventual competition betweenthese organs for sucrose.

Materials and methods

Biological materials and growth conditions

Seeds of two lines of Medicago ciliaris were providedby the Laboratory of Interaction between Legumes andMicroorganisms in the Center of Biotechnologies at thetechnopole of Borj Cedria in Tunis. These lines are

originated from local populations from the edge of salinedepressions of Enfidha (TNC 1.8) and non-saline habit inMateur (TNC 11.9). Their hard seed coat strongly limitsgermination, and treatment of the seeds with concen-trated H2SO4 for 40min was necessary to obtain amaximal rate of germination. The seeds were thenwashed 10 times with sterile distilled water and placedon sterile agar medium at 25 1C in the dark. Three dayslater, germinated seeds were transferred into pots filledwith sterile vermiculite and were inoculated with 1 mL(about 108/mL) of Sinorhizobium medicae CI 1.12/E22strain suspension (Zribi et al., 2007), which was kindlyprovided by the same laboratory.

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I. Ben Salah et al.478


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Experiments were performed in the greenhouse fromMay to June under controlled conditions: 27/19 1Ctemperature and at relative humidity of 4080% day/night; day length ranged from 13 to 14 h. Plants wereregularly irrigated with N-free nutrient solution (Hewitt,1966). Salt treatment (100 mM) was applied a monthlater. The plants were harvested 3 weeks after the startof salt treatment (at late vegetative stage). Plants wereuprooted carefully, washed with distilled water andseparated into shoots and roots. In addition, noduleswere carefully separated from the roots and all plantparts were oven dried at 60 1C for 3d, and dry weightswere determined.

Nitrogen-fixation assay and nitrogen determination

Nitrogen fixation was estimated by measuring insitu the acetylene reduction activity (ARA) at the end ofthe vegetative stage. The ARA assay in closed systemsmay underestimate the nitrogenase activity due toacetylene-induced inhibition of this enzyme. However,

this assay is useful for comparative purposes. A total of10% C2H2 was added to the root atmosphere. After 20minof incubation, formed ethylene was measured using a gaschromatograph (Cromatix KNK-2000) (Hardy et al.,1968). N was determined as described by Kjeldahl(1983). Nitrogen fixation was estimated as the differencebetween total N quantities (g plant1) before and aftersalt treatment.

Chlorophyll content

Chlorophyll concentration (mg g1 FW) of fully ex-panded leaves was determined according to the method

of Torrecillas et al. (1984). Five mL of acetone 80% wasadded to fresh leaf samples (approximately 100 mg) cutinto discs. The total extraction took place after 72 h indarkness at 4 1C. Absorbance of extracts was measuredat 649 and 665 nm.

Nutrient determination

The dried and ground samples (100mg) of leaves,stems, roots and nodules were digested in micro-wave with HNO3:H2O2 (5:3) according to the methodof Oliva et al. (2003), and the mineral concentrationwas determined by inductively coupled plasma spectro-metry.

14C-sucrose application and radioactivity

determination

One day before the harvest, an area of two selected insitu source leaves (fully expanded) was gently abraded toenhance [U-14C]-sucrose (625 mCi/mmol) entry throughthe cuticle. A volume of 10 mL of 14C-sucrose (15 kBq) wasapplied on each leaf and it was then covered withlanoline. Nodules, root tips and young leaves (near theapex of stems) were harvested 1, 2, 3 and 24h after14C-sucrose application and were cut in small pieces. The

radioactivity of the solid samples was determined usingOptiPhase HiSafe 3 cocktail (WallacPerkin Elmer) byliquid scintillation spectroscopy with a resulting countingefficiency of 97.8%.

Enzyme extraction and assay

After harvest, nodules were immediately frozen inliquid nitrogen and stored at 80 1C until analysis.Samples containing polyvinylpirrolydone and Fontaine-bleau sand were homogenized in 1 mL of extractionbuffer containing 50mM HEPES (pH 7), 10mM MgCl2,1 mM EDTA(2H2O), 2.6 mM dithiothreitol (DTT), 10%ethylene glycol and 0.02% Triton X-100 (Pelleschiet al., 1997). After centrifugation at 20,000g, thesupernatant was desalted on a G25-Sephadex columnpre-equilibrated with 4 mL of the reaction buffercontaining 50 mM HEPES, pH 7, 2 mM MgCl2, 1 mMNa2EDTA, 2.6mM DTT and 0.1% bovine serum albumin.Sucrose synthase, alkaline, and soluble and insoluble acid

invertases were assayed by an enzyme-linked assaymonitoring NADH formation at 340nm (Balibrea et al.,2003).

