The removal of nitrate reductase phosphorylation enhances tolerance to ammonium nitrogen deficiency in rice

Nitrate reductase (NR) is a key enzyme for nitrogen assimilation in plants, and its activity is regulated by posttranslational phosphorylation. To investigate the effects of dephosphorylation of the NIA1 protein on the growth and the physiological and biochemical characteristics of rice under different forms of nitrogen supplies, the phenotypes, nitrogen metabolism and reactive oxygen metabolism were measured in NIA1 phosphorylation site-directed mutant lines (S532D and S532A), an OsNia1 over-expression line (OE) and Kitaake (wild type, WT). Compared with WT and OE, S532D and S532A have stronger nitrogen assimilation capacities. When ammonium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and chlorophyll (Chl) contents of S532D and S532A were lower than those of the WT and OE, whereas hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and nitrite contents were higher. When potassium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and Chl contents of S532D and S532A were higher than those of the WT and OE, there were no significant differences in the contents of H₂O₂ and MDA in the leaves of the test materials, and the difference in nitrite contents among different lines decreased. When ammonium sulfate served as the nitrogen source, there were no significant differences in the physiological indexes of the test materials, except NR activity. Compared with ammonium nitrate and ammonium sulfate, the content of NH₄⁺-N in the leaves of each plant was lower when potassium nitrate was used as the nitrogen source. The qPCR results showed that OsGS and OsNGS1 were negatively regulated by downstream metabolites, and OsNrt2.2 was induced by nitrate. In summary, when ammonium nitrate served as the nitrogen source, the weak growth of NIA1 phosphorylation site-directed mutant lines was due to the toxicity caused by the excessive accumulation of nitrite. When potassium nitrate served as the nitrogen source, the assimilation rates of nitrate, nitrite and ammonium salt were accelerated in NIA1 phosphorylation site-directed mutant lines, which could provide more nitrogen nutrition and improve the tolerance of rice to ammonium nitrogen deficiency. These results could provide a possible method to improve the efficiency of nitrogen utilization in rice under low-nitrogen conditions.     

施氮量和种植密度对优质晚稻稻米品质的调控效应及其机制

以‘玉针香’为材料,设计4个施氮量处理,分别为N1(不施氮)、N2(尿素180 kg/hm2)、N3(尿素126 kg/hm2)、N4(180 kg/hm2尿素+生物炭200 t/hm2),2个种植密度处理,分别为D1(18 cm×25 cm)、D2(14 cm×25 cm),于2020—2021年开展大田试验,研究施氮量、种植密度及其互作对稻米品质与叶片氮代谢特征的影响。结果表明：种植密度对稻米出米率、直链淀粉含量、胶稠度、蛋白质组分均无显著影响,D2处理各指标均略高于D1处理;施氮提高了出米率与各蛋白质组分含量,降低了垩白粒率和垩白度,以N4处理的效果最好;施氮量与种植密度互作对稻米品质影响显著,N4D2处理的稻米加工品质与外观品质最优,胶稠度最长,蛋白质组分含量最高;种植密度对叶片含氮量、硝态氮含量以及硝酸还原酶(NR)、亚硝酸还原酶(NiR)活性均无显著影响;D2处理的叶片含氮量以及NR、NiR活性较高;施氮提高了叶片全氮含量、硝态氮含量以及NR、NiR活性,以N4处理的最高;相关性分析结果表明,叶片全氮含量、NR活性、NiR活性与整精米率、直链淀粉含量、胶稠度、蛋白组分含量呈显著或极显著正相关,与垩白粒率和垩白度呈显著或极显著负相关;增施生物炭可显著提高整精米率,显著降低垩白度和垩白粒率。本试验条件下,N4D2处理对优质稻稻米品质的调控效应最好,其调控机制在于提高了叶片全氮含量和氮代谢酶活性。     

Changes in the activities of key enzymes and the abundance of functional genes involved in nitrogen transformation in rice rhizosphere soil under different aerated conditions

