J.PlantPhysiol. Vol. 135.pp. 719-724(1990)

Introduction

Nitrogen Assimilation in Germinating Phaseolus aureus Seeds Under Saline Stress

N EELAM MISRA and U. N. DWIVEDI'~ Department of Biochemistry, Lucknow University, Lucknow-226 007, India

Received July 7,1989· Accepted October 23,1989

Summary

The developmental patterns of nitrate reductase, glutamate dehydrogenase, glutamate synthase, glut­amine synthetase and soluble protein were investigated and compared in root and shoot portions of ger­minating green gram seeds both in the absence and presence of different concentrations of sodium chlo­ride. The activity of all these enzymes initially increased gradually until day 4 of germination and thereafter declined under all conditions in both root and shoot tissues. In the presence of NaCI the activ­ity levels each of nitrate reductase, glutamine synthetase and glutamate synthase were found to increase over that of the control (in absence of NaCl). Increasing the concentration of NaCI caused a further in­crease in the levels of these enzymes throughout the germination period in a concentration-dependent manner. On the other hand, glutamate dehydrogenase activity decreased in the presence of NaCI, and in­creasing concentrations of NaCI caused a further decline in the level of enzyme throughout. Saline treat­ment inhibited the rate of protein depletion in both shoot and root tissues throughout germination. The results indicate that the glutamine synthetase/glutamate synthase pathway of ammonia assimilation be­comes operative under conditions of saline stress.

Key words: Phaseolus aureus, glutamate dehydrogenase, glutamate synthase, glutamine synthetase, nitrate reductase, saline stress, nitrogen assimilation.

Abbreviations: NR = Nitrate reductase; GS = Glutamine synthetase; GOGAT = Glutamate synthase; GDH = Glutamate dehydrogenase; SD = Standard deviation.

Currently, two enzyme systems are considered of primary importance in ammonia assimilation in plants (Miflin and Lea, 1980). The GS/GOGAT pathway (Tempest et al., 1970; Lea and Miflin, 1974; Dougall and Bloch, 1976) involves transfer of ammonia to glutamate to form glutamine by the enzyme, glutamine synthetase. GOGAT, in turn, removes the amide group from glutamine in the presence of a-keto­glutarate to form two moles of glutamate. The second and al­ternative one is the GDH pathway which mediates the re­ductive amination of a-ketoglutarate to yield glutamic acid. The GS/GOGAT pathway is operative not only under con­ditions of ammonia limitation but also under a variety of

physiological and environmental conditions, including stress. (Miflin and Lea, 1976; Miranda-Ham and Loyola-Var­gas, 1988). Moreover, the real physiological role and signifi­cance of these pathways of ammonia assimilation are still a matter of debate and therefore need further investigation. Thus, Miranda-Ham and Loyola-Vargas (1988) have shown that there is a switch from one pathway of ammonia assimi­lation to another depending on the nature of the stress and the tissue in which the process takes place. GDH has been suggested to be the key enzyme involved in ammonia assimi­lation in roots from non-stressed plants (Loyola-Vargas et aI., 1988; Miranda-Ham and Loyola-Vargas, 1988).

Salinity in sailor water presents a stress condition for crop plants that are predominantly sensitive to the presence of high concentrations of salts (Staples and Toenniessen, 1984). Germination of seeds, one of the most critical phases of plant * To whom correspondence should be addressed.

© 1990 by Gustav Fischer Verlag, Stuttgart

720 NEELAM MISRA and U. N. DWiVEDI

life, is greatly influenced by salinity. Both growth as well as metabolism is reported to be altered under saline stress (Sheoran and Garg, 1978; Ramana and Das, 1978; Rao and Das, 1979; Dubey, 1982, 1984). Barring few fragmentory re­ports on the effect of salt stress on some of the nitrogen as­similating enzymes in plants (Austenfeld, 1974; Rakova et al., 1978; Hever and Plant, 1979; Klyshev et al., 1979; Kasymbekov et al., 1980), a detailed study of nitrogen as­similating enzymes in relation to their development under saline stress during seed germination is missing, especially with reference to the pathway of ammonia assimilation under saline stress.

