Regulation of the nitrate assimilation pathway in cultured tobacco cells

The mhsm underying the sharp increase in activity of nitrate reductase (EC 1.6.6.1) in Chlorefi vulgars forma terdis (strain 211 8k) during the first hour of the 7 hours/5 hours Light/dark cycle was investigated. Using the method of density labelng and isopycnic centaton, It could be trated that this rapi increase in activity is based on ligt-mediated activation ratber than denovo synthesis of the enzyme. The problematic nature of cycloheximide spechfcity and models of nitrate reductase activation are discussed.The reduction of nitrate to ammonia, requiring a total of eight electrons, is catalyzed during the first two steps by the enzymes nitrate reductase (EC 1.6.6.1) and nitrite reductase (EC 1.6.6.4); both enzymes have been frequently investigated in a variety of organisms. During the cell cycle of Chlorella vulgarisforma tertia (20), it was found that the activities of both enzymes exhibited a 28-fold increase in specific activity during the 1st hr of illumination. RESULTSConditions of Density Labling. The usual way of performing density labeling studies is to apply the label of heavy amino acids together with the change in physiological conditions which are to be studied (12). This strategy could not be used during the course of this study because introductory experiments showed that even a small amount of external amino acids completely suppressed the increase in nitrate reductase activity after onset of the light. In addition, it was found that supplementing the growth medium with amino acids (4 mM) did not result in an increase in the amino acid pool of the organism (data not shown). Therefore the labeling scheme had to be reversed. The cells used in our experiments had to be pregrown in the presence of deuterated amino acids, then transferred to the standard medium and illuminated without heavy label. Inasmuch as the activity increase of nitrate reductase was repressed by any external amino acids, the heavy amino acids should be depleted from the pool before the expected synthesis of nitrate reductase. Any protein being synthesized before illumination thus should contain deuterated amino acids, later produced proteins should be composed mainly by light amino acids.Intermal Markers. As intemal density markers and indicators for over-all protein synthesis, NAD-glutamate dehydrogenase (EC 1.4.1.2) and acid phosphatase (EC 3.1.3.2) were chosen which both could be found as single bands in the density gradients. Furthermore, these specific activities behave in a way considerably different from those of nitrate reductase (Table I). Assuming the 1st hr of the light cycle, where nitrate reductase exhibits an increase in activity of 2,850%, both glutamate dehydrogenase and acid phosphatase decrease slightly in their specific activities. The same behavior was observed in the presence of actidion, whereas the nitrate reductase was inhibited in its activity of 98%. The residual enzyme activity was much higher in the case of the two other enzymes. These differences in the behavior of the specific activiti...

Section: Discussionsupporting

confidence: 70%

Light-mediated Activation of Nitrate Reductase in Synchronous Chlorella

Tischner¹,

Hüttermann²

1978

“…Because induction of NR activity normally follows the influx of NO3-into the tissue (12,13), these data support the contention that increased levels of CO2 in some way suppresses the movement of nitrate into the leaf. Conversely, it is possible that the higher CO2 level enhanced assimilation rates and thus assimilation could be the major factor regulating NO3-accumulation.…”

Section: B Rate Of No3-accumulation and Induction Of Nr In Leafsupporting

confidence: 68%

“…This suggests that an apparent threshold level of NO3-accumulation in the whole leaf must be reached before initiation of enzyme induction. However, these measurements did not distinguish between "mobile" and "storage" pools of NO3-as proposed by Heimer and Filner, (12).…”

Section: B Rate Of No3-accumulation and Induction Of Nr In Leafmentioning

confidence: 73%

“…Plants exposed to 300, 400, and 600 of ul/l CO2 for 72 hr had NR activities of 12, 9, and 7.5 ,imoles of NO2-produced g fresh wt-1 hr-', respectively. Other work (12,13,20) suggests that the level of activity is a reflection of the rate of NO:-movement into the leaf. The second was that the net change in total reduced N per shoot decreased slightly with increasing CO2 concentrations during the 72 hr period.…”

Section: B Rate Of No3-accumulation and Induction Of Nr In Leafmentioning

confidence: 97%

See 1 more Smart Citation

Effect of Carbon Dioxide on Nitrate Accumulation and Nitrate Reductase Induction in Corn Seedlings

Purvis

Peters²,

Hageman³

1974

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric C02 levels ranging from 100 to 800 ,ul/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with C02 in the dark and maintained in an atmosphere containing 100 ,ul/l C02 accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 ,l/l C02, after 5 hours of illumination.

“…Nitrite accumulates under such conditions and the anaerobic production of nitrite has been used to assay nitrate reductase in situ in a variety of plant tissues (4, 11-13, 17, 22, 24, 25). The reaction was found to be linear for more than 1 hr and dependent upon the addition of exogenous nitrate (4,13,24 contain more than one pool of a compound (20,21), and there is evidence for more than one pool of nitrate in cultured tobacco cells (10). Suspensions of tobacco XD cells were used to test the idea.…”

mentioning

confidence: 99%

Anaerobic Nitrite Production by Plant Cells and Tissues: Evidence for Two Nitrate Pools

Ferrari¹,

Yoder²,

Filner³

1973

Self Cite

178

Nitrite reduction is inhibited by anaerobiosis in barley aleurone layers (5) and wheat roots (18), whereas the reduction of nitrate to nitrite by nitrate reductase is enhanced (4). Nitrite accumulates under such conditions and the anaerobic production of nitrite has been used to assay nitrate reductase in situ in a variety of plant tissues (4, 11-13, 17, 22, 24, 25). The reaction was found to be linear for more than 1 hr and dependent upon the addition of exogenous nitrate (4,13,24