Soil microorganisms play important roles in nitrogen transformation.  The aim of this study was to characterize changes in the activity of nitrogen transformation enzymes and the abundance of nitrogen function genes in rhizosphere soil aerated using three different methods (continuous flooding (CF), continuous flooding and aeration (CFA), and alternate wetting and drying (AWD)).  The abundances of amoA ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), nirS, nirK, and nifH genes, and the activities of urease, protease, ammonia oxidase, nitrate reductase, and nitrite reductase were measured at the tillering (S1), heading (S2), and ripening (S3) stages.  We analyzed the relationships of the aforementioned microbial activity indices, in addition to soil microbial biomass carbon (MBC) and soil microbial biomass nitrogen (MBN), with the concentration of soil nitrate and ammonium nitrogen.  The abundance of nitrogen function genes and the activities of nitrogen invertase in rice rhizosphere soil were higher at S2 compared with S1 and S3 in all treatments.  AWD and CFA increased the abundance of amoA and nifH genes, and the activities of urease, protease, and ammonia oxidase, and decreased the abundance of nirS and nirK genes and the activities of nitrate reductase and nitrite reductase, with the effect of AWD being particularly strong.  During the entire growth period, the mean abundances of the AOA amoA, AOB amoA, and nifH genes were 2.9, 5.8, and 3.0 higher in the AWD treatment than in the CF treatment, respectively, and the activities of urease, protease, and ammonia oxidase were 1.1, 0.5, and 0.7 higher in the AWD treatment than in the CF treatment, respectively.  The abundances of the nirS and nirK genes, and the activities of nitrate reductase and nitrite reductase were 73.6, 84.8, 10.3 and 36.5% lower in the AWD treatment than in the CF treatment, respectively.  The abundances of the AOA amoA, AOB amoA, and nifH genes were significantly and positively correlated with the activities of urease, protease, and ammonia oxidase, and the abundances of the nirS and nirK genes were significantly positively correlated with the activities of nitrate reductase.  All the above indicators were positively correlated with soil MBC and MBN.  In sum, microbial activity related to nitrogen transformation in rice rhizosphere soil was highest at S2.  Aeration can effectively increase the activity of most nitrogen-converting microorganisms and MBN, and thus promote soil nitrogen transformation.      

Effect of Salt Stress on Nitrogen Assimilation of Functional Leaves and Root System of Rice in Cold Region

The aims were to investigate the effect of salt stress on key enzyme activity of nitrogen metabolism and the concentration of nitrate nitrogen and ammonium nitrogen response to salt stress.Two rice cultivars,Mudanjiang 30 (sensitive cultivar) and Longdao 5 (salt-tolerant cultivar),were treated with different salt concentrations (CK 0%,S1 0.075%,S2 0.15%,S3 0.225% and S4 0.3%).The results showed that the activities of nitrate reductase (NR),glutamine synthase (GS),glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in the functional leaves and roots of rice in cold region presented a single peak curve change and the peak occurred in the heading stage;compared with those of the CK,the activities of NR,GS and GOGAT of rice in cold region decreased,but the activity of GDH increased in the heading stage under salt stress.The variation for key enzyme activity of nitrogen metabolism was the highest under S4 treatment.The activities of NR,GS and GOGAT in the functional leaves significantly decreased compared with those in roots;the concentrations of nitrate nitrogen and ammonium nitrogen in the functional leaves and roots of rice in cold region presented a single peak curve change and the peak occurred in the heading stage;compared with that of the CK,the concentration of nitrate nitrogen decreased in leaves and roots,the concentration of ammonium nitrogen decreased and the concentration of ammonium nitrogen in roots increased under salt stress.The variations for the activities of NR,GS and GOGAT in the functional leaves and roots of Longdao 5 were less than those of Mudanjiang 30 under the same concentration of salt stress.     

稻草型生物有机肥对茄子产量与品质的影响

为推广应用稻草型生物有机肥,采用田间小区试验研究了稻草型生物有机肥对茄子产量、品质及其根系特性的影响。结果表明:稻草型生物有机肥显著提高了茄子的产量,增加了茄子中维生素C、可溶性糖含量,降低了茄子中硝酸盐和亚硝酸盐含量,增强了茄子生育中后期的根系活力,提高了茄子根系硝酸还原酶的活性。     

植物硝酸还原酶功能的研究进展

硝酸还原酶(Nitrate Reductase,简称NR)是高等植物氮素同化的限速酶,可直接调节硝酸盐还原.从而调节氮代谢,并影响到光合碳代谢.发掘NR新的生理功能并探讨其作用机理具有重要的理论和实际意义.随着分子生物学技术的迅猛发展.可以从分子水平上深入了解NR的生理功能及作用机制;此外还可能由此了解氮代谢及其与其他生理过程之间的关系;同时也深入了解NR、NO与植物抗逆性的关系;以及研究硝酸还原酶的展望.     

