Seeds of Zea mays L., cv. Ganga safed-2 were surface sterilized with 0.1% HgCl₂ for 1 - 2 min and then washed thoroughly with distilled water. Seedlings were raised in small plastic pots containing acid washed sand in continuous dark for 7 - 8 d at 25˚C ± 3˚C. They were watered on alternate days with half strength Hoagland’s solution, which was modified to exclude nitrogen. For various treatments, primary leaves from uniformly grown seedlings were cut into about 0.5x0.5 cm² pieces and floated on 1/4^th strength Hoagland’s solution (pH 6.0) containing the desired compounds for 24 h in continuous light of intensity about 40 W∙m⁻² at 25˚C ± 2˚C. Treated material was used for enzymatic analyses.

Glutamine synthetase was extracted from the treated material in cold using pestle and mortar in the presence of a pinch of sand and the extraction medium, 0.1 M potassium phosphate buffer pH 7.8 containing 0.4 M sucrose, 10 mM DTT, 10 mM KCl, 1 mM MgCl₂ and 10 mM EDTA. The ratio of leaf material to extraction medium was 1:4 (w/v). The crude homogenate of the enzyme was desalted by running through a Sephadex G-25 column using the eluent buffer consisting of 1 M immidazole/HCl buffer, pH 7.0 and used for assay. Enzyme activity was assayed by the method described in Shapiro and Stadtman [16] . The reaction mixture contained 1.0 ml 1 M Imidazole-HCl buffer; 2.5 ml 0.12 M ATP solution; 2.0 ml 2 M L-Glutamate; 1.0 ml 1 M NH₄Cl solution; 0.6 ml 1.67 M MgCl₂; 2.9 ml distilled water. For assay 0.2 ml of the reaction mixture, 0.1 ml of distilled water and 0.1 ml of enzyme preparation was incubated at 37˚C for 10 minutes followed by the addition of 1.8 ml of 29 mM FeSO₄ prepared in 0.3 N H₂SO₄ to stop the reaction and 0.15 ml of the coloring reagent, 5.3 mM ammonium molybdate prepared in 7.5 N H₂SO₄. The blank was prepared by reversing the sequence of the addition of enzyme and FeSO₄ solution. The absorbance of blue colored solution was measured at 660 nm against blank. One unit of enzyme activity was defined as μmoles Pi formed min⁻¹ g⁻¹ fr. wt.

For Fd-GOGAT analysis the extraction and assay was carried out by the method of Hecht et al. [17] . NADH-GDH and NADH-GOGAT were extracted as per method described in Duke and Ham [18] . The crude extract was desalted by using Sephadex G-25 column. The activities of desalted preparations were measured spectrophotometrically by monitoring the change in absorbance at 340 nm according to the method of Duke and Ham [18] . To calculate specific activity of the enzymes, the protein content of the preparations were determined by the method of Lowry et al. [19] after precipitation with 10 % TCA followed by dissolving the ppt in 0.1 N NaOH.

Data presented in the paper are average of at least four independent experiments with ± S.E. Significance of difference obtained for various treatments was tested by student’s t test and p values denoted are: *p < 0.05, **p < 0.01, ***p < 0.001.

Supply of 0.1 to 0.5 mM CdCl₂ in the presence of 5 mM NH₄NO₃ to excised etiolated maize leaf segments during greening affected NADH-GDH activity in a concentration dependent manner being marginally reduced at 0.1 mM, remained unaffected at 0.2 mM, but increased at 0.5 mM (Table 1). The specific activity of the enzyme remained unaffected at lower Cd concentration, but increased gradually at higher concentrations. Cd exerted opposing effects on NADH-GOGAT activity being decreased with increasing concentrations of Cd (Table 1). The specific activity remained unaffected at 0.1 mM Cd, while decreased at 0.2 and 0.5 mM Cd being more significant for the later.

Supply of 0.1 to 0.5 mM CdCl₂ to excised etiolated maize leaf segments during greening increased substantially the total as well as specific activity of NADH- GDH with the increase in specific activity being more pronounced than total activity (Table 2). Also Cd treatment increased both the total and specific activities of NADH-GOGAT with the more prominent effect at 0.2 mM Cd (Table 2).

Supply of 0.5 mM CdCl₂ in the presence of 5 mM NH₄NO₃ to excised etiolated maize leaf segments during greening decreased the total and specific GS activity by 44 % and 33%, respectively (Table 3). While treatment with 0.5 mM Cd increased the total as well as specific activity of Fd-GOGAT with the effect being more prominent for the later (Table 3).