Sucrose, malate, total soluble sugars and

starch determination

Sucrose and malate were analyzed according toBalibrea et al. (1997). The ethanol extracts (1:3, W:V)were directly filtered through 13mm diameter and0.45 mm pore size Millex filters (Millipore Co., Milford,MA, USA) and analyzed by HPLC (Shimadzu Co. Ltd.,Kyoto, Japan). Sucrose was determined by using a

column Waters Spherisorb NH2 5 mm (Waters Co., Milford,MA, USA) and RI detection, with acetonitrilewater(63:31, V:V) as a phase mobile at a flow rate of1mLmin1 and at 45 1C. Malate was determined byusing an ion exchange column Interaction ORH-801(Interaction Chromatography Inc., San Jose, CA, USA),an UV detector at 210nm, a mobile phase with H2SO40.01 N at a flow rate of 0.6mLmin1 and at 451C.Quantification was performed by the external standardmethod by using a data analysis Chrompass program(Jasco Co. Ltd., Tokyo, Japan). Total soluble sugarswere quantified with the anthrone reagent according toYemm and Willis (1954). After washing the pellet

resulting from the soluble sugars with 80% ethanol andhydrolyzing with perchloric acid 35%, starch was deter-mined with the same method using glucose as thestandard (Yemm and Willis, 1954).

Statistical analysis

Analysis of variance was used for the statisti-cal analysis of data. Mean separation procedureswere carried out using the multiple range testswith Fishers least significant difference procedure(Po0.05).

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Nitrogen fixation under salt stress 479


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Results

Growth and chlorophyll content

The effect of salt stress on growth parameters isshown in Table 1. In the absence, as in the presenceof 100 mM NaCl, TNC 1.8 showed higher production

of whole plant biomass. The adverse effect ofsalinity was significant in both lines of Medicagociliaris, but total plant growth inhibition provedgreater in TNC 11.9 than TNC 1.8 (reduction of 42%compared to 19% in the latter). This reduction wasassociated with a decline in shoot and root biomassin the two lines, but TNC 1.8 exhibited the higherpotentialities of biomass production of theseorgans. The chlorophyll content was poorlydependent on the presence of NaCl in the mediumin TNC 1.8. On the contrary, salt induced a largedecrease in the chlorophyll of TNC 11.9 leaves

(Table 2).The results showed that nodules are the most

sensitive organs to salt in the two lines, with thissensitivity always higher in TNC 11.9 (reduction of45% compared to 25% in TNC 1.8). Specificnitrogenase activity, estimated by the acetylenereduction assay, also decreased in the two lines,and was more sensitive to salt than nodule growth(Table 1). TNC 1.8 consistently exhibited higher

values of nitrogen fixation (40% of control incomparison to 14% of control in TNC 11.9). Withrespect to the amount of fixed nitrogen (Table 1), adecline was observed in both lines under salt stress,with TNC 1.8 showing higher values of N-fixed incomparison with the other line.

Nutrient determination

In the absence of, as in the presence of salt andin the leaves, roots and nodules of the two lines,potassium was preferentially accumulated; it wasfollowed by magnesium and then calcium. Undersalt stress, only the calcium content of TNC 11.9leaves was diminished, that of the tolerant line wasnot affected. Potassium and magnesium contents inthe leaves increased in both lines (Table 3). Parallelto this increase, stem and root potassium contentof both lines decreased. Both lines accumulated

more sodium in leaves and stems in comparisonwith roots. TNC 1.8 accumulated more sodium in itsleaves in comparison with the sensitive line. Saltstress reduced potassium and calcium content innodules of the two lines to the same extent,whereas the magnesium content was not affected.Salt stress induced an increase in nodule sodiumcontent without any significant differences be-tween the two lines (Table 3).

