Salinity induced behavioural changes in malate dehydrogenase and
glutamate dehydrogenase activities in rice seedlings of differing salt
tolerance
Ritambhara G. Kumar, Kavita Shah, R.S. Dubey *
Department of Biochemistry,Faculty of Science,Banaras Hindu Uni6ersity,Varanasi 221 005, India
Received 13 October 1999; received in revised form 31 January 2000; accepted 16 February 2000
Abstract
The activities of malate dehydrogenase in whole tissue extract (NAD+-MDH) as well as in mitochondrial (NAD+-MDH) and
chloroplastic (NADP+-MDH) preparations of aminating (NADH-GDH) and deaminating(NAD+-GDH) glutamate
dehydroge-nases were studied in two sets of rice cultivars differing in salt tolerance grown under moderate (7 dS m−1) and high (14 dS m−1)
NaCl salinity levels. A contrasting response to salinity on enzyme activities was found between the sensitive and tolerant cultivars during a 5 – 20-day growth period of study. NaCl salinity in situ caused increase in all three MDH activities in salt tolerant cvs. CSR-1 and CSR-3 whereas in salt sensitive cvs. Ratna and Jaya 16 – 100% inhibition in activities was noted. Chloroplastic MDH was extremely sensitive to NaCl. In seedlings of salt tolerant cultivars concomitant increase in both aminating and deaminating GDH activities was observed with increase in salinity level, whereas in sensitive cultivars under higher salinity level decrease in GDH activity was noted. Under in vitro conditions NaCl concentration in the range 1 – 1000 mM caused gradual inhibition in MDH activity. With 400 mM NaCl in vitro, complete loss of mitochondrial and chloroplastic MDH activities was observed. GDH activity increased with increasing concentration of NaCl up to 200 mM NaCl and other salts in vitro and was inhibited thereafter. However 800 mM NaCl caused complete loss of deaminating GDH activity from sensitive cultivar but not from tolerant cultivar. Results suggest varying behaviour of MDH and GDH in two sets of rice cultivars differing in salt tolerance and that inhibition in the activities of dehydrogenases in salt sensitive rice cultivars due to salinity may be one of the possible reasons for decreased growth of rice plants under saline conditions. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Salinity; Malate dehydrogenase; Glutamate dehydrogenase; Chloroplast; Mitochondria; Salt tolerance; Rice;Oryza sati6a
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1. Introduction
Soil salinity is a major limitation to agricultural productivity in many parts of the world, specially in arid and semi-arid areas [1]. Salinity inhibits plant growth by lowering soil water potential, causing ion toxicity and ionic imbalance within the tissues [1,2]. The degree to which each of these factors affects growth depends on the plant geno-types, environmental conditions and the extent of salinity [1,2].
Salt tolerance is not conferred by a single trait but is the consequence of complex gene interac-tions [3]. Genotypes of crop species differing in salt tolerance, when grown under increasing levels of NaCl salinity, show distinct morphological dif-ferences as well as alterations in behaviours of key enzymes of various metabolic pathways [1,4 – 7]. Rice species vary widely in their response to salt stress ranging from extremely sensitive to tolerant [4,5,8] and morphological changes of salt effects have been correlated with metabolic abnormalities in growing plants [5,7].
Dehydrogenases play important roles in plant metabolism and catalyze the oxidation or reduc-tion of substrates by removal or addireduc-tion of two
* Corresponding author. Tel.: +91-542-317219; fax: + 91-542-317074.
E-mail address:[email protected] (R.S. Dubey)
hydrogen atoms. The enzymes malate dehydroge-nase (NAD+-MDH, NADP+-MDH) and gluta-mate dehydrogenase (NAD+-GDH) have been purified and characterized from different plant species and their subcellular localization has been investigated in detail [9 – 11]. Malate dehydroge-nases catalyze the interconversion of oxaloacetic acid and malate and exist in various isoforms [12]. The isoforms localized in subcellular organelles like peroxisomes, mitochondria and cytosol are NAD+dependent whereas the chloroplastic one is NADP+dependent [12]. Differential expression of MDH isoforms and changes in its activity have been reported in many plant species under abiotic stresses [11,13]. Activation of NAD+-MDH has often been associated with adaptation to drought in C3 plants [11]. Chloroplastic MDH is very
important in C3 plants, as it shuttles reducing
equivalents between the chloroplast and cytosol [14].
