In Vitro Stability of Nitrate Reductase from Wheat Leaves

ABSTRACITCultures of Lemna gibba L. G3 were maintained at a constant, Nlimited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day-'. Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemma is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.Assimilation ofNO3-in higher plants is dependent on a variety of environmental factors, the availability of NO3-being one of the most obvious (2, 6). The flux rather than the tissue content of NO3-was shown to be decisive for the NRA2 in maize (22).However, although NRA fluctuates in response to the flux or availability of NO3-in barley (8) and pea (19), the actual N03-reduction (estimated from the tissue N03-balance) is better correlated to net NO3-uptake than to the NRA in vitro (8,19 complicated by the labile nature of NR in most in vitro preparations and several precautions must be taken to stabilize the enzyme (5, 16, 21). Proteinases more or less specific for NR have been isolated from both roots (28, 31) and shoots ( 11) of higher plants although their importance in regulating the amount or activity of NR is not as yet thoroughly understood. If extracted and assayed in an appropriate way, however, the in vitro NR assay indicates the potential NRA in the tissue (29) which might be essential for the understanding of the regulation of NR.The aim of the present work was to study the relationship between NO3-fluxes and NO3-reduction, and to compare actual NO3-reduction with NRA assayed in vitro, taking into consideration especially the stability ofthe enzyme towards proteolysis. These parameters were investigated in N-limited Lemna gibba growing at a stable suboptimal growth rate controlled by exponentially increasing the daily doses of NO3-, while keeping other nutrients at a sufficient level (13,14,19). In plants maintained in this ...

Section: Discussionsupporting

confidence: 68%

Nitrogen Utilization in Lemna

Ingemarsson

1987

“…Of the four hybrids, D had the lowest NRA/g root, an observation consistent with the observation that low root NRA permits greatest root growth (16). For each hybrid pair, the relative rankings with respect to level of leaf NRA was the same with both assays; however, greater differences in NRA were obtained with the in vitro than the in vivo assay for genotypes A and D. For both hybrid pairs, these differences could be due to in vitro assay problems, such as enzyme stability (21), to the presence of inhibitors (6,23), or to the fact that availability of reductant limits the in vivo reduction (14).…”

Section: Resultsmentioning

confidence: 86%

Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.)

Reed

Hageman²

1980

The study presented here was an extension of a preceding field project concerned with changes in N metabolism of four maize hybrids during grain development. The objectives were to relate uptake, flux, and reduction of nitrate to accumulation of reduced N in growth-chamber-grown seedlings of the same four hybrids and to compare these results with those obtained in the field study.Hybrid D took up more nitrate than the other three hybrids, primarily because of a larger root system. The correlations between total N (nitrate plus reduced N plant-) accumulated by harvest and root dry weight or shoot to root ratios were r = +0.97 and -0. In wheat and corn, significant correlations between the estimated amount of reduced N supplied to the plant (by the in vitro or in vivo NR3 assay) and the actual amount accumulated by the plant (2,5,7,8) support the concept that nitrate reduction is the rate-limiting step in the assimilation of nitrate to reduced N. Significant correlations were found between integrated seasonal leaf NRA and grain yield, plant reduced N, and grain reduced N for maize. In these and other experiments, the correlation values were low, indicating that factors other than the level of leaf NRA affect these relationships.Dalling et al. (5) showed that transport of vegetative N to the grain of wheat was one of these factors. They also found that wheat cultivars with comparable levels of NRA could accumulate different amounts of reduced N. Deckard et al. (6) identified one maize genotype with a relatively low NRA but a high capacity to accumulate reduced N. The fact that this genotype had a high leaf nitrate concentration suggests that the availability of nitrate to NR in the leaf, as well as the level of enzyme, may affect the rate of nitrate reduction (3). This possibility is supported by (a) the stimulation of the in vivo NR assay with nitrate (10), and (b) the correlation found between nitrate uptake and accumulation of reduced N in several maize genotypes (4).Although nitrate flux to the leaves of maize regulates the level of NR (21), it has not been shown that nitrate flux and NR are directly related among genotypes. Since the nitrate flux provides both substrate for and inducer of NR, genotypic differences in the partitioning of nitrate and/or responsiveness of the induction mechanism to nitrate could explain the discrepancy observed for the one genotype in the study by Deckard et al. (7).The study presented here was an extension of a preceding field project that utilized the same four maize hybrids (19). The field study was concerned with changes in N metabolism (enzymes and N components) in various plant parts during grain development.The objectives of the current study were: (a) to relate nitrate uptake, nitrate flux, shoot to root partitioning of nitrate reduction, and NRA to the accumulation of reduced N by the plants during early vegetative growth; and (b) to compare these results with seedlings to those obtained in the preceding field study.

“…NRs are also inactivated by specific NRinactivating proteins isolated from corn roots (14,15), rice-cell cultures (17,18), and soybean leaves (4). Two types of uncharacterized protein-like factors have recently been identified in wheat leaves (9,10). One of these factors reduced the stability, whereas the others seemed to confer stability on NR.…”

mentioning

confidence: 99%

Activation of Nitrate Reductase by Extracts from Corn Scutella

Yamaya

Oaks²

1980

NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A x W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex I (acetate) and Dowex 50 (H+) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH2-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 x 38-11) and rice cell cultures.NADH-NR4 is considered to be the key enzyme in the nitrate assimilation pathway, and the rapid changes in its activity in response to changes in environmental conditions indicate the presence of effective regulatory systems in plant tissues. The rapid fluctuations could result from changes in relative rates of synthesis or degradation of the enzyme or from its activation or inactivation. For example, upon transfer of Chlorella from ammonium to nitrate medium, Johnson (3) recently showed by density-labeling technique that there was a rapid increase in NADH-NR activity, but only part of this was the consequence of increased de novo synthesis; there was apparently considerable activation of existing enzyme. Many factors can affect the NR level in plant tissues and some of these have recently been reviewed by Hewitt et aL (2).There is increasing evidence relating to the activation or inactivation of NR. For example, NRs isolated from Chlorella (12), spinach leaves (8), and corn scutella (15) are inactivated by NAD(P)H and/or cyanide and the inactivated NRs can be reactivated by ferricyanide. NRs are also inactivated by specific NRinactivating proteins isolated from corn roots (14,15), rice-cell cultures (17, 18), and soybean leaves (4). Two types of uncharacterized protein-like factors have recently been identified in wheat leaves (9, 10). One of these factors reduced the stability, whereas the others seemed to confer stability on NR. There are also reports on the effects of low mol wt components, EDTA, cysteine, FAD, or even N02, on the activation of NR (5,11,13 After 3 days at 26 C, scutella were separated from the seedlings for the isolation of the activator. In some experiments, the seedlings were transferred to liquid medium containing the same nutrients, but without agar, and grown for another 3 days at 26 C with a 12-h photoperiod.Extraction of the Activator. One g of corn scutella was quickly frozen in liquid N2 and then extracted with 10 ml 50 mm Kphosphate buffer (pH 7.5), containing 0.5 mm EDTA and 5 mM L-cysteine. The homogenates were filtered through 3 layers of Miracloth and centrifuged at 30,000g for 20 min. The supernatant was treated at 50 C for 10 min to remove NR activity and recentrifuged. The supernatant solution (crude extract) then was mixed with ethanol ...