ammonia assimilation and proline

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AMMONIA ASSIMILATION AND PROLINEACCUMULATION IN YOUNG CASHEW PLANTS

DURING LONG TERM EXPOSURE TO NaCl-SALINITY1

Ricardo Almeida Vigas2 and Joaquim Albensio Gomes da Silveira3

Laboratrio de Metabolismo e Fixao do Nitrognio (LABFIX), Departamento deBioqumica e Biologia Molecular, Universidade Federal do Cear, C.P. 6020, Fortaleza,Cear, 60451-970, Brazil. E-mail: [email protected]

ABSTRACT - In spite of years of research, the biochemical basis for the accumulation of nitrogenouscompounds, mainly proline, in plants growing under saline conditions still remains unresolved. To studythis process, 35-day-old young cashew plants (Anacardium occidentaleL.), were cultured with 0 (control),50 or 100 mol m-3 NaCl in the nutrient solution for 30 days. The shoot dry mass was 23 and 52% lower inplants growing with 50 and 100 mol m-3 NaCl, respectively, while root dry mass was only reduced in the

highest level of NaCl as compared to control. Nitrate reductase (NR) activity both in leaf and root wasdecreased about 70% in relation to the control in both salt treatments. Contrarily, leaf glutamine synthetase(GS) activity and soluble proteins increased two-fold in the highest NaCl level. On the other hand, the saltstress decreased GS activity and soluble protein in the root. The total free amino acid, free proline, andammonium concentrations increased in the leaf whereas in the root they were nearly unchanged inresponse to salinity. These results indicate that salinity treatment induced a differential behavior forammonium assimilation and protein metabolism among roots and leaves of cashew. Thus, we concludethat proline accumulation in the leaf of cashew plants seems to be related to glutamate from the GS/GOGAT cycle, which could be explained, at least in part, by a higher availability of NH

3in salt treated

cashew, probably originated from increases in photorespiration and leaf protein catabolism.

Additional index terms: glutamine synthetase, nitrate reductase, nitrogen metabolism, salinity, saltstress.

ASSIMILAO DE AMNIA E ACUMULAO DE PROLINA EM PLANTASJOVENS DE CAJUEIRO SUBMETIDAS SALINIDADE DURANTE LONGA

DURAO

RESUMO- A despeito de vrios anos de pesquisa, ainda no foram esclarecidos os mecanismosbioqumicos da acumulao de compostos nitrogenados solveis, particularmente prolina, em plantassubmetidas ao estresse salino. Como objetivo de estudar tais relaes, plantas jovens de cajueiro(Anacardium occidentaleL.), uma espcie arbrea sensvel salinidade, foram tratadas durante 30 diascom soluo nutritiva contendo zero; 50 ou 100 mol m-3 de NaCl. A massa seca da parte area dasplantas estressadas foi reduzida em 23 e 52%, nos dois nveis de NaCl, respectivamente, enquantoque a massa das razes foi reduzida somente pelo maior nvel de NaCl, em aproximadamente 33%. A

atividade in vivode redutase de nitrato (RN), em folhas e razes, foi reduzida de maneira similar emaproximadamente 70%, tanto em 50 quanto em 100 mol m-3 de NaCl. Ao contrrio, a atividade total deglutamina sintetase (GS) nas folhas aumentou cerca de duas vezes nas plantas estressadas, observan-do-se variao semelhante para a concentrao de protenas solveis. Entretanto, os tratamentos comNaCl induziram significativas redues na atividade de GS e concentrao de protenas solveis nasrazes. As concentraes de aminocidos livres totais, prolina livre e amnia foram aumentadas nasfolhas por efeito do NaCl, enquanto que nas razes permaneceram praticamente inalteradas. Os resulta-dos demonstraram que os efeitos da salinidade induziram respostas diferentes na assimilao de amnia

1Received 05/17/1999 and accepted 08/10/1999.2Professor Assistente, Universidade Federal da Paraiba/

Campus de Patos, Bolsista de Doutorado da CAPES.

