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Journal of Integrative Agriculture  2022, Vol. 21 Issue (3): 631-643    DOI: 10.1016/S2095-3119(20)63473-6
Special Issue: 水稻遗传育种合辑Rice Genetics · Breeding · Germplasm Resources
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The removal of nitrate reductase phosphorylation enhances tolerance to ammonium nitrogen deficiency in rice
HAN Rui-cai1, 2, XU Zhi-rong1, LI Chen-yan1, Adnan Rasheed1, PAN Xiao-hua1, SHI Qing-hua1, WU Zi-ming1
1 Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education/College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, P.R.China 
2 Rice Research Institute, Jiangxi Academyof Agricultural Sciences/Jiangxi Provincial Key Laboratory for Physiology and Genetics of Rice, Nanchang 330200, P.R.China
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硝酸还原酶(Nitrate reductase, NR)是植物体内同化硝态氮的关键酶,其活性受翻译后磷酸化修饰调控。通过分析硝酸还原酶NIA1磷酸化位点定向突变株系(S532DS532A)、OsNia1过表达株系(OE)及野生型(WT)的表型、氮代谢和活性氧代谢的差异,探究不同形态氮素营养下NIA1蛋白的去磷酸化对水稻生长和生理生化的影响。研究表明,与WT和OE相比,S532DS532A具有更强的氮素同化能力。以硝酸铵作为氮源时,S532DS532A的株高、地上部干重和叶绿素含量均低于WT和OEH2O2、MDA和亚硝酸盐含量则较高;以硝酸钾作为氮源时,S532DS532A的株高、地上部干重和叶绿素含量高于WT和OE,所有株系叶片中的H2O2和MDA含量无明显差异,各株系间亚硝酸盐含量差异减小;以硫酸铵作为氮源,除NR活性外,各株系间的其它生理指标均无显著差异。相较于硝酸铵和硫酸铵,以硝酸钾作为氮源时各株系叶片中NH4+-N的含量较低。q-PCR分析表明OsGSOsNGS1基因表达受下游代谢产物的负调控,OsNrt2.2受硝酸盐诱导表达。综上,硝酸铵作为氮源时NIA1磷酸化位点定向突变株系长势较弱是由于过量积累的亚硝酸盐对自身的毒害;硝酸钾作为氮源时NIA1磷酸化位点定向突变株系对硝酸盐、亚硝酸盐和铵盐的同化速率加快,能够提供较多的氮素营养,提高了水稻对铵态氮缺乏的耐受性。

Abstract  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.  

Keywords:  Oryza sativa L.       nitrate reductase       phosphorylation       NR activity       nitrogen utilization       nitrite  
Received: 27 May 2020   Accepted: 28 October 2020
Fund: The research was supported by the National Natural Science Foundation of China (31560350, 31760350 and 31660431), the National Key Research and Development Program of China (2018YFD0301102), the Jiangxi Natural Science Foundation, China (20202BABL205020), the Key Research and Development Program of Jiangxi Province, China (20171ACF60018 and 20192ACB60003), the Jiangxi Agriculture Research System, China (JXARS-18) and the Training Program for Academic and Technical Leaders of Major Discipline in Jiangxi Province, China (20204BCJL22044). 
About author:  HAN Rui-cai, E-mail:; WU Zi-ming, Tel: +86-791-83828113, Fax: +86-791-83813877, E-mail:

Cite this article: 

HAN Rui-cai, XU Zhi-rong, LI Chen-yan, Adnan Rasheed, PAN Xiao-hua, SHI Qing-hua, WU Zi-ming. 2022. The removal of nitrate reductase phosphorylation enhances tolerance to ammonium nitrogen deficiency in rice. Journal of Integrative Agriculture, 21(3): 631-643.

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