Supply of CdCl 2 in the presence of NH 4 NO 3 to excised etiolated maize leaf segments during greening decreased the glutamine synthetase and nicotinamide adenine dinucleotide reduced (NADH) dependent glutamate synthase activities, while the ferredoxin (Fd) dependent glutamate synthase and glutamate dehydrogenase activities were increased. Inclusion of inorganic nitrogen, metabolites and the inhibitor influenced the effect of Cd on glutamine synthetase activity. The % inhibition of activity caused by Cd was higher with NO<sub>3</sub><sup style="margin-left:-6px;">-</sup> but lower with NH<sub>4</sub><sup style="margin-left:-6px;">+</sup> . Glutamine, 2-oxoglutarate, glutathione and sucrose decreased the % inhibition by Cd with the more prominent effect with glutamine and sucrose. Methionine sulfoximine exerted a more prominent effect for + Cd enzyme at lower concentration. The results indicate the involvement of reciprocal effects of Cd on glutamine synthetase and glutamate dehydrogenase activities and also on NADH- and Fd-glutamate synthase activities. For the inhibitory effect of Cd on glutamine synthetase activity, NH<sub>4</sub><sup style="margin-left:-6px;">+</sup> , glutamine, 2-oxoglutarate, glutathione and sucrose exerted a protective effect with the sucrose being most effective.
The environmental pollution with heavy metals is becoming an increasing problem in day to day life. The source of heavy metals in plant is the environment in which they grow and their growth medium (soil) from where heavy metals are then taken up by roots or foliage of plants. In general, heavy metal toxicity is attributed to binding of heavy metal to enzymes, resulting in inhibition and alteration of metabolism. Cadmium (Cd) is a common environmental contaminant introduced into the soil through anthropogenic activities. It is released into the environment by power stations, heating systems, metal-working industries, waste incinerations, urban traffic, cement factories and as a by-product of phosphate fertilizers [
Nitrogen is one of the nutrients that most commonly limit plant growth. Higher plants take up principally nitrate from the soil and reduce it to ammonium by the plant enzymes, nitrate reductase and nitrite reductase. Since
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 1/4th 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 [
For Fd-GOGAT analysis the extraction and assay was carried out by the method of Hecht et al. [
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 (
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 (
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 (
CdCl2, Conc., mM | NADH-GDH activity, units g−1 fr.wt. mg−1 protein | NADH-GOGAT activity, units g−1 fr.wt. mg−1 protein |
---|---|---|
0.0 | 188 ± 16 (100) 20.8 ± 2 (100) | 171 ± 40 (100) 18.7 ± 4.0 (100) |
0.1 | 176 ± 18 (94) 20.7 ± 2 (99) | 161 ± 21 (94) 19.0 ± 2.5 (101) |
0.2 | 191 ± 27 (102) 22.4 ± 3 (108) | 139 ± 43 (81) 15.0 ± 4.0 (80) |
0.5 | 206 ± 65 (110) 27.7 ± 9 (133) | 63 ± 12* (37) 8.0 ± 28* (43) |
CdCl2, Conc., mM | NADH-GDH activity, units g−1 fr.wt. mg−1 protein | NADH-GOGAT activity, units g−1 fr.wt. mg−1 protein |
---|---|---|
0.0 | 36 ± 6 (100) 2.7 ± 0.8 (100) | 13 ± 5 (100) 1.0 ± 0.3 (100) |
0.1 | 47 ± 5 (130) 4.3 ± 0.5 (159) | 21 ± 8 (161) 2.0 ± 0.8 (200) |
0.2 | 48 ± 10 (133) 5.5 ± 1* (204) | 24 ± 9 (185) 3.2 ± 1 (320) |
0.5 | 65 ± 8*(180) 8.3 ± 1*** (307) | 22 ± 6 (169) 2.9 ± 0.8 (290) |
CdCl2, Conc., mM | GS activity, units g−1 fr.wt. mg−1 protein | Fd-GOGAT activity, units g−1 fr.wt. mg−1 protein |
---|---|---|
0.0 | 18.5 ± 1.5 (100) 1.2 ± 0.1 (100) | 536 ± 106 (100) 24.4 ± 2 (100) |
0.5 | 10.4 ± 1.0*** (56) 0.8 ± 0.1** (67) | 771 ± 183(137) 56.0 ± 14* (229) |
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 (
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 (
In vitro inclusion of the competitive inhibitor of GS, methionine sulfoximine
Treatments | GS activity, units g−1 fr.wt. -Cd +Cd | % Inhibition | |
---|---|---|---|
Control | 18.5 ± 2 (100) | 13.6 ± 2 (100) | 27 |
KNO3, 10 mM | 18.1 ± 4 (98) | 5.9 ± 2* ( 43) | 67 |
NH4Cl, 10 mM | 13.9 ± 3 (75) | 10.4 ± 1 (76) | 25 |
NH4NO3, 5 mM | 18.5 ± 1.5 (100) | 10.4 ± 1 (76) | 44 |
NH4NO3, 5 mM + KNO3, 10 mM | 18.7 ± 5 (101) | 12.3 ± 3 (90) | 34 |
NH4NO3, 5 mM + NH4Cl, 10 mM | 19.7 ± 3 (106) | 14.8 ± 4 (109) | 25 |
Metabolites | GS activity, units g−1 fr.wt. -Cd +Cd | % Inhibition | |
---|---|---|---|
None | 18.6 ± 1.5 (100) | 10.4 ± 1 (100) | 44 |
Glutamine | 17.3 ± 1 (93) | 14.6 ± 2* (140) | 16 |
Glutamate | 22.9 ± 4 (123) | 11.6 ± 2 (112) | 49 |
2-Oxoglutarate | 28.0 ± 2*** (151) | 16.9 ± 3* (163) | 40 |
GSH | 24.3 ± 2* (131) | 16.8 ± 1* (162) | 31 |
Sucrose | 28.1 ± 2*** (151) | 23.2 ± 2*** (223) | 17 |
(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 (
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 [
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 (
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 (
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 [
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 [
Importance of 2-oxoglutarate (2-OG) in higher plant ammonia assimilation has been reviewed by Lancien et al. [
Supply of metallic toxicant, Cd in the presence of NH4NO3 decreased the GS and NADH-GOGAT activities, while increased the Fd GOGAT and NADH-GDH activities. Percent inhibition of GS activity caused by Cd is influenced by the inclusion of inorganic nitrogenous compounds,
GS―Glutamine synthetase; NADH―Nicotinamide adenine dinucleotide; Fd― Ferredoxin; MSO―Methionine sulfoximine; GOGAT―glutamate synthase. GDH― Glutamate dehydrogenase.
Sarangthem, J., Dhamgaye, S. and Gadre, R. (2017) Cadmium Effects on Enzymes of Ammonia Assimilation in Excised Etiolated Maize Leaf Segments during Greening: A Mechanistic Approach. American Journal of Plant Sciences, 8, 1399-1410. https://doi.org/10.4236/ajps.2017.86095