NaCl stress effects on enzymes involved in nitrogen assimilation pathway in tomato "Lycopersicon esculentum" seedlings

Tomato plants (Lycopersicon esculentum Mill, cv. Chibli F1) grown for 10 days on control medium were exposed to differing concentrations of NaCl (0, 25, 50, and 100mM). Increasing salinity led to a decrease of dry weight (DW) production and protein contents in the leaves and roots. Conversely, the root to shoot (R/S) DW ratio was increased by salinity. Na(+) and Cl(-) accumulation were correlated with a decline of K(+) and NO(3)(-) in the leaves and roots. Under salinity, the activities of nitrate reductase (NR, EC 1.6.6.1) and glutamine synthetase (GS, EC 6.3.1.2) were repressed in the leaves, while they were enhanced in the roots. Nitrite reductase (NiR, EC 1.7.7.1) activity was decreased in both the leaves and roots. Deaminating activity of glutamate dehydrogenase (GDH, EC 1.4.1.2) was inhibited, whereas the aminating function was significantly stimulated by salinity in the leaves and roots. At a high salt concentration, the nicotinamide adenine dinucleotide reduced (NADH)-GDH activity was stimulated concomitantly with the increasing NH(4)(+) contents and proteolysis activity in the leaves and roots. With respect to salt stress, the distinct sensitivity of the enzymes involved in nitrogen assimilation is discussed.     

Effects of NaCI stress on nitrogen and phosphorous metabolism in a true mangrove Bruguiera parviflora grown under hydroponic culture

The influence of varying levels of salinity (0, 100, 200 and 400 mM) on the activities of nitrate reductase (NR, E.C. 1.6.6.1), acid phosphatase (ACP, E.C. 3.1.3.2), and alkaline phosphatase (ALP, EC 3.1. 3.1) as well as on nitrate and phosphate uptake and total nitrogen levels in leaves of a true mangrove Bruguiera parviflora was investigated under hydroponic culture conditions. NR activity increased in 100 mM NaCl treated plants, whereas it decreased gradually in 200 and 400 mM treated plants, relative to the controls. Decreased activity of NR by NaCl stress was also accompanied by a decrease in total nitrogen level and nitrate uptake. Decreases in NR activity, nitrate (NO₃⁻), and total nitrogen level due to high salinity may be responsible for a decrease in growth and biomass production in this plant. However, salinity caused an increase in both ACP and ALP activity. Activity staining of ACP by native polyacrylamide gel electrophoresis revealed three isoforms: ACP-1, ACP-2, and ACP-3. We observed a preferential enhancement in the ACP-3 isoform by salinity. In order to understand whether the salinity-induced increase in phosphatase activity was due to inhibition in phosphate uptake, we monitored phosphate (Pi) levels in leaves and noted that phosphate levels decreased significantly under salinity. These results suggest that the induction of acid and ALP under salt stress may be due to a phosphorous deficiency.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Changes in growth and activity of enzymes involved in nitrate reduction and ammonium assimilation in tomato seedlings in response to NaCl stress

BACKGROUND AND AIMS: In Tunisia, salt water is largely used for tomato irrigation. In this work, a study was made of the changes in the nitrate reduction and ammonium assimilation into amino acids in tomato seedlings under salinity in order to providee further insight into the salt effects on plant growth. Methods Ten-day-old tomatoes (Solanum lycopersicum) were subjected to 100 mm NaCl stress, and nitrogen metabolism in leaves and roots was studied. KEY RESULTS: The concentrations of Na+ and Cl- rapidly increased in the leaves and in the roots following exposure of tomato seedlings to NaCl stress. In contrast, the NO3- concentrations were lowered first in the roots and later in the leaves. From 5 to 10 d of treatment, salt ions provoked a decrease in the dry weight and an increase in the NH4+ concentrations in the leaves. Inhibition was observed in the leaves for the activities of nitrate reductase (NR, EC 1.6.6.1), ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and deaminating glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2). NaCl affected these enzyme activities less in the roots than in leaves. This was in accordance with the pronounced decrease of dry weight by salt in leaves compared with that in the roots. CONCLUSIONS: NaCl stress effects on growth, metabolite concentrations and enzyme activities depended on the duration of salt treatment and the plant tissue.     

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.     

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.     

