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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.

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.

Xanthine dehydrogenase (XDH) is a crucial enzyme involved in purine metabolism. To evaluate the effect of XDH deficiency on rice growth during dark treatment, wild type (WT) Nipponbare (Oryza sativa L.) and two independent transgenic lines with severe RNAi suppression (xdh3 and xdh4) were used in the present experiment. Under normal growth conditions, chlorophyll levels and biomass were indistinguishable between WT and the two RNAi transgenic lines, but XDH enzyme activity and ureide levels were suppressed in XDH RNAi transgenic lines. When XDH RNAi transgenic lines were subjected to dark treatment, chlorophyll content and biomass were significantly decreased, while O2–· production rate and malonaldehyde (MDA) were significantly increased compared to WT. The spraying test of exogenous allantoin raised chlorophyll content and biomass and reduced O2–· production rate and MDA in WT and both transgenic lines, and it also simultaneously reduced differences between RNAi and WT plants caused by XDH deficiency in growth potential and anti-oxidative capacity under dark treatment. These results suggested that fully functional purine metabolism plays an important role in reducing the sensitivity of rice seedlings to dark stress.