The enzyme glutamate dehydrogenase repre-sents an alternative route to the usual GS/
GOGAT pathway of ammonia assimilation in plants and mediates the reductive amination of
a-ketoglutarate to yield glutamic acid [15]. Al-though, due to high Km of GDH for NH4+ [16],
certain workers have argued against its assimila-tory role [17], in many plant species under growth, differentiation and abiotic stresses, increased activ-ity of GDH and its significant role in the synthesis of glutamate from ammonia released either due to photorespiration or due to nitrate reduction, have been reported [16,18,19]. It is agreed that there is a switch from one pathway of ammonia assimilation to another depending on the nature of stress and the tissue in which the process takes place [9]. GDH catalyses both the amination of a -ketoglu-tarate (aminating or synthetic reaction) and the deamination of glutamate (deaminating or catabolic reaction). During deamination it cataly-ses the oxidation of glutamate ensuring sufficient carbon skeleton for effective functioning of tricar-boxylic acid (TCA) cycle [16]. The enzyme thus serves as an important link between TCA cycle and metabolism of amino acids and appears to have significant role in provision of carbon skele-tons under conditions of C limitation [17]. The enzyme is primarily a mitochondrial one, however it has also been found in cytosol and chloroplasts [20].
Influence of NaCl salinity stress on the oxidative status of growing plants and more specially the abilities of the tissues to generate reducing power under stressful environment of salinity remain to be understood. Besides, seedling stage is one of the most critical stages for salt damage during the life cycle of rice plants [21]. Dehydrogenases are con-sidered important in generating reducing powers which are utilized in various metabolic activities including reductive biosynthesis of amino acids, fatty acids in growing tissues and also replenish the mitochondrial compartment with reducing powers in the event of metabolic limitations [22]. Considering the key role of MDH in generating and shuttling reducing equivalents amongst differ-ent subcellular organelles and the important role played by GDH in amination and deamination reactions in the tissues, the present study was undertaken to study the behaviours of these en-zymes in seedlings of rice genotypes differing in salt tolerance grown under increasing levels of NaCl salinity in order to specify the possible roles of these enzymes in rice plants in the stressful condition of salinity and to get more insight into the mechanism of salt tolerance in rice.
2. Materials and methods
2.1. Plant materials and salinity conditions
Two sets of rice (Oryza sati6a L.) cultivars
differing in salt tolerance were used.
Cultivars CSR-1 and CSR-3 were salt tolerant whereas cvs. Ratna and Jaya were salt sensitive. Salt sensitivity and tolerance of these cultivars were established in our laboratory [5]. Seeds were surface sterilized with 1% sodium hypochlorite solution and then imbibed in water for 24 h. Seedlings were raised in sand cultures in plastic pots saturated with either Hoagland nutrient tion [23] which served as control, or nutrient solu-tions supplemented with NaCl to achieve electrical conductivity of 7 and 14 dS m−1, which served as
treatment solutions. Seedlings were maintained in a growth chamber for 20 days at 2891°C, 80% relative humidity and 12-h light/dark cycle (irradi-ance of 40 – 50mmol m−2s−1). On alternate days,
5-day intervals and experiments were carried out in triplicate.
