Effects Of OsRGA1 On Nitrogen-Use And Yield In Rice And It’s Mechanism

Nitrogen is essential macronutrient for plant growth and grain yield.However,excessive nitrogen fertilizer addition will add to environmental insecurity.Consider that the world’s population continue to grow,improving N-use efficiency(NUE)and yield are still in urgency.Studies have shown that OsRGA1 is involved in nitrogen signaling pathway in rice.However,the mechanism of OsRGA1 regulates nitrogen use is still very limited.Therefore,studying OsRGAl effects on nitrogen use and yield is of importance to reveal OsRGA1 function and increase yield.In this study,two OsRGAl mutants stably inherited,named R5 and R8,were obtained from japonica rice Nipponbare(NIP)by CRISPR-Cas9.Two treatment,hydroponics and field experiment were conducted.Phenotypes of wild type and mutants and related physiological and molecular indexes were observed.Combined with omics analysis,OsRGAl effects on nitrogen use and yield were further studied.The main results were as follows(1)Compared with wild-type,the plant height,length of panicle,flag leaf and internode were significantly reduced in mutants.The primary structure of OsRGA1R5 and OsRGA1R8 was shortened,and some C-terminal fragment of ras-like domain on the tertiary structure was lost,resulting in the deletion of binding sites I,EBR(3),RBR and GTP conserved region.(2)OsRGA1 deficiency reduced the response of rice seedlings to nitrogen.The seedling height of wild-type increased with availible nitrogen,while the seedling height and root length of mutants were not significant change.The enzymes activity related to carbon and nitrogen metabolism,the content of NO3-,total amino acid and glutamate of leaves in mutants were significantly lower than those in wild type.(3)Under either zero nitrogen rate(0 kg hm-2,ON)or normal nitrogen rate(270 kg hm-2,NN)conditions,compared with NIP,the grain yield of R5 and R8 significantly decreased by 45.16%and 43.72%,38.78%and 35.14%,respectively.Under ON condition,panicle number of R5 and R8 significantly increased by 49.56%and 48.89%,seed setting rate and 1000-grain weight of R5 and R8 significantly decreased by 33.81%and 31.37%,43.93%and 44.84%,compared with NIP,respectively;under NN condition,panicle number of R5 and R8 significantly increased by 22.85%and 29.27%,seed setting rate and 1000-grain weight of R5 and R8 significantly decreased by 14.47%and 13.43%,40.67%and 41.34%,compared with NIP,respectively.(4)OsRGAl deficiency significantly increased highest and effective tiller number,significantly decreased leaf area index and total dry weight per plant and increased SPAD value and photosynthetic rate.At the same time,the lacking of this gene reduced the response of rice flag leaves to nitrogen-promoted enzyme activity and gene expression levels related to carbon and nitrogen metabolism,resulting in significantly lower carbon and nitrogen content of mutants at grain filling stage than that of wild type.(5)OsRGA1 deficiency significantly reduced NUE in rice.Compared with NIP,the agronomic nitrogen use efficiency,nitrogen physiologicale efficiency and partial nitrogen productivity of R5 and R8 decreased by 28.50%and 21.27%,26.93%and 29.39%,38.78%and 35.08%,respectively.(6)Metabolome and transcriptome analysis showed that the contents of metabolites and gene expression levels related to carbon and nitrogen metabolism of the mutants were not significant difference at the grain-filling stage under 0N condition,compared to those of the wild type.Compared with NIP,the mutants exhibited lower gene expression levels related to carbon fixation and nitrogen assimilation,and higher gene expression levels related to sugar transport and glycolysis of flag leaves at the grain-filling stage.Thus,the mutants had lowercontent of 2-OG related to carbon metabolism and Glu,Gln,Asp and Asn related to nitrogen metabolism.In this study,the mechanism of OsRGA1 on NUE and grain yield in rice was discussed.It was confirmed that OsRGA1 deficiency increased rice tiller number,decreased enzymes activity and gene expression levels related to carbon and nitrogen metabolism,resulting in lower content of 2-OG related to carbon metabolism and Glu,Gln,Asp and Asn related to nitrogen metabolism.Thus,the mutants exhibited lower NUE,seed setting rate and 1000-grain weight,causing lower grain yield.Therefore,OsRGAl promotes nitrogen use efficiency and grain yield,which could provide a theoretical basis for improving nitrogen use efficiency and yield in rice.