Revista Brasileira de FisiologiaVegetal, 11(3):153-159, 1999.

AMMONIA ASSIMILATION AND PROLINE ACCUMULATION 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 de Bioqumica e Biologia Molecular, Universidade Federal do Cear, C.P. 6020, Fortaleza, Cear, 60451-970, Brazil. E-mail: labfix@ufc.br
ABSTRACT - In spite of years of research, the biochemical basis for the accumulation of nitrogenous compounds, mainly proline, in plants growing under saline conditions still remains unresolved. To study this process, 35-day-old young cashew plants (Anacardium occidentale L.), 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 in plants 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 was decreased 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 salt stress decreased GS activity and soluble protein in the root. The total free amino acid, free proline, and ammonium concentrations increased in the leaf whereas in the root they were nearly unchanged in response to salinity. These results indicate that salinity treatment induced a differential behavior for ammonium assimilation and protein metabolism among roots and leaves of cashew. Thus, we conclude that 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 NH3 in salt treated cashew, probably originated from increases in photorespiration and leaf protein catabolism. Additional index terms: glutamine synthetase, nitrate reductase, nitrogen metabolism, salinity, salt stress.

ASSIMILAO DE AMNIA E ACUMULAO DE PROLINA EM PLANTAS JOVENS DE CAJUEIRO SUBMETIDAS SALINIDADE DURANTE LONGA DURAO
RESUMO - A despeito de vrios anos de pesquisa, ainda no foram esclarecidos os mecanismos bioqumicos da acumulao de compostos nitrogenados solveis, particularmente prolina, em plantas submetidas ao estresse salino. Como objetivo de estudar tais relaes, plantas jovens de cajueiro (Anacardium occidentale L.), uma espcie arbrea sensvel salinidade, foram tratadas durante 30 dias com soluo nutritiva contendo zero; 50 ou 100 mol m-3 de NaCl. A massa seca da parte area das plantas estressadas foi reduzida em 23 e 52%, nos dois nveis de NaCl, respectivamente, enquanto que a massa das razes foi reduzida somente pelo maior nvel de NaCl, em aproximadamente 33%. A atividade in vivo de redutase de nitrato (RN), em folhas e razes, foi reduzida de maneira similar em aproximadamente 70%, tanto em 50 quanto em 100 mol m-3 de NaCl. Ao contrrio, a atividade total de glutamina sintetase (GS) nas folhas aumentou cerca de duas vezes nas plantas estressadas, observando-se variao semelhante para a concentrao de protenas solveis. Entretanto, os tratamentos com NaCl induziram significativas redues na atividade de GS e concentrao de protenas solveis nas razes. As concentraes de aminocidos livres totais, prolina livre e amnia foram aumentadas nas folhas por efeito do NaCl, enquanto que nas razes permaneceram praticamente inalteradas. Os resultados demonstraram que os efeitos da salinidade induziram respostas diferentes na assimilao de amnia

