Aluminium is a potent toxicant in acidic soils. The present study was taken up to analyze the effects of Al on enzymes of nitrogen assimilation in excised bean ( Phaseolus vulgaris) leaf segments so as to gain an insight of the mechanism involved. Supply of 0.001 to 0.1 mM AlCl3 to excised bean leaf segments affected the in vivo nitrate reductase activity differently in the presence of various inorganic nitrogenous compounds, being inhibited with 5 mM ammonium nitrate and 10 mM ammonium chloride but enhanced with 10 mM potassium nitrate. Al effect with 50 mM KNO 3 varied with time, showing an increased activity at shorter duration, but decreased at longer duration. Al effect on in vivo NRA was dependent upon the nitrate concentration, thus, inhibiting it at 0, 1 and 50 mM KNO 3, while increasing at 2 and 10 mM. Further, saturating and non-saturating effects were observed in the absence and presence of Al. Al supply influenced the in vitro NRA also, being increased at 10 mM, but decreased at 50 mM KNO 3. Supply of Al to excised leaf segments substantially inhibited the glutamate dehydrogenase activity in the absence as well as presence of 5 mM NH 4NO 3 but increased the glutamate synthase activity. Inhibition of specific glutamate dehydrogenase activity by Al supply was also observed. However, specific glutamate synthase activity was increased in the presence of NH4NO3 only. The experiments demonstrated that effect of supply of aluminium on in vivo nitrate reductase activity depended upon nitrogenous source as well as nitrate concentration and it exerted reciprocal regulation of glutamate dehydrogenase and glutamate synthase activities, which depended upon N supply too.
Aluminium (Al), one of the most abundant metals, is not regarded as an essential nutrient for plants, but low concentrations can sometimes increase plant growth or induce other desirable effects [
Nitrate reductase (NR, EC 1.6.6.1) is a substrate inducible key enzyme of nitrate assimilation. It is regulated by a number of nutritional and environmental factors [
Seeds of Phaseolus vulgaris cv. Rajmah purchased from a local dealer were surface sterilized with 0.1% HgCl2 for 1 - 2 minutes followed by thorough washing with distilled water. The seedlings were raised in plastic pots containing acid washed sand for 7 - 8 days in continuous light of intensity 30 Wm−2 supplied by fluorescent tubes at 28˚C ± 3˚C. They were watered with 1/2 strength Hoagland’s solution (pH 6.0) containing no nitrogen. For various treatments primary leaves from uniformly grown seedlings were cut into about 0.5 × 0.5 cm segments and floated on 1/4 strength Hoagland’s solution containing desired compounds, as mentioned in the tables, for required time period in continuous light supplied by fluorescent tubes.
In vivo NRA was assayed by colorimetric estimation of nitrite according to the method of Srivastava [
Results expressed are the average values of at least four independent experiments with ± SE. Difference between means obtained for various treatments was tested by Student’s t test at level of significance―a: p < 0.05, b: p < 0.01, c: p < 0.001.
Supply of 0.001 to 0.1 mM AlCl3 to excised bean leaf segments in the presence of 10 mM KNO3 gradually increased in vivo NRA (
Supply of 0.1 mM Al in the presence of 50 mM KNO3 for short interval up to 4 h maintained a higher level of in vivo NRA over control ranging from 15% to 36% (
When leaf segments were treated with Al in presence of varying concentrations of KNO3, the in vivo NRA was inhibited in the absence of nitrate and at 1 and 50 mM KNO3 (
Treatment of leaf segments with 0.1 mM Al in the presence of 10 mM KNO3 caused an increase in total as well as specific in vitro activity of NR (
Supply of 0.1 mM AlCl3 to leaf segments inhibited the NADH-GDH activity significantly (
Treatment | NRA, nmoles NO2 h−1∙g−1 fr. wt. | ||
---|---|---|---|
AlCl3 conc., mM | KNO3, 10 mM | NH4NO3, 5 mM | NH4Cl, 10 mM |
0.000 | 1610 ± 78 (100) | 1283 ± 110 (100) | 597 ± 79 (100) |
0.001 | 1660 ± 197 (103) | 1020 ± 135 (80) | 520 ± 94 (87) |
0.010 | 1836 ± 195 (114) | 1038 ± 122 (81) | 481 ± 44 (81) |
0.100 | 1827 ± 148 (114) | 1040 ± 152 (81) | 461 ± 60 (77) |
Leaf segments were floated on 1/4 strength Hoagland’s solution containing the desired nitrogenous compounds in the presence of varying concentrations of AlCl3 for 24 h at continuous light intensity of 30 Wm−2 and temperature 26˚C ± 2˚C. Values relative to control are given in parentheses.
Treatment | In vivo NRA, nmoles NO2 h−1∙g−1 fr. wt. | ||
---|---|---|---|
KNO3 conc., mM | ?Al | +Al | % Increase/Decrease |
00 | 774 ±20 (100) | 606 ± 31c (100) | 22% Decrease |
01 | 957 ± 99 (124) | 894 ± 75 (147) | 7% Decrease |
02 | 1134 ± 82 (146) | 1242 ± 112 (205) | 9% Increase |
10 | 1610 ± 78 (208) | 1827 ± 148 (301) | 14% Increase |
50 | 2434 ± 110 (314) | 1920 ± 74c (317) | 21% Decrease |
Leaf segments were floated on 1/4 strength Hoagland’s solution containing the desired concentrations of KNO3 in the absence and presence of 0.1 mM AlCl3 for 24 h at continuous light intensity of 30 Wm−2 and temperature 26˚C ± 2˚C. Values relative to control are given in parentheses. Level of significance―c: p < 0.001.