2.2. Total malate dehydrogenase assay
The activity of total malate dehydrogenase (NAD+-MDH, EC 1.1.1.37) was assayed in roots and shoots of growing seedlings at 5-day intervals up to 20 days. First, 200-mg fresh samples were homogenized in 5 ml of 100 mM Tris – HCl buffer (pH 7.8) containing 20 mM MgCl2, 1 mM dithiothrietol, 1 mM EDTA and 1
mM PMSF using a chilled pestle and mortar. The homogenate was then centrifuged at 4°C at 22 000×g for 15 min. The supernatant was di-alysed in cold for 3 h against the extraction buffer. Total malate dehydrogenase (NAD+ -MDH, EC 1.1.1.37) activity was assayed as de-scribed in Ref. [13] with some modifications. Reaction mixture in a total volume of 3 ml con-tained 100 mM Tris – HCl buffer (pH 7.8), 20 mM MgCl2, 1 mm EDTA, 0.1 mM NADH, 0.5
mM oxaloacetate and 200 ml enzyme. Reaction was initiated at 25°C with the addition of the enzyme and oxidation of NADH was monitored at 340 nm in a Baush and Lomb Spectronic-20 spectrophotometer. Enzyme specific activity is expressed as nmol NADH oxidized s−1 mg
protein−1.
2.3. Preparation of mitochondria and mitochondrial MDH assay
Mitochondria were isolated from roots and shoots of rice seedlings according to the method of Ref. [24]. First, 500-mg fresh samples were ground in 5 ml of grinding medium consisting of 0.5 M mannitol, 70 mM Tris – HCl buffer (pH 7.5), 1 mM EDTA, 4 mM cysteine and 1 mg ml−1 BSA using chilled mortar and pestle. The
extract was then filtered through four layers of muslin cloth and then centrifuged at 8000×g for 10 min. The supernatant liquid was discarded and the sediment was resuspended in 3 ml of a washing medium consisting of 0.3 M mannitol, 50 mM Tris – HCl (pH 7.2) and 1 mg ml−1 BSA.
Starch, nuclei, cell wall and other debris were removed by low speed centrifugation at 250×g
for 10 min. Supernatant was further centrifuged at 12 000×g for 5 min and the sediment resus-pended in 3 ml of the washing medium, and
again centrifuged at 12 000×g for 5 min. The sedimented organelles were suspended in 1 ml of a medium consisting of 0.25 M mannitol, 50 mM Tris – HCl (pH 7.2) and 4 mM MgCl2. All
operations were performed at 4°C. Integrity of mitochondrial preparation was assessed using cy-tochrome c oxidase as marker enzyme [25]. For the extraction of mitochondrial MDH, freeze-thaw extracts of mitochondria were prepared by freezing at −20°C for 24 h followed by thawing at room temperature. After repeating this proce-dure twice, the thawed suspension was cen-trifuged at 22 000×g for 30 min. The clear supernatant served as enzyme extract in which NAD+-MDH was assayed as described earlier.
2.4. Chloroplast isolation and MDH assay
Chloroplasts were isolated from leaves of seedlings uprooted at 5-, 10-, 15- and 20-day in-tervals according to Ref. [26]. First, 500 mg fresh leaves were thoroughly washed with water and then cut into 5-mm wide strips. The strips were then homogenized in a prechilled mortar and pestle in 15 ml of a medium contain-ing 20 mM Tris – HCl buffer (pH 7.8), 400 mM sucrose and 10 mM NaCl. The slurry was filtered through four layers of muslin cloth and the filtrate was centrifuged at 3000×g for 5 min to sediment the chloroplasts. The pellet was washed twice with 10 ml of the same medium and then resuspended in 1 ml of suspension buffer containing 20 mM HEPES (pH 7.5), 100 mM sucrose, 10 mM NaCl and 2 mM MgCl2.
All operations were carried out at 4°C. Integrity of chloroplasts was checked using ferricyanide assay [25]. Freeze-thaw extracts of chloroplasts were prepared as described for mitochondria and assay of chloroplastic MDH (NADP+-MDH; EC 1.1.1.82) was done as described earlier except that reaction mixture contained NADPH in place of NADH.
