Responses of sweet potato peroxidases to sodium nitroprusside-mediated nitric oxide

To ascertain the response of sweetpotato peroxidases (PODs) to nitric oxide (NO), we treated the leaves of sweet potato with the NO generator sodium nitroprusside (SNP) and the NO scavenger carboxyl-PTIO (cPTIO). Exogenous application of more than 5 mM SNP caused damage to sweetpotato leaves at 24 h after treatment. The accumulation of NO in leaves was positively correlated with the SNP dose. The specific activity of PODs in sweet potato leaves was markedly increased by treatment with greater than 1 mM SNP for 24 h, whereas POD activity and accumulated NO content decreased to low levels by treatment with cPTIO. Expression analysis of POD genes in response to treatment with SNP and cPTIO revealed that major stress-inducible acidic genes, such as swpa1, swpa2, swpa3, and swpa4, were specifically regulated. These results indicate that increased NO levels in sweet potato leaves are closely linked to an improved defense capability mediated by stress-inducible PODs.     

Characterization of melanin isolated from a dark septate endophyte (DSE), Exophiala pisciphila

Melanin produced by a dark septate endophyte, Exophiala pisciphila, was isolated and purified. The solubility properties, Ultraviolet–visible and Fourier transform infrared spectra of the purified E. pisciphila melanin were similar to those of typical melanin. Inhibition of melanin production was observed when colonies exposed to tricyclazole (1,8-dihydroxynaphthalene (DHN) melanin inhibitor), but not to kojic acid (3,4-dihydroxyphenylalanine melanin inhibitor). Thus the E. pisciphila melanin was a member of DHN melanin family. In addition, the antioxidant activities of E. pisciphila melanin were evaluated in vitro by 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay. E. pisciphila melanin exhibited a strong antioxidant activity. Addition of 50–350 mg L⁻¹ Cd(II) to the medium increased the melanin production in E. pisciphila.     

Mechanism underlying the response of fungi and their Fusarium symbiotic networks to the rotations of soybean and corn

Fusarium oxysporum and Fusarium solani are the main soybean root rot pathogens in northern China. We investigated the distribution and driving factors of Fusarium under different cropping systems to evaluate and regulate soil health. The factors affecting Fusarium in soybean cropping systems were assessed using high-throughput sequencing of ITS1 to identify soil microbial population diversity, and then the soil physicochemical properties were assessed to determine the levels of various elements present in the environment. According to the results, the abundance of Fusarium was obviously reduced in the corn–soybean rotation and uncultivated soil systems. The relative abundance of Fusarium in the soil and the abundance and diversity of fungal communities were significantly positively associated with the abundance of Ascomycota. Additionally, the relative abundance of Fusarium was significantly positively correlated with the zinc (Zn) content. When the Zn content was high, the abundance of Fusarium increased, and the correlations with Chaetomium, Cryptococcus, Penicillium and Trichoderma significantly decreased. Soybean yield was significantly negatively correlated with fungal community abundance and diversity. Based on our results, the uncultivated soil and corn–soybean rotation cropping systems improved the organizational structure of the soil fungal community and were conducive to the health and production of soybean.     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity

Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.     

Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells

Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd²⁺). Cd²⁺ is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd²⁺-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 μM CdCl₂ underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd²⁺ concentration was measured subsequently. SNP led more Cd²⁺ content than Cd²⁺ treatment alone. By contrast, the prevention of NO by l-NAME decreased Cd²⁺ accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd²⁺ fluxes. This analysis revealed the promotion of Cd²⁺ influxes into cells by application of SNP, while l-NAME and cPTIO reduced the rate of Cd²⁺ uptake or even resulted in net Cd²⁺ efflux. Based on these founding, we concluded that NO played a positive role in CdCl₂-induced PCD by modulating Cd²⁺ uptake and thus promoting Cd²⁺ accumulation in BY-2 cells.     

Nitric oxide (NO) counteracts cadmium induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H₂O₂ thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25 μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5 mM SNP for 6 h when exposed to 25 μM Cd for 24 h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100 μM cPTIO pretreatment to roots for 1 h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.     