Since nitrogen metabolism is directly related to yield po­tentials of crop plants, the present endeavour was aimed at understanding the metabolism of nitrogen in germinating seeds of green gram, in the absence as well as presence of var­ious levels of salinity, by investigating the developmental patterns of nitrate reductase, glutamate dehydrogenase, glut­amine synthetase and glutamate synthase.

Material and Methods

Plant material

Green gram (Phaseolus aureus CV T-44) seeds were well-screened for their saline tolerance and thus found to be fairly tolerant to saline stress. After surface sterilization with 1 % sodium hypo­chlorite solution for 10 min followed by rinsing with water, seeds were imbibed for 4h in distilled water (or in NaCI solution of spe­cified concentration) and spread over washed neutral sand covered with filter paper in petri dishes. Seeds were allowed to germinate for 5 days at 30± 1 °C in an incubator with a 12 h light/12h dark photo­period using uniform quantities of triple-distilled water (control) and respective salt solutions. Four concentrations of NaCl, viz. 50, 100, 150 and 200 mM, were used. The beds were adequately moist­ened with distilled water. In the case of nitrate reductase activity de­terminations the seeds were germinated in the presence of an addi­tional 10 mM KN03 both in controls as well as in treated seeds in order to induce the enzyme. The rest of the treatments were similar to those of other sets. Starting with the 4-h soaked seeds (zero h of germination), the germinated seeds were taken out at 24 h intervals up to 5 days; roots and shoots (along with cotyledons) were sep­arated from the seeds and protein estimation and enzyme assays were performed in triplicate.

Enzyme Extraction

A crude enzyme extract was prepared by homogenizing 500 mg of tissue (root or shoot along with cotyledons) in 0.1 M Tris HCl buffer, pH 7.5, at 4°C. The homogenate was centrifuged at 18,000 x g for 30 min. The supernatant was used as the crude en­zyme preparation.

Enzyme assays

Glutamine synthetase

The biosynthetic assay procedure of Boyer et al. (1959) was used for GS activity determination. The assay mixture contained 100 mM Tris HCl buffer, pH 7.0, 50 mM NH4Cl, 10 mM ATP, 50 mM MgCh and 100 mM glutamate in a final volume of 0.2 ml. Reaction was started by addition of the enzyme extract and after incubation for 15 min at 37°C was stopped by adding 1.8 ml 1.1 % FeS04 in

0.3N H 2S04. Colour was developed by adding 0.15ml 6.6% (N~hMo04 in 7.5 N H2S04 and the absorbance recorded at 540 nm. Simultaneously, a control was run where glutamate was omitted from the otherwise complete reaction system. The contents ?f the tubes were also centrifuged, if necessary, before taking read­mgs.

Glutamate synthase

GOGAT activity was determined after the method of Sodek and DaSilva (1977) by following the decrease in absorbance at 340nm due to NADH oxidation. The final assay volume of 1 ml contained 100 mM Tris HCl buffer, pH 7.5, 20 mM a-ketoglutarate, 50 mM glutamine and 0.2 mM NADH. Reaction was started by adding the enzyme extract.

Glutamate dehydrogenase

GDH activity was determined after the method of Pahlich and Joy (1971) by following the decrease in absorbance at 340 nm due to NADH oxidation. The final assay volume of 1 ml contained 100 mM Tris-HCl buffer, pH 7.5, 10 mM a-ketoglutarate, 100 mM NH4CI, and 0.1 mM NADH. Reaction was started by adding the enzyme extract.

The activity patterns of GOGAT (Fig. 5) and GDH (Fig. 3) have been corrected for NADH oxidase activity.

All the enzyme activities were calculated in terms of katal.