Supply of inorganic nitrogenous compounds during Cd treatment to excised etiolated maize leaf segments affected the inibitory effect on GS activity. Thus, 10 mM KNO₃ decreased the enzyme activity significantly in the presence of 0.5 mM CdCl₂ and thereby increasing the % inhibition prominently (Table 4). With the inclusion of 10 mM NH₄Cl, GS activity was found to be reduced to same extent in the absence as well as presence of Cd, thus % inhibition remained unaffected (Table 4(a)). Supply of 5 mM NH₄NO₃ decreased the GS activity in Cd treated material causing more of % inhibition. When along with 5 mM NH₄NO₃, 10 mM KNO₃ and 10 mM NH₄Cl were also included during treatment, the inhibitory effect of Cd was reduced in both (Table 4).

Varying effects of metabolites, such as, glutamine, glutamate, 2-oxoglutarate (2-OG), reduced glutathione (GSH) and sucrose were observed in relation to inhibition of GS activity by cadmium. Thus, inclusion of 5 mM glutamine along with 5 mM NH₄NO₃, increased the GS activity in the presence of 0.5 mM CdCl₂ and thereby decreased strongly the inhibitory effect of Cd (Table 5). Addition of 5 mM glutamate increased the enzyme activity in the absence of Cd to greater extent causing more of % inhibition. When 5 mM each of 2-OG, GSH and sucrose were included, the enzyme activity was increased in the absence as well as presence of Cd with the increase being more prominent for later (Table 5). The % inhibition of enzyme activity by Cd was also reduced in presence of these metabolites with more prominent effect with GSH and Sucrose.

In vitro inclusion of the competitive inhibitor of GS, methionine sulfoximine

(MSO) at concentrations 0.02 and 0.1 µM during assay of enzyme activity from Cd treated (+Cd enzyme) and untreated (−Cd enzyme) material caused significant inhibition in both (Figure 1). However, more prominent inhibitory effect was observed with +Cd enzyme at lower concentration of MSO.

The enzymes of ammonia assimilation are glutamine synthetase, glutamate synthase and glutamate dehydrogenase. Glutamine synthetase plays a central role in nitrogen metabolism of higher plants. GS is active in a variety of plant organs and functions to assimilate ammonia generated by a number of physiological processes. Analysing the effect of metallic toxicant, Cd, on GS activity in different plants have demonstrated varying effects. Thus, decrease in GS activity has been reported in nodules and roots of soybean plants [13] , tomato seedlings [14] and leaves of Solanum nigrum at high concentration [15] while it remained unaffected in oat [12] and leaves of Solanum nigrum at low concentration [15] . The

results of present study demonstrate an inhibitory effect of Cd in the presence of NH₄NO₃ on GS activity in excised etiolated maize leaf segments during greening, while increasing NADH-GDH activity (Table 1 and Table 3). Increased NADH-GDH activity during Cd supply only is also observed (Table 2). This suggests that Cd stress increases toxicity and to overcome it stress responsive GDH activity is increased. Increased GDH activity under Cd stress conditions has been reported in several systems [14] [15] [20] [21] [22] [23] . Further, NADH-GOGAT activities in the absence and presence of NH₄NO₃ are differently affected by Cd showing increase with former and decrease with later (Table 1 and Table 2). This indicates that NADH-GOGAT may be responsible for maintaining glutamate level under the condition of Cd toxicity in the absence of nitrogen. Increased NADH-GOGAT activity by Cd in tomato seedlings has also been reported [14] . Moreover, reciprocal effects for NADH-GOGAT activity in radish cotyledons under light and dark conditions have been reported, where it was increased by NH₄NO₃ in dark, but decreased in light [24] . Differential effects for Fd-GOGAT and NADH-GOGAT activities by Cd in the presence of NH₄NO₃ is observed showing increase for former and decrease for later (Table 1 and Table 3). Thus, it is likely that Fd-GOGAT is involved in maintaining glutamate level during Cd stress when supplied with inorganic nitrogen. However, Cd supply to tomato seedlings decreased Fd- GOGAT activity [14] .

