Functional Analysis Of Rice Isochorismate Synthase OsICS1 In Salicylic Acid Synthesis Pathway

Salicylic acid is a plant hormone and has many important biological functions.As the key enzyme for SA synthesis,isochorismate synthase(ICS)has been well studied in dicotyledons.And ICS is the main pathway for SA synthesis in Arabidopsis.Rice is one of the most important cereal crops in the world and the level of SA is much higher than that in Arabidopsis thaliana or other dicotyledons,the high level of SA may play a more essential role.However,the synthesis pathway of SA in rice is still unclear.So the function of rice isochorismate synthase gene was studied by means of reverse genetics,biochemistry,enzymology,metabonomics,plant physiology and agronomy.The main results are as follows:(1)OsICS1 can convert chorismic acid to isochorismic acid in vitro was determinate.Based on the existing research foundation,in this study,OsICS1 was expressed and purified in a heterologous system and it was proved that OsICS1 can convert chorismate to isochorismate in vitro was determinate by enzyme activity test and the catalytic activity is about 81.7% of AtICS1.(2)The tissue expression characteristics and protein localization of OsICS1 were studied.To elucidate the function of OsICS1,the expression pattern of OsICS1 in different tissues was studied.It was found that OsICS1 was mainly expressed in leaves but had no significant differences in different periods.Besides,OsICS1 expression was significantly induced in the rice germs after 24 h immersion,and increased more significantly in germination 24 h and 48 h of germs.By hormone treatment we found that OsICS1 was not induced by SA,IAA,6-BA,tZ,ACC and ABA,and it was weakly induced by JA.OsICS1 was not induced by P6 of Xanthomonas Oryzae.The proICS1::GUS transgenic rice materials were also used in this study.The results exclaimed that OsICS1 gene was highly expressed in rice leaves,germinated seed germs and young spike anthers,the expression was weak in roots,but not in stems.Subcellular localization observation results showed OsICS1 was localized in the cytoplasm.(3)Analysis of SA metabolism in osics1 mutants at different stages.According to the expression pattern of OsICS1,the SA metabolism of osics1 was analyzed.There was no significant differences between the SA levels of NIP and osics1 in dry seed germs,48 h soaking germs,24 h germination germs,leaves and panicles at different stages,implying that OsICS1 was not obviously involved in SA synthesis or there were other compensation pathways for SA synthesis.(4)Analysis of SA metabolism in OsICS1 overexpression material at different periods.To confirm the biological function of OsICS1 in rice,the level of SA in OsICS1 OE plants was detected.There was no significant differences in Free SA,SAG and 2,5-DHBA between OsICS1 over-expressed and wild-type rice,which indicated that OsICS1 did not obviously participate in SA biosynthesis in rice.(5)The osics1 mutant has delayed germination type.Further study of osics1 showed that it had an obvious delayed germination phenotype compared with NIP.The analysis of hormone metabolism in NIP and osics1 germs after soaking for 24 hours showed that the ABA contents in osics1 were increased by 52% to 110% while the contents of JA were significantly increased by 46% to 215%.However,IAA,ACC and cytokinins had no significant differences.(6)The osics1 mutant with large-grain phenotype.Statistical analysis of agronomic traits in field were conducted so as to elucidate the function of OsICS1 in rice.The results showed that the grain length,width and 1000-grain weight were increased significantly,implying that the mutation of OsICS1 affects grain type.The results above showed that OsICS1 highly expresses in leaves,germs and yong spike anthers,and the subcellular localization of OsICS1 is in cytoplasm.However,while OsICS1 can convert chorismic acid into isochorismic acid in vitro,it was not obviously involved in SA synthesis in vivo.Thus,we speculated that IC is not the major pathway of SA synthesis in rice.We also observed an evident delay phenotype of germinating in the seeds of osics1 mutants,and the content of ABA and JA in these seeds germs elevated significantly,suggesting that OsICS1 may have an impact on seed germination.Statistical analysis of agronomic traits showed that OsICS1 increased grain width,grain length and 1000-grain weight significantly,which proved that OsICS1 was involved in regulating rice grain type.These results suggest that IC pathway may not be the main pathway for SA synthesis in rice.OsICS1 may play an important role in seed germination and grain size regulation.The results are of great significance to elucidate the synthetic pathway of SA in rice and the physiological function of rice OsICS1.