高等植物叶片中的硝酸还原酶活性稳定因子

从小麦、油菜、浮萍、番茄、烟草的叶片中分离得到NR-SF。不同植物材料中NR及NR-SF能起交叉反应;不同NR-SF影响NR酶动力学性质相同;不同NR-SF的凝胶电泳谱带显示蛋白和糖蛋白性质。NR-SF广泛存在于植物细胞中。     

Nitrate reductase from <Emphasis Type="Italic">Triticum aestivum</Emphasis> leaves: Regulation of activity and possible role in production of nitric oxide

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation. In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR. The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule. Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue.     

Preliminary Studies on the Acting Mechanism of Stabilizing Factor of Nitrare Reductase Activity (NR-SF)

Nitrate reductase (NR, E. C. 1.6.6.1), which was purified for 500-fold from crude extract of wheat leaves through the Blue-Dexdran Sepharose 4B affinity colume, could be still increased obviously by NR-SF in vitro. The results demonstrated that NR-SF increasedmainly activity of Cytochrome-C reductase (CytcR) in NR complex, but not affected activity of reduced methylviologen-nitrate reductase (MVH-NR). NR-SF was not similar to glutathione (GSH) which ieversed the inhibition of P-chloromercuribenzoate (PCMB) on activity of NR. So that the action of NR-SF was not in protecting sulfhydryl group of NR protein from oxidation alone.     

曼陀罗细胞中6-BA结合蛋白与硝酸还原酶活力的关系

曼陀罗单倍体细胞,在无水层情况下,经紫外线诱变,在氯酸盐培养基上筛选得到一株缺少硝酸还原酶活力的突变细胞株。经3年培养证明突变性状稳定。对它进行生理生化分析,看到它核内细胞分裂素结合蛋白减少。结合蛋白与细胞分裂素结合后能够促进核内 RNA 聚合酶活力,加速基因转绿。在正常细胞中,细胞分裂素能够促进硝酸还原酶诱导活力,而在这些缺少核内细胞分裂素结合蛋白的突变细胞中,外源细胞分裂素对酶活力的诱导没有作用。从而猜测,在突变细胞中,由于激素不能充分与结合蛋白结合,降低了细胞中转绿的总水平,可能是硝酸还原酶活力提不高的原因。本文还讨论了激素与受体结合后在调控基因表达上的可能途径。     

Stabilizing factor(s) of nitric oxide (NO) synthetase.

We found that the cytosol of rat peritoneal polymorphonuclear neutrophils contains factor(s) that can stabilize an unstable enzyme, nitric oxide synthetase, in the cytosol. This enzyme has been purified to a single protein from the cytosol. Its half-life was 3 hours at 4 degrees C and was prolonged to greater than 24 hours by the stabilizing factor in the cytosol. The molecular weight of the stabilizing factor was greater than 100,000. Its activity was lost by the treatment with heating or alkali for 1 min or with acid for 5 min. It did not adhere to the carboxymethyl or diethylaminoethyl column at neutral pH. This stabilizing factor(s) may play a role in the regulation of the nitric oxide synthetase.     

Effect of Some Environmental Conditions on the Activity of the Nitrate Reductase-Stabi-lizing Factor (NRsf.)

The purpose of this work is to examine the relation between NR and NRSF in the leaves of the wheat plants under the different environmental conditions. The leaves of wheat plants which were grown in the nutritional solution containing NO3-, present 15 times higher of NR activity than that grown in the nutritional solution containing NH4+, but NRSF, which activated NO3-NR of the former increased only 20% as compared with that of the latter, indicating that NR is independent of NRSF. In addition, in the yellowing leaves dark grown in NO3-N solution and those in the same solution containing W with no nitrogen and Mo, the NRSF is quite stable, and keep higher activity, but no NR activity can be determined. So the NRSF is not considered as a major factor to regulate activity of NR in the leaves.     

不同氮源对小麦幼苗谷氨酰胺合成酶的影响

利用ＤＥＡＥ－纤维素柱层析、酶活性测定、Ｎｏｒｔｈｅｒｎ 分子杂交等技术,研究了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）幼苗的根、叶和离体叶在不同氮源培养条件下谷氨酰胺合成酶（ＧＳ）活性和同工酶变化, 以及不同氮源对ＧＳ基因转录－ＧＳ－ｍ ＲＮＡ 的影响． 同时与硝酸还原酶（ＮＲ）活性进行比较, 结果表明∶当以ＮＨ＋４ 作唯一氮源时,小麦幼苗根谷氨酰胺合成酶（ＧＳｒ）和叶细胞质谷氨酰胺合成酶（ＧＳ１）活性要比以ＮＯ－３ 作唯一氮源的高．当以ＮＯ－３ 为唯一氮源时, ＮＯ－３ 则促进完整叶片和离体叶片叶绿体谷氨酰胺合成酶（ＧＳ２）活性． 从转录水平上看,ＮＨ＋４ 促进根ＧＳ－ｍ ＲＮＡ 的合成,而ＮＯ－３ 促进叶ＧＳ－ｍ ＲＮＡ 的合成     

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (P_i). Addition of Ca²⁺ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO₂ fixation, indicating that NR activation follows saturation kinetics with respect to CO₂ fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO₂ fixation.     

