Nitrate Reductase Transcript Levels Research Articles

Thiamine primed defense provides reliable alternative to systemic fungicide carbendazim against sheath blight disease in rice (Oryza sativa L.)

A novel pathogen defense strategy by thiamine priming was evaluated for its efficacy against sheath blight pathogen, Rhizoctonia solani AG-1A, of rice and compared with that of systemic fungicide, carbendazim (BCM). Seeds of semidwarf, high yielding, basmati rice variety Vasumati were treated with thiamine (50 mM) and BCM (4 mM). The pot cultured plants were challenge inoculated with R. solani after 40 days of sowing and effect of thiamine and BCM on rice growth and yield traits was examined. Higher hydrogen peroxide content, total phenolics accumulation, phenylalanine ammonia lyase (PAL) activity and superoxide dismutase (SOD) activity under thiamine treatment displayed elevated level of systemic resistance, which was further augmented under challenging pathogen infection. High transcript level of phenylalanine ammonia lyase (PAL) and manganese superoxide dismutase (MnSOD) validated mode of thiamine primed defense. Though minimum disease severity was observed under BCM treatment, thiamine produced comparable results, with 18.12 per cent lower efficacy. Along with fortifying defense components and minor influence on photosynthetic pigments and nitrate reductase (NR) activity, thiamine treatment significantly reduced pathogen-induced loss in photosynthesis, stomatal conductance, chlorophyll fluorescence, NR activity and NR transcript level. Physiological traits affected under pathogen infection were found signatory for characterizing plant's response under disease and were detectable at early stage of infection. These findings provide a novel paradigm for developing alternative, environmentally safe strategies to control plant diseases.

A Model for the Circadian Oscillations in Expression and Activity of Nitrate Reductase in Higher Plants

Nitrate is the major nitrogen source for many plants. The first step of the nitrate assimilation pathway is the reduction of nitrate to nitrite, catalysed by nitrate reductase (NR). Circadian oscillations in expression and activity of NR have been demonstrated in many plant species. The pathway by which this circadian behaviour is regulated remains to be elucidated. In this study, based on recent experimental observations, a mathematical model is proposed to explain the origin of diurnal and circadian oscillations in NR gene expression and enzyme activity. The dynamic model is based on the feedback interconnections between NR and its substrate, nitrate. In the model, NR activity is regulated at the transcriptional level, in response to the balance between nitrate influx and reduction, and at the post-translational levels in response to signals from carbon assimilation. Conditions for the model system to generate self-sustained circadian oscillations are investigated by numerical simulations. Under light/dark cycles, the simulation results are in agreement with the observed diurnal pattern of changes in leaf nitrate concentration, NR transcript level and NR activity. Within a range of kinetic parameter values, circadian oscillation behaviour persists even under constant light, with periods of approx. 24 h. These simulation results suggest that sustained circadian oscillations can originate from the feedback interactions between NR and its substrate, nitrate, without the need to postulate the existence of an endogenous 'circadian clock'.

Regulation of nitrate reductase expression in leaves by nitrate and nitrogen metabolism is completely overridden when sugars fall below a critical level

ABSTRACTThese experiments use Nia30(145), a tobacco nia1nia2 double null mutant transformed with a NIA2 construct, to define when sugar supply plays the dominating role in the regulation of nitrate reductase (NIA) expression. The null alleles of Nia30(145) are transcribed and translated to produce non‐functional NIA transcript and NIA protein, providing an endogenous reporter system to track NIA expression at the transcript and protein level. The re‐introduced NIA2 construct is expressed at low efficiency, providing a background in which the response to changes in sugar status is not complicated by simultaneous changes in the rate of nitrate assimilation and the levels of nitrate and glutamine. In an alternating light–dark regime, Nia30(145) contained high levels of nitrate and low levels of glutamine and other amino acids. This drives constitutive overexpression of NIA. After transfer of Nia30(145) to continuous darkness, nitrate remains high and glutamine low, but the NIA transcript level and NIA protein decreased significantly within 24 h and were undetectable from 48 h onwards. The decrease of the NIA transcript level was fully reversed and the decrease of NIA protein was partly reversed when leaves were detached from the pre‐darkened plants and supplied with sucrose in the dark. The decrease was not reversed by nitrate or cytokinin. The NIA transcript disappeared when the leaf sugar content fell below 4 μmol hexose equivalents g−1 FW, and recovered when sugars rose above 8 μmol hexose equivalents g−1 FW. It is concluded that low sugar represses NIA, completely overriding signals derived from nitrate and nitrogen metabolism.