烟草硝酸还原酶的生物信息学分析

为深入研究烟草中硝酸还原酶(nitrate reductase,NR)的功能,采用生物信息学方法,对烟草、拟南芥和水稻中的NR氨基酸序列进行了预测和分析。通过构建系统进化树,发现烟草与拟南芥NR的遗传距离较近,而与水稻中NR的遗传距离较远;蛋白质二级结构分析表明,烟草、拟南芥和水稻NR序列的C末端较为相似,而N末端差别较大;所有NR序列中都预测到5个结构域,且磷酸化位点主要位于N末端。但烟草中NR预测到的磷酸化位点少于拟南芥中预测到的,说明拟南芥中NR的活性受到更复杂的调控。     

甜菜硝酸还原酶的研究进展

施氮不当是影响甜菜产量、品质和生产效益的主要原因之一,而硝酸还原酶是氮素同化的关键酶,影响甜菜对氮吸收。文章分析了硝酸还原酶的特性及调控机理,阐述了甜菜硝酸还原酶的提取、纯化及一些基本特性、硝酸还原酶活力的测定方法和影响因素及其与产量质量的关系,并表述了结构基因表达调控的最新进展,这些研究内容对于提高氮肥利用率,提高甜菜的产量和品质具有重要意义。     

开放式大气CO2浓度增高对水稻土反硝化活性的影响

利用江都市小记镇的稻-麦轮作FACE平台,研究大气CO2浓度增高对农田反硝化活性的影响.在2005年水稻生长季研究不同施肥(施常规氮量和低氮量)、不同秸秆还田(秸秆半还田、秸秆不还田)处理土壤中的反硝化细菌数量、硝酸还原酶和亚硝酸还原酶活性在大气CO2浓度升高(FACE)条件下随时间的变化情况,并且在拔节期测定对照及FACE土壤的反硝化潜势.结果表明:CO2浓度增高对水稻生长各时期的土壤反硝化细菌数量具有一定的抑制作用,这种抑制作用在常规氮肥施用及秸秆不还田情形下最为显著(P＜0.01).FACE对硝酸还原酶和亚硝酸还原酶活性几乎没有影响.对反硝化潜势的研究发现,在低氮与常氮施用条件下,FACE土壤的反硝化潜势分别为对照土壤的84.21%和97.46%.通过与土壤理化性质的相关性研究,认为主要是因为FACE促进植物生长和土壤中的微生物活性,反硝化微生物在竞争中受到抑制,使得反硝化活性降低.     

大豆叶片硝酸还原酶活性动态研究

试验采用框栽和砂培两种方法,框栽土壤为黑土,试验品种为东农42、东农47、龙选1号和秣食豆;砂培试验品种为东农47,分别对不同品种及不同施氮处理大豆叶片的硝酸还原酶(NR)活性进行了研究。结果表明,在大豆叶片中都检测到了NR活性,框栽与砂培试验测得的NR活性都呈单峰曲线,框栽大豆峰值出现在R2期(秣食豆为R1期),砂培大豆出现在R2和R4期;NR活性上部叶片明显高于中、下部;NR活性随氮水平提高而升高,施氮水平明显影响NR活性。     

遮光下外源水杨酸对韭菜硝酸盐还原同化效应的研究

【目的】通过研究不同光强下外源水杨酸（SA）对韭菜叶片硝酸盐（NO3-）累积、氮代谢关键酶活性、光系统II电子传递速率（ETR）及主要氨基酸和可溶性蛋白合成的调控效应,以期明确遮光水平下SA对硝酸盐还原同化的影响。【方法】以韭菜品种‘新世纪雪韭’为供试材料,在前期试验工作基础上,采用显著降低韭菜硝酸盐累积的3.0 mmol•L-1 SA对韭菜叶面进行喷施处理,设自然光（900-1 050 μmol•m-2•s-1）和遮光（40%自然光）2种光照强度,分析SA对韭菜硝酸盐累积及NO3-还原同化的影响。【结果】SA处理缓解了遮光水平下韭菜叶片氮代谢关键酶硝酸还原酶（NR）、谷氨酰胺合成酶（GS）、谷氨酸草酰乙酸转氨酶(GOT)和谷氨酸丙酮酸转氨酶（GPT）活性的降低,显著降低了硝酸盐的累积,其中,与弱光对照相比,SA处理下GOT活性和GPT增幅较明显,分别为23.7%和12.3%;SA处理还显著提高了弱光下叶片的全氮含量和干物率,但叶绿素含量和光系统间电子传递速率ETR的增幅不大;另外,SA处理增加了弱光下韭菜叶片多数游离氨基酸组分的含量,效果最为显著的为色氨酸和丝氨酸,分别比对照提高了89.8%和50.6%;并且提高了游离氨基酸总量和可溶性蛋白含量,但降低了游离氨基酸与可溶性蛋白比值A/P。【结论】弱光降低了韭菜氮素同化能力和物质生产能力,而SA处理促进了弱光下韭菜叶片氮代谢中硝酸盐的还原和铵的同化,同时调动转氨作用的积极协同配合,促进了硝态氮转化为游离氨基酸和可溶性蛋白,这可能是遮光下SA降低韭菜硝酸盐累积的一个重要原因。     

