The role of nitrate reduction in the anoxic metabolism of roots II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity

The effects of root anoxia on a tobacco (Nicotiana tabacum) wild type (WT) and a transformant (LNR-H) lacking root nitrate reductase were compared. LNR-H plants were visibly more sensitive to oxygen deprivation than WT, showing rapid and heavy wilting symptoms. LNR-H roots also produced substantially more ethanol and lactate than WT roots under anoxia, and their sugar and sugar-P content, as well as their ATP levels, remained higher. The fermentation rates of WT and LNR-H roots were unaffected by sugar feeding and the higher fermentation rate in the LNR-H roots was associated with a greater acidification of the cytoplasm under anoxia. From these observations it is concluded: (i) that the absence of NR activity in the LNR-H roots does not necessarily limit NADH recycling; and (ii) that nitrate reduction in the WT roots results in a more acidifying metabolism. It is the higher metabolic rate in the LNR-H roots that leads to the greater cytoplasmic acidification under anoxia despite the absence of a contribution from the metabolism of nitrate. Competition for NADH cannot explain this difference in metabolic rate, and it remains unclear why the NR-free LNR-H, and tungstate-treated WT roots, had much higher fermentation rates than WT roots. The difference in anaerobic metabolism could still be due to the presence or absence of nitrate reductase and the possibility that this could occur through the production of nitric oxide is discussed.     

植物氮代谢硝酸还原酶水平调控机制的研究进展

氮代谢是植株体内最基本的物质代谢之一,硝酸还原酶是植物氮代谢的关键酶。主要对植物氮代谢在硝酸还原酶水平上调控的研究新进展,尤其是其合成／降解及活性调控机制进行了较为系统的综述。硝酸还原酶合成的调控主要发生在转录水平和翻译水平上,硝酸还原酶降解的调控主要发生在翻译后水平上,同时NO3^-及光在硝酸还原酶转录水平调控上的作用重大,硝酸还原酶编码基因转录的mRNA的稳定性强弱影响植物的氮代谢,而影响mRNA稳定性的因素很多,机理复杂;磷酸化／去磷酸化在硝酸还原酶活性调控中占举足轻重的地位,研究也比较深入。钝化蛋白也能够影响硝酸还原酶活性,许多小分子物质对硝酸还原酶活性有影响。     

Regulation of secondary metabolism by the carbon-nitrogen status in tobacco: nitrate inhibits large sectors of phenylpropanoid metabolism

Interactions between nitrogen and carbon metabolism modulate many aspects of the metabolism, physiology and development of plants. This paper investigates the contribution of nitrate and nitrogen metabolism to the regulation of phenylpropanoid and nicotine synthesis. Wild-type tobacco was grown on 12 or 0.2 mm nitrate and compared with a nitrate reductase-deficient mutant [Nia30(145)] growing on 12 mm nitrate. Nitrate-deficient wild-type plants accumulate high levels of a range of phenylpropanoids including chlorogenic acid, contain high levels of rutin, are highly lignified, but contain less nicotine than nitrogen-replete wild-type tobacco. Nia30(145) resembles nitrate-deficient wild-type plants with respect to the levels of amino acids, but accumulates large amounts of nitrate. The levels of phenylpropanoids, rutin and lignin resemble those in nitrogen-replete wild-type plants, whereas the level of nicotine resembles that in nitrate-deficient wild-type plants. Expression arrays and real time RT-PCR revealed that a set of genes required for phenylpropanoid metabolism including PAL, 4CL and HQT are induced in nitrogen-deficient wild-type plants but not in Nia30(145). It is concluded that nitrogen deficiency leads to a marked shift from the nitrogen-containing alkaloid nicotine to carbon-rich phenylpropanoids. The stimulation of phenylpropanoid metabolism is triggered by changes of nitrate, rather than downstream nitrogen metabolites, and is mediated by induction of a set of enzymes in the early steps of the phenylpropanoid biosynthetic pathway.     

Nitrite and nitrate reduction by molybdenum centers of the nitrate reductase type: Computational predictions on the catalytic mechanism

Nitrate reductases (NRs) are enzymes that catalyze reduction of nitrate to nitrite using a molybdenum cofactor. In an alternative reaction, plant NRs have also been shown to catalyze reduction of nitrite to nitric oxide, and this appears to be a major source of nitric oxide synthesis in plants, although other pathways have also been shown. Here, density functional theory (DFT) results are shown, indicating that although nitrate is thermodynamically the preferred substrate for the NR active site, both nitrite and nitrate are easily reduced to nitrite and NO, respectively. These mechanisms require a Mo(IV) state. Additionally, in the case of the nitrite, linkage isomerism is at work and controlled by the metal oxidation state, and reduction is, unlike in the nitrate case, dependent on protonation. The data may be relevant to other molybdenum enzymes with similar active sites, such as xanthine oxidase.     

