Functional Analysis Of Rice Transcription Factor OsbZIP18 And OsBCAT Family Genes

Valine(Val),leucine(Leu)and isoleucine(Ile)are essential amino acids that cannot be synthesized in human body and can only be obtained from food.Because of ?-carbon contains branched aliphatic hydrocarbon chain,it is called branched chain amino acid(BCAAs).The synthetic pathway of branched chain amino acids in plants has been well known,but the regulation and physiological function of BCAAs are still poorly understood.In this study,we preliminarily explored the physiological function of BCAAs in rice and identified a transcription factor Osb ZIP18 that regulates the natural variation of BCAAs content in rice leaves.Branched chain amino acid aminotransferase(BCAT)is a key enzyme in the biosynthesis of BCAAs,which plays an important role in the synthesis of BCAAs.Homologous sequence analysis showed that five rice BCAT members Os BCAT1-Os BCAT5,heterologous expression of Os BCAT1,2,4 and 5 could partially restore the BCAAs deficiency phenotype of in yeast mutant,indicating that these four genes had the function of branched chain amino acid transferase.Both Os BCAT4 and Os BCAT5 were located in chloroplasts.Overexpression of Os BCAT4 and Os BCAT5 could significantly increase the contents of Val and Ile in roots and shoots,and knockout Os BCAT4 and Os BCAT5 genes significantly decreased the contents of Val and Ile in roots,indicating that Os BCAT4 and Os BCAT5 were mainly responsible for the synthesis of Val and Ile in rice.In the sufficient nutrition condition,compared with the wild-type ZH11,the root growth of the osbcat4/osbcat5 double mutant plant was slower,showing the phenotype of shorter main root length and less adventitious root number.Meanwhile,the contents of mineral elements in shoot and root of osbcat4/osbcat5 double mutant were significantly higher than those of wild type.While in low concentration nutrition,there was no significant difference in root length and adventitious root number between osbcat4/osbcat5 double mutant and wild type.The element content analysis showed that the accumulation of mineral elements in the roots of osbcat4/osbcat5 double mutants returned to the wild type level.These results show that the excessive accumulation of mineral elements was the main reason for the root growth retardation of osbcat4/osbcat5 double mutants.Furthermore,the phenotype of inhibition of root growth and excessive increase of mineral element content in osbcat4/osbcat5 double mutant plants can be alleviated by applying BCAAs exogenously.In addition,we found that exogenous Ile inhibited the main root elongation,while exogenous Val decreased the main root elongation and increased the number of adventitious roots,which revealed that BCAAs(Val and Ile)had the same and unique biological functions in regulating root development.Together,these results suggest that Os BCAT4 and Os BCAT5 regulate BCAAs homeostasis,as well as the mineral accumulation,which play an important role on root development in rice.We measured the content of BCAAs in leaves of 520 rice germplasm materials with wide genetic diversity in our laboratory,and found that the content of BCAAs in leaves of these materials had great variation.The content of BCAAs in leaves of Japonica rice was significantly higher than that of indica rice.A transcription factor Osb ZIP18,which affects the accumulation of BCAAs in leaves,was identified by m GWAS.Osb ZIP18 was mainly expressed in leaves and subcellular localized in nucleus.Knockout of Osb ZIP18 gene significantly reduced the content of BCAAs in rice leaves.The results of EMSA and LUC experiments showed that Osb ZIP18 could directly bind to the promoter of Os BCAT1 and Os BCAT2,and activate the expression levels of Os BCAT1 and Os BCAT2.We constructed CRISPR material of Os BCAT1.Compared with wild type,the content of Leu in leaves of Os BCAT1 CRISPR mutant was significantly lower,suggesting that Os BCAT1 is involved in BCAAs synthesis.These results suggest that Osb ZIP18 regulates the accumulation of BCAAs in rice leaves by activating the expression of BCAAs synthesis genes.The natural variation analysis of Osb ZIP18 allele showed that a SNP site in Osb ZIP18 promoter region had a high correlation with BCAAs content;q RT-PCR results showed that the expression level of Osb ZIP18 gene was significantly positively correlated with BCAAs content,and the expression level of indica rice genotype was higher than that of Japonica rice genotype.The natural variation of BCAAs content may be caused by the change of transcription level caused by Osb ZIP18 promoter polymorphism.The expression of Osb ZIP18,Os BCAT1 and Os BCAT2 were significantly induced by nitrogen deficiency(initial stage),and the content of BCAAs in rice leaves increased with the increase of these gene expression levels.However,there was no significant difference in the expression of Os BCAT1 and Os BCAT2 and BCAAs content between Osb ZIP18 CRISPR mutant and wild type under nitrogen deficiency condition.These results indicate that the accumulation of BCAA induced by N starvation is mainly dependent on Osb ZIP18.In conclusion,we preliminarily explored the biological functions of Os BCAT4 and Os BCAT5 and the genetic basis of natural variation of BCAAs in rice leaves.We identified Osb ZIP18,a transcription factor affecting BCAAs content in rice leaves by GWAS,and explored the molecular mechanism of Osb ZIP18 regulating BCAAs accumulation in rice.