Nitrate reductase

The in vivo NR activity was estimated after the method of Kadam et al. (1980) with some modifications. A known amount of the tissue was placed in a Thunberg tube containing 100 mM sodium phosphate buffer, pH 7.5, and 20 mM sodium nitrate in a final volume of 2 ml. The tubes were evacuated for 2 min to facilitate in­filtration of the substrate into the tissue cells. The evacuated tubes were incubated for 1 h at 30°C in the dark. At the end of the in­cubation period the vacuum was released and the tubes were placed for 5 min in a boiling water bath for complete extraction of nitrite. The tubes were centrifuged and nitrite was determined in the super­natant in a suitable aliquot by adding 1 ml each of 1 % sulfanilamide in 1 N HCI and 0.Q1 % N-(l-naphthyl)-ethylene-diamine dihydro­chloride. Colour was allowed to develop for 10 min and recorded at 540 nm. In case the supernatant was not clear 0.2 ml zinc acetate (1 M) was added to an aliquot of 1 ml and then centrifuged. In the clear supernatant, nitrite was determined as above. To rule out the possibility of further nitrite utilization by nitrite reductase a control was run where sodium nitrate was replaced by sodium nitrite (20 mM) in the reaction system mentioned above. All other condi­tions were kept the same. It was observed that under the experi­mental conditions used for the assay of nitrate reductase, no further utilization of nitrite occurred.

Since the NR enzyme assay was performed using the in vivo system wherein continuous turnover of proteins could be visual­ized, it was not feasible to express the NR activity in terms of katall mg protein, i.e. specific activity. Hence NR activity was calculated in terms of katal/g fresh wt.

Protein was determined by the method of Lowry et al. (1951).

Results

Nitrate reductase

Fig. 1 shows the developmental profile of nitrate reductase activity in the root portion of germinating green gram seeds

> 1-U

<l:

0::: :2

No. of days of germination

Fig. 1: Developmental patterns of nitrate reductase activity in root during seed germination under conditions of absence and presence of different concentrations of sodium chloride. 0--0 in absence of NaCl; e--e, 6--6, A--A, x--x, in presence of 50, 100, 150 and 200 mM NaC!, respectively. Enzyme activity per mg fresh wt. of the tissue from 3 replicates were averaged and SD de­termined. The corresponding percentages were calculated based on activity from day 4 in presence of 200 mM NaCl (Maximal activity) taken as 100%.

c QJ

1-o '-

Cl.

2

O~ ____ ~ ____ L-____ ~ ____ ~~

1 2 3 4 5 No. of days of germination

Fig. 2: Soluble protein pattern in root during seed germination under conditions of absence and presence of different concentra­tions of sodium chloride. 0--0, in absence of NaC!; e--e, 6--6, A--A, x--x in presence of 50, 100, 150 and 200 mM NaC!, respectively. Protein per mg fresh weight of the tissue from 3 replicates were averaged and SD determined.

Nitrogen assimilation under saline stress 721

in the absence and presence of different concentrations of NaCl. Activity initially increased until day 4 and thereafter declined under all conditions. The presence of NaCI in­creased the enzyme activity throughout the germination pe­riod in comparison to the control (i.e. in absence of NaCl). Further, with increasing concentrations of NaCI the activity continued to increase throughout in a concentration-depend­ent manner. Note that on day 4, the day of peak level activ­ity, 200 mM NaCI brought about a five-fold increase in root nitrate reductase activity over that of the control (i.e. in absence of NaCl). We also investigated the developmental profile of NR in shoot tissue. The results obtained with shoot tissue were found to be similar in all respects to those of root in the absence as well as presence of all concentra­tions of NaCI (data not shown). Both root and shoot main­tained more or less the same level of NR throughout under all conditions.

Soluble protein

The changes in protein content in root tissue during ger­mination in the absence and presence of different concentra­tions of NaCI is shown in Fig. 2. Note that with progress of germination the protein content gradually decreased in both the absence and presence of all concentrations of NaCI used. With increasing salinity the tissue was found to maintain in­creased levels of protein throughout the germination period. Investigation of the protein profile of shoot tissue during seed germination also revealed a similar result to that obtain­ed with root tissue under the conditions of absence as well as presence of all concentrations of NaCI (data not shown). However, in comparison to root, shoot maintained two- to three-fold more protein content throughout the germination period, under all conditions.