3Professor Adjunto, Doutor, Universidade Federal do Cear.Bolsista de Pesquisa do CNPq.


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e metabolismo de protenas entre folhas e razes. Aocontrrio, a atividade de RN foi reduzida pelo NaCl comintensidade similar nessas duas partes da planta. pos-svel que o intenso incremento no acmulo de prolinanas folhas das plantas estressadas possa ter sido rela-

cionado com a maior produo de glutamato atravs dociclo GS/GOGAT, a partir da maior disponibilidade deNH

3e glutamato provenientes de aumentos nos proces-

sos de fotorrespirao e catabolismo de protenas,ambos induzidos pela salinidade.

Termos adicionais para indexao: estresse salino,metabolismo de nitrognio, redutase de nitrato,salinidade, sintetase de glutamina.

INTRODUCTION

Nitrogen assimilation has assumed centralimportance in plants exposed to saline and other stress

conditions (Rao & Gnanam, 1990). In view of thedecreased productivity of crop plants in saline soils,uptake and reduction of nitrate assume pivotal roles inplants exposed to saline and other stress conditions(Krishna & Gnanam,1990). Thus, it seems to be correctto suggest that a high plant sensitivity to changes imposedby salinity on NO-

3assimilation leads to more severe,

deleterious salt-induced effects on plant growth (Vigaset al., 1999), since several nitrogenous compoundsderived from N assimilation such as proteins, amines andamino acids, are believed to be related to salinity andother forms of environmental stress tolerance (Rabe,1993; Winicov, 1998).

Molecules known as compatible solutes constitute agroup which includes some amino acids (particularlyproline) and quaternary compounds of ammonium, andtheir accumulation has been a common response to saltstress in both cell suspensions and intact plants(Greenway & Munns, 1980; La Rosa et al., 1991;Kuznetsov & Shevyakova, 1997). These compoundsshare the property of being uncharged at neutral pH, arehighly soluble in water and, when at high concentration,they have little or no effect on macromolecule-solventinteractions (Yancey, 1994). The compatible solutes tendto be excluded from the hydration sphere of proteins andstabilize folded protein structure, whereas the inorganicions such as Na+ and Cl- readily enter the hydration sphere

of proteins, favoring unfolding (Kuznetsov & Shevyakova,1997).

Proline accumulation is a common metabolic responseof higher plants to water deficits and salinity stress (Taylor,1996) and it has been proposed to be part of the processof osmotic adjustment that contributes to the cellularadaptation of many plant species to drought, salinity, andother stresses (Stewart, 1981). This highly water solubleamino acid is accumulated by leaves of many halophyticplant species grown in saline environments (Briens &Larher, 1982). Proline may play a role in protectingmembranes and proteins against the adverse effects ofhigher concentrations of inorganic ions and temperature

extremes (Santoro et al., 1992). Of the two knownbiochemical pathways leading to proline (Thompson,

1980), the one starting with glutamate has been shownto be functional under water stress in tomato cells (Rhodset al., 1986).

A substantial supply of glutamate is needed whenthere are high rates of proline synthesis in salt stressed

plants (Berteli et al., 1995). Under conditions of osmoticstress the glutamine synthetase (GS)-glutamatesynthase (GOGAT) cycle has been shown to be the mostimportant source of glutamate (Rhods et al., 1986).However, studies with tobacco cells expressing salttolerance and over-expression of the P5CR enzyme,which catalyzes the last step in proline synthesis, showthat enzyme was not correlated with differential salinitytolerance (La Rosa et al., 1991). They conclude that otherreactions in the glutamate pathway seem(s) to be mostlimiting for the increase in proline synthesis. On the otherhand, tomato plants cultured in saline conditionspresented higher GOGAT activity whereas the GS activitywas slightly reduced (Berteli et al., 1995). These results

are related to tissue proline accumulation, suggesting thatproline synthesis was dependent on glutamate availability.