2.5. Effect of NaCl in 6itro on MDH acti6ity
2.6. Glutamate dehydrogenase assay
The activities of aminating (NADH-GDH) and deaminating (NAD+-GDH) glutamate dehydroge-nase (EC 1.4.1.2) were assayed in enzyme prepara-tions from roots and shoots of rice seedlings growing under increasing levels of NaCl salinity. First, 200 mg fresh samples were homogenized in 5 ml of 100 mM Tris – HCl buffer (pH 8.0) containing 3.3 mM MgCl2, 1 mM EDTA, 1 mM b-mercaptoethanol, 1 mM DTT and 1 mM PMSF. Contents were then centrifuged at 22 000×g for 10 min at 4°C. Supernatants after dialysis against the extraction buffer in cold for 4 h served as enzyme extracts. Aminating (NADH-GDH) and deaminating (NAD+-GDH) activities of the en-zymes were assayed according to Ref. [27] based on the change in absorbance at 340 nm due to NADH oxidation or NAD+ reduction. Assay medium for amination reaction contained 76 mM Tris – HCl buffer (pH 8.1), 20 mMa-ketoglutarate, 150 mM (NH4)2SO4, 0.2 mM NADH, 1 mM
MgCl2 and 200 ml enzyme in a total volume of 3
ml. Oxidation of NADH was followed by record-ing change in absorbance at 340 nm in a Baush and Lomb Spectronic-20 Spectrophotometer. En-zyme specific activity is expressed as nmol NADH oxidized s−1 mg protein−1.
The assay mixture for deamination reaction (NAD+-GDH) consisted of 100 mM Tris – HCl buffer (pH 9.0), 0.6 mM NAD+, 50 mM
L -gluta-mate and 200ml enzyme in a total volume of 3 ml. All assays were done at 25°C and in triplicate. Enzyme specific activity is expressed as nmol NAD+ oxidized s−1 mg protein−1.
2.7. Effect of NaCl in 6itro on GDH acti6ity
To study the effect of increasing concentrations of NaCl in vitro on aminating and deaminating GDH activities, 15-day grown non-salinized seedlings of salt sensitive cv. Ratna and tolerant cv. CSR-3 were used. Enzyme extracts were pre-pared as earlier and were dialysed against 100 mM Tris – HCl buffer (pH 8.3) in cold with three to four changes of buffer. Assay mixture in addition to all normal ingredients of the medium contained increasing concentrations (0 – 1000 mM) of NaCl. All enzymatic determinations were carried out in triplicate.
2.8. Protein estimation
In all enzyme preparations, protein was esti-mated according to Ref. [28] using bovine serum albumin (BSA, Sigma) as standard.
3. Results
3.1. Effect of NaCl salinity in situ on MDH acti6ity in rice seedlings
Figs. 1 and 2 as well as Tables 1 and 2 show the activities of total (NAD+) mitochondrial (NAD+) and chloroplastic (NADP+) malate dehydroge-nases in enzyme preparations from roots and shoots of seedlings of salt tolerant rice cvs. CSR-1, CSR-3 and the salt sensitive cvs. Ratna and Jaya during 5 – 20 days of growth, when the seedlings were raised under increasing levels of NaCl salin-ity in the growth medium. As is evident, in the salt tolerant cvs. CSR-1 (Fig. 1) and CSR-3 (Table 1) NaCl salinity in situ caused marked increase in total as well as mitochondrial MDH activities whereas either increase or almost no alteration in chloroplastic MDH activity was noted in these cultivars due to salinity. In 20-day grown seedlings of salt tolerant rice cultivars under 14 dS m−1
NaCl salinity 21 – 90% increase in mitochondrial MDH and 12 – 126% increase in total MDH activ-ity was observed. In the salt sensitive cvs. Ratna (Fig. 1) and Jaya (Table 2), due to a moderate in situ salinity level of 7 dS m−1NaCl either increase
or similar level of MDH activity was observed in roots as well as shoots compared to controls whereas under higher salinity level of 14 dS m−1
NaCl a marked inhibition in total, mitochondrial as well as chloroplastic MDH (NADP+-MDH) activity was noted. In salt sensitive cv. Ratna, almost complete inhibition in chloroplastic MDH activity was observed in seedlings grown for 20 days under 14 dS m−1 NaCl.