Glutamate dehydrogenase Fig. 3 shows the developmental profile of GDH activity in

root tissue in the absence and presence of different concen­trations of NaCl. In all cases the activity initially increased attaining a maximal level (peak level) on day 4 and thereafter declined. In the presence of NaCI at all the concentrations used, the GDH activity was found to decline throughout the germination period. Furthermore, this decrease in GDH activity level was a function of NaCl concentration, such that with increasing NaCI concentration GDH activity level showed a gradual decrease. Note that 200 mM NaCI brought a six-fold decrease in the GDH enzyme level in root tissue on day 4 of germination (day of peak level of activity) as com­pared to control. The developmental profile of GDH was also investigated in shoot tissue, and the results obtained were found to be similar in all respects to that of root tissue in the absence as well as presence of all concentrations of NaCI used (data not shown). Root, however, showed a high specific activity compared to shoot under all conditions throughout germination.

Glutamine synthetase

The developmental profile of GS in the root portion of germinating green gram seeds in the absence and presence of

722 NEELAM MISRA and U. N. DWIVEDI

100

~ 0

80 >-.... > .... u

60 « u

u QJ 40 Q. l/)

::t: Cl l:l 20

No . of days of germination

Fig. 3: Developmental patterns of glutamate dehydrogenase activity in root during seed germination under conditions of absence and presence of different concentrations of sodium chloride. 0--0 in absence of NaCl; e--e, 6--6, .A.--.A., and x--x in presence of 50, 100, 150 and 200 mM NaCl, respectively. Enzyme activity per mg of protein from 3 replicates were averaged and SD determined. The corresponding percentages were calculated based on activity on day 4 in absence of NaCl (Maximal activity) taken as 100%.

different concentrations of NaCl is shown in Fig.4. In all cases the activity initially increased reaching a peak value on day 4 and declined thereafter. The presence of NaCl caused an increase in GS-specific activity throughout the germina­tion period. Increasing levels of NaCI caused an increase in GS activity in a concentration-dependent manner. Thus, on day 4, 200 mM NaCI brought a 1.2S-fold increase in GS­specific activity over that of the control. Investigation of the developmental profile of GS activity in shoot tissue also re­vealed a pattern similar to that obtained in root tissue both in the absence as well as presence of all concentrations of NaCI (data not shown). However, the GS-specific activity was high in root as compared to shoot throughout germina­tion under all conditions.

Glutamate synthase

Fig. S shows the developmental profile of GOGAT activ­ity in the root portion of germinating green gram seeds in the absence and presence of different concentrations of NaCl. Under all conditions the activity gradually increased until day 4 and thereafter declined. Saline treatment caused an increase in GOGAT-specific activity throughout the ger­mination period. Further, with increasing NaCI concentra­tion the GOGAT-specific activity increased gradually throughout the germination period in a concentration-de­pendent manner. Note that 200 mM NaCl brought about a two-fold increase in GOGAT activity in root tissue on day 4 of germination over that of the control. The developmental

~ 0

>-.... > .... u « u .... u QJ Q. l/)

l/) l!)

No . of days of germ ination

Fig. 4: Developmental patterns of glutamine synthetase activity in root during seed germination under conditions of absence and pres­ence of different concentrations of sodium chloride. Enzyme activ­ity per mg protein from 3 replicates were averaged and SD de­termined. The corresponding percentages were calculated based on activity on day 4 in presence of 200 mM NaCl (Maximal activity) taken as 100%. 0--0 in absence of NaCl; e--e, 6--6, .A.--.A. and x--;x in presence of 50, 100, 150 and 200 mM NaCl, respectively.