In the other study NaCl salinity has been shown tostimulate GS activity (Roosens et al., 1998), suggestingthat the GS/GOGAT cycle should play a key role insupplying glutamate for proline synthesis in plants growingunder salt stress (Berteli et al., 1995; Peng et al., 1996).Recently, Costa (1999), working with Vigna unguiculataplants subjected to water stress, observed a parallelincrease in the GS activity and proline concentration inleaves. On the other hand, it is possible that someenvironmental stress conditions such as water deficitand salinity could lead to increases in leaf protein

catabolism (Rabe, 1993), yielding high amounts ofammonia and free amino acids. The ammonia producedunder these conditions might be re-assimilated throughthe GS/GOGAT cycle (Lea, 1997).

The photorespiratory nitrogen cycle present in plantswith C3 photosynthetic mode is responsible for highamounts of NH3 recycling. The amount of ammoniarecycling during photorespiration is about twenty foldhigher than that from nitrate assimilation (Kumar & Abrol,1990). In addition there is some evidence that watershortage and salinity (Kriedemann & Downton, 1981) leadto increases in photorespiration, particularly underconditions of high temperature and full sunlight (Canvin,1990). Under stress conditions, high concentrations ofpoliamines, free amino acids and ammonium ion occurin plant tissue (Silveira & Crocomo, 1989; Rabe, 1993)which, in turn, favors proline synthesis, particularly underreduced demand for amino acids protein synthesis andgrowth in stressed plants (Rabe, 1993).

In a previous study with young cashew plantssubjected to salinity-shock with 100 mol m-3 NaCl, weobserved a steady increase in leaf free amino acids andproline concentration parallel to an increase in solubleprotein concentration 24 hours after the salt treatment(Vigas et al., 1999). In spite of a high free prolineconcentration in leaves of young cashew plants, it wasnot possible to establish accurately the effective role of

proline in osmotic adjustment as well as its metabolicorigin in cashew plants growing under conditions of

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salinity. Thus, the present study was carried out toestablish the relationship between proline accumulationand glutamine synthetase activity in young cashew plantsgrowing under NaCl-salinity conditions in a long termexperiment.

MATERIALS AND METHODS

Plant growth conditions and harvest

Cashew (Anacardium occidentaleL.) seeds, CP 1001clone, were surface sterilized for 10min in 0.5% (v/v)sodium hypochlorite and further washed with anabundance of distilled water, after which they weregerminated in vermiculite saturated with 0.1 mol m -3CaSO4 at environmental temperature. After 20 days fromgermination, the plants were allowed to acclimatize toone-tenth ionic strength of Hoagland and Arnons nutritivesolution, modified according to Vigas et al. (1999), over

a 10-day period in individual vessels containing 5L ofnutrient solution continuously aerated and changed at 5day intervals during the experimental period, presentingthe following composition - mol m-3 (0.4 KNO3; 0.3Ca(NO3)2; 0.1 CaCl2; 0.1 MgSO4; 0.1 K2HPO4; andmicronutrients - mmol m-3 (4 H

3BO

3; 0.9 Mn2+; 1.8 Cl-;

0.03 Cu2+; 0.07 Zn2+; 5 Na2MoO4; 10 Fe-EDTA). After thisacclimatization period, the ionic strength of the nutrientsolution was increased tenfold and salinity treatmentstarted 10 days later, when seedlings were exposed tono NaCl addition (control) or 50 or 100 mol m -3 NaClsupplied in the nutrient solution. Each treatment wasreplicated 4 times in a complete randomized design. The

pH of the solution was adjusted daily to values between5.5 and 6.0. Plants were grown under greenhouseconditions under full sunlight and natural 12-h photoperiodof summer in Fortaleza, Brazil, with temperatures rangingfrom 28C to 36C during the day and from 24C to 27Cduring the night. Relative humidity means in thegreenhouse were about 45 and 82% during day and night,respectively. After 30 days of salinity treatment, 4 plantsper treatment were harvested and separated into roots,stem, and leaves. Fresh mass was then determined andplant parts were both frozen in liquid nitrogen and kept at-20C until measurement of glutamine syntethase (GS)activities or lyophilized for determination of chemicalcomposition.