Relationship of nitrate supply with growth rate, plasma membrane-bound and cytosolic nitrate reductase, and tissue nitrate content in tobacco plants

cNR, cytosolic nitrate reductase
PM-NR, plasma membrane-bound nitrate reductase

Activities of plasma membrane-bound nitrate reductase (PM-NR) and cytosolic nitrate reductase (cNR) in tobacco (Nicotiana tabacum L. cv. Samsun) are regulated differently, depending upon the nitrate supply to the culture medium (in sand culture). The cNR activity of roots was higher at low nitrate concentrations with the maximum at 5 mM nitrate supply and declined to low values beyond 5 mM . In contrast, the PM-NR activity of roots increased with higher nitrate concentrations with the maximum at 25 mM nitrate and clearly decreased only at 40 mM . This high PM-NR activity correlated with a low growth rate and might be one of the responses to excess nitrate. Internal nitrate and total nitrogen content of the tissues, however, showed a relative minimum in shoots and in roots of between 15 and 25 mM external nitrate. With declining PM-NR activities beyond 25 mM external nitrate, the nitrate content in the tissue increased indicating an inverse relationship between tissue nitrate content and root PM-NR activity. In leaves both NR activities (cNR and PM-NR) correlated with the internal nitrate content, but with a different response at low nitrate.     

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

The responses of nitrate reductase (NR) activity and levels of NR-mRNA to environmental nitrate and exogenous cytokinins are characterised in roots and shoots of barley ( Hordeum vulgare L., cv. Golf), using a chemostate-like culture system for controlling nitrate nutrition. Experiments were mainly performed with split root cultures where nitrate-N was supplied at a constant relative addition rate of 0.09 day⁻¹, and distributed between the subroots in a ratio of 20%:80%. The subroot NR-mRNA level and NR activity, as well as the endogenous level of zeatin riboside (ZR), increased when the local nitrate supply to one of the subroots was increased 4-fold by reversing the nitrate addition ratio (i.e. from 20%:80% to 80%:20%). Also shoot levels of ZR, NR-mRNA and NR activity increased in response to this treatment, even though the total nitrate supply remained unaltered. External supply of ZR at 0.1 μ M caused an approximately 3-fold increase in root ZR levels within 6 h. which is comparable to the nitrate-induced increase in root ZR. External application of ZR. zeatin. isopentenyl adenine or isopentenyl adenosine at 0.1 μ M caused from insignificant to 25% increases in NR-mRNA and activity in roots and up to 100% stimulation in shoots, whereas adenine or adenosine had no effect. No synergistic effects of perturbed nitrate supply and cytokinin application were detected in either roots or shoots. The translocation of nitrate from the root to the shoot was unaffected by application of ZR or switching the nitrate distribution ratio between subroots. The data give arguments for a physiological role of cytokinins in the response of root and shoot NR to environmental nitrate availability. The nature and limitations of the physiological role of cytokinins are discussed.     

Multiple forms of nitrate reductase and their role in nitrate assimilation in roots of wheat at low temperature or high salinity

NADPH-, NADH-, and KNO₃-eluted fractions of nitrate reductase (NR) were isolated from roots of winter wheat ( Triticum aestivum L. cv. Mironovskaya 808) grown under low temperature or high salinity. All three fractions exerted activity with either NADPH or NADH as electron donor. The NADPH-eluted fraction showed the highest activity with NADPH, whereas the NADH- and KNO₃-eluted fractions were most active with NADH. The NADH- and NADPH-dependent activities in the NADH- and KNO₃-eluted fractions were the ones that changed the most in response to low temperature. The inhibitory effect of salt stress was the same for both activities in each of the NADH- and KNO₃-eluted fractions. The NADPH-eluted NR was the one least affected by the growth conditions.     