3.2. Effect of NaCl in 6itro in the reaction
medium on MDH acti6ity
When dialysed enzyme extracts prepared from roots and shoots of 15-day grown non-salinized seedlings of a salt tolerant cv. CSR-1 and a sensi-tive cv. Ratna were assayed for total,
(NADP+-MDH) enzyme activities incorporating increasing concentrations of NaCl (0 – 1000 mM) in the reaction medium, it was observed that in enzyme preparations from both the rice cultivars NaCl from 50 to 1000 mM in the reaction medium caused a gradual inhibition in the activities of total, mitochondrial as well as chloroplastic MDH enzymes in a concentration dependent manner (Fig. 2). In enzyme preparations from the salt tolerant cultivar all three MDH activities were 96 – 100% inhibited due to 1000 mM NaCl in the reaction medium (Fig. 2). Maximum inhibition was observed with chloroplastic MDH, where the activity was completely lost with 1000 mM NaCl. The MDH preparations from salt sensitive rice cv. Ratna were extremely sensitive to the in vitro effects of NaCl compared to the enzyme prepara-tions from the tolerant cultivars. NaCl beyond 50 mM in the reaction medium caused gradual
inhibi-tion in the MDH activities in the enzyme prepara-tions from sensitive cultivar and the activity was completely lost in the presence of 600 mM NaCl in the reaction medium (Fig. 2). Like the salt tolerant cultivar, in the sensitive cultivar as well, chloro-plastic MDH appeared to be more sensitive to NaCl than total or mitochondrial MDH.
3.3. Effect of NaCl salinity in situ on GDH acti6ity in rice seedlings
When seedlings of salt sensitive rice cv. Ratna and tolerant cv. CSR-1 were grown under increas-ing levels of NaCl salinity in the medium and aminating as well as deaminating GDH activities were determined in roots and shoots at increasing days of growth, a gradual increase in aminating (NADH-GDH) activity was observed in both roots and shoots of the two cultivars up to 15 days
Fig. 1. Activities of total, mitochondrial and chloroplastic malate dehydrogenases in roots and shoots of seedlings of salt sensitive rice cv. Ratna and salt tolerant cv. CSR-1 at different days of growth under increasing levels of NaCl salinity (filled rectangle, control; lined rectangle, 7 dS m−1NaCl; dotted rectangle, 14 dS m−1NaCl). Values are mean9S.D. based on three replicates
Fig. 2. Effect of increasing concentration of NaCl in vitro on the activities of malate dehydrogenases in the enzyme preparations from roots (filled rectangle) and shoots (lined rectangle) of 15-day grown non-salinized seedlings of salt tolerant rice cvs. CSR-1 and CSR-3 and salt sensitive cvs. Ratna. and Jaya. Values are mean9S.D. based on three replicates and bars indicate standard deviations.
of growth and thereafter decline in enzyme activity was noted in the sensitive cultivar but not in the tolerant one (Fig. 3). The activity of aminating (NADH-GDH) increased in both roots and shoots of salt sensitive cv. Ratna with moderate salinity level of 7 dS m−1 NaCl whereas the enzyme
activity decreased under high level of salinization. However in salt tolerant cv. CSR-1, aminating GDH activity increased under salinization. The activity of deaminating enzyme (NAD+-GDH) de-clined steadily during 5 – 20-day growth of
Table 1
A comparison of total (NAD+), mitochondrial (NAD+) and chloroplastic (NADP+) malate dehydrogenase specific activities
(nmol NADH/NADPH oxidized s−1mg protein−1) in enzyme preparations from roots and shoots of salt tolerant rice cv. CSR-3
at 5, 10, 15, and 20 days of growth under increasing levels of NaCl salinitya
Conduc- Enzyme Root Shoot
extract tivity of
NaCl 5 10 15 20 5 10 15 20
5592.7 10096.1 9695.2 9994.3
Control Total 5692.2 5091.6 5592.1 5391.9 Mitochond- 3.8290.12 3.9690.16 3.5290.17 3.2090.11 4.5490.14 5.1290.31 5.6290.31 5.6890.33 rial
Chloro- 2.1290.13 2.3290.11 2.4690.21 2.4890.26 plastic
6795.2 120911 11099.9 10899.6
7 dS m−1 Total 7096.9 5594.1 8495.2 10096.4
Mitochond- 4.5490.35 4.9990.36 5.2090.31 5.1190.37 5.5890.33 5.9190.21 6.1290.41 6.3490.43 rial
Chloro- 2.3490.17 2.3990.19 2.3990.20 2.3490.19 plastic
14 dS m−1 Total 8894.50 200 146 11198.90 8596.10 8097.10 11091.30 12091.00
911.20
919.20
Mitochond- 6.5090.33 6.7690.51 6.1290.42 6.1190.58 6.6290.58 6.7790.42 6.9090.51 6.8990.66 rial
Chloro- 2.4690.98 2.4290.67 2.5190.07 2.4890.01 plastic
aValues are mean9S.D. based on three independent determinations.