Solute measurements

Samples of lyophilized plant tissue were extracted in10 ml of distilled, deionized water and then placed in aboiling water bath to estimate the free amino acid pool.An aliquot of extract was reacted with ninhydrin reagentand its absorbance read at 510 nm as previouslydescribed (Vigas et al., 1999). Free proline wasestimated according to Bates et al. (1973), Bradfordsmethod (1976) was utilized to estimate soluble proteincontent and ammonia concentration was assayedaccording to the phenolate colorimetric method describedin Silveira & Crocomo (1989). Total chlorophyll content

was measured according to Arnons method describedby Costa (1999).

Glutamine syntethase (GS) activity

Frozen leaves and roots were separately ground in achilled mortar in the presence of liquid nitrogenand thenextracted, with 6 mL/g fresh mass of an extraction buffercontaining Tris-HCl 25 mol m-3 pH 7.6; EDTA 5 mol m-3;

5% (m/v) polyvinypolypyrrolidone; 10 mol m -3mercaptoethanol, for five minutes, at 4C. Thehomogenate was slowly filtered through two layers ofcheese cloth and cleared by centrifuging at 20,000g for30 minutes at the same extraction temperature. Thesupernatant was collected and utilized to evaluateglutamine synthetase activity according to thehydroxamate biosynthesis method described previously(Silveira et al., 1998a). An aliquot of enzyme extract (0.2mL) was added to the reaction medium then reactedwith 0.6 mL 250 mol m-3 Tris-HCl, 0.2 mL 300 mol m -3 Na-glutamate, 0.2 mL 30 mol m-3 ATP, 0.2 mL 500 mol m -3MgSO4. The reaction was initiated by adding 0.2 mL 1:1

(v/v) NH2OH.HCl 1.0 mmol.cm-3

: NaOH 1.0 mmol.cm-3

mixture in the reaction medium. After 30 minutesincubation, at 32C, the reaction was stopped by adding0.5 mL 1:1:1 FeCl3.6H2O 10% (m/v) in HCl 0.2 mmol.cm

-

3; TCA 24% (v/v); HCl 50% (v/v) in the assay medium.Subsequently, the reaction mixture was centrifuged at7,000g for 10 minutes at room temperature. Aftercentrifugation the supernatant was read at 540 nm in aspectrophotometer. The results were expressed as mmolgamma-glutamyl hydroxamate formed h-1 kg fresh mass-1.

NR-activityin vivo assayThe second leaf (the youngest completely expanded)

or root apical tissue (200 mg fresh mass) were harvested

4 h after onset of light period and placed in the 5 mL ofan assay medium containing 100 mol m-3 potassiumphosphate buffer pH 7.5, 50 mol m-3 KNO3 and 1% (v/v) iso-propanol, at 30C for 30 minutes in anaerobicconditions under darkness. The NO 2

- formed wasdetermined by the colorimetric method describedpreviously by Silveira et al. (1998b). The results wereexpressed as mmol NO-2 produced h

-1 kg fresh mass-1.