固氮施氏假单胞菌（Pseudomonas stutzeri）A1501同化硝酸盐代谢基因簇分布及调控研究

固氮施氏假单胞菌（Pseudomonas stutzeri）A1501在有氧条件下能够利用硝酸盐/亚硝酸盐为唯一氮源生长,表明该菌除了具有固氮和反硝化等氮循环系统外还有同化硝酸盐/亚硝酸盐系统。为进一步阐明该菌同化硝酸盐的代谢机制,利用生物信息学手段分析了硝酸盐同化相关基因的组成及分布情况,并初步研究了同化硝酸盐途径特异性调控关系。结果表明, P. stutzeri A1501中存在两个硝酸盐同化基因簇nasST-nasA-nirBDnasBcobA和nasR-nasFED,分布于基因组不同部位。第一个基因簇中nasS-nasT编码二元抗转录终止因子,nasA编码NarK/NasA家族硝酸盐转运蛋白,nirBD编码亚硝酸盐还原酶,nasB编码同化硝酸盐还原酶,cobA编码参与西罗血红素合成的尿卟啉-Ⅲ C-甲基转移酶;第二个基因簇中nasR编码单一组分抗转录终止因子,nasFED编码ABC型（ATP依赖）硝酸盐/亚硝酸盐转运蛋白。NasS-NasT双组分蛋白调控硝酸盐/亚硝酸盐还原酶基因的转录,NasR调控硝酸盐/亚硝酸盐转运蛋白基因的转录。     

烘烤过程中烟叶蛋白质与硝态氮代谢规律研究

对烘烤过程中烤烟烟叶蛋白质与硝酸盐代谢规律的研究表明,蛋白质含量随烘烤进程发展逐渐减少,氨基酸含量逐渐增加,且均在变黄中期和定色其有一定个快速变化阶段,烤后烟叶中氨基酸和可溶性蛋白与鲜叶中可溶性蛋白的呈呈高度正相关;蛋白酶活性在烤烤开始后２４ｈ有一个高峰,硝酸的酶活性从烘烤开始就逐渐升高,２４ｈ达到最大值,之后迅速失活,ＮＯ３和ＮＯ２的含量均在烘烤上升,变黄期达到 最大值,在定色阶段快速下降,但烤     

棉花叶片硝酸还原酶活性的测定方法

本研究应用磺胺比色法测定棉花叶片硝酸还原酶活性(NRA),探讨了体内法测定棉花叶片NRA的最适条件。通过分析比较硝酸盐诱导、2,4-二硝基苯酚、三氯乙酸及煮沸等因素对NRA测定结果的影响,建立了体内法测定棉花叶片NRA的最佳条件,即用50 mmol/L的KNO3溶液诱导棉花叶片48 h、抽气前后分别加入2,4-二硝基苯酚和1,6-二磷酸果糖、暗保温35 min后煮沸10 min可以使NRA的活性达到最高。棉花叶片NRA测定方法的建立为进一步研究棉花的氮素代谢奠定了基础。     

耕作方式和大田水分管理对水稻氮素吸收利用的影响

[目的]探讨不同耕作方式和大田水分管理模式对水稻氮素吸收利用、氮代谢酶活性和土壤氮素的影响,为优化水稻栽培技术提供参考.[方法]以水稻品种金优253和桂旱1号为材料,2008年早季和晚季采用湿润灌溉、交替灌溉、水层灌溉3种水分管理模式和免耕、常耕两种耕作方式进行田间试验,于拔节期、抽穗期和成熟期测定水稻氮素吸收和利用和各种生理指标.[结果]在相同耕作方式下,水层灌溉相对有利于提高早季水稻全氮积累量和氮素回收率,提高抽穗期早季水稻叶片GP转氨酶活性、拔节期和抽穗期晚季水稻叶片GS活性以及成熟期早晚季常耕水稻叶片GS活性.采用湿润灌溉处理相对有利于提高水稻叶片硝酸还原酶、成熟期水稻叶片GPT转氨酶活性以及水稻氮素稻谷生产效率、土壤碱解氮含量、晚季水稻氮素回收率,但降低晚季水稻产量.采用交替灌溉处理相对有利于提高拔节期早晚季水稻叶片GPT转氨酶活性、抽穗期晚季水稻叶片CP转氨酶活性及拔节期和抽穗期早季水稻叶片GS活性.在相同水分管理模式下,与常耕水稻相比,免耕利于提高交替灌溉处理水稻氮素积累量以及早、晚季土壤全氮和碱解氮含量.[结论]耕作方式和水分管理对水稻氮素吸收利用有重要的调控作用;免耕和节水互作条件下,有利于土壤供氮能力的提高,从而提高氮素利用率.     