Table 2
A comparison of total (NAD+), mitochondrial (NAD+) and chloroplastic (NADP+) malate dehydrogenase specific activities
(nmol NADH/NADPH oxidized s−1mg protein−1) in enzyme preparations from roots and shoots of salt sensitive rice cv. Jaya
at 5, 10, 15, and 20 days of growth under increasing levels of NaCl salinitya
Conduc- Enzyme Root Shoot
extract tivity of
NaCl 5 10 15 20 5 10 15 20
Total 7295.10 9198.3 3292.1 5093.9 6595.1 7595.3 8294.6
Control 9098.3
3.6290.29 3.2290.1 4.5290.36 3.8090.26 3.5290.22
4.5590.31 2.1190.13
4.3890.39
Mitochond-rial
Chloro- 2.290.15 2.390.18 2.690.01 3.290.27 plastic
120910
7 dS m−1 Total 7595.9 10099.1 11099.6 4793.2 4393.9 2592.2 2291.4
4.7890.34 3.1290.26 4.6092.60 4.1290.28 4.3490.03
4.9690.40 4.0190.32
4.8490.36
Mitochond-rial
2.1090.16 2.3090.12 2.4090.16
Chloro- 2.6090.19
plastic
23.591.80 2.0692.10 2392.10 2491.80
2592.10 2392.20 2191.60
2292.10 Total
14 dS m−1
3.6290.25 3.6090.29 2.5890.15 2.1290.2 2.0690.18 2.1190.13 2.5890.17 2.0090.05
Mitochond-rial
0.2090.01 0.890.03 0.990.06
Chloro- 1.1090.06
plastic
Fig. 3. Aminating (NADH-GDH) and deaminating (NAD+-GDH) glutamate dehydrogenase activities in roots ( — ) and shoots
(----) of salt tolerant rice cv. CSR-3 and salt sensitive cv. Ratna at different days of growth under increasing levels of NaCl salinity (, control; , 7 dS m−1 NaCl; ,14 dS m−1 NaCl). Values are mean9S.D. based on three replicates and bars indicate
standard deviations.
both sets of rice cultivars. Further, NADH-GDH (aminating) always showed higher activity in roots than shoots.
3.4. Effect of NaCl in 6itro in the reaction medium
on GDH acti6ity
When dialysed enzyme extracts prepared from 15-day grown non-salinized seedlings of salt sensi-tive rice cv. Ratna and the tolerant cv. CSR-3 were assayed for NADH-GDH (aminating) and NAD+-GDH (deaminating) activities incorporat-ing increasincorporat-ing concentrations of NaCl in the reac-tion medium, in enzyme preparareac-tions from both the rice cultivars NADH-GDH (aminating) activ-ity increased gradually up to 400 mM NaCl in the medium and beyond that up to 1000 mM a grad-ual inhibition in enzyme activity was observed (Fig. 4). The extent of inhibition in NADH-GDH activity due to NaCl was more in enzyme from salt sensitive cultivar than the tolerant one. With 1000 mM NaCl 62% inhibition in NADH-GDH ac-tivity was observed in enzyme preparations from roots and 52% inhibition from the shoots of sensi-tive cultivars.