Identification of a Protein That Inhibits the Phosphorylated Form of Nitrate Reductase from Spinach (Spinacia oleracea) Leaves

The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted from its separation from an approximately 110-kd nitrate reductase inhibitor protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this hypothesis, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR.     

硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。     

Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.     

Two endogenous proteins that induce cell wall extension in plants.

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.     

外源钙离子对小麦幼苗氮素代谢的影响

以普通小麦豫麦34为材料,研究了不同浓度的外源Ca2 对小麦幼苗氮素代谢的影响.在小麦第一片叶完全展开后,开始外源Ca2 处理,设0 (对照)、2、4 mmol · L-1 和8 mmol · L-1 4个Ca2 浓度梯度.处理5d后,测定氮同化酶活性、氮同化量及其它相关代谢物含量.结果表明,小麦幼苗叶片中硝酸还原酶(NR)和谷氨酰胺合成酶(GS)在2 mmol · L-1 Ca2 处理下活性比对照有显著增加,4 mmol · L-1 Ca2 处理的NR活性增加明显,但GS活性增加不显著;8 mmol · L-1 Ca2 处理下NR和GS活性比对照均明显降低.谷氨酸脱氢酶(NADH-GDH)活性在2 mmol · L-1 Ca2 处理下活性增加不明显,而在4、8 mmol · L-1 Ca2 处理下活性显著增加.小麦幼苗氮同化量以4 mmol · L-1处理最大,2 mmol · L-1处理与4 mmol · L-1之间差异不显著;Ca2 浓度为8 mmol · L-1时,氮素同化量明显降低.结果揭示了小麦幼苗不同氮同化途径对Ca2 的响应不同,GS途径比GDH途径对小麦氮素同化量的增加作用更大;4 mmol · L-1对小麦幼苗的氮素利用可能是最有效的Ca2 浓度.     

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     

Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels.

A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase.     

某些环境条件对硝酸还原酶活性稳定因子的影响

小麦 Triticum aestivum L.苗在 NO_3~--N 完全营养液中培养比在 NH_4~ -N 完全营养液中培养,它们叶细胞内的硝酸还原酶(NR)即 NO_3-NR 比 NH_4-NR 活性增高了15倍,而它们叶片中的稳定因子(NR_(SF)),即 NO_3-NR_(SF)比 NH_4-NR_(SF)活化 NO_3-NR 的能力仅增加0.2倍,表明 NR 与 NR_(SF)不是依存关系;另外在 NO_3~--N 培养的黄化小麦叶片,及黄化缺氮、缺铝,加(?)的叶片中,所有的 NR_(SF)都十分稳定,并且保持较高活性,但这些叶片中没有测出NR 活性,因而认为,在植物叶细胞中,NR_(SF)不是调节 NR 活性的主要条件。     

高温胁迫下番茄幼苗对冷激的响应

高温逆境是影响夏秋季蔬菜设施集约化育苗质量的主要因素之一,利用温度逆境诱导植物产生交叉适应是植物获得抗逆性的一种有效手段.为探索冷激强度对番茄幼苗高温胁迫的缓解效应,试验采用人工气候箱模拟夏季设施中的高温胁迫,研究了不同冷激温度(5、10、15 ℃)和冷激持续时间(10、20、30 min)对番茄幼苗生长、生物膜保护系统的影响,并研究了单次适宜冷激处理对番茄小分子热激蛋白LeHSP23.8和CaHSP18基因表达的影响.结果表明： 在高温胁迫前对番茄幼苗进行冷激处理可以抑制其下胚轴的伸长和株高的生长.冷激缓解番茄幼苗高温胁迫的效应在不同冷激温度下表现出不同的变化趋势;5 ℃冷激处理抑制了番茄幼苗抗氧化酶活性的升高,使细胞膜透性增大,对幼苗产生伤害;10 ℃冷激处理对番茄幼苗高温胁迫的缓解效应随冷激时间的延长呈降低趋势;而15 ℃冷激处理缓解番茄幼苗高温胁迫的效应随冷激时间的延长呈增加趋势.适宜冷激温度和冷激持续时间能够诱导番茄幼苗对高温逆境的交叉适应性,在高温胁迫前将番茄幼苗进行温度为10 ℃、持续10 min的冷激处理效果最佳,与对照相比,显著提高了高温胁迫下番茄幼苗植株单株干质量和壮苗指数,降低了番茄幼苗叶片相对电导率和丙二醛含量,促进了脯氨酸和可溶性蛋白的积累,提高了番茄幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT) 3种抗氧化酶活性,并诱导了小分子热激蛋白基因LeHSP23.8和CaHSP18在常温条件下的上调表达.
     

cDNA encoding a wheat (Triticum aestivum cv. Chinese Spring) glycine-rich RNA-binding protein

A wheat cDNA encoding a glycine-rich RNA-binding protein, whGRP-1, was isolated. WhGRP-1 contains two conserved domains, the RNA-binding motif (RNP motif) combined with a series of glycine-rich imperfect repeats, characteristic of a conserved family of plant RNA-binding proteins. Northern analysis revealed that whGRP-1 mRNA accumulates to high levels in roots and to lower levels in leaves of wheat seedlings. whGRP-1 mRNA accumulation is not enhanced by exogenous abscisic acid in seedlings and accumulates to very high levels during wheat embryo development, showing a pattern different from that of the ABA-inducible wheat Em gene.