Alena Gaudinová

Regulation of Glutamate Dehydrogenase and Nitrate Reductase Levels in Excised Pea Roots by Exogeneously Supplied Sugar

Summary The activities of NADH 2 dependent glutamate dehydrogenase (GDH) and nitrate reductase were followed in excised pea roots cultured in liquid medium with or without sucrose. Whilst the persistance of nitrate reductase activity in excised pea roots depends directly on the presence of sugar in the medium and whilst in its absence nitrate reductase activity drops to the initial value before the induction with nitrate, both NADH 9 dependent GDH and NADPH2 dependent GDH activities rise in excised pea roots in the absence of sugar in the medium, and vice versa, their activities are maintained at approximately the same level as in corresponding parts of roots on intact seedlings in the presence of sugar in the medium. The increase in NADH 2 dependent GDH activity is inhibited by actidione which indicates that de novo synthesis of the enzyme is probably responsible for the increase. Glucose and fructose are in their effects equivalent to sucrose. The omission of sugar from the medium causes also a change in the ratio of the activities of individual isoenzymes of NAD + dependent GDH. Physiological concentrations of ammonium and nitrate ions neither influence GDH level in the presence of sugar nor in the absence of it. The above mentioned sugars are apparently involved in the regulation of these enzymes directly and not indirectly by means of their metabolites formed during respiration. This is indicated by the results obtained in the experiments with exogenously supplied metabolites of glycolysis, pentose phosphate cycle and Krebs cycle, which did not show any significant effect on GDH and nitrate reductase levels. The increase in GDH level observed after the addition of sublethal concentrations of some respiratory inhibitors to the medium containing sugar was probably caused by decreased sugar uptake and transport. The regulation of GDH level controlled by sugars is in isolated pea roots independent of some other regulatory mechanisms (controlled by acids, nitrite and hydroxylamine), and vice versa. On the other hand, the results of our experiments indicate that the increase in the level of NADH 2 dependent GDH observed by other authors in the roots of whole intact plants after their exposure to high concentrations of ammonium salts, may be, at least partly, caused indirectly by changes in the level of sugars in these roots.

Effects of synthetic cytokinins on levels of endogenous cytokinins and respiration patterns ofBeta vulgaris cells in suspension

Respiration patterns and growth of cytokinin-dependent cell suspensions ofBeta vulgaris L., precultured in media with or without three different synthetic cytokinins [benzyladenine (BA), kinetin (KIN), and thidiazuron (TDA)], were compared. The content of endogenous cytokinins, especially zeatin and isopentenyladenine, as well as the dry mass yield, were dependent on the kind of synthetic cytokinin present in the culture medium and decreased in the following order: thidiazuron, kinetin, benzyladenine, no cytokinin. The apparent capacity of the alternative pathway, as measured after blocking of the cytochrome pathway by cyanide, was inversely proportional to the content of endogenous cytokinins. Some synthetic cytokinins (e.g., benzyladenine), when exogenously applied, are known to inhibit selectively the alternative pathway. However, this does not necessarily imply that the mechanism of action of endogenous cytokinins on the respiration pattern is limited to a single effect on the alternative pathway. Multiple effects on oxidative processes cannot be excluded.

Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulatedPisum sativum L.

Effect of nitrate on the nitrogenase (C2H2-reduction) activity, growth of nodule tissue accumulation of nitrate and nitrate reductase activity in 4-weeks-old nodulated peas (Pisum sativuml.) was investigated. A relatively slow decrease of the total nitrogenase activity (μmol C2H4 per root per h), as compared with plants cultivated without nitrate, was due to both retardation of further growth of the nodule tissue and to a decrease of their specific nitrogenase activity (μmol C2H4 per gf.wt. per h). However, an absolute and pronounced decrease of both nitrogenase activities occurred only 4 or 7 d after the application of nitrate. The addition of nitrate led to its rapid accumulation in the nodule and leaf tissue with a simultaneous induction of the nitrate reductase activity. The nitrogenase activity was not completely inhibited even after a 7-d cultivation with 280 ppm NO3−-N in the nutrient medium and after accumulation of up to 180 ppm NO3−-Nf.wt. in the nodule tissue. The results obtained indicate that the “photosynthate deprivation” reflects competition between assimilation of nitrate and fixation of dinitrogen.

The effect of cytokinins on nitrate reductase activity.

Cytokinins in addition to nitrate induce nitrate reductase activity (NRA) in some plants. Effects of cytokinins onNRA was investigated in stem pith parenchyma of kale, intact wheat and barley seedlings and isolated cucumber cotyledons.The most profound effect onNRA was found in barley and wheat seedlings.NRA in seedlings sprayed with 100 μM 6-benzylaminopurine (BAP) for three subsequent days was increased in leaves and decreased in roots. These changes were further enhanced in seedlings grown in nutrient solution lacking nitrate:NRA in wheat and barley leaves was increased by 57% and 202%, respectively, in plants supplied with nitrate theNRA increase was not significant: in wheat and barley leaves by 22% and 9%, respectively.Similar effect of BAP and kinetin was found in kale stem parenchyma and cucumber cotyledons. The cytokinin kinetin or BAP alone increasedNRA about twice in kale and three times in cucumber. Addition of nitrate to the medium enhanced the effect of kinetin in kale discs, but the two effects were not additive. Additive effect of nitrate and BAP onNRA was found in cucumber cotyledons in light.In general NRA was more affected by cytokinins in intact seedlings of wheat and barley as compared to explanted tissue of kale and cucumber, and lack of nitrogen made their effect more expressive.