The activity of NAD+-GDH (deaminating) showed a gradual increase with 0 – 50 mM NaCl in
the reaction medium, however beyond 50 mM NaCl inhibition in the activity was observed in a concentration dependent manner (Fig. 4). Like NADH-GDH, the activity of NAD+-GDH was more sensitive to NaCl in enzyme preparations from salt sensitive cultivar than the tolerant one. In the former (sensitive cultivar), complete loss of NAD+-GDH activity was observed at 800 mM NaCl in the medium, whereas in the later (tolerant cultivar) even in presence of 1000 mM NaCl 50 – 80% residual activity was observed.
4. Discussion
Results of the present study suggest different activity behaviours of total, mitochondrial and chloroplastic malate dehydrogenases as well as of aminating and deaminating glutamate dehydroge-nases in growing seedlings of rice cultivars differ-ing in salt tolerance, when raised under increasdiffer-ing levels of NaCl salinity.
[1,4]. Although NaCl is effectively transported to the vacuole, its concentrations as high as 150 mM have been reported from the cytoplasm [29]. Many cytoplasmic enzymes are strongly inhibited at these salt levels in vitro [21].
Dehydrogenases occupy key positions in plant metabolism as they generate reducing power for various biosynthetic processes and support redox cycling in the cell. These events become more important to sustain the effect of stress [30]. Malate dehydrogenases catalyze the interconversion of ox-aloacetic acid (OAA) and malate and have diver-sified roles in plant cell metabolism which is evident from their variety of cellular locations and cofactor specificities [12].
In our experiments, in growing rice seedlings a marked decrease in the activities of total, mitochon-drial and chloroplastic MDH was noted under high salinity level whereas in the tolerant cultivars en-zyme activities in seedlings increased under saliniza-tion. In cytosol NAD+-MDH catalyzes the formation of malate from OAA. This malate enters in mitochondria through the dicarboxylate trans-porter where mitochondrial MDH (NAD+-MDH) catalyzes conversion of malate to OAA [12,31]. This OAA is channelized in TCA cycle. A high level of NAD+-MDH activity in cytosol and mitochondria thus causes better functioning of TCA cycle as OAA produced in the reaction by mitochondrial MDH is able to react with another molecule of acetyl CoA in order to start another turn of TCA cycle [32]. Thus optimum level of cytosolic and mitochondrial MDH activities are very important for growth processes as they allow TCA cycle to continue [32]. Further, OAA serves as amino acid precursor in plants [32]. Increased MDH activity under in situ salinization thus appears to be an adaptational feature of salt tolerant rice cultivars in maintaining higher activity of TCA cycle and possibly helping the plant cells in more synthesis of amino acids. Some of the key physiological changes that occur during adaptation of plants to salt stress include increased synthesis of certain amino acids and soluble nitrogenous compounds which act as os-molytes [30].
Though total and mitochondrial MDH activities increased at moderate salinity level at some stages in seedlings of salt sensitive cultivars, higher salinity level was always inhibitory to the enzymes, whereas in tolerant cultivars, even a higher salinity level of 14 dS m−1 NaCl caused increase in total and
mitochondrial MDH activities. This suggests differ-ent behaviours of MDHs in the two sets of rice cultivars differing in salt tolerance and that salt tolerance ability in rice is correlated with increased MDH activity under salinization. Increase in MDH activity in the leaves of certain plants exposed to salinity [33] and drought [34] has been reported whereas its differential expression during high and low temperature stresses [35] has been observed. Among the three MDHs studied, in situ salinity led to more inhibition of chloroplastic MDH in salt sensitive cultivars than other MDHs, whereas in salt tolerant cultivars increased activity of the enzyme was noted under salinization. This shows greater sensitivity of chloroplastic MDH to NaCl in salt sensitive varieties. Chloroplastic MDH is
Fig. 4. Effect of increasing concentration of NaCl in vitro on the activities of aminating (NADH-GDH) and deaminating (NAD+-GDH) glutamate dehydrogenase in the dialyzed
NADP+ dependent enzyme which is present in mesophylls as well as guard cells of all C3 and C4
plants and catalyzes malate synthesis from ox-aloacetate [14]. Optimum level of malate produc-tion is an essential requirement in the light induced swelling of guard cells and inhibition in the activity of this enzyme has been suggested to be one of the causes for stomatal closure under stressful conditions [14]. Further, it is suggested that chloroplastic MDH along with PEP carboxy-lase fixes certain amount of CO2, during day and
night in all plants, into malate [32]. Malate pro-duction in chloroplasts is essential for growth processes as it replenishes organic acids converted into amino acids and other larger molecules and allows the Kreb’s cycle to continue [32]. Besides, optimal NADP+-MDH activity is also needed to maintain a favourable stromal status under stress-ful conditions [4,30]. Therefore, NaCl led inhibi-tion of chloroplastic MDH might limit stomatal opening, Kreb’s cycle and influence redox state, thus altering both photosynthesis and respiration in salt sensitive cultivars. Our earlier work sug-gested decreased photosynthetic efficiency at 14 dS m−1salinity level in salt tolerant rice cultivars but
not in the tolerant [1]. Our results thus suggest that unlike salt sensitive rice cultivars, in the toler-ants, higher NADP+-MDH as well as total and mitochondrial MDH activities under salinization might be helpful for such plant species to maintain optimum photosynthesis and respiration rate un-der stressful condition of salinity.