Soaking seeds of Lens culinaris with 28-homobrassinolide increased nitrate reductase activity and grain yield in the field in India

Surface‐sterilised seeds of Lens culinaris cv. Pusa‐6 were soaked in 0 M, 10^?6 M, 10^?8 M or 10^?10 M aqueous solutions of 28‐homobrassinolide (HBR) for 4 h, 8 h or 12 h and planted in the field in a sandy loam soil. Plants were sampled 60, 90 and 120 days after sowing (DAS). Soaking with HBR decreased root length and nodule number per plant but increased nitrate reductase activity (E.C.1.6.6.1). Soaking with HBR also increased grain yield at the final harvest 140 DAS. The greatest increase was obtained with an HBR concentration of 10^?8 M.     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

The effect of root temperature on the induction of nitrate reductase activities and nitrogen uptake rates in arctic plant species

We investigated whether six arctic plant species have the potential to induce nitrate reductase (NR) activity when exposed to NO₃ ^--nitrogen under controlled environment conditions, using an in vivo assay that uses the rate of NO₂ ^--accumulation to estimate potential NR activity. We also assessed the effect of low root temperatures on NR activity, growth and nitrogen uptake (using ¹⁵N applications) in two of the selected species. Five of the six species (Cerastium alpinum, Dryas intergrifolia, Oxyria digyna, Saxifraga cernua and Salix arctica) were capable of inducing NR activity when exposed to solutions containing 0.5 mM NO₃ ^- at 20°C for 10 days. Although in vivo NR activity was not induced in Saxifraga oppositifolia under controlled conditions, we conclude that it was capable of growing successfully on NO₃ ^-, due to the presence of moderate rates of NR activity observed in both NH₄ ⁺-grown and NO₃ ^--treated plants. Exposure of O. digyna and D. integrifolia to 3°C root temperatures for two weeks, with the shoots kept at 20°C, resulted in root and leaf NR activity rates of NO₃ ^--treated plants being reduced to rates exhibited by NH₄ ⁺-grown plants. Although these decreases in NR in both species appeared to be due to limitations in NO₃ ^--uptake and growth rate (rather than direct low-temperature inhibition of NR synthesis per se), direct low-temperature inhibition of root NR synthesis could not be ruled out. In contrast to the temperature insensitivity of NH₄ ⁺ uptake in D. integrifolia, NO₃ ^--uptake in D. integrifolia was inhibited by low root temperatures. We conclude that the selected arctic species have the genetic potential to utilize NO₃ ^--nitrogen, and that low root temperatures, in conjunction with other environmental limitations, may be responsible for the lack of induction of NR in D. integrifolia and Salix arctica under field conditions.     

Novel growth characteristics and high rates of nitrate reduction of an Escherichia coli strain, LCB2048, that expresses only a periplasmic nitrate reductase

Escherichia coli strain LCB2048 is a double mutant defective in the synthesis of the two membrane-associated nitrate reductases A and Z. This strain can grow anaerobically on a non-fermentable carbon source, glycerol, in the presence of nitrate even in media supplemented with high concentrations of tungstate. This growth was totally dependent upon a highly active, periplasmic nitrate reductase (Nap). Due to the presence of a previously unreported narL mutation, synthesis of the periplasmic nitrate reductase by this strain was induced during anaerobic growth by nitrate. We have also demonstrated that methyl viologen is an ineffective electron donor to Nap: its use leads to an underestimation of the contribution of Nap activity to the rate of nitrate reduction in vivo.     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

A comparative study between two citrus rootstocks: Effect of nitrate on the root morpho-topology and net nitrate uptake

Root morpho-topology and net nitrate uptake of two citrus seedlings, Volkamer Lemon and Carrizo Citrange, grown at two nitrogen supplies (NO₃-N 5 M and 1000 M, respectively) were studied. Root morphological and topological parameters were gauged by an image-specific analysis system (WinRHIZO). Net nitrate uptake was estimated using the nitrate depletion method. The main findings showed that Carrizo seedlings had a dichotomous branching root system characterized by high root tip numbers and long 2^nd order lateral roots. Conversely, Volkamer root systems had a herringbone structure with a long tap root and 1^st order lateral root. Nitrate treatment did not seem to affect the pattern of the two genotypes, except for the 2^nd order lateral roots (Carrizo more than Volkamer) and root/shoot ratio and root mass ratio (Volkamer more than Carrizo) that were significantly different at low nitrate supply. Nitrate treatments induced a diverse net nitrate uptake regulation between citrus rootstocks. Indeed, at low nitrate supply, Carrizo showed a more efficient nitrate acquisition process in terms of: 1) higher net nitrate uptake maximum of the inducible high affinity transport system or full induction (A), (2) higher cumulative nitrate uptake (A_t) and (3) lower t₁ parameter defined as the half time of the net nitrate uptake rate of the inducible transport system during the induction phase, compared to Volkamer. Conversely, at the high nitrate level, only the genotypical difference of the t₁ parameter was maintained. The results suggested that, at the low nitrate level, the morphological root traits such as higher 2^nd order lateral roots and greater root tip numbers of the Carrizo compared with Volkamer seedlings, enhance the capacity to absorb nitrate from nutrient solution.