Stimulatory and period-specific effect of nitric oxide on in vitro caulogenesis in Albizzia lebbeck (L.) Benth.

The paper reports stimulatory effect of nitric oxide (NO) on in vitro caulogenesis in Albizzia lebbeck, a tree legume. Exogenously supplied NO donor, sodium nitroprusside (SNP) stimulated shoot differentiation from hypocotyl explants of Albizzia lebbeck, excised from its in vitro seedlings. Potassium ferrocyanide, a structural analog of SNP incapable of releasing NO, did not promote shoot organogenesis. Likewise, metabolic products of NO, NO₂ ⁻ and NO₃ ⁻, provided as NaNO₂ and NaNO₃ did not enhance shoot differentiation. The NO scavenger, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO), supplemented along with SNP, at equimolar concentration, reversed the stimulatory effect of the latter, thus, confirming the role of NO in promotion of in vitro caulogenesis. The transfer of explants cultured on the basal medium (BM) to the same containing SNP and vice versa after different time intervals revealed that for its enhancing effect, SNP was required only during the initial phase (5 days) of culture. Its presence or administration beyond 5 days neither promoted nor inhibited the caulogenic response.     

Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency

Cymbidium shoot buds grown under Mg²⁺ deficiency without naphthalene acetic acid (NAA) showed knotted appearance. Ultrastructure of the cortical cells showed a progressive disorganization and disintegration of chloroplast membranes. The growth of shoots was resumed with the addition of 10 μM NAA. Specific NO scavenger, cPTIO induced deformation in shoot growth in 80 % of cultures. In longitudinal sections of shoots treated with cPTIO, depositions of densely stained particles in cells were observed. These inhibitory responses of cPTIO were ameliorated by 10 μM NAA. The NO donor, sodium nitroprusside (SNP), treated shoot buds displayed rapid senescence followed by necrosis of leaves. Ultrastructure of cortical cells at this stage revealed the endocytosis of mitochondria along with membrane bound cytoplasmic inclusions in the vacuole. A sharp increase in H₂O₂ generation was observed with a little change in the activity of antioxidant glutathione disulfide (GSSG), suggesting NO mediated oxidative stress. Surprisingly, after 4 weeks these necrotic shoots were converted into a globular, embryo like shoot tip with numerous structures termed here as ‘neomorph’ in its base. Neomorphs were different from protocorm like bodies both anatomically and morphologically. Ultrastructure of the rhizome tip exhibited numerous amyloplast and round mitochondria. At this stage, the generation of high rate of H₂O₂ was masked by GSSG, and the generation of GSSG was proportional with the concentrations of SNP, and not observed in the control (without SNP). The neomorphs were further sub-cultured to medium with different concentrations of SNP or cPTIO. After 4 weeks of culture, only the neomorphs sub-cultured on medium with SNP developed into shoots and approximately ten shoots were observed to emerge from the axils of each rhizome. Ultrastructure of cells of regenerating green neomorphs showed different shapes of mitochondria and chloroplasts and presence of active dictyosomes. The obtained shoots subjected to the acclimatization in polyhouse, expressed good growth with 85 % survival. Therefore it is reasonable to suggest that the process of de-differentiation and re-differentiation leading to rhizome formation under the condition of Mg²⁺ deficiency is NO mediated.     

Nitric oxide promotes in vitro organogenesis in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

