Persulfidation of Nitrate Reductase 2 Is Involved in l-Cysteine Desulfhydrase-Regulated Rice Drought Tolerance.

Hydrogen sulfide (H2S) is an important signaling molecule that regulates diverse cellular signaling pathways through persulfidation. Our previous study revealed that H2S is involved in the improvement of rice drought tolerance. However, the corresponding enzymatic sources of H2S and its regulatory mechanism in response to drought stress are not clear. Here, we cloned and characterized a putative l-cysteine desulfhydrase (LCD) gene in rice, which encodes a protein possessing H2S-producing activity and was named OsLCD1. Overexpression of OsLCD1 results in enhanced H2S production, persulfidation of total soluble protein, and confers rice drought tolerance. Further, we found that nitrate reductase (NR) activity was decreased under drought stress, and the inhibition of NR activity was controlled by endogenous H2S production. Persulfidation of NIA2, an NR isoform responsible for the main NR activity, led to a decrease in total NR activity in rice. Furthermore, drought stress-triggered inhibition of NR activity and persulfidation of NIA2 was intensified in the OsLCD1 overexpression line. Phenotypical and molecular analysis revealed that mutation of NIA2 enhanced rice drought tolerance by activating the expression of genes encoding antioxidant enzymes and ABA-responsive genes. Taken together, our results showed the role of OsLCD1 in modulating H2S production and provided insight into H2S-regulated persulfidation of NIA2 in the control of rice drought stress.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

[The changes of metabolism in myocardium at ischemia-reperfusion and activating of the ATP-sensitive potassium channels].

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) it was investigated the changes of biochemical processes in the different areas of heart (intact, risk and necrotic zone) during intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible cardioprotective mechanisms of flocalin action at ischemia-reperfusion conditions: the preservation of sufficient levels of de novo (by cNOS) NO synthesis, an inhibition of de novo (by iNOS) and salvage (by NADH-dependent nitratreductase) NO synthesis, an inhibition of L-arginine degradation by arginase, an inhibition of oxidizing metabolism due to limitation of ROS and RNS generation, inhibition of free arachidonic acid and eicosanoids synthesis, inhibition of ATP and GTP degradations and, possibly, stimulation of protective haem degradation. These changes may prevent formation of toxic peroxynitrite and suggest the possibility of participating in flocalin-mediated cardioprotective effects of warning a mitochondrial permeability transition pore (MPTP) opening and inhibition of apoptosis and/or necrosis of cardiomyocytes induced by it.

[Arginase-nitric oxide synthase system changes due to adaptation to swimming in adult and aged rat hearts].

We tested the physiological indices of adult rat heart beat for the adaptation to prolonged physical exercise (swimming). It was shown that the stimulation of NO-production in the heart mitochondria of trained adult rats improves both systolic and diastolic heart function. In adult rats trained by swimming the activity of both de novo and salvage enzymes of nitric oxide synthesis studied (iNOS, cNOS, nitratreductase) were increased in heart mitochondria, whereas in the old rats only the activity of oxidative de novo enzymes. Training reduced the nitrate pools only in the mitochondria from adult rats and the urea pools in mitochondria from old rats. Intramitochondrial nitrite pools were unchanged. In adult rats, mitochondrial H2O2 pools increased after training, whereas in the old rats they were reduced, the level of uric acid (a marker ofxanthinoxydase activity) in ageing rats after training period was declined. Swimming training resulted in a significant increase in the value of "oxygenation index" in mitochondria of adult rats and decreased the activity of mitochondrial arginase II. The results suggest that swimming is one of the methods of physical load stimulates NO production in the mitochondria of adult and old rats and therefore could be considered as an effective non-pharmacological tool for correction of mitochondrial dysfunction in adults and aging heart.

UV-B induced differential effect on growth and nitrogen metabolism in two cyanobacteria under copper toxicity.