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Table 1. Effect of NaCl (mM) treatment on whole plant (WPDW, mg plant1), roots (RDW, mg plant1) and nodule dry

weight (NDW, mg plant1

), specific acetylene-reduction activity (ARAs, mmol C2H4 h1

mg1

NDW) and nitrogen fixation(N-fixed, mg plant1) of Medicago ciliaris lines inoculated with Sinorhizobium medicae CI 1.12/E22 strain

Line WPDW SDW RDW NDW ARAs N-fixed

TNC 1.8

0 mM NaCl 27637155c 20537137c 667769c 49.577.1b 0.48170.097 7.270.4c100 mM NaCl 22327327b 15427426b 540748b 37.176.3a 0.19370.039b 5.071.5b

TNC 11.9

0 mM NaCl 21707191b 16897167b 528739b 51.977.1b 0.50670.118c 11.770.8d100 mM NaCl 12547147a 10467143a 260739a 28.473.5a 0.07070.018a 2.670.1a

Number of plants 7 7 7 7 5 3

Mean values followed by the same letter are not significantly different at Po0.05.

Table 2. Chlorophyll concentration changes in two Medicago ciliaris lines inoculated with Sinorhizobium medicae CI1.12/E22 strain after 21 d of salt stress (100 mM)

NaCl (mM) Chl a Chl b Chl total

TNC 1.8 0 0.6270.21a 0.3970.15ab 1.0170.36ab100 0.5870.22ab 0.4170.15ab 0.9970.37ab

TNC 11.9 0 0.8370.11b 0.3870.08b 1.2270.18b100 0.3870.08a 0.2470.06a 0.6270.14a

Mean values followed by the same letter are not significantly different at Po0.05 (n 5).



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14C-sucrose transport

14C-sucrose transport to young leaves, root tipsand nodules in control and treated plants of the twolines occurred even after 1 h of its application onfully expanded source leaves. Generally, no differ-ences between sink organs or treatments wereobserved (Figure 1). Interestingly, in the absence of

salt, specific radioactivity in nodules increasedsignificantly and reached a maximum after 3 h andthen declined after 24h; this was not the case foryoung leaves and root tips, in which specific radio-activity remained unchanged and increased onlyafter 24 h. The two lines displayed the same trend.

The increase in specific radioactivity observed incontrol nodules was not recorded in treated nodules.In fact, no change in specific radioactivity wasobserved even after 24 h of 14C-sucrose applicationon fully expanded source leaves in TNC 1.8; itincreased slightly in TNC 11.9 but remained sig-nificantly lower than that in control nodules. Understress conditions, young leaves and root tips had thesame specific radioactivity as in control plants. Itincreased only after 24 h, but values were inferior tocontrol values in root tips of the two lines and youngleaves of TNC 11.9. The specific radioactivity inyoung leaves of TNC 1.8 was not affected by salt.

Nodule sucrolytic activities

The activity of all sucrose-cleaving enzymes(sucrose synthase, alkaline/neutral, cell-wall and

vacuolar invertases) in control and salt-stressednodules was investigated (Figure 2). In controlnodules, important sucrose synthase and alkaline/neutral invertase activities were found; the activityof cell-wall invertase was too low, whereas vacuo-lar invertase activity was not detectable (Figure 2).With respect to salt stress-induced perturbations innodule sucrolytic activities in the two lines, we

found that sucrose synthase was inhibited, but theinhibition was more accentuated in TNC 11.9.Alkaline/neutral activity was not altered in TNC1.8, whereas it decreased by more than half in TNC11.9. Cell-wall invertase was increased in TNC 1.8and diminished slightly in TNC 11.9. Total sucrolyticactivities, calculated as the sum of sucrosesynthase, alkaline/neural, cell-wall and vacuolarinvertases, are shown in Figure 3. Under controlconditions, TNC 11.9 nodules showed higher totalsucrolytic activities. In nodules, the negative effectof salt stress on total sucrolytic activities was more

pronounced in TNC 11.9.