1Received 05/17/1999 2Professor

and accepted 08/10/1999. 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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Vigas & Silveira 1980), the one starting with glutamate has been shown to be functional under water stress in tomato cells (Rhods et al., 1986). A substantial supply of glutamate is needed when there are high rates of proline synthesis in salt stressed plants (Berteli et al., 1995). Under conditions of osmotic stress the glutamine synthetase (GS)-glutamate synthase (GOGAT) cycle has been shown to be the most important source of glutamate (Rhods et al., 1986). However, studies with tobacco cells expressing salt tolerance and over-expression of the P5CR enzyme, which catalyzes the last step in proline synthesis, show that enzyme was not correlated with differential salinity tolerance (La Rosa et al., 1991). They conclude that other reactions in the glutamate pathway seem(s) to be most limiting for the increase in proline synthesis. On the other hand, tomato plants cultured in saline conditions presented higher GOGAT activity whereas the GS activity was slightly reduced (Berteli et al., 1995). These results are related to tissue proline accumulation, suggesting that proline synthesis was dependent on glutamate availability. In the other study NaCl salinity has been shown to stimulate GS activity (Roosens et al., 1998), suggesting that the GS/GOGAT cycle should play a key role in supplying glutamate for proline synthesis in plants growing under salt stress (Berteli et al., 1995; Peng et al., 1996). Recently, Costa (1999), working with Vigna unguiculata plants subjected to water stress, observed a parallel increase in the GS activity and proline concentration in leaves. On the other hand, it is possible that some environmental stress conditions such as water deficit and salinity could lead to increases in leaf protein catabolism (Rabe, 1993), yielding high amounts of ammonia and free amino acids. The ammonia produced under these conditions might be re-assimilated through the GS/GOGAT cycle (Lea, 1997). The photorespiratory nitrogen cycle present in plants with C3 photosynthetic mode is responsible for high amounts of NH3 recycling. The amount of ammonia recycling during photorespiration is about twenty fold higher than that from nitrate assimilation (Kumar & Abrol, 1990). In addition there is some evidence that water shortage and salinity (Kriedemann & Downton, 1981) lead to increases in photorespiration, particularly under conditions of high temperature and full sunlight (Canvin, 1990). Under stress conditions, high concentrations of poliamines, free amino acids and ammonium ion occur in plant tissue (Silveira & Crocomo, 1989; Rabe, 1993) which, in turn, favors proline synthesis, particularly under reduced demand for amino acids protein synthesis and growth in stressed plants (Rabe, 1993). In a previous study with young cashew plants subjected to salinity-shock with 100 mol m-3 NaCl, we observed a steady increase in leaf free amino acids and proline concentration parallel to an increase in soluble protein concentration 24 hours after the salt treatment (Vigas et al., 1999). In spite of a high free proline concentration in leaves of young cashew plants, it was not possible to establish accurately the effective role of proline in osmotic adjustment as well as its metabolic origin in cashew plants growing under conditions of

e metabolismo de protenas entre folhas e razes. Ao contrrio, a atividade de RN foi reduzida pelo NaCl com intensidade similar nessas duas partes da planta. possvel que o intenso incremento no acmulo de prolina nas folhas das plantas estressadas possa ter sido relacionado com a maior produo de glutamato atravs do ciclo GS/GOGAT, a partir da maior disponibilidade de NH3 e glutamato provenientes de aumentos nos processos 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 central importance in plants exposed to saline and other stress conditions (Rao & Gnanam, 1990). In view of the decreased productivity of crop plants in saline soils, uptake and reduction of nitrate assume pivotal roles in plants exposed to saline and other stress conditions (Krishna & Gnanam,1990). Thus, it seems to be correct to suggest that a high plant sensitivity to changes imposed by salinity on NO-3 assimilation leads to more severe, deleterious salt-induced effects on plant growth (Vigas et al., 1999), since several nitrogenous compounds derived from N assimilation such as proteins, amines and amino acids, are believed to be related to salinity and other forms of environmental stress tolerance (Rabe, 1993; Winicov, 1998). Molecules known as compatible solutes constitute a group which includes some amino acids (particularly proline) and quaternary compounds of ammonium, and their accumulation has been a common response to salt stress in both cell suspensions and intact plants (Greenway & Munns, 1980; La Rosa et al ., 1991; Kuznetsov & Shevyakova, 1997). These compounds share the property of being uncharged at neutral pH, are highly soluble in water and, when at high concentration, they have little or no effect on macromolecule-solvent interactions (Yancey, 1994). The compatible solutes tend to be excluded from the hydration sphere of proteins and stabilize folded protein structure, whereas the inorganic ions such as Na+ and Cl- readily enter the hydration sphere of proteins, favoring unfolding (Kuznetsov & Shevyakova, 1997). Proline accumulation is a common metabolic response of higher plants to water deficits and salinity stress (Taylor, 1996) and it has been proposed to be part of the process of osmotic adjustment that contributes to the cellular adaptation of many plant species to drought, salinity, and other stresses (Stewart, 1981). This highly water soluble amino acid is accumulated by leaves of many halophytic plant species grown in saline environments (Briens & Larher, 1982). Proline may play a role in protecting membranes and proteins against the adverse effects of higher concentrations of inorganic ions and temperature extremes (Santoro et al., 1992). Of the two known biochemical pathways leading to proline (Thompson,

R. Bras. Fisiol. Veg., 11(3):153-159, 1999.

Ammonia assimilation and proline accumulation in young cashew plants . . . salinity. Thus, the present study was carried out to establish the relationship between proline accumulation and glutamine synthetase activity in young cashew plants growing under NaCl-salinity conditions in a long term experiment.

