In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG immunoglobulin G - NR nitrate reductase - PEPCase phosphoenolpyruvate carboxylase This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F.     

Stabilization of nitrate reductase in maize roots by chymostatin

Nitrate reductase (NR) in maize (Zea mays cv W64A × W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO⁻₂ produced per hour per gram fresh weight was measured at 4 days).     

Light-modulation of nitrate reductase activity in leaves and roots of maize

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash ( Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G₁ with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.     

Inhibition of nitrate reductase induction by canavanine in maize roots

The induction of nitrate reductase activity in maize root tips was inhibited by canavanine and the inhibition increased with increasing concentration of canavanine between 0·1 and 1 mM. Addition of canavanine to the induced enzyme had little effect on the disappearance of the enzyme when nitrate was removed, and it is likely that the canavanine reduces the activity of the nitrate reductase by inhibiting its synthesis rather than by accelerating its breakdown.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.     

The miRNA-mediated post-transcriptional regulation of maize response to nitrate

Stress responses depend on the correct regulation of gene expression. The discovery that abiotic as well as biotic stresses can regulate miRNA levels, coupled with the identification and functional analyses of stress-associated genes as miRNA targets, provided clues about the vital role that several miRNAs may play in modulating plant resistance to stresses. Nitrogen availability seriously affects crops productivity and environment and the understanding of the miRNA-guided stress regulatory networks should provide new tools for the genetic improvement of nitrogen use efficiency of crops. A recent study revealed the potential role of a number of nitrate-responsive miRNAs in the maize adaptation to nitrate fluctuations. In particular, results obtained suggested that a nitrate depletion might regulate the expression of genes involved in the starvation adaptive response, by affecting the spatio-temporal expression patterns of specific miRNAs.     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.     

Differential regulation of nitrate reductase induction in roots and shoots of cotton plants

The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.     

Intercellular localization of nitrate reductase in roots

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO₃⁻ for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO₃⁻ likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO₃⁻, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO₃⁻ occurs during uptake and reduction of exogenous NO₃⁻. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO₃⁻. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO₃⁻, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO₃⁻ is taken up by cells of the cortex and stele, and/or (c) NO₃⁻ is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO₃⁻ reduction.     

The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 mM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.     

Synthesis and turnover of nitrate reductase in corn roots

The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.     

Proliferation of maize (Zea mays L.) roots in response to localized supply of nitrate

Maize (Zea mays L.) plants with two primary nodal root axes were grown for 8 d in flowing nutrient culture with each axis independently supplied with NO3-. Dry matter accumulation by roots was similar whether 1.0 mol m-3 NO3- was supplied to one or both axes. When NO3- was supplied to only one axis, however, accumulation of dry matter within the root system was significantly greater in the axis supplied with NO3-. The increased dry matter accumulation by the +N-treated axis was attributable entirely to increased density and growth of lateral branches and not to a difference in growth of the primary axis. Proliferation of lateral branches for the +N axis was associated with the capacity for in situ reduction and utilization of a portion of the absorbed NO3-, especially in the apical region where lateral primordia are initiated. Although reduced nitrogen was translocated to the -N axis, concentrations in the -N axis remained significantly lower than in the +N axis. The concentration of reduced nitrogen, as well as in vitro NO3- reductase activity, was greater in apical than in more basal regions of the +N axis. The enhanced proliferation of lateral branches in the +N axis was accompanied by an increase in total respiration rate of the axis. Part of the increased respiration was attributable to increased mass of roots. The specific respiration rate (micromoles CO2 evolved per hour per gram root dry weight) was also greater for the +N than for the -N axis. If respiration rate is taken as representative of sink demand, stimulation of initiation and growth of laterals by in situ utilization of a localized exogenous supply of NO3- establishes an increased sink demand through enhanced metabolic activity and the increased partitioning of assimilates to the +N axis responds to the difference in sink demand between +N and -N axes.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.