Nodule carbohydrates and malate content

In the absence of salt, total soluble sugars andsucrose were higher in TNC 11.9 in comparison withTNC 1.8. The response to salt stress was differentbetween the two lines; the nodule content of thesecompounds increased in TNC 1.8, while it de-creased in TNC 11.9. The starch content in TNC 1.8control nodules was higher compared with TNC

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Table 3. Changes in ions content (mgg1 DM) in leaves and nodules of Medicago ciliaris lines cultivated in theabsence or presence of 100 mM NaCl

Ions content (mgg1 DM) NaCl (mM) K Ca Mg Na

TNC 1.8

Leaves 0 23.270.2a 7.271.2a 7.170.2a 1.070.1a100 31.071.6b 6.370.8a 10.170.6b 54.371.9c

Stems 0 30.771.3c 5.171.0ab 2.770.1a 1.570.2a100 15.372.7a 3.770.7a 4.370.4c 42.272.6c

Roots 0 21.973.8b 4.570.3c 12.574.3b 1.870.1a100 8.173.8a 2.470.4a 8.970.2ab 13.071.1b

Nodules 0 28.271.8b 3.470.3b 4.570.3a 1.470.1a100 18.971.6a 2.170.2a 6.371.8a 13.670.4b

TNC 11.9

Leaves 0 24.571.5a 8.870.9b 7.170.1a 1.070.1a100 35.172.4c 7.470.5ab 10.971.2b 47.671.6b

Stems 0 35.272.0d 5.770.2ab 3.570.2b 1.470.1a100 19.973.3b 7.073.1b 3.670.2b 37.671.6bRoots 0 33.373.5c 5.470.5d 7.571.3a 2.970.4a

100 24.071.4b 3.070.1b 6.270.3a 23.871.8c

Nodules 0 36.173.2c 3.170.4c 4.670.6a 1.170.2a100 26.071.5b 2.570.2b 6.271.2a 13.870.9b

Mean values followed by the same letter are not significantly different at Po0.05 (n 3).



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11.9. However, this content decreased by a third inTNC 1.8, but was not changed in TNC 11.9. Themalate content of nodules of both lines decreased,

but this decrease was more accentuated in thesensitive line: the reduction was of about 66%against 31% in the tolerant line (Figure 3).

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Young leaves

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1 2 3 24 1 2 3 24

Figure 1. Specific radioactivity (calculated on the basis of the organ weight) found in young leaves, root tipsand nodules of control (~) and salt-stressed () Medicago ciliaris lines, in symbiotic nitrogen fixation,after 14C-sucrose application on two source leaves. Mean values followed by the same letter are not significantlydifferent at Po0.05 (n 3).

b

Total sucrolytic activities Sucrose synthase Cell-wall invertaseAlkaline / neutral invertase

nmolNADHmn-1mg-1FW

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Figure 2. Total sucrolytic activities, sucrose synthase, alkaline/neutral, cell-wall and vacuolar invertase activities ofcontrol (&) and salt-stressed ( ) nodules of two Medicago ciliaris lines after 21 d of treatment with 100 mM of NaCl.Mean values followed by the same letter are not significantly different at Po0.05 (n 3).



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Discussion

In the present work, the effect of 100 mM NaCl ontwo lines of Medicago ciliaris, inoculated withSinorhizobium medicae, applied at the vegetativegrowth stage, was investigated. Our results showedthat salt stress reduced the growth of the two lines,but TNC 1.8 was more tolerant than TNC 11.9.Nodules were more sensitive to salt than were thevegetative organs in the two lines, with TNC 1.8

always exhibiting better biomass production. Thisline also showed better activity of nitrogenfixation, expressed as acetylene reduction on adry weight basis. The data showed that nodulegrowth was less affected by salt than was nodulefunctioning; in fact, while the inhibition of nodulegrowth was 25% and 45%, nitrogen fixation was

depressed by 60% and more than 86%, in TNC 1.8and TNC 11.9, respectively. Some authors havereported that the reduction of nodule biomassproduction and functioning under salt stress is dueto an inhibition of infection resulting from alimitation in root and nodule growth followingrestriction of photoassimilates supply (Saadallahet al., 2001). In our two lines, however, nodulenumber was not affected by salt (data not shown);this could suggest that salt stress did not inhibit thedevelopment of new nodule generation in the twolines.