RESULTS AND DISCUSSION

Effects of NaCl-salinity on plant growth and NR activity

In the current study young cashew plants were cultured

over long-term exposure to NaCl (0, 50 or 100 mol m-3).Under these conditions, the root dry mass was lessaffected by salinity than the shoot(Figure 1A). This is atypical response pattern of non-halophytic species tosalinity (Munns & Termaat, 1986; Allarcn et al., 1993;Nabil & Coudert, 1995). Furthermore, salinity treatmentled to reduction in total leaf area and the root extensionwas slowed and followed by swelling (data not shown). Inaddition, visible senescence and necrosis symptoms werefound on the basal, oldest leaf of young cashew plantsafter ten days of salt treatment. These symptoms couldbe a result of excess Na+ and Cl- ions which inducechlorosis and then death of the oldest leaf (Nabil &

Coudert,1995). While, on the one hand, these senescencesymptoms clearly showed a salt ionic toxicity, they could

Ammonia assimilation and proline accumulation in young cashew plants . . .


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DRY MASS A

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FIGURE 1-Dry matter accumulation in shoot and root (A), leafchl a and total chl content (B) and leaf-nitrate reductase activity(C)of cashew plants submitted to 0, 50 or 100 mol m-3NaClfor 30 days. The data are expressed as % of control (0 molm-3 NaCl); 100% of shoot and root dry matter accumulationwere 8.82 and 2.24 g plant-1 , respectively; 100% of chl a andtotal chl content were 4.5 and 6.7 g kg-1 dry mass, respectively;and 100 % of leaf and root NR activity were 0.50 and 1.08

mmol NO-2 kgfresh mass-1 h-1, respectively. The coefficient ofvariation varied from 8 to 12%.

provide, on the other, a convincing argument that therewas a substantial reduction in the photosynthetic CO2assimilation, indicated by a decrease in total chlorophyllcontent (Figure 1B) and an intense decrease intranspiration rate (Vigas et al., 1999).

According to Munns & Termaat (1986), during longterm exposure to salt, plants experience ionic stress whichcan lead to premature senescence of mature leaves andthus a reduction in the photosynthetic area available tosupport further growth. On account of reducedtranspiration flux and lower nitrate uptake in the salttreated plants, the leaf NO

3- content was one half of the

control (Vigas et al., 1999). In the present study, leafand root nitrate reductase activity in salt treated plantswas about 70% lower than control (Figure 1C).Nevertheless, it is not clear if the NR activity or the in situnitrate assimilatory reduction process are limiting stepsfor the young cashew plant growth under these conditionsof restricted amino acid supply for protein synthesis. Most

likely, salinity treatment led to intense NR proteindegradation and decreased protein synthesis. Moreover,NR protein turnover as well as NR synthesis and activityare strongly dependent on nitrate flux from the metabolicpool in water stressed plants (Foyer et al., 1998). Aprevious study has shown that in vivoNR activity wasgreatly reduced due to salinity 24 h after salt addition tocashew plants (Vigas et al., 1999).

Visual symptoms of salt induced leaf senescence incashew plants were associated with decrease inchlorophyll content (Figure 1B) and increase in leaf solubleprotein (Figure 2A), free amino acids (Figure 2B) andammonium content (Figure 2C). In roots a decrease in

soluble protein was observed (Figure 2A) and only slightchanges in free amino acids (Figure 2B) and ammoniumcontent occurred (Figure 2C). In this manner, salinitystress seemed to induce early, pronounced senescencein the leaf, estimated from chlorophyll degradation andaccumulation of soluble nitrogenous fraction.

Protein turnover, photorespiration and prolineaccumulation

The total leaf GS activity (on a fresh mass basis)increased about twofold in plants growing with 100 molm-3 NaCl whilst in the roots it was about 20 and 40%reduced with 50 and 100 mol m-3 NaCl, respectively (Fi-gure 3A). These results are similar to those of solubleprotein content (Figure 2A), suggesting a differentialbehavior for the NaCl effect on protein metabolism andGS activity in leaf and root. As briefly stated above,salinity-induced senescence was mainly seen in leavesand not in roots. Thus, it is expected that increasing leafprotease activity induces a concomitant increase insoluble nitrogenous fractions.