In the present study, impact of low (UV-B(L): 0.1 µmol m(-2) s(-1)) and high (UV-BH: 1.0 µmol m(-2) s(-1)) fluence rates of ultraviolet-B on growth and nitrogen metabolism in two cyanobacteria: Phormidium foveolarum and Nostoc muscorum under copper toxicity (2 and 5 µM) was investigated after 24 and 72 h of experiments. Copper and UV-BH treatment suppressed growth but more in N. muscorum which was accompanied by significant accumulation of Cu. Nitrate and nitrite uptake rates and activities of nitrogen assimilating enzymes i.e. nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS) and glutamate synthase (GOGAT) except glutamate dehydrogenase activity (GDH; aminating) were decreased following treatments of Cu and UV-BH, and under combined treatments the effect was greater. On contrary, UV-BL declined Cu toxicity significantly. The study concludes that Cu and UV-BH suppressed the activity of NR, NiR, GS and GOGAT (except GDH) hence decreased growth. However, UV-BL showed cross tolerance in test organisms against Cu toxicity up to certain extent. Phormidium foveolarum is comparatively less sensitive against UV-BH and excess Cu, a situation likely exists in nature, hence it may be used as a biofertilizer for sustainable agriculture.

Hydrogen peroxide-mediated activation of MAP kinase 6 modulates nitric oxide biosynthesis and signal transduction in Arabidopsis.

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological and developmental processes in plants, including lateral root development. In this study, we used biochemical and genetic approaches to analyze the function of Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) in the regulation of NO synthesis in response to hydrogen peroxide (H2O2) during lateral root development. In both mpk6 mutants studied, H2O2-induced NO synthesis and nitrate reductase (NR) activity were decreased dramatically. Furthermore, one NR isoform, NIA2, was required for the MPK6-mediated production of NO induced by H2O2. Notably, NIA2 interacted physically with MPK6 in vitro and in vivo and also served as a substrate of MPK6. Phosphorylation of NIA2 by MPK6 led to an increase in NR activity, and Ser-627 was identified as the putative phosphorylation site on NIA2. Phenotypical analysis revealed that mpk6-2 and mpk6-3 seedlings produce more and longer lateral roots than wild-type plants did after application of the NO donor sodium nitroprusside or H2O2. These data support strongly a function of MPK6 in modulating NO production and signal transduction in response to H2O2 during Arabidopsis root development.

Kinetic analysis of 14-3-3-inhibited Arabidopsis thaliana nitrate reductase.

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in k(cat) and a decrease in the apparent K(M)(nitrate) (from 280 to 141 µM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1.

Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in a barley mutant lacking NADH-dependent nitrate reductase activity.

Plant growth, photosynthesis and leaf constituents were examined in the wild-type (WT) and mutant nar1 of barley (Hordeum vulgare L. cv. Steptoe) that contains a defective structural gene encoding NADH-dependent nitrate reductase (NADH-NAR). In controlled environment experiments, total biomass, rates of photosynthesis, stomatal conductance, intercellular CO(2) concentrations and foliar non-structural carbohydrate levels were unchanged or differed slightly in the mutant compared with the WT. Both genotypes displayed accelerated plant growth rates when the CO(2) partial pressure was increased from 36 to 98 Pa. Total NADH-NAR activity was 90% lower in the mutant than in the WT, and this was further decreased by CO(2) enrichment in both genotypes. Inorganic nitrate was greater in the mutant than in the WT, whereas in situ nitrate assimilation by excised leaves was two-fold greater for the WT than for the mutant. Foliar ammonia was 50% lower in the mutant than in the WT under ambient CO(2). Ammonia levels in the WT were decreased by about one-half by CO(2) enrichment, whereas ammonia was unaffected by elevated CO(2) in mutant leaves. Total soluble amino acid concentrations in WT and mutant plants grown in the ambient CO(2) treatment were 30.1 and 28.4 micromol g(-1) FW, respectively, when measured at the onset of the light period. Seven of the twelve individual amino acids reported here increased during the first 12 h of light in the ambient CO(2) treatment, leading to a doubling of total soluble amino acids in the WT. The most striking effect of the mutation was to eliminate increases of glutamine, aspartate and alanine during the latter half of the photoperiod in the ambient CO(2) treatment. Growth in elevated CO(2) decreased levels of total soluble amino acids on a diurnal basis in the WT but not in mutant barley leaves. The above results indicated that a defect in NADH-NAR primarily affected nitrogenous leaf constituents in barley. Also, we did not observe synergistic effects of CO(2) enrichment and decreased foliar NADH-NAR activity on most N-containing compounds.