活力素对菠菜产量及硝酸盐含量的影响

利用微区试验研究了不同氮磷钾配比条件下,喷施活力素对菠菜产量及硝酸盐含量的影响。结果表明:氮磷钾配合施用能显著提高菠菜产量,并能提高硝酸还原酶活性、降低硝酸盐含量。在单施氮肥条件下,喷施活力素有明显的增产效果;喷施活力素也明显提高了硝酸还原酶活性,降低了硝酸盐和亚硝酸盐含量,改善了菠菜的食用品质。     

黄瓜硝酸还原酶cDNA片段的克隆及其在缺氮胁迫下的表达

以黄瓜叶片总RNA为模板,应用逆转录—聚合酶链式反应(RT-PCR),首次扩增出黄瓜硝酸还原酶(NR)cDNA部分片段。经过测序和同源性分析表明:该基因片段长度为395 bp,与GenBank上所登录的笋瓜NR基因同源性达89%,根据所测序列推导该片段由131个氨基酸残基组成。进一步检测其在缺氮胁迫下的活性和表达变化,结果表明缺氮胁迫下硝酸还原酶的活性和表达都明显下降,推测黄瓜NR在转录水平上调节其活性。     

氮胁迫对水稻营养生长期氮代谢及相关基因表达量的影响

通过对水稻品种日本晴进行0、1h和1、3、7d的缺氮胁迫,以及缺氮7d后,恢复供氮生长2h和1d,研究氮素同化相关酶的基因表达及其活性的动态变化情况。结果表明:缺氮胁迫下,根部NH4+、NO3-含量显著下降。短期缺氮胁迫下,地上部NR1、NR2、NiR2、GS2、Fd-GOGAT、GDH2、GDH3以及根部NR1、NR2、GDH4的表达量均有增加;随着缺氮胁迫时间延长,上述基因的表达量均大幅下降。缺氮胁迫下,植株GS、Fd-GOGAT、地上部NR和根部NADH-GOGAT的活性下降,地上部NADPH-GDH活性增加,根部NR、GDH活性先增加后下降,地上部NiR活性先下降后增加。植株缺氮7d后,恢复供氮生长1d时,NR、NiR、GS、GOGAT、GDH的基因表达量及活性基本趋于恢复正常水平,部分基因表达量有所增加。     

钼对甘蔗氮代谢的影响

本试验选用巴西固氮甘蔗品种B 1和B 8为供试材料,在温室条件下桶栽砂培甘蔗施以含不同钼水平的营养液(0m g/L、0.01m g/L、0.02m g/L、0.04m g/L),在不同时期测定甘蔗叶片中硝酸还原酶、G S酶活性及硝态氮、氨态氮含量。试验结果表明,在大棚含氮条件下,三个钼处理都提高了两个甘蔗品种的硝酸还原酶活性而降低了硝态氮含量。0.01m g/L钼处理在拔节期后提高了B 1叶片的G S酶活性,而在幼苗期提高了B 8叶片的G S酶活性,降低了氨态氮含量,促进了氮代谢。在大棚无氮条件下,三个钼处理在拔节期后都提高了两个甘蔗品种的NR酶活性,降低硝态氮含量;提高了B 1的G S酶活性,降低氨态氮含量;在分蘖期也提高了B 8叶片的G S酶活性而降低了其氨态氮含量。因而在大棚里,无论是含氮或无氮条件下,在不同时期不同钼处理对甘蔗氮代谢和氨同化过程的作用也不同,但是相对以较低水平钼处理较能提高两个甘蔗品种的硝酸还原酶活性而降低硝态氮含量;提高了B 1与B 8的G S酶活性,降低氨态氮含量,促进了甘蔗的氮代谢和氨同化。