The inhibitory effect of Cd on GS activity seems to depend upon inorganic nitrogenous compound included. Thus, nitrate inclusion causes higher % inhibition of GS activity by Cd, while ammonium causes lower % inhibition and NH₄NO₃ causing an intermediary effect (Table 4). Hence, it is likely that Cd supply inhibits nitrate acquisition and/or assimilation severely thereby reducing ammonia assimilation. More severe inhibition of nitrate reductase activity compared to GS activity by Cd in leaves of Solanum nigrum has been demonstrated [15] . Moreover, % inhibition of activity by Cd is more with excess of nitrate compared to excess of ammonium (Table 4). Thus, Cd may also influence and transporter activities to different extent and thereby inhibit GS activity differently. Greater sensitivity to Cd in relation to growth of nitrate-fed bean plants as compared to ammonium-fed plants has been observed [23] . Differential response of GS towards these nitrogenous compounds in light and dark has been reported in radish cotyledons [24] . In dark KNO₃ inhibited it, while NH₄Cl promoted it and NH₄NO₃ exerted intermediary effect. In light all the three sources significantly promoted it. Although the different isoforms of GS have not been analysed in the present study, possible involvement of these in differential effects of nitrogenous compounds is likely. Hence, it is worth to discuss the regulation of different isoforms of GS1 with the supply of inorganic nitrogen as reported in Arabidopsis [25] and rice roots [26] . In Arabidopsis GLN1:2 is significantly upregulated by ammonium and exhibited low affinity to ammonium, while GLN1:3 was not stimulated by ammonium but significantly inhibited by high concentration of glutamate. The high affinity form GLN1:1 accumulated during nitrogen starvation and was down regulated by ammonium excess [25] . In rice roots cytosolic GS1 isoform, OsGLN1:1 accumulated in nitrogen limited condition, while OsGLN1:2 was abundantly expressed under nitrogen sufficient conditions [26] . Further, NADH-GOGAT accumulation in root tips have been observed after ammonium treatment [27] [28] , which was identical in localization with OsGLN1:2. This indicated that the two enzymes cooperated to assimilate ammonia taken up by ammonium inducible forms of AMTs (OsAMT1:1 and OsAMT1:2) [29] .

Inclusion of key metabolites of plant tissue during Cd treatment influences the inhibitory effect of Cd on GS activity. Glutamine, a key metabolite, may act as a regulator of overall nitrogen metabolism. It is required as a precursor of many amino acids, nucleic acids, alkaloids and polysaccharides, as well as secondary metabolites like polyamine [30] . Various amino acids (Asp, Asn, Glu, Gln) antagonizing the inductive effect of sucrose on GS has been shown in Arabidopsis [31] . In this study, Glutamine (Gln), one of the product of GS reaction exerted strong protective effect against Cd inhibition of GS activity (Table 5). Hence, it is likely that inhibitory effects of Gln/Cd are not sensed in presence of the other. So Cd may release such an effect of Gln resulting in increased GS activity in its presence. Another organic nitrogenous compound, Glu, one of the substrate of GS reaction caused stimulation of activity to lesser extent with Cd (Table 5). Further, MSO, a competitive inhibitor of GS more strongly inhibits the enzyme from Cd treated material (Figure 1) suggesting that glutamate binding to enzyme and its subsequent phosphorylation is affected by Cd. Moreover, in Medicago truncatula phosphorylation of GS1a mediated by Ca²⁺ independent kinases was found to increase the affinity of the enzyme for the substrate glutamate [32] .

Reduced glutathione is an important protectant against metal toxicity, may function as a precursor for phytochelatins or as an antioxidant. Decline in GSH level upon exposure to Cd in the tomato cell suspension culture [33] and maize seedlings [34] has been reported. In this study, the supply of GSH, a ubiquitous thiol being responsible for maintaining reducing environment within tissue also causes an increase in GS activity to greater extent in presence of Cd (Table 5). Thus, Cd toxicity appears to affect thiol groups of enzyme and/or reducing environment of the system. Alternatively, it may overcome Cd toxicity by mediating synthesis of heavy metal binding peptides, phytochelatins, which are involved in metal detoxification.

Importance of 2-oxoglutarate (2-OG) in higher plant ammonia assimilation has been reviewed by Lancien et al. [35] . Plant cell 2 OG level is dependent on inorganic and organic N content and their related metabolism as well as nitrate and sugar levels [36] [37] and thus could play a signaling role in coordination of C- and N- metabolism. In the present study, 2 OG, the TCA intermediate and the metabolite linking C- and N- metabolism, more prominently increases GS activity during Cd supply (Table 5). Thus, Cd stress may influence the availability of TCA intermediates and thereby affecting ATP production to inhibit GS activity. Further, Cd supply may affect the cytosolic form of GS, GS1, as 2-OG has been reported to induce specifically cytosolic GS1 in Arabidopsis [31] . Moreover, increased NADH-GDH activity during Cd stress (Table 2 and Table 3) seems to utilize more of 2OG reducing its level in the system. Other important carbon compound and also the photosynthate, sucrose, significantly increased the GS activity with a more profound effect in the presence of Cd (Table 5). Such a stimulation suggest reduced photosynthetic C- assimilation during Cd toxicity. Further, dramatic induction of chloroplastic GS2 resulted by light in Arabidopsis [31] and sucrose supply mimicked the light effect partly. Hence it is likely that GS2 isoform of the enzyme is affected to greater extent with the supply of Cd.