Relationships between nitrate level, nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO3-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - axb) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO3-R activity correlated well with thein vitro activity up to the 50 ppm NO3-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.

The effect of NO3- and NH4+ ions on enzymes involved in nitrogen assimilation inPisum sativum L

Nitrate reductase level in leaves of pea plants is higher than in roots despite of the lower content of endogenous nitrate. Addition of ammonium ions to nutrient solution containing nitrate decreases nitrate reductase level in leaves estimatedin vivo while its level estimatedin vitro is increased.Glutamine synthetase (GS) level in roots decreases during short (24 and 48 h) and long (14 d) term cultivation of seedlings in solutions containing ammonium ions. This decrease occurs in leaves only after the long term influence of ammonium ions. Level of this enzyme is higher in plants grown in the presence of nitrogen (ammonium and nitrate) as compared to those grown without the nitrogen.Level of glutamate dehydrogenase in roots is increased after both short and long term cultivation of plants in the presence of ammonium ions.

Fluctuations of nitrogenase and cytosol glutamine synthetase activity in pea (Pisum sativum L.) nodules in the course of vegetation

Changes in glutamine synthetase activity located in the cytosol of root nodules were followed in pea (Pisum sativum L.) plants in relation to their nitrogenase activity. The highest glutamine synthetase activity was found in young nodules (15 days after inoculation) and its changes in 17-to 45-day-old plants showed a positive correlation with nitrogenase activity. In contrast to nitrogenase activity, changes in glutamine synthetase activity during the day and night period could not be unequivocally interpreted in terms of diurnal fluctuation.

Behaviour of nodulatedPisum sativum L. under short-term nitrate stress conditions

Nitrate (20 mM) applied to the root medium of 28-day-old nodulated pea plants (Pisum sativum L., cv. Jupiter) immediately retarded nodule growth and inhibited root nodulation. Acetylene-reducing and H2-evolving nitrogenase activities were also significantly inhibited. The inhibitory effect of nitrate on nodule respiration was less pronounced while the respiration of roots was increased after the addition of nitrate. The levels of cytosol glutamine synthetase and nitrate reductase in nodule cytosol were permanently decreased from the 4th day after nitrate application. These results indicate that the inhibitory effect of high nitrate concentration on whole nodule metabolism is nonspecific in nature.

The effect of mineral nutrition on nitrogen assimilation by intact plants ofPisum sativum L

The effect of macroelements on nitrogen assimilation, level of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and content of nitrate nitrogen were studied in 17 day-old pea seedlings. The highest inhibition of NR was found at deficiencies of Mg and Ca while deficiency of K was less effective. The level of GS was decreased only in roots exclusively at deficiency of Mg. On the other hand, the deficiency of Mg and Ca increased the level of GDH. The lowest level of endogenous nitrate was found at deficiency of K. Present results show that assimilation of nitrate by plants was decreased by Mg and Ca deficiency while assimilation of ammonium was not affected.

Enhancement of Glutamate Dehydrogenase Activity in Excised Pea Roots by Exogenously Supplied Acids

Summary The addition of acids to the nutrient medium and the increase in the concentration of hydrogen ions in the external medium and within the roots therein obviously the determining factor causes an increase in GDH activity in excised pea roots. This increase is dependent on a proteosynthesis indicated by the complete inhibition of the increase by actidione; it is not, however, the result of generally enhanced proteosynthesis. Nitrate reductase activity was not induced by the increase in the concentration of H + ions; the activity of aspartate aminotransferase was slightly decreased. The increase in GDH activity caused by the addition of the acids was abolished by the addition of bases (incl. NH 4 OH), if the pH of the medium was shifted by this treatment nearer to neutral region (pH from 5 to 7). The shifting of pH into the alkaline region resulted in the inhibition of that increase in GDH level caused by the omission of sugar from the medium. The GDH level was not influenced significantly by this treatment in roots cultured with sugar. Among ammonium salts, NH 4 Cl (10 meq NH 4 + ) caused a statistically significant increase in GDH activity and simultaneously a statistically significant increase in the concentration of H + ions in roots cultured with sugar, whereas (NH 4 ) 2 HPO 4 (10 meq NH 4 + ) tendend to bring about an opposite, even though statistically insignificant, effect. These results indicate that the effect of higher concentrations of ammonium salts on GDH activity observed in the roots of intact plants by other authors does not reside in the direct influence of NH 4 + ions on GDH synthesis, but in the combination of (1) indirect influence of physiologically acid ammonium salts mediated by the increase in the concentration of H + ions inside the roots and of (2) another indirect influence mediated by the mechanism controlled by sugars (which was described and discussed in our previous paper - Sahulka et al., 1975). The GDH level could be increased up to four times during a 22 h incubation even in excised pea roots by the combined effects of the addition of acids and of the omission of sugar from the medium.