Lithium inhibits phytochrome-stimulated nitrate reductase activity and transcript level in etiolated maize leaves

Our earlier work indicated an involvement of signals generated via polyphosphoinositide turnover in phytochrome (Pfr) mediated light stimulation of nitrate reductase (NR) activity. We now show that lithium, an inhibitor in the cycling of inositol phosphates, inhibits red-light stimulated nitrate induced NR activity. The effect of lithium was more pronounced if it was given immediately after red light irradiation than if supplied1 h after. The inhibition of NR activity by lithium was partially reversed by myo-inositol and totally reversed by calcium. Red light stimulated NR transcript level was also inhibited by lithium, however, calcium could only partially reverse this effect. In the present system we show that lithium decreased the level of phosphoinositide-bis-phosphate as well as other phosphoinositides in red light irradiated tissues. These results further suggests an involvement of calcium and signals produced via turnover of polyphosphoinositides in signal transduction in Pfr regulation of NR transcript level and NR activity.

ZmcPKC70, a Protein Kinase C-type Enzyme from Maize: BIOCHEMICAL CHARACTERIZATION, REGULATION BY PHORBOL 12-MYRISTATE 13-ACETATE AND ITS POSSIBLE INVOLVEMENT IN NITRATE REDUCTASE GENE EXPRESSION

The crucial enzyme in diacylglycerol-mediated signaling is protein kinase C (PKC). In this paper we provide evidence for the existence and role of PKC in maize. A protein of an apparent molecular mass of 70 kDa was purified. The protein showed kinase activity that was stimulated by phosphatidylserine and oleyl acetyl glycerol (OAG) in the presence of Ca2+. Phorbol 12-myristate 13-acetate (PMA) replaced the requirement of OAG. [3H]PMA binding to the 70-kDa protein was competed by unlabeled PMA and OAG but not by 4alpha-PMA, an inactive analog. The kinase phosphorylates histone H1 at serine residue(s), and this activity was inhibited by H-7 and staurosporine. These properties suggest that the 70-kDa protein is a conventional serine/threonine protein kinase C (cPKC). Polyclonal antibodies raised against the polypeptide precipitate the enzyme activity and immunostained the protein on Western blots. The antibodies also cross-reacted with a protein of expected size from sorghum, rice, and tobacco. A rapid increase in the protein level was observed in maize following PMA treatments. In order to assign a possible role of PKC in gene regulation, the nitrate reductase transcript level was investigated. The transcript level increased by PMA, not by 4alpha-PMA treatments, and the increase was inhibited by H-7 but not by okadaic acid. The data show the existence and possible function of PKC in higher plants.

Impact of elevated atmospheric CO2 on nitrate reductase transcription and activity in leaves and roots of Plantago major

Vegetative plants of an inbred line, A4, of Plantage major ssp. pleiosperma (L.) Pilger were grown at 350 mu l 1(-1) or at elevated (700 mu l l(-1)) CO2 in non-limiting nutrient solution with nitrate. Both the relative growth rate (RGR) and the root to total plant weight ratio (RWR) were increased by elevated CO2. However, the stimulation of both RGR and RWR was transient and did not last longer than 8 days. To investigate the physiological mechanisms involved in this stimulation, related changes in C/N metabolism were examined. In the roots soluble sugar concentration increased during the transient period of RGR stimulation (up to 23%), as did the root respiration rate. Changes in nitrogen metabolism were also restricted to this period and consisted of an increase in (1) in vivo and in vitro root nitrate reductase (EC 1.6.6.1) activity, (2) in vitro leaf nitrate reductase activity, (3) leaf and root nitrate reductase mRNA and (4) reduced nitrogen concentration in the roots. The elevated CO2-related signal for the increase in nitrate reductase transcript levels in the roots is discussed in terms of the increased availability of soluble sugars. The results suggest that the short-term enhancement of root carbon and nitrogen metabolism may be responsible for the transient effect of elevated CO, on whole plant RGR.