It is assumed that salt stress causes an imbalanceof the cellular ions leading to nutritional perturba-tions. In this study, our two lines accumulatedhigher amounts of sodium in aerial parts incomparison with roots (Table 3). Photosyntheticorgans were well supplied in nutrients with theexception of calcium, which decreased in thesensitive line. Interestingly, under stress condi-tions, leaf potassium and magnesium contentsincreased (in the case of both lines) and leafcalcium was unchanged (in the case of the tolerantline). However, potassium, magnesium (in bothlines) and calcium (in the tolerant line) amounts

allocated to these organs were maintained con-stant under stress conditions, and this maintenanceoccurred at the expense of stems and roots(Figure 4). The tolerant line accumulated moresodium in its leaves in comparison with thesensitive line (Table 3); this line could replacepotassium by this ion for the osmotic adjustment,and use potassium for specific functions such asenzyme activation, photosynthesis and stomatamovements (Maeser et al., 2002).

Although the tolerant line accumulated higheramounts of sodium in its leaves, chlorophylls seem

to be stable. The loss of chlorophyll is oftenconsidered as a marker of a cellular component ofsalt stress (Singh and Dubey, 1995). Therefore, itseems that TNC 1.8 preserved better photosyn-thetic productivity, given that its chlorophyllcontent was not influenced by salt stress, as shownin chickpea (Mahmoudi et al., 2005). In thesensitive line (TNC 11.9), sucrose flow from leavesto nodules could fall following the salt-induceddecrease in photosynthetic activity as reported byLopez et al. (2007) in Medicago truncatula andLotus japonicus. In TNC 1.8, in spite of the possible

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TNC 1.8 TNC 11.9

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Figure 3. Sucrose, total soluble sugars (TSS), starch andmalate content changes in control (&) and salt-stressed() nodules of two Medicago ciliaris lines after 21 d ofsalt treatment with 100 mM of NaCl. Mean valuesfollowed by the same letter are not significantly differentat Po0.05 (n 3).



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maintenance of photosynthetic activity, nodulegrowth and nitrogen fixation were drasticallyreduced. Thus, the relative tolerance of nitrogen

fixation in this line could not be entirely explainedby its better photosynthetic production. It hasgenerally been assumed that nitrogen fixation ismore sensitive to abiotic stresses than is photo-synthesis (Soussi et al., 1998; Galvez et al., 2005).The question arises, therefore, whether salt stressaffected the step that follows photosynthatesproduction, namely the allocation of these pro-ducts to nodules. There have been no reports onthe effect of this constraint on sucrose transporttowards nodules.

As illustrated in Figure 1, 14C-sucrose transport

towards control nodules of the two lines increasedduring the first 3 h, following its application on fullyexpanded source leaves, and declined after 24 h.The opposite was observed for other sink organs:14C-sucrose transport remained unchanged for thefirst 3 h and then increased. This led us to suggestthat nodules had more priority to sucrose incomparison with vegetative organs. Other authorshave argued the contrary; they have suggested thatphotoassimilates are allocated to the strongest sink(sink strength size activity) and that nodulesare very active sinks but they represent a relatively

small proportion of the total plant biomass.Consequently, photoassimilate distribution to ve-getative apices and new leaves has priority over

distribution to nodules (Boller and Heichel, 1983;Cralle and Heichel, 1985; Kouchi et al., 1985). Thedecline recorded in transported 14C-sucrose to-wards nodules of our two lines could be explainedby the fact that it is instantaneously used forbuilding, maintaining, functioning of nodule ma-chinery and the export of nitrogenous compoundstowards organs including young leaves and roottips. Salt stress reduced 14C-sucrose in nodules ofthe two lines 2 h after 14C-sucrose application onsource leaves. Interestingly, after 24 h, the sensi-tive line (TNC 11.9) showed higher nodule 14C-

sucrose content, which was less affected by saltthan in TNC 1.8. In spite of the drastic decrease in14C-sucrose in nodules of the two lines, youngleaves and root tips had the same 14C-sucrosecontent in comparison with control plants: itincreased only after 24 h. Thus, it can be hypothe-sized that the decrease of 14C-sucrose transporttowards nodules is not due to competition betweennodules and other sink organs for sucrose (youngleaves and root tips), given that the 14C-sucrosetransport towards these later organs was notincreased under salt.