Differential expression of the two Arabidopsis nitrate reductase genes

The differential regulation of the two nitrate reductase (NR, EC 1.6.6.1) genes of Arabidopsis thaliana L. Heynh was examined. cDNAs corresponding to each of the NR genes (NR1 and NR2) were used to measure changes in the steady-state levels of NR mRNA in response to nitrate, light, circadian rhythm, and tissue specificity. Although nitrate-induction kinetics of the two genes are very similar, NR1 is expressed in the absence of nitrate at a higher basal level than NR2. Nitrate induction is transient both in the roots and leaves, however the kinetics are different: the induction and decline in the roots precede that in the leaves. Light induces the expression of each of the genes with significantly different kinetics: NR2 reached saturation more rapidly than did NR1. Both genes showed similar diurnal patterns of circadian rhythm, with NR2 mRNA accumulating earlier in the morning.     

盐胁迫下荒漠共生植物红砂与珍珠的根茎叶中离子吸收与分配特征

西北荒漠地区C3小灌木红砂(Reaumuria soongorica)和C4半灌木珍珠猪毛菜(Salsola passerina)在特定环境下混生在一起,分布面积广阔。以采自腾格里沙漠边缘荒漠地带的天然野生珍珠猪毛菜和红砂群落的幼苗为材料,经0、100、200、300、400mmol/L NaCl盐溶液共同胁迫10 d,检测它们的含水量、主要矿质离子在根茎叶的含量与分布,揭示二者耐盐的共生协同的离子平衡适应机制。试验结果发现,珍珠猪毛菜叶片具有"吸钾排钠的"的耐盐特征,红砂叶片具备"吸钠排钾"的特征,吸收利用无机矿质离子具备互补效应。二者耐盐Cl、Ca和Si离子吸收与累积能力存在很大差异:随着盐胁迫程度加剧,红砂的根茎叶中Cl离子含量持续增加,并且为珍珠猪毛菜的2—5倍;珍珠猪毛菜根中Ca离子含量为红砂的2—3倍,但含量变化不显著;红砂根中Si离子含量迅速降低后稳定,并且是珍珠猪毛菜根的3—5倍,其他器官变化差异较小。因此,红砂与珍珠猪毛菜的共培养盐胁迫下根中吸收的离子侧重不同,红砂以Na、Cl、Si为主,珍珠猪毛菜以K、Ca为主。随着盐胁迫的程度加强,离子选择吸收系数S k,Na的变化趋势降低,表明二者叶部对Na的选择性减小,K的选择性吸收积累增大,增强了它们的抗盐性,最终使叶片所受盐害减小。总之红砂与珍珠猪毛菜共生的耐盐离子稳态机制显著不同,离子吸收与分布具有互补互利的效应。     

外源脯氨酸对盐胁迫下甜瓜幼苗硝酸还原的影响

采用营养液水培方法,以＂雪美＂品种甜瓜（Cucumis melo L.）为材料,研究了外源脯氨酸（Proline）对盐胁迫下甜瓜幼苗叶片和根系硝酸还原的影响。结果表明：（1）盐胁迫提高了甜瓜幼苗叶片和根系内铵态氮（NH4＋-N）和可溶性蛋白含量;降低了硝态氮（NO-3-N）含量和硝酸还原酶（nitrate reductase,NR）活性。（2）外源施用脯氨酸明显地提高了盐胁迫下甜瓜幼苗叶片和根系内NO-3-N和可溶性蛋白含量;降低了盐胁迫下甜瓜幼苗叶片和根系内NH＋4-N含量;增强了盐胁迫下甜瓜幼苗体内NR活性。研究结果表明,外源脯氨酸可以通过调节甜瓜幼苗体内硝酸还原酶活性和氮化合物含量来缓解盐胁迫对甜瓜幼苗植株的伤害。     

Foliar and fine root nitrate reductase activity in seedlings of four forest tree species in relation to nitrogen availability

Summary Seedlings of red maple, white pine, pitch pine and red pine were fertilized with nutrient solutions containing 4 levels of nitrate or ammonium additions. These levels corresponded to approximately 0.5, 1, 2 and 4 times normal availability of nitrogen in northeastern forests. Nitrate reductase (NR) activity was assayed in roots and leaves. Red maples treated with nitrate showed much higher leaf activities and higher ratios of leaf NR activity to root NR activity than any other species. Ammonium additions to red maple and white pine appeared to inhibit NR activity in leaves. With high nitrate additions, NR activity was induced in roots and leaves of pine species, but activity in roots remained much higher than in leaves.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance

The plant growth, nitrogen absorption, and assimilation in watermelon (Citrullus lanatus [Thunb.] Mansf.) were investigated in self-grafted and grafted seedlings using the salt-tolerant bottle gourd rootstock Chaofeng Kangshengwang (Lagenaria siceraria Standl.) exposed to 100 mM NaCl for 3 d. The biomass and NO₃⁻ uptake rate were significantly increased by rootstock while these values were remarkably decreased by salt stress. However, compared with self-grafted plants, rootstock-grafted plants showed higher salt tolerance with higher biomass and NO₃⁻ uptake rate under salt stress. Salinity induced strong accumulation of nitrate, ammonium and protein contents and a significant decrease of nitrogen content and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in leaves of self-grafted seedlings. In contrast, salt stress caused a remarkable decrease in nitrate content and the activities of GS and GOGAT, and a significant increase of ammonium, protein, and nitrogen contents and NR activity, in leaves of rootstock-grafted seedlings. Compared with that of self-grafted seedlings, the ammonium content in leaves of rootstock-grafted seedlings was much lower under salt stress. Glutamate dehydrogenase (GDH) activity was notably enhanced in leaves of rootstock-grafted seedlings, whereas it was significantly inhibited in leaves of self-grafted seedlings, under salinity stress. Three GDH isozymes were isolated by native gel electrophoresis and their expressions were greatly enhanced in leaves of rootstock-grafted seedlings than those of self-grafted seedlings under both normal and salt-stress conditions. These results indicated that the salt tolerance of rootstock-grafted seedlings might (be enhanced) owing to the higher nitrogen absorption and the higher activities of enzymes for nitrogen assimilation induced by the rootstock. Furthermore, the detoxification of ammonium by GDH when the GS/GOGAT pathway was inhibited under salt stress might play an important role in the release of salt stress in rootstock-grafted seedlings.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

外源CaCl2对NaCl胁迫下酸枣幼苗氮代谢的影响

为了研究CaCl₂对NaCl胁迫下酸枣幼苗根、茎、叶的氮代谢影响,探索钙缓解幼苗NaCl胁迫的作用途径。该研究以酸枣幼苗为试验材料,检测不同浓度CaCl₂(0、5、10、20 mmol/L)对NaCl(150 mmol/L)胁迫下幼苗叶片H₂O₂、O^-·₂含量,根、茎、叶中硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)活性及游离氨基酸、可溶性蛋白、硝态氮含量的影响,并采用主成分分析法筛选出评价CaCl₂缓解NaCl胁迫效应的生理指标。结果表明：与NaCl胁迫相比,盐胁迫幼苗叶片的H2O₂、O^-·₂积累量在5、10 mmol/L CaCl₂处理下显著减少;GOGAT活性在5、10 mmol/L CaCl₂处理下的植株根和茎内以及各浓度 CaCl₂处理的叶内均显著升高, GS、NR活性在10、20 mmol/L CaCl₂处理的根内和10 mmol/L CaCl₂处理的茎内以及5、10、20 mmol/L CaCl₂处理的叶内均显著升高;可溶性蛋白含量在5、10、20 mmol/L CaCl₂处理的根、茎、叶内均显著升高,游离氨基酸含量在10、20 mmol/L CaCl₂处理的根和茎内以及10 mmol/L CaCl₂处理的叶内均显著升高,硝态氮含量在10 mmol/L CaCl₂处理的根和茎内以及5、10、20 mmol/L CaCl₂处理的叶内均显著升高。研究发现,150 mmol/L NaCl胁迫对酸枣幼苗造成明显过氧化伤害,抑制了体内氮代谢;外源CaCl₂可通过促进幼苗根和茎内GS/GOGAT循环对NH₄⁺的同化作用,提高叶片NR活性,加快硝态氮的转化速率,从而增强幼苗对NaCl胁迫的适应性,并以10 mmol/L CaCl₂处理缓解效果最佳;游离氨基酸、GOGAT、NR可以作为CaCl₂缓解幼苗NaCl胁迫伤害的评价指标。     

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light-dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation.     

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.