Accumulation of soluble nitrogenous fractions underconditions of high photorespiratory activity should leadto an increase in amino acid turnover parallel to ammoniaproduction (Feller, 1990; Kumar & Abrol, 1990). It isimportant to mention that the present study was carriedout under conditions of high temperature and high

photosynthetic radiation which, in turn, are believed toincrease photorespiration rates in C3

plants (Canvin,

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SOLUBLE PROTEIN A

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FIGURE 2 - Soluble protein (A), total free amino acid (B) andfree ammonia (C)in leaf and root of cashew plants submittedto 0, 50 or 100 mol m-3 NaCl during 30 days. The data areexpressed as % of control (0 mol m -3 NaCl); 100% of leaf androot soluble protein content were 7.78 and 2.84 g kg -1 freshmass, respectively; 100% of leaf and root total amino acidcontent were 47.0 and 110.5 mmol kg-1 dry mass, respectively;and 100% of leaf and root ammonia content were 1.50 and6.25 mmol kg-1 dry mass, respectively. The coefficient ofvariation varied from 7 to 10%.

1990). Some evidence has suggested a closerelationship between photorespiratory rates and the levelof chloroplastic glutamine synthetase isoforms in manyhigher plant species (Lea, 1993). The level of leafglutamine synthetase isoforms partly reflects the extent

to which ammonia metabolism is dominated by thephotorespiratory nitrogen cycle (Stewart et al., 1987).Plants with a C

3photosynthetic system have 60-100% of

their total GS activity as GS2, the chloroplastic isoform.This GS isoform synthesis could be induced by thephotorespiratory activity (Lea, 1997).

In the present study, the higher leaf GS activity ofsalt stressed plants was related to higher free amino acidsand ammonium content (Figure 2B, 2C), and prolineaccumulation (Figure 3B). Recently, an increase in activity

GS ACTIVITY A

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FIGURE 3 - Glutamine synthetase activity (A) and free proline(B) and in leaf and root of cashew plants submitted to 0, 50or 100 mol m-3NaCl during30 days. The data are expressedas % of control (0 mol m-3 NaCl); 100% of leaf and root prolinecontent were 3.52 and 2.21 mmol kg-1 dry mass, respectively;and 100% of leaf and root GS activity were 9.52 and 10.64mmol -glutamil hydroxamate kg-1 fresh mass h-1, respectively.The coefficient of variation varied from 6 to 9%.



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of the glutamate pathway enzymes was observed forsalt treated plants (Roosens et al., 1998). As there is noagreement with respect to the mechanism of the GSactivity increase under conditions of salinity, we havepostulated that it is a result of imbalances in other

metabolic processes. Therefore, both the sudden fall inthe transpiration rate (Vigas et al., 1999), and theobserved increase in the soluble N fractions induced bysalinity treatment have pointed towards to a strong declinein the photossynthetic CO2 assimilation and to aconsiderable increase in protease activity, respectively.The latter process, through an increase in proteincatabolism, provides amino acids that could contributeto the osmotic adjustment and may play a key role inmitigating nitrogen deficiency caused by decreased nitratereduction (Figure 1C) as an additional source of glutamateand NH3. On the other hand, since CO2 and O2 arecompetitive substrates for RUBISCO (Canvin, 1990), asmaller CO

2to O

2ratio, due to an expected decrease in

CO2 availability, provoked by salinity induced stomatalclosure, will favor the enzyme oxygenase activity, therebyincreasing photorespiration. Evidence suggests that NH

3formed in photorespiration is re-assimilated via a cytosolicglutamine synthetase (Lea, 1997). This would produceglutamine that would move into the chloroplast forconversion to glutamate via glutamate synthase (Kumar& Abrol, 1990).