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34%

46%

18%2%

64%

27%

8% 1%

33%

44%

20%3%

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18%2%

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25%

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65%16%

17%2%

51%

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56%37%

7%0%

54%32%

13%1%

48%

34%

17% 1%

58%29%

12% 1%

53%32%

14% 1%

52%37%

10%1%

K+

Ca2+

Mg2+

Na+

TNC 11.9TNC 1.8

70 44 64 30

1015 14 7

18 17 12 8

4289

100mM NaCl0mM NaCl0mM NaCl 100mM NaCl

Figure 4. Potassium, calcium, magnesium and sodium repartition within plant organs in control and salt-treated plantsof Medicago ciliaris. Values in the center correspond to the total absorbed amount of ion (mg organ1). Leaves;stems; roots; nodules.



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In these latter organs and after 24 h, values wereless than the control in root tips of the two linesand young leaves of TNC 11.9. The 14C-sucrosecontent in young leaves of TNC 1.8 was not affectedby salt. These results show a probable ability ofyoung leaves of this tolerant line to attractphotoassimilates under salt stress in comparison

with other vegetative organs in the same plant andin the sensitive line. This likely allows the main-tenance of leaf growth and the continuation ofsucrose synthesis and transport, not only towardsnodules but also to other growing organs or storedas starch. In the two lines, nodules showed lower14C-sucrose content in comparison with othersink organs. However, we cannot confirm thatnodules have less priority for assimilates, assuggested by Gordon et al. (1985) and Janezet al. (1997), given that the sucrose contentrepresents the instantaneous sum of a number of

dynamic processes including import from thesource leaves, respiratory loss as 14CO2, re-fixationof 14CO2 and re-export of

14C-labelled nitrogenproducts newly formed in nodules towards otherorgans.

In nodules of our two lines, important sucrosesynthase and alkaline/neutral invertase activitieswere found; the activity of cell-wall invertase wastoo low, whereas vacuolar invertase activity wasnot detectable (Figure 2). This is partially inagreement with data presented by Flemetakiset al. (2006) and Morell and Copeland (1984);cytoplasmic alkaline/neutral invertase was found

to be the predominant invertase activity in nitro-gen-fixing mature nodules. Both sucrose synthaseand alkaline/neutral invertase activity werereduced by salt in the sensitive line (TNC 11.9).The decline in total sucrolytic activities in thetolerant line (TNC 1.8) was attributed only to adrop in sucrose synthase activity; this inhibitionwas less pronounced than it was in the sensitiveone. These results could explain the higher sucrosecontent in nodules of the sensitive line. It wasdemonstrated previously that the alkaline/neutralinvertase activity was not affected by stresses that

reduced nitrogen fixation and could not compen-sate for the lack of sucrose synthase activity inthe pea rug4 mutant (Gordon et al., 1999). Thedecrease in sucrose synthase activity led to areduction in the malate content in both lines, butthis reduction was less pronounced in the tolerantline (Figure 3). This decline could lead to a shortageof substrates for bacteroid respiration and probablya disturbance in the regulation of oxygen perme-ability (Galvez et al., 2000). Consequently, it couldexplain the observed decline in nitrogenase activityin both lines and provide additional evidence for

the central role of sucrose synthase in the regula-tion of nodule carbon metabolism and nitrogenfixation, and explain the relative tolerance of TNC1.8 to salt stress. Sucrose cleavage activity ofsucrose synthase, apart from its role in providinghexoses for malate synthesis, is also linked to cell-wall biosynthesis by providing UDP-glucose for the

cellulose synthase complex and substrates forstarch synthesis in sink organs (Koch, 2004) and soensured better maintenance of nodule growthunder salt stress in the tolerant line.

