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     

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.     

Arsenic-induced oxidative stress in the common bean legume,Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

The adverse effects of arsenic (As) toxicity on seedling growth, root and shoot anatomy, chlorophyll and carotenoid contents, root oxidizability (RO), antioxidant enzyme activities, H₂O₂ content, lipid peroxidation and electrolyte leakage (EL%) in common bean (Phaseolus vulgaris L.) were investigated. The role of exogenous nitric oxide (NO) in amelioration of As-induced inhibitory effect was also evaluated using sodium nitroprusside (100 μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200 μM PTIO) as NO scavenger in different combinations with 50 μM As. As-induced growth inhibition was associated with marked anomalies in anatomical features, reduction in pigment composition, increased RO and severe perturbations in antioxidant enzyme activities. While activity of superoxide dismutase and catalase increased, levels of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased significantly and guaiacol peroxidase remained normal. The over-accumulation of H₂O₂ content along with high level of lipid peroxidation and electrolyte leakage indicates As-induced oxidative damage in P. vulgaris seedlings with more pronounced effect on the roots than the shoots. Exogenous addition of NO significantly reversed the As-induced oxidative stress, maintaining H₂O₂ in a certain level through balanced alterations of antioxidant enzyme activities. The role of NO in the process of amelioration has ultimately been manifested by significant reduction of membrane damage and improvement of growth performance in plants grown on As + SNP media. Onset of oxidative stress was more severe after addition of PTIO, which confirms the protective role of NO against As-induced oxidative damage in P. vulgaris seedlings.     

Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.)

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.     

Accumulation and reduction of nitrate in cereal plants dependent on N supply

Summary In pot experiments the NO ₃ ^– accumulation and the occurrence of nitrate reductase (NR) capacity of wheat plants were investigated depending on late N applications at tillering, shooting and heading. NO ₃ ^– is preferentially accumulated in the stems, while NR dominates in the leaves. NO ₃ ^– accumulation is enhanced by late N treatments especially if N supply at seeding is sufficient. NR capacity of the plants is stimulated by late nitrogen supply, but its increment rates decrease with increasing NO ₃ ^– accumulation.     

Control of NO level in rhizobium-legume root nodules: Not only a plant globin story

Nitric oxide (NO) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1 h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H₂O₂), superoxide (O₂^•−) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.     

Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn.Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO₄ (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO⁻).Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species.Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Involvement of nitrate reductase in auxin-induced NO synthesis

It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.¹ Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N^G-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.^2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.^4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation.Key words: atnoa1, indole-3-butyric acid, nia1, nia2 double mutant, nitric oxideLateral roots are formed from root pericycle cells postembryonically which process is promoted by indole-acetic acid (IAA). It was recognized that IAA share common steps with NO in the signal transduction cascade towards the auxin induced adventitious and lateral root formation.^6–8 Previously it was suggested that besides IAA, indol-3-butyric (IBA) is a true endogenous auxin in Arabidopsis, which acts in adventious and lateral root development.^9,10 Our results showed that IBA induced LR initials emitted intensive NO fluorescence in Arabidopsis. This increased level of NO was present only in the LR initials in contrast to primary root (PR) sections where it remained at the control level.In plants NO can be produced by a number of enzyme systems and non-enzymatic ways. In roots, the most likely candidates of NO synthesis are NR enzymes (cytoplasmic and plasma membrane-bounded isoenzymes, cNR and PM-NR). Recently a new type of enzyme, the PM-bounded nitrite:NO reductase (Ni:NOR) was identified as a possible source of NO in roots.¹¹ Because of the several formation potentials of NO, the identification of its source in plant tissues under different conditions is complicated. Using diverse mutants proved to be a good opportunity to investigate the possible sources of NO. In our experiments wild-type (Col-1), Atnoa1 (nitric oxide synthase associated 1 deficient) and nia1, nia2 (NR deficient) seedlings were applied in order to determine the enzymatic source of NO induced by auxin. In roots of these plants, different NO levels were measured in their control state (i.e., without IBA treatment). The NO content in Atnoa1 roots was similar to that of wild-type, while nia1, nia2 showed lower NO fluorescence than the other groups of plants. This result suggests that NR activity is needed to NO synthesis in roots. Further on, it was demonstrated that IBA induced NO generation in both the wild type and Atnoa1 root primordia, but this induction failed in the NR-deficient mutant. This reveals that the NO accumulation in root primordia induced by auxin requires NR activity. These observations were evidenced also by biochemical manner. On the one part, we applied L-NMMA, which is a specific inhibitor of mammalian NOS, on the other part, the inhibitor of NR enzyme tungstate was used and we monitored NO fluorescence in wild-type roots. The NOS inhibitor displayed no effect on NO levels neither at control state nor during auxin treatment, while tungstate inhibited NO synthesis in lateral roots and primary roots of control plants. The effect of tungstate was similar in auxin-treated roots, since application of this NR enzyme inhibitor decreased NO levels in PRs and LRs (Fig. 1).Open in a separate windowFigure 1NO fluorescence in lateral roots (white columns) and primary roots (grey columns) of control, control + 1 mM tungstate, IBA and IBA + 1 mM tungstate-treated wild-type Arabidopsis thaliana. Vertical bars are standard errors.Some speculations can be made on these results. Although more efforts are needed to make the scene clear, now we can predict that auxin somehow may induce NR isoenzymes, which produce nitrite in root cells. From this point, two further scenarios are possible: as the result of accumulated nitrite, either the NO-producing activity of NR or Ni:NOR activity are promoted, hereby NO is generated from nitrite reduction. NO formed in these two possible ways modulates the expression of certain cell cycle regulatory genes contributing to division of pericycle cells in LR primordia, as was published in tomato.¹²Nowadays research in the “NO-world” of plants is running very actively. Nevertheless, lot of more work is needed to reveal all the unknown faces of this novel multipurpose signaling molecule.     

Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.

     

Physiological and biochemical roles of nitric oxide against toxicity produced by glyphosate herbicide in <Emphasis Type="Italic">Pisum sativum</Emphasis>

The present study assessed the response of pea plants exposed to herbicide induced oxidative stress in the plants present in agriculture field. We analysed the effect of exogenous nitric oxide (NO) regulated chlorophyll and protein content, nitrate reductase enzyme activity and antioxidant enzyme activity in herbicidetreated green pea (Pisum sativum L.). Glyphosate (0.25 mM) treatment alone or in combination with 250 μM sodium nitroprusside (SNP, 250 μM with glyphosate) was given to pea and we observed the changes in biophysical and biochemical parameters. During oxidative stress ion leakage is the first step of cellular damage. Supplementation of SNP with glyphosate significantly reduced ion leakage and moderately reduced H2O2 and malondialdehyde (MDA) content. SNP also increased chlorophyll content and antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (POD) activity as compared to herbicide treatment alone. The present result suggests that NO protects pea plants from damage caused by glyphosate.