155

Glutamine syntethase (GS) activity

Frozen leaves and roots were separately ground in a chilled mortar in the presence of liquid nitrogen and then extracted, with 6 mL/g fresh mass of an extraction buffer containing Tris-HCl 25 mol m-3 pH 7.6; EDTA 5 mol m-3; 5% (m/v) polyvinypolypyrrolidone; 10 mol m -3 mercaptoethanol, for five minutes, at 4 C. The homogenate was slowly filtered through two layers of cheese cloth and cleared by centrifuging at 20,000g for 30 minutes at the same extraction temperature. The supernatant was collected and utilized to evaluate glutamine synthetase activity according to the hydroxamate biosynthesis method described previously (Silveira et al., 1998a). An aliquot of enzyme extract (0.2 mL) was added to the reaction medium then reacted with 0.6 mL 250 mol m-3 Tris-HCl, 0.2 mL 300 mol m-3 Naglutamate, 0.2 mL 30 mol m-3 ATP, 0.2 mL 500 mol m-3 MgSO4. 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 minutes incubation, at 32C, the reaction was stopped by adding 0.5 mL 1:1:1 FeCl3.6H2O 10% (m/v) in HCl 0.2 mmol.cm3 ; TCA 24% (v/v); HCl 50% (v/v) in the assay medium. Subsequently, the reaction mixture was centrifuged at 7,000g for 10 minutes at room temperature. After centrifugation the supernatant was read at 540 nm in a spectrophotometer. The results were expressed as mmol gamma-glutamyl hydroxamate formed h-1 kg fresh mass-1.

MATERIALS AND METHODS

Plant growth conditions and harvest
Cashew (Anacardium occidentale L.) seeds, CP 1001 clone, were surface sterilized for 10min in 0.5% (v/v) sodium hypochlorite and fur ther washed with an abundance of distilled water, after which they were germinated in vermiculite saturated with 0.1 mol m-3 CaSO4 at environmental temperature. After 20 days from germination, the plants were allowed to acclimatize to one-tenth ionic strength of Hoagland and Arnons nutritive solution, modified according to Vigas et al. (1999), over a 10-day period in individual vessels containing 5L of nutrient solution continuously aerated and changed at 5 day intervals during the experimental period, presenting the following composition - mol m-3 (0.4 KNO 3; 0.3 Ca(NO3)2; 0.1 CaCl 2; 0.1 MgSO4; 0.1 K2HPO4; and micronutrients - mmol m-3 (4 H3BO3; 0.9 Mn2+; 1.8 Cl-; 0.03 Cu2+; 0.07 Zn2+; 5 Na2MoO4; 10 Fe-EDTA). After this acclimatization period, the ionic strength of the nutrient solution was increased tenfold and salinity treatment started 10 days later, when seedlings were exposed to no NaCl addition (control) or 50 or 100 mol m-3 NaCl supplied in the nutrient solution. Each treatment was replicated 4 times in a complete randomized design. The pH of the solution was adjusted daily to values between 5.5 and 6.0. Plants were grown under greenhouse conditions under full sunlight and natural 12-h photoperiod of summer in Fortaleza, Brazil, with temperatures ranging from 28C to 36C during the day and from 24C to 27C during the night. Relative humidity means in the greenhouse were about 45 and 82% during day and night, respectively. After 30 days of salinity treatment, 4 plants per treatment were harvested and separated into roots, stem, and leaves. Fresh mass was then determined and plant parts were both frozen in liquid nitrogen and kept at -20C until measurement of glutamine syntethase (GS) activities or lyophilized for determination of chemical composition.