The effects of nitric oxide (NO) on caulogenesis, shoot organogenesis and rhizogenesis from hypocotyl explants of Linum usitatissimum were investigated. Exogenously supplied NO donors, 5 μM sodium nitroprusside (SNP), 2 μM S-nitroso-N-acetylpenicillamine (SNAP) and 2 μM 3-morpholinosydnonimine (SIN-1), significantly promoted shoot differentiation from the hypocotyl explants of L. usitatissimum excised from its in vitro raised seedlings. Potassium ferrocyanide, a structural analogue of SNP, lacking NO group, did not promote shoot organogenesis. Likewise, products of NO, \textNO₂ ^- {\text{NO}}_{2}^{ - } and \textNO₃ ^- {\text{NO}}_{3}^{ - } supplied as 5 μM NaNO₂ and 5 μM NaNO₃ did not enhance shoot differentiation. Another source of NO, a mixture of sodium nitrite (SN) provided along with ascorbic acid (AsA), also caused significant promotion in the average number of shoots per responding explant. SNP also augmented the rhizogenic response of the microshoots in terms of percentage of responding explants, number of roots per responding explant and average root length. The NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB), provided along with SNP, SNAP, SIN-1 or SN + AsA, at concentrations equimolar to the optimum concentration of the donors, reversed the promotory influence, thereby, confirming the role of NO in promotion of in vitro morphogenesis. However, NO scavengers individually did not affect the observed morphogenic processes. Morphological and histological studies of hypocotyl segments cultured on BM or BM + SNP for 4, 8 and 12 days demonstrated that SNP enhanced shoot differentiation by inducing a higher number of shoot primordia, each of which develops into a single shoot.     

植物病原真菌黑色素与致病性关系的研究进展

黑色素是一类生物聚合分子的总称,不同来源的黑色素种类不同,其中报道较多的是DOPA黑色素和DHN黑色素。DOPA黑色素和DHN黑色素具有相似的理化性质但其合成底物和途径不同。DHN黑色素在植物病原直菌中广泛存在,与病原菌致病能力密切相关。病原菌侵染寄主时黑色素沉积存附着胞细胞壁的内层,防止了形成膨压的溶质渗透到细胞外,产生很大的机械压力,保证病原菌侵入寄主。结合作者的研究结果综述了黑色素的种类、性质及黑色素与病原菌致病性关系等方面的研究进展。     

Cytosolic acidification precedes nitric oxide removal during inhibition of ABA-induced stomatal closure by fusicoccin

The role of nitric oxide (NO) and the relationship between NO and cytosolic pH during inhibition of ABA effect by fusicoccin (FC) in guard cells of Vicia faba were analyzed. ABA induced NO generation and stomatal closure, but FC inhibited the effects of ABA. Treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide (cPTIO) and N^G-nitro-L-Arg-methyl ester (L-NAME) mimicked the effects of FC. These data suggest that inhibition of ABA effect by FC is possibly related to the decreasing in the NO level. Furthermore, like cPTIO, FC not only suppressed stomatal closure and NO level in guard cells treated with NO donor sodium nitroprusside (SNP), but also reopened stomata, which had been closed by ABA, and reduced the level of NO in guard cells that had been produced by ABA, indicating that FC caused NO removal. Butyric acid simulated the effects of FC on the stomatal aperture and increased NO levels in guard cells treated with SNP and had been closed by ABA, and both FC and butyric acid surely reduced cytosolic pH, which demonstrates that cytosolic acidification mediates FC-induced NO removal. Taken together, our results show that FC induces NO removal and reduces NO level via cytosolic acidification in guard cells, thus inhibiting ABA effect.     

Nitric Oxide Functions as a Positive Regulator of Root Hair Development

The root epidermis is composed of two cell types: trichoblasts (or hair cells) and atrichoblasts (or non-hair cells). In lettuce (Lactuca sativa cv. Grand Rapids var. Rapidmor oscura) plants grown hydroponically in water, the root epidermis did not form root hairs. The addition of 10 µM sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in almost all rhizodermal cells differentiated into root hairs. Treatment with the synthetic auxin 1-naphthyl acetic acid (NAA) displayed a significant increase of root hair formation (RHF) that was prevented by the specific NO scavenger carboxy-PTIO (cPTIO). In Arabidopsis, two mutants have been shown to be defective in NO production and to display altered phenotypes in which NO is implicated. Arabidopsis nos1 has a mutation in an NO synthase structural gene (NOS1), and the nia1 nia2 double mutant is null for nitrate reductase (NR) activity. We observed that both mutants were affected in their capacity of developing root hairs. Root hair elongation was significantly reduced in nos1 and nia1 nia2 mutants as well as in cPTIO-treated wild type plants. A correlation was found between endogenous NO level in roots detected by the fluorescent probe DAF-FM DA and RHF. In Arabidopsis, as well as in lettuce, cPTIO blocked the NAA-induced root hair elongation. Taken together, these results indicate that: (1) NO is a critical molecule in the process leading to RHF and (2) NO is involved in the auxin-signaling cascade leading to RHF.Key Words: auxin, nitric oxide, root hair, lettuce, arabidopsis, nos1 mutant, nia1, nia2 mutant     