Treatment | In vitro NRA | Cyt c reductase | |
---|---|---|---|
nmoles NO2 h−1∙g−1 fr. wt. | nmoles NO2 h−1∙mg−1 protein | ∆A550 min−1∙g−1 fr. wt. | |
KNO3, 10 mM | 664 ± 149 (100) | 20 ± 4 (100) | 0.882 ± 0.067 (100) |
KNO3, 10 mM +AlCl3, 0.1 mM | 874 ± 289 (132) | 25 ± 8 (125) | 0.844 ± 0.061 (96) |
KNO3, 50 mM | 1239 ± 220 (100) | 38 ± 6 (100) | 0.900 ± 0.035 (100) |
KNO3, 50 mM + AlCl3, 0.1 mM | 995 ± 442 (80) | 27 ± 9 (71) | 0.797 ± 0.061 (89) |
Leaf segments were floated on 1/4 strength Hoagland’s solution containing 10 and 50 mM KNO3 in the absence and presence of 0.1 mM AlCl3 for 24 h at continuous light intensity of 30 Wm−2 and temperature 26˚C ± 2˚C. Values relative to control are given in parentheses.
NADH-GDH activity | NADH-GOGAT activity | |||
---|---|---|---|---|
Treatment | Units ml−1 Enzyme | Units mg−1 Protein | Units ml−1 Enzyme | Units mg−1 Protein |
Control (-N) | 63.1 ± 6.3 (100) | 34.2 ± 3.9 (100) | 8.4 ± 2.4 (100) | 4.4 ± 1.3 (100) |
AlCl3, 0.1 mM | 31.6 ± 1.9b (50) | 19.7 ± 2.9a (58) | 14.6 ± 5.2 (174) | 4.2 ± 1.6 (96) |
Leaf segments were floated on 1/4 strength Hoagland’s solution in either, the absence (–N Control) or presence of 0.1 mM AlCl3 for 18 h at continuous light intensity of 40 Wm−2 and temperature 26˚C ± 2˚C inside “Newtronics” growth chamber. Values relative to control are given in parentheses. Level of significance―a: p < 0.05, b: p < 0.01.
Treatment | NADH-GDH activity | NADH-GOGAT activity | ||
---|---|---|---|---|
Units ml−1 Enzyme | Units mg−1 Protein | Units ml−1 Enzyme | Units mg−1 Protein | |
Control (+N) | 96.3 ± 11.0 (100) | 65.8 ± 7.4 (100) | 15.3 ± 3.2 (100) | 8.3 ± 1.9 (100) |
AlCl3, 0.1 mM | 19.7 ± 2.2c (20) | 26.3 ± 2.9c (40) | 19.8 ± 6.0 (129) | 11.2 ± 3.1 (135) |
Leaf segments were floated on 1/4 strength Hoagland’s solution in either, the absence (+N Control) or presence of 0.1 mM AlCl3 for 18 h at continuous light intensity of 40 Wm−2 and temperature 26˚C ± 2˚C inside “Newtronics” growth chamber. Values relative to control are given in parentheses. Level of significance―c: p < 0.001.
Al supply increased the NADH-GOGAT activity substantially (
When leaf segments were treated with 0.1 mM AlCl3 containing 5 mM NH4NO3, severe inhibition of NADH- GDH activity was observed (
The results demonstrate a differential effect of Al supply on in vivo nitrate reductase activity in bean leaf segments depending upon the nitrogenous compound included and nitrate concentration as well. The enzyme activity is increased by Al in the presence of KNO3, but decreased with NH4NO3 as well as NH4Cl (
Plants have multiple nitrate carriers with distinct kinetic properties and regulation. Thus, there are at least three distinct
Reciprocal regulation of NADH-GDH and NADH-GOGAT during supply of Al with and without NH4NO3 in excised bean leaf segments was demonstrated. Thus, Al stress severely inhibits NADH-GDH activity but activates NADH-GOGAT activity (
In the present investigation, the inhibitory effect of Al on NADH-GDH activity is dependent on the supply of nitrogen in the incubation medium. Thus, stronger inhibition of enzyme activity results in the presence of NH4NO3, as N-supply increases the activity in the absence of Al only (
Effect of aluminium supply on in vivo NRA depends upon nitrogenous source as well as nitrate concentration and it exerts reciprocal regulation of NADH-GDH and NADH-GOGAT activities, which depends upon N supply too.
Priyanka Gupta,Juliana Sarengthem,Sonal Dhamgaye,Rekha Gadre, (2016) Differential Effect of Aluminium on Enzymes of Nitrogen Assimilation in Excised Bean Leaf Segments. Advances in Biological Chemistry,06,106-113. doi: 10.4236/abc.2016.63009