NR-activity in vivo assay The 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 of an assay medium containing 100 mol m-3 potassium phosphate buffer pH 7.5, 50 mol m-3 KNO3 and 1% (v/ v) iso-propanol, at 30C for 30 minutes in anaerobic conditions under darkness. The NO 2- formed was determined by the colorimetric method described previously by Silveira et al. (1998b). The results were expressed 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 less affected by salinity than the shoot (Figure 1A). This is a typical response pattern of non-halophytic species to salinity (Munns & Termaat, 1986; Allarcn et al., 1993; Nabil & Coudert, 1995). Furthermore, salinity treatment led to reduction in total leaf area and the root extension was slowed and followed by swelling (data not shown). In addition, visible senescence and necrosis symptoms were found on the basal, oldest leaf of young cashew plants after ten days of salt treatment. These symptoms could be a result of excess Na+ and Cl- ions which induce chlorosis and then death of the oldest leaf (Nabil & Coudert,1995). While, on the one hand, these senescence symptoms clearly showed a salt ionic toxicity, they could

Solute measurements
Samples of lyophilized plant tissue were extracted in 10 ml of distilled, deionized water and then placed in a boiling water bath to estimate the free amino acid pool. An aliquot of extract was reacted with ninhydrin reagent and its absorbance read at 510 nm as previously described (Vigas et al ., 1999). Free proline was estimated according to Bates et al. (1973), Bradfords method (1976) was utilized to estimate soluble protein content and ammonia concentration was assayed according to the phenolate colorimetric method described in Silveira & Crocomo (1989). Total chlorophyll content was measured according to Arnons method described by Costa (1999).

R. Bras. Fisiol. Veg., 11(3):153-159, 1999.

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DRY MASS
120 100 Root

Vigas & Silveira provide, on the other, a convincing argument that there was a substantial reduction in the photosynthetic CO2 assimilation, indicated by a decrease in total chlorophyll content (Figure 1B) and an intense decrease in transpiration rate (Vigas et al., 1999). According to Munns & Termaat (1986), during long term exposure to salt, plants experience ionic stress which can lead to premature senescence of mature leaves and thus a reduction in the photosynthetic area available to support fur ther growth. On account of reduced transpiration flux and lower nitrate uptake in the salt treated plants, the leaf NO3- content was one half of the control (Vigas et al., 1999). In the present study, leaf and root nitrate reductase activity in salt treated plants was about 70% lower than control (Figure 1C). Nevertheless, it is not clear if the NR activity or the in situ nitrate assimilatory reduction process are limiting steps for the young cashew plant growth under these conditions of restricted amino acid supply for protein synthesis. Most likely, salinity treatment led to intense NR protein degradation and decreased protein synthesis. Moreover, NR protein turnover as well as NR synthesis and activity are strongly dependent on nitrate flux from the metabolic pool in water stressed plants (Foyer et al., 1998). A previous study has shown that in vivo NR activity was greatly reduced due to salinity 24 h after salt addition to cashew plants (Vigas et al., 1999). Visual symptoms of salt induced leaf senescence in cashew plants were associated with decrease in chlorophyll content (Figure 1B) and increase in leaf soluble protein (Figure 2A), free amino acids (Figure 2B) and ammonium content (Figure 2C). In roots a decrease in soluble protein was observed (Figure 2A) and only slight changes in free amino acids (Figure 2B) and ammonium content occurred (Figure 2C). In this manner, salinity stress seemed to induce early, pronounced senescence in the leaf, estimated from chlorophyll degradation and accumulation of soluble nitrogenous fraction.

A
Shoot

% of control

80 60 40 20 0 0 50 100 lllll ;;;;

CHLOROPHYLL
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B
total

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NR - ACTIVITY
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Protein turnover, photorespiration and proline accumulation

The total leaf GS activity (on a fresh mass basis) increased about twofold in plants growing with 100 mol m-3 NaCl whilst in the roots it was about 20 and 40% reduced with 50 and 100 mol m-3 NaCl, respectively (Figure 3A). These results are similar to those of soluble protein content (Figure 2A), suggesting a differential behavior for the NaCl effect on protein metabolism and GS activity in leaf and root. As briefly stated above, salinity-induced senescence was mainly seen in leaves and not in roots. Thus, it is expected that increasing leaf protease activity induces a concomitant increase in soluble nitrogenous fractions. Accumulation of soluble nitrogenous fractions under conditions of high photorespiratory activity should lead to an increase in amino acid turnover parallel to ammonia production (Feller, 1990; Kumar & Abrol, 1990). It is important to mention that the present study was carried out under conditions of high temperature and high photosynthetic radiation which, in turn, are believed to increase photorespiration rates in C3 plants (Canvin,