A substantial body of evidence has been built up toshow that the GS/GOGAT pathway is the sole port ofentry of NH3 into amino acids in higher plants (Lea et al.,1990). Generally in higher plants both glutamate andornithine are recognized as possible precursors of proline

(Delauney et al., 1993). However, for the salt treatedplants, free proline increase seemed to be due to theactivity of the enzymes of the glutamate pathway(Roosens et al., 1998). A recent study has shown increaseboth in the Fd-GOGAT activity and proline accumulationin tomato leaves although the protein content and GSactivity did not significantly change by imposition ofsalinity stress (Berteli et al., 1995). The authors suggestthat synthesis or transamination rather than other eventsis likely the source of glutamate for proline synthesis.Indeed, when mulberry (Morus alba) plants weresubjected to salt stress, there was a significant increasein GS, GDH and aminotransferase activities and in freeamino acid concentration (Ramanjulu et al., 1994).

The accumulation of proline (Figure 3B) and theincrease in leaf GS activity (Figure 3A) induced by salinityseem to support this this hipothesis. In addition, itreinforces our suggestion that proline accumulationappears to be, to a large extent, a result of the saltinhibitory effect on the photosynthetic CO2 assimilationand increase in protein catabolism, which, in turn, induceincrease of glycine-NH3 recycling from photorespiration.In addition, other events such as decrease in the prolineoxidation to glutamate (Stewart et al., 1977), decreasedutilization of proline in protein synthesis (Peng et al.,1996), and enhancement of protein degradation(Fukutoku & Yamada, 1984) should, to a considerable

extent, account for the high content of proline in leavesof salt treated plants.

It is possible that a higher level of the free ammoniumand glutamate or from protein catabolism andphotorespiratory nitrogen cycle induced a higher GSconcentration and/or activity in leaves of salt treatedplants. Thereafter, higher availability of glutamate could

increase the intensity of glutamate pathway the for prolinesynthesis (Thompson, 1980; Lea et al., 1990; Berteli etal., 1995). In addition, the photorespiratory nitrogen cycleshould also increase free ammonium and amino acidcontent (Kumar & Abrol, 1990). On the other hand, somestudies have shown that NaCl-salinity led to an increaseboth in the activity and concentration of GOGAT (Berteliet al., 1995) while another study has shown an increasein enzyme activity of the glutamate pathway (Roosens etal., 1998). Indeed, under these metabolic conditionsproline synthesis is stimulated from the increase inglutamate and ornithine (Delauney et al., 1993).

This study suggests that protein catabolism and GSactivity were differently affected by NaCl in leaves and

roots. Furthermore, root proline content was not changedin response to salinity (Figure 3B) while in the leaf it wasabout 19 fold increased in plants growing with 100 molm-3 NaCl. It is probable that an increase of this order ofmagnitude was not only dependent on higher availabilityof the ammonia and glutamate from leaf proteincatabolism and from the photorespiration induced byNaCl-salinity. In addition, gene induction and over-expression of enzyme synthesis of proline pathwayinduced by salt stress are expected (Petrusa & Winicov,1997; Winicov, 1998). Thus, it is important to observethat the root was not able to accumulate proline inresponse to salt stress. The effectiveness of leaf proline

accumulation for osmotic adjustment in young cashewplants seems to be questionable. Nevertheless, moreresearch is necessary to discover the role of proline inthe osmotic adjustment and salt tolerance in youngcashew plants (Vigas et al., 1999).

In conclusion, our results lead us to suggest thatintense leaf proline accumulation in young cashew plantscultured under salinity conditions was closely related toincreased GS activity. On the other hand, the increase inGS activity appeared to be induced by the increase inammonia and glutamate levels from photorespiration andfrom protein catabolism processes, leading to stimulationof the glutamate pathway for proline synthesis.

ACKNOWLEDGEMENTS

To Financiadora de Estudos e Projetos (FINEP),Conselho de Desenvolvimento Cientfico e Tecnlogico(CNPq) and Fundao de Amparo Pesquisa do estadodo Cear (FUNCAP) for financial support. To CNPq forResearch Fellowship to Dr. J.A.G. Silveira.

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