Impact of elevated atmospheric CO2 on nitrate reductase transcription and activity in leaves and roots of Plantago major

Vegetative plants of an inbred line, A4, of Plantago major ssp. pleiosperma (L.) Pilger were grown at 350 μl I−1 or at elevated (700 μl I−1) CO2 in non‐limiting nutrient solution with nitrate. Both the relative growth rate (RGR) and the root to total plant weight ratio (RWR) were increased by elevated CO2. However, the stimulation of both RGR and RWR was transient and did not last longer than 8 days. To investigate the physiological mechanisms involved in this stimulation, related changes in C/N metabolism were examined. In the roots soluble sugar concentration increased during the transient period of RGR stimulation (up to 23%), as did the root respiration rate. Changes in nitrogen metabolism were also restricted to this period and consisted of an increase in (1) in vivo and in vitro root nitrate reductase (EC 1.6.6.1) activity. (2) in vitro leaf nitrate reductase activity, (3) leaf and root nitrate reductase mRNA and (4) reduced nitrogen concentration in the roots. The elevated CO2‐related signal for the increase in nitrate reductase transcript levels in the roots is discussed in terms of the increased availability of soluble sugars. The results suggest that the short‐term enhancement of root carbon and nitrogen metabolism may be responsible for the transient effect of elevated CO2 on whole plant RGR.

Expression of enzyme activities and transcripts required for nitrate assimilation in maize ( Zea mays L. A188 × BMS) suspension cell cultures

The response of a rapidly growing, transformable maize suspension cell culture line ( Zea mays L. A188 × BMS) to changes in media N and NO 3 − treatment was examined. Growth, N content, enzyme activities and transcript levels were measured in cells transferred to N-rich media (N6) or to media lacking N (N-free). Transferring cells to N-free media had little impact on cell growth (fresh and dry weight). However, reduced N, soluble protein, and total RNA per g dry weight were about 50% lower than in cells transferred to N6 media. Cellular NO 3 −, nitrate reductase (NR) and nitrite reductase (NiR) activity declined rapidly when cells were transferred to N-free media. Within 24 h after transfer to N-free media, treatment with 1 mM KNO 3 increased NADH: NR, NAD(P)H: NR, and NiR activities, and NR transcript levels. Cells transferred to N6 media did not respond to KNO 3 treatment. Three days after transfer to N-free media, NR transcript levels increased rapidly and transiently in response to low concentrations of NO 3 −. This response was not prevented by the cytoplasmic protein synthesis inhibitor, cycloheximide. The similarity between the response of the N-starved A188 × BMS cell culture and maize roots to environmental NO 3 − indicated that the cell cultures provide a uniform, easily manipulable system for the study of NO 3 − signal transduction processes.

Phosphorylation/dephosphorylation steps are key events in the phytochrome-mediated enhancement of nitrate reductase mRNA levels and enzyme activity in maize.

We provide evidence to show that the increase in nitrate reductase (NR) transcript level stimulated by red light is mediated via a phosphorylation-dependent step. The light-stimulated enhancement of NR transcript level was significantly inhibited by H-7, a protein kinase inhibitor, whereas okadaic acid (OKA), a phosphatase inhibitor, had no effect. Phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) enhanced the NR transcript level in dark-grown leaves. No correlation between changes in NR transcript level and NR activity (NRA) was observed. Inhibition of NRA by OKA and stimulation by H-7 indicated that NRA is increased by dephosphorylating the enzyme. We have identified a protein kinase (C type) that can phosphorylate the purified NR in vitro without the involvement of other accessory proteins. By in vivo labelling with 32P and immunoprecipitation of NR with NR antibodies it was found that in the presence of OKA most NR protein (NRP) was present in phosphorylated state, while with H-7 the reverse was seen. The red (R) and far-red (FR) light reversible experiments suggested that phytochrome (Pfr, an active form) stimulation of NRA is mediated by dephosphorylation of the enzyme, suggesting that Pfr regulates both NR transcription and NRA via phosphorylation/dephosphorylation steps controlled by separate signal transduction pathways.

Evidence for some common signal transduction events for opposite regulation of nitrate reductase and phytochrome-I gene expression by light.

We have explored the possible involvement of the phosphoinositide (PI) cycle and protein kinase C (PKC) in the phytochrome (Pfr)-mediated light signal transduction pathway using nitrate reductase (NR) and phytochrome-I (PhyI) genes as model systems. We have shown earlier that phorbol myristate acetate (PMA) completely replaces the red light effect in stimulating nitrate reductase activity and transcript levels in maize. In this paper, we present detailed evidence to show that PMA mimics the red light effect and follows similar kinetics to enhance NR steady-state transcript accumulation in a nitrate-dependent manner. We also show that PMA inhibits phyI steady-state transcript accumulation in a manner similar to red light, indicating that a PKC-type enzyme(s) may be involved in mediating the light effect in both cases. Serotonin or 5-hydroxytryptamine (5-HT), a stimulator of PI turnover, was also found to mimic the red light effect in enhancing NR transcript levels and inhibiting phyI transcript accumulation, indicating the role of the PI cycle in generating second messengers for regulating the two genes. These results indicate that phytochrome-mediated light regulation of NR and phyI gene expression may involve certain common steps in the signal transduction pathway such as the PI cycle and protein phosphorylation by a PKC-type enzyme.