It has been suggested that the decrease insucrolytic activities would lead to an accumulationof sucrose (Gordon et al., 1997; Galvez et al.,2005). But in this study, in spite of higher activitiesof enzymes of sucrose breakdown and the reductionof sucrose transport towards nodules of thetolerant line (Figure 1), this latter accumulatedunder salt stress more sucrose in nodules in

comparison with the sensitive one, which, incontrast, showed a decrease in the sucrose contentof its nodules (Figure 3). On the other hand, thestarch content was reduced in the tolerant line, butnot in the sensitive line. We can thereforehypothesize that starch (in which synthesis wouldbe better in the tolerant line owing to greateractivity of sucrose synthase) mobilization couldaccount for the increase in total soluble sugarsnamely sucrose and then contributes in osmoticadjustment under salt stress as it was suggested inPhaseolusnodules subjected to water stress (Ramoset al., 1999). The accumulation of organic solutes

could constitute an adaptive response to salt stress,given that this mechanism is involved in therestoration of turgor, the reduction of oxidativedamage induced by free radicals and also mem-branes structure and enzymes stabilization (Chenand Murata, 2002).

Under this constraint, potassium and calciumcontent in the nodules decreased, probably follow-ing competition with sodium on the absorption site.This decrease may alter the physiological activityof nodules. In fact, it was proposed by Minchinet al. (1994) that calcium and magnesium contents

are involved in the operation of the corticaldiffusion barrier. In parallel, increased levels ofNa+ and Cl in the cytoplasm could affect a varietyof metabolic activities. In our two lines, nodulesaccumulated the same amount of Na+; these lineslikely presented differential distributions of this ionacross nodule cell layers (uninfected and infectedcells) as shown by Abd-Alla et al. (2001). Under saltstress, Na+ enters cells and would be sequesteredin vacuoles (Tester and Davenport, 2003). Thissequestration could induce cytoplasmic acidifica-tion (Katsuhara et al., 1989) unless cytoplasm

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regulates its pH by H+ extrusion to the mediumand/or metabolic buffering by H+-consumingreactions (Fan et al., 1989) such that it is catalyzedby malate dehydrogenase (Smith and Raven, 1979).This enzyme shows greater activity in comparisonwith sucrose synthase in nodules (80, 64 and 56times in soybean, Medicago truncatula and pea

nodules, respectively) (see Gordon, 1992; Gonzalezet al., 1998; Lopez et al., 2007). The activity of thisenzyme could have decreased under salt stress, asshown by Soussi et al. (1999) and Lopez et al.(2007), and likely induced intracellular pH changescausing likely the observed inhibition of sucrolyticenzymes in our two lines.

In conclusion, on the basis of the data relative to14C-sucrose transport, salt stress decreased theallocation of sucrose towards nodules, which havepriority to sucrose. The depressive effect of saltstress on sucrose allocation towards these organs

did not seem to be linked to competition betweennodules and other sink organs for sucrose. Theresults presented here also allow us to suggestthat the relative tolerance of TNC 1.8 to saltstress is related to its ability to conserve photo-synthetic activity and to maintain higher nodulesucrolytic activity. The data provide further evi-dence for the importance of nodule carbohydratemetabolism in nitrogen-fixation maintenance understress conditions.

Acknowledgments

This work was funded by the Tunisian Ministry ofHigher Education, Scientific Research and Techno-logy (LR02CB02) and by the Fundacion Seneca(Communidad Autonoma de Murcia, Spain, project03011/PI/05). We wish to thank the Laboratory ofInteraction between Legumes and Microorganismsin the Center of Biotechnologies at the technopoleof Borj Cedria in Tunis for the generous gift ofSinorhizobium medicae CI 1.12/E22 strain andMedicago ciliarisseeds. Besides, the technical helpof Dr. Maria Dolores Alcazar Fernandez in the

Servicio de Radioproteccion y Residuos in theServicio de Apoyo a las Ciencias Experimentales(SACE, Campus de Espinardo, Universidad deMurcia) is gratefully acknowledged.

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+++++Response of Nitrogen Fixation in Relation to Nodule Carbohydrate Metabolism in Medicago Ciliaris Lines Subjected to Salt Stress