100

NaCl (mol m )

FIGURE 1- Dry matter accumulation in shoot and root (A), leaf chl a and total chl content (B) and leaf-nitrate reductase activity (C) of cashew plants submitted to 0, 50 or 100 mol m-3NaCl for 30 days. The data are expressed as % of control (0 mol m-3 NaCl); 100% of shoot and root dry matter accumulation were 8.82 and 2.24 g plant-1 , respectively; 100% of chl a and total 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 kg fresh mass-1 h-1, respectively. The coefficient of variation varied from 8 to 12%.

R. Bras. Fisiol. Veg., 11(3):153-159, 1999.

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

157

SOLUBLE PROTEIN
200 160 Leaf Root

120 80 40 0 0 50 100

1990). Some evidence has suggested a close relationship between photorespiratory rates and the level of chloroplastic glutamine synthetase isoforms in many higher plant species (Lea, 1993). The level of leaf glutamine synthetase isoforms partly reflects the extent to which ammonia metabolism is dominated by the photorespiratory nitrogen cycle (Stewart et al., 1987). Plants with a C3 photosynthetic system have 60-100% of their total GS activity as GS2, the chloroplastic isoform. This GS isoform synthesis could be induced by the photorespiratory activity (Lea, 1997). In the present study, the higher leaf GS activity of salt stressed plants was related to higher free amino acids and ammonium content (Figure 2B, 2C), and proline accumulation (Figure 3B). Recently, an increase in activity

% of Control

AMINO ACIDS
300 250 Leaf Root

B
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GS ACTIVITY

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150

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1500

Root

1000

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FIGURE 2 - Soluble protein (A), total free amino acid (B) and free ammonia (C) in leaf and root of cashew plants submitted to 0, 50 or 100 mol m-3 NaCl during 30 days. The data are expressed as % of control (0 mol m-3 NaCl); 100% of leaf and root soluble protein content were 7.78 and 2.84 g kg-1 fresh mass, respectively; 100% of leaf and root total amino acid content were 47.0 and 110.5 mmol kg-1 dry mass, respectively; and 100% of leaf and root ammonia content were 1.50 and 6.25 mmol kg-1 dry mass, respectively. The coefficient of variation varied from 7 to 10%.

FIGURE 3 - Glutamine synthetase activity (A) and free proline (B) and in leaf and root of cashew plants submitted to 0, 50 or 100 mol m-3 NaCl during 30 days. The data are expressed as % of control (0 mol m-3 NaCl); 100% of leaf and root proline content were 3.52 and 2.21 mmol kg-1 dry mass, respectively; and 100% of leaf and root GS activity were 9.52 and 10.64 mmol -glutamil hydroxamate kg-1 fresh mass h-1, respectively. The coefficient of variation varied from 6 to 9%.

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Vigas & Silveira It is possible that a higher level of the free ammonium and glutamate or from protein catabolism and photorespiratory nitrogen cycle induced a higher GS concentration and/or activity in leaves of salt treated plants. Thereafter, higher availability of glutamate could increase the intensity of glutamate pathway the for proline synthesis (Thompson, 1980; Lea et al., 1990; Berteli et al., 1995). In addition, the photorespiratory nitrogen cycle should also increase free ammonium and amino acid content (Kumar & Abrol, 1990). On the other hand, some studies have shown that NaCl-salinity led to an increase both in the activity and concentration of GOGAT (Berteli et al., 1995) while another study has shown an increase in enzyme activity of the glutamate pathway (Roosens et al., 1998). Indeed, under these metabolic conditions proline synthesis is stimulated from the increase in glutamate and ornithine (Delauney et al., 1993). This study suggests that protein catabolism and GS activity were differently affected by NaCl in leaves and roots. Furthermore, root proline content was not changed in response to salinity (Figure 3B) while in the leaf it was about 19 fold increased in plants growing with 100 mol m-3 NaCl. It is probable that an increase of this order of magnitude was not only dependent on higher availability of the ammonia and glutamate from leaf protein catabolism and from the photorespiration induced by NaCl-salinity. In addition, gene induction and overexpression of enzyme synthesis of proline pathway induced by salt stress are expected (Petrusa & Winicov, 1997; Winicov, 1998). Thus, it is important to observe that the root was not able to accumulate proline in response to salt stress. The effectiveness of leaf proline accumulation for osmotic adjustment in young cashew plants seems to be questionable. Nevertheless, more research is necessary to discover the role of proline in the osmotic adjustment and salt tolerance in young cashew plants (Vigas et al., 1999). In conclusion, our results lead us to suggest that intense leaf proline accumulation in young cashew plants cultured under salinity conditions was closely related to increased GS activity. On the other hand, the increase in GS activity appeared to be induced by the increase in ammonia and glutamate levels from photorespiration and from protein catabolism processes, leading to stimulation of the glutamate pathway for proline synthesis.