Investigations on the nature of the phytochrome-induced transmitter for the regulation of nitrate reductase in etiolated leaves of maize

A lag phase of 30 min for phytochrome-mediated stimulation in the induction of nitrate reductase (NR) in etiolated leaves of Zea mays var. 'Ganga 5', was eliminated by a 2 h pre-irradiation with red light, supplied in the absence of nitrate. Earlier, photoreversibil-ity of NR activity was found to be lost completely by 2 h and a transmitter with a life span of 12 h was proposed to mediate the phytochrome effect on NR (Sharma and Sopory, 1984). Low temperature (0°C) treatment prevented the loss of the photoreversibility. Almost complete photoreversibility was observed after 2 h, suggesting that the formation of the transmitter requires active metabolism. Tungstate, given 2 h after red light treatment, inhibited the increase in the enzyme activity by red light, suggesting that the transmitter is not inactive NR itself, which becomes activated by nitrate. Analysis of steady-state levels of the NR transcript revealed that NR mRNA is not induced in response to Pfr formation in the absence of nitrate treatment, suggesting that it could not be the transmitter mediating the phytochrome effect on NR activity. Red light was found to increase the uptake of 45Ca2+ by isolated maize protoplasts. When supplied exogen-ously calcium increased NR activity by 48% of that obtained by red light irradiation. However, exogenous addition of phorbol myristate acetate (PMA), an activator of protein kinase C, was found to increase the NR transcript level to the same extent obtained after 5 min of red light irradiation. The results suggest that phytochrome may be acting through calcium along with messengers like diacylglycerol, generated through the phosphoinositide (PI) cycle to stimulate the induction of NR.

Transcriptional Regulation of Nitrate Reductase mRNA Levels by Cytokinin-Abscisic Acid Interactions in Etiolated Barley Leaves

To investigate the mechanisms by which the expression of a specific gene may be modulated by multiple hormones, the regulation of nitrate reductase (NR) mRNA levels by cytokinin and abscisic acid (ABA) was studied in etiolated barley (Hordeum vulgare L. cv Robust) leaves using a barley NR cDNA as a probe. Northern blot analyses of the levels of NR mRNA indicate that accumulation of the mRNA was enhanced by the cytokinin, benzyladenine (BA), and suppressed by ABA and that the hormonal effects on NR gene expression were responsive to dose concentrations (10(-7) to 10(-4) molar). The NR mRNA levels were influenced by the BA/ABA concentration ratios, and the inhibition of the NR transcript levels by ABA was not totally reversed by addition of equal concentrations (10(-7) to 10(-4) molar) of BA. Nuclear runoff transcription studies showed that the level of transcription was suppressed by ABA, and addition of an equal concentration of BA partially reversed the ABA action. Thus, the interaction of cytokinin and ABA on NR gene expression is, at least in part, at the level of transcription.

Cytokinin enhancement of the light induction of nitrate reductase transcript levels in etiolated barley leaves

To investigate the molecular mechanism of cytokinin regulation of nitrate reductase (NR) activity, the influence of benzyladenine (BA) on the level of NR transcript was studied in etiolated barley leaves using a barley NR cDNA as a probe. Northern blot analyses of the levels of NR poly (A)+ RNA indicate that the amount present is proportional to the concentration of BA (2 x 10(-8) to 2 x 10(-4) M) applied to the leaves. Enhancement of NR mRNA by 2 x 10(-5) M BA was clearly detected after 15 minutes of exposure of the leaves to light. The enhancement is cytokinin-specific and adenine is ineffective. Brief treatment with the protein synthesis inhibitor, cycloheximide, inhibited BA-enhanced NR activity but did not inhibit BA-enhanced NR transcript level, thus the enhancement was independent of concurrent protein synthesis. Nuclear runoff transcription studies showed that the enhancement of NR mRNA was at least partially due to increased transcription rates.