Signaling Effects of Nitric Oxide, Salicylic Acid, and Reactive Oxygen Species on Isoeuphpekinensin Accumulation in Euphorbia pekinensis Suspension Cells Induced by an Endophytic Fungal Elicitor

Nitric oxide (NO), salicylic acid (SA), and reactive oxygen species (ROS) are important signal molecules that mediate plant resistance reactions and play important roles in secondary metabolism. To research the signal transduction pathway of the endophytic fungal elicitor from Fusarium sp. E5 promoting secondary metabolism in Euphorbia pekinensis suspension cells, the changes in NO, SA, ROS, and isoeuphpekinensin contents in the cells were investigated after elicitor addition to the cell suspension culture. The elicitor did not change H₂O₂ or O₂ ^? contents notably, whereas NO and SA contents were enhanced. Both the NO donator sodium nitroprusside (SNP) and SA enhanced isoeuphpekinensin content in the absence of the fungal elicitor, whereas the NO scavenger cPTIO and SA biosynthesis inhibitor cinnamic acid (CA) inhibited isoeuphpekinensin accumulation in the presence of the elicitor. In addition, cPTIO inhibited SA production induced by the fungal elicitor. CA did not inhibit NO production, but it significantly inhibited isoeuphpekinensin accumulation. The results demonstrated that in Euphorbia pekinensis suspension cells the endophytic fungal elicitor induced increased NO content and SA production, which promoted isoeuphpekinensin accumulation. ROS are clearly not involved in the endophytic fungus–host interaction signaling pathway.     

The role of melanin in the antagonistic interaction between the apple scab pathogen Venturia inaequalis and Microsphaeropsis ochracea

The objective of this study was to examine the role of melanin in the interaction between the mycoparasite Microsphaeropsis ochracea and the apple scab pathogen Venturia inaequalis. Melanin was extracted from the cell wall of the pathogen and its chemical and physical properties determined on the basis of biochemical tests and visible and infrared spectra. The physical and chemical characteristics of V inaequalis melanin were similar to the those of synthetic dihydroxyphenylalanine (DOPA) melanin. Precursors of the four known melanin biosynthetic pathways were tested for their ability to restore the pigmentation of an albino strain of V inaequalis. Scytalone, an intermediate of the 1,8-dihydroxynaphthalene (DHN) pathway, was the only precursor to restore the dark-brown pigmentation. Tricyclazole and pyroquilon, two antipenetrant fungicides, specific inhibitors of DHN melanin synthesis in Pyricularia oryzae, were used to confirm the melanin pathway in V. inaequalis wild type. A reddish-brown pigment was obtained due to the accumulation of shunt products of the DHN melanin pathway instead of a dark-brown pigment, suggesting that the melanin extracted from V inaequalis was a DHN melanin. Furthermore, growth of an albino mutant of V. inaequalis on scytalone-amended medium resulted in the formation of dark granules similar to those seen in wild-type isolates. Transmission electron microscopic observations of M. ochracea grown in the presence of melanin showed that the granules accumulated gradually along fungal cell walls to form a uniform dark coating.     

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

Background and Aims

Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum).

Methods

NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings.

Key Results

Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N₂ flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate.

Conclusions

The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein.Key words: Nitric oxide, nitrate, nitrate reductase, post-translational regulation, tomato, Solanum lycocarpum