of the glutamate pathway enzymes was observed for salt treated plants (Roosens et al., 1998). As there is no agreement with respect to the mechanism of the GS activity increase under conditions of salinity, we have postulated that it is a result of imbalances in other metabolic processes. Therefore, both the sudden fall in the transpiration rate (Vigas et al., 1999), and the observed increase in the soluble N fractions induced by salinity treatment have pointed towards to a strong decline in the photossynthetic CO 2 assimilation and to a considerable increase in protease activity, respectively. The latter process, through an increase in protein catabolism, provides amino acids that could contribute to the osmotic adjustment and may play a key role in mitigating nitrogen deficiency caused by decreased nitrate reduction (Figure 1C) as an additional source of glutamate and NH3. On the other hand, since CO2 and O2 are competitive substrates for RUBISCO (Canvin, 1990), a smaller CO2 to O2 ratio, due to an expected decrease in CO2 availability, provoked by salinity induced stomatal closure, will favor the enzyme oxygenase activity, thereby increasing photorespiration. Evidence suggests that NH3 formed in photorespiration is re-assimilated via a cytosolic glutamine synthetase (Lea, 1997). This would produce glutamine that would move into the chloroplast for conversion to glutamate via glutamate synthase (Kumar & Abrol, 1990). A substantial body of evidence has been built up to show that the GS/GOGAT pathway is the sole port of entry of NH3 into amino acids in higher plants (Lea et al., 1990). Generally in higher plants both glutamate and ornithine are recognized as possible precursors of proline (Delauney et al., 1993). However, for the salt treated plants, free proline increase seemed to be due to the activity of the enzymes of the glutamate pathway (Roosens et al., 1998). A recent study has shown increase both in the Fd-GOGAT activity and proline accumulation in tomato leaves although the protein content and GS activity did not significantly change by imposition of salinity stress (Berteli et al., 1995). The authors suggest that synthesis or transamination rather than other events is likely the source of glutamate for proline synthesis. Indeed, when mulberry ( Morus alba ) plants were subjected to salt stress, there was a significant increase in GS, GDH and aminotransferase activities and in free amino acid concentration (Ramanjulu et al., 1994). The accumulation of proline (Figure 3B) and the increase in leaf GS activity (Figure 3A) induced by salinity seem to support this this hipothesis. In addition, it reinforces our suggestion that proline accumulation appears to be, to a large extent, a result of the salt inhibitory effect on the photosynthetic CO2 assimilation and increase in protein catabolism, which, in turn, induce increase of glycine-NH3 recycling from photorespiration. In addition, other events such as decrease in the proline oxidation to glutamate (Stewart et al., 1977), decreased utilization 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 leaves of salt treated plants.

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

REFERENCES
ALLARCN, J. J.; SNCHEZ BLANCO, M. J.; BOLARN, M. C. & TORRICELIS, A. Water relations and osmotic adjustment in Lycopersicon esculentum and L. pennellii during short-term salt exposure and recovery. Physiologia Plantarum, 89:441-447, 1993. BATES, L. S.; WALDREN, R. P. & TEARE, I. D. Rapid determination of

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