100

~ 0

80 >-.... > .... u « 60 u .-.... u

~ 40 l/)

I-« l!J

20 0 l!J

No . of days of germination

Fig. 5: Developmental patterns of glutamate synthase activity in root during seed germination under conditions of absence and pres­ence of different concentrations of sodium chloride. Otherwise as Fig. 4.

profile of GOGAT in shoot tissue was also investigated and the results obtained were found to be similar to that of root in all respects, in the absence as well as presence of NaCI

(data not shown). However, root tissue exhibited a higher GOGAT activity level than that of shoot throughout the germination period.

Discussion

With increasing saline level the NR activity was found to increase throughout the germination period in both root and shoot tissues. Similar enhancement of NR by saline treat­ment has been reported from corn seedling and Salicomia eu· ropaea (Aliva and Klyshev, 1975; Austenfeld, 1974).

However, NR is also reported to be inhibited by saline stress (Rakova et al., 1978; Hever, 1979; Lal and Bharadwaj, 1987). The data suggest that the tolerant seeds exhibit effi­cient nitrate reduction under saline stress. This seems to be an adaptation to saline stress.

Salinity treatment caused a decreased rate of protein deple­tion from seed parts during germination. This decrease was more pronounced with increasing levels of salinity. Similar results have been reported from rice seeds (Dubey, 1982). The decreased depletion of protein from different parts of seeds, i.e. root and shoot during germination on saline treat­ment, might be helping in maintaining osmolarity in the cells during saline stress (Steward and Larher, 1980 and Rani, 1988).

Glutamine synthetase activity was also found to increase in a concentration-dependent manner with saline treatment. Rakova et al. (1978) reported that GS is one of the most salt­resistant enzymes tested in vitro among the nitrogen assimi­lating enzymes (i.e. NR, GS and GDH).

GOGAT activity was also found to increase with saline treatment in a concentration-dependent manner. Miranda­Ham and Loyla-Vargas (1988) reported a high GOGAT activity under saline stress.

The developmental patterns of GS and GOGAT were identical in both root and shoot tissues and under all condi­tions of treatment. The identical activity profiles of these enzymes reflect the interdependence of these enzymes since they are known to work in conjunction (Miflin and Lea, 1976).

In contrast to GS and GOGAT, the GDH activity de­creased in the presence of all NaCI concentrations used. GDH has been reported to be inhibited by saline treatment from a number of sources (Rakova et aI., 1978; Kasym­bekov et al., 1980). Moreover, GDH has been suggested to be the key enzyme involved in ammonia assimilation in roots from non-stressed plants (Loyala-Vargas et aI. , 1988 and Miranda-Ham and Loyala-Vargas, 1988).

Thus, the nitrogen assimilation in salt tolerant germinating green gram seeds during saline stress was found to be charac­terized by high levels of NR, GS and GOGAT activity concomitant with a low GDH activity.

An overview of the data presented here (GS, GOGAT and GDH) suggests that during saline treatment, when nitrate re­duction is occurring very efficiently, ammonia is probably assimilated by the GS/GOGAT pathway rather than GDH, as evident by high enzyme activity levels of GS and GOGAT and a simultaneous low level of GDH. However, whether the shift in enzyme level would also imply a shift in NH4 +

Nitrogen assimilation under saline stress 723

assimilation from GDH to the GS/GOGAT pathway dur­ing salt stress can not be predicted with certainty at this stage of study since high levels of enzyme activity as such are not sufficient to permit NH4 + assimilation. Other factors such as concentrations of ATP, reductants, ex-ketoglutarate and glutamine pool etc. may also be critical in controlling the actual rate of ammonia assimilation (Givan, 1979).

Acknowledgements

The financial assistance (in the form of J.R.F. to Neelam Misra) of the Council of Science and Technology, U.P. is gratefully ac· knowledged. The work was partially supported by grants from the University Grants Commission, New Delhi, under the Special As­sistance Programme and COSIST Programme to this Department. Also we are thankful to Prof. G. G. Sanwal for encouragement.

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