Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (1): 15-25.DOI: 10.1016/S2095-3119(20)63386-X

所属专题： 水稻遗传育种合辑Rice Genetics · Breeding · Germplasm Resources

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  • 收稿日期:2020-05-06 接受日期:2020-08-01 出版日期:2022-01-01 发布日期:2022-01-01

Reducing phosphorylation of nitrate reductase improves nitrate assimilation in rice

HAN Rui-cai1, 2, LI Chen-yan1, Adnan Rasheed1, PAN Xiao-hua1, SHI Qing-hua1, WU Zi-ming1   

  1. 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 Academy of Agricultural Sciences/Jiangxi Provincial Key Laboratory for Physiology and Genetics of Rice, Nanchang 330200, P.R.China
  • Received:2020-05-06 Accepted:2020-08-01 Online:2022-01-01 Published:2022-01-01
  • About author:HAN Rui-cai, E-mail: hrc1988113@163.com; Correspondence WU Zi-ming, Tel: +86-791-83828113, Fax: +86-791-83813877, E-mail: wuzm@jxau.edu.cn
  • Supported by:
    This research was supported by the National Natural Science Foundation of China (31560350 and 31760350) and the Key Research and Development Program of Jiangxi Province, China (20171ACF60018 and 20192ACB60003).

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摘要：

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。通过分析磷酸化位点定向突变株系（S532D 和S532A）、OsNia1过表达株系（OE）及野生型（WT）的表型、NR蛋白I（NIA1）及其磷酸化水平、NR活性、硝态氮代谢和活性氧代谢的差异，探究NIA1蛋白的去磷酸化对NR活性、氮代谢和水稻生长的影响。研究表明，S532D和S532A株系中的外源NIA1蛋白不能被磷酸化，其NR活性、NR活性状态和NO3--N的同化效率均高于WT和OE。OE中以上生理生化指标均小于S532D和S532A。表明解除转录水平控制对N代谢没有太大影响，而翻译后修饰的解除对N代谢水平有深刻的影响。随着NIA1蛋白磷酸化的解除和硝酸盐同化能力的增强，S532D 和 S532A的株高和叶绿素含量均降低，过氧化氢（H2O2）和丙二醛（MDA）的含量升高，这可能与硝酸盐代谢的中间产物亚硝酸盐的过度积累有关。以上结果表明NR的磷酸化可能是水稻的一种自我保护机制，NIA1磷酸化水平的降低能够促进硝酸盐的同化，而磷酸化水平的提高则会降低亚硝酸盐的积累，降低活性氧积累对水稻的毒害作用。

Abstract: Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and post-translational phosphorylation regulates NR activity.  To evaluate the impact of the dephosphorylation of nitrate reductase 1 (NIA1) protein on NR activity, nitrogen metabolism and plant growth, NIA1 phosphorylation site directed mutant lines (S532D and S532A) and an OsNia1 over-expression line (OE) were constructed, and the phenotype, NIA1 protein and its phosphorylation level, NR activity, nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed.  Exogenous NIA1 protein was not phosphorylated in S532D and S532A mutant lines, and their NR activities, activity states of NR and assimilation efficiencies of NO3–-N were higher than those in Kitaake (WT) and OE.  The changes in these physiological and biochemical indexes in the OE line were less than in S532D and S532A compared to WT.  These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism, but the removal of post-translational modification had a profound effect on it.  With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency, the plant height and chlorophyll content of S532D and S532A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased, which may be related to the excessive accumulation of nitrite as an intermediate metabolite.  These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice.  The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate, and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice. 

Key words: rice (Oryza sativa L.) , nitrate reductase ,  phosphorylation ,  directed mutation ,  nitrogen metabolism