Though the enzyme GDH catalyzes both the amination of alpha-ketoglutarate and the deami-nation of glutamate, its aminating role has been more realised under conditions of ample ammonia supply or adverse environmental conditions [18,36]. Its deaminating role helps in ensuring sufficient carbon skeleton for operation of TCA cycle, especially under conditions of C limitation [36]. Our findings that increased activity of both aminating and deaminating GDH occurs at mod-erate salinity level in both sets of rice cultivars suggests that under stressful condition of salinity GDH possibly plays an important role in assimila-tion and reassimilaassimila-tion of ammonia. These find-ings are in agreement with the increased GDH activity observed by other workers under stressful conditions of darkness [37,38], senescence [20], salinity [18] and heavy metal toxicity [19]. Various isoenzymes of GDH are found in plants [39] and
these could be involved either in the assimilation of ammonia [39] or could catalyze oxidative deam-ination of glutamate to provide carbon skeletons depending on nutrient status of plants and stress conditions [40]. It is advocated that plant GDH offers a means for improved diagnosis of the nutrient status of crops and functions as a sensor in the monitoring of environmentally induced stress [40]. Increased deaminating GDH activity under salinization, as observed in our studies, suggests that under stressful condition of salinity, GDH could play a catabolic role producing 2-ox-oglutarate to sustain general carbon metabolism by supplying intermediates to the TCA cycle [41]. A relationship between catabolic GDH activity and carbon starvation has been proposed in higher plants [36].
Studies related to in vitro effects of NaCl salin-ity on MDH and GDH activities revealed enzyme as well as genotype specific inhibition. The enzyme activities were inhibited more by NaCl in vitro in preparations from sensitive cultivars compared to the tolerant ones. In salt tolerant cultivars, com-paratively lesser inhibition in MDH activity with 200 – 400 mM NaCl compared to the enzyme from sensitive cultivars appears to be significant from physiological point of view, as, like many halo-phytic plants, salt tolerant plant species are often exposed to high salt concentrations [42]. Earlier studies conducted in our laboratory have revealed differences in inhibition patterns of proteolytic, oxidative and nitrogen assimilatory enzymes both due to in situ as well as in vitro NaCl salinity in enzyme preparations from rice cultivars differing in salt tolerance [2,6,7].
The present study suggests different behaviours of total, mitochondrial and chloroplastic MDHs as well as of aminating and deaminating GDHs in the two sets of rice cultivars differing in salt tolerance and that salt tolerance ability in rice is associated with increased MDH and GDH activi-ties under salinization. The differential behaviour of MDH and GDH in the two sets of rice cultivars differing in salt tolerance, thus reflects the possible association of these enzymes with salt tolerance in rice.
Acknowledgements
This work was financially supported by grants from the Ministry of Environment and Forests, Government of India, New Delhi in the form of a project. One of the authors (K.S.) is thankful to CSIR for providing Research Associateship.
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