Denitrification by the fungus Fusarium oxysporum involves NADH-nitrate reductase.

Fusarium oxysporum JCM11502 expresses a denitrifying (nitrate (NO(3)(-))-respiring) mechanism and can thrive under oxygen (O(2)) limitation. The fungus reduces NO(3)(-) to nitrite at the initial step of denitrification. In this study, we cloned the gene coding NADH-NO(3)(-) reductase (NADH-Nar) (niaD) from F. oxysporum JCM11502. The niaD gene complemented the defective NO(3)(-) assimilation by mutant strain M10, indicating that the fungus reduced NO(3)(-) through NADH-Nar activity and assimilated it like other fungi. We found that the transcription of niaD and the production of NADH-Nar activity were enhanced under O(2)-limited denitrifying conditions relative to aerobic conditions. Strain M10 produced less NADH-Nar activity and less denitrified product than the wild-type strain. Introducing niaD into the mutant also restored these defects, indicating that niaD is involved in denitrification. These results indicate that the fungus denitrified NO(3)(-) through NADH-Nar activity in addition to the ubiquinol-Nar mechanism.

Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana.

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 degrees C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 degrees C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 degrees C, respectively, with only 33 and 23% of maximal activities being retained at 10 degrees C. In presence of 2 microM flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 degrees C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 degrees C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 degrees C (Q10) and apparent activation energy (E(a)) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 degrees C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.

Salinity-induced tissue-specific diurnal changes in nitrogen assimilatory enzymes in tomato seedlings grown under high or low nitrate medium.

We studied the salt stress (100 mM NaCl) effects on the diurnal changes in N metabolism enzymes in tomato seedlings (Lycopersicon esculentum Mill. cv. Chibli F1) that were grown under high nitrogen (HN, 5 mM NO(3)(-)) or low nitrogen (LN, 0.1 mM NO(3)(-)). NaCl stress led to a decrease in plant DW production and leaf surface to higher extent in HN than in LN plants. Total leaf chlorophyll (Chl) content was decreased by salinity in HN plants, but unchanged in LN plants. Soluble protein content was decreased by salt in the leaves from HN and LN plants, but increased in the stems-petioles from LN plants. Nitrate reductase (NR, EC 1.6.1.6) showed an activity peak during first part of the light period, but no diurnal changes were observed for the nitrite reductase (NiR, EC 1.7.7.1) activity. Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activities increased in HN plant leaves during the second part of the light period, probably when enough ammonium is produced by nitrate reduction. NR and NiR activities in the leaves were more decreased by NaCl in LN than in HN plants, whereas the opposite response was obtained for the GS activity. Fd-GOGAT activity was inhibited by NaCl in HN plant leaves, while salinity did not shift the peak of the NR and Fd-GOGAT activities during a diurnal cycle. The induction by NaCl stress occurred for the NR and GS activities in the roots of both HN and LN plants. Glutamate dehydrogenase (GDH, EC 1.4.1.2) activity shifted from the deaminating activity to the aminating activity in all tissues of HN plants. In LN plants, both aminating and deaminating activities were increased by salinity in the leaves and roots. The differences in the sensitivity to NaCl between HN and LN plants are discussed in relation to the N metabolism status brought on by salt stress.

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.