Characterization And Functional Analysis Of Isochorismate Synthase And Salicylic Acid Hydroxylase In Rice

Salicylic acid(SA)is an important plant hormone,which is a product of phenylpropanoid secondary metabolism.SA plays critical roles in plant defense,senescence in a direct or indirect manner.The content of SA varies from different plants.At present,biosynthesis,metabolism and biological function of SA are well understood in Arabidopsis thaliana,but in rice,these have not been studied.In order to explore the biosynthetic metabolism of SA in rice,we carried out experiments of biochemistry,genetics to characterize and study the biological functions ofisochorismate synthase(OsICS1)and two SA hydroxylase(OsS5H-1 and OsS5H-2).The results are listed as below:(1)Bioinformatics analysis revealed that,protein sequences of OsICS1 contain the domain of isochorismate synthase;OsS5H-1 and OsS5H-2 contain the domain of 2-OG-Fe(?)oxygenase.(2)Using the enyme activity measurement,protein were induced and the enzyme activity were determined.OsS5H-1 and OsS5H-2 can catalyze SA into 2,5-DHBA.(3)Using transgenic technology of Arabidopsis thaliana,we obtained complementary plants OsICS1-C-1,OsIC1,-C-2;OsS5H-1-C-1,OsS5H-1-C-2,OsS5H-1-C-3;OSS5H-2-C-1,OsS5H-2-C-2 successfully.(4)Using CRISPR Cas9 technology,we obtained homozygous mutants icsl-1,icsl-2,icsl-3,s5h-1-1,s5h-1-2,s5h-1-3,s5h-2-1,s5h-2-2 and overexpression lines OsS5H-10E-1,OsS5H-10E-2,OsS5H-1OE-3,OsS5H-1OE-1,OsS5H-1OE-2,OsS5H-10E-3.(5)Using Q-PCR technology,we detected the relevant gene expression in different senescencing times,after treatment with different hormones and in icsl mutants at 15day.OsICSl and OsS5H-1 expressed higher in young and mature leaves,different to OsS5H-2;(OSICS1 and OsS5H-2 are not induced by SA;OsS5H-1 increased by 95%after SA treatement;PR1#051 and PR10a,which could response to SA,expressed significantly lower than Nip.(6)Genetic analysis of the transgenic lines showed that,1)OsICS1-C-1 and OsICS1-C-2 changed the delayed aging phenotype of atsid2;2)OsS5H-1-C-1,OsS5H-1-C-2,OsS5H-1-C-3,OsS5H-2-C-1,OsS5H-2-C-2 changed the dwarf and premature phenotype of ats3hs5h;3)mutants icsl-1,ics1-2,ics1-3 at 15 day showed the disease spot resulted from oxidative stress,similar to NahG transgenic rice,which contains little SA;4)the height of s5h-1-1,s5h-1-2,s5h-1-3 are shorter than Nip,but the height of OsS5H-1OE-1,OsS5H-1OE-2 and OsS5H-10E-3 are significantly taller than Nip;5)all overexpression plants performed enhanced susceptibility,but s5h-1 and s5h-2 all mutants show no enhanced disease resistance after inoculation of Xoo,which is similar to Nip.(7)Metabolic analysis of transgenic plants showed that OsICS1-C-1 and OsICS1-C-2 changed the phenotype of atsid2,which is short of SA,because of the increased SA and 2,5-DHBA;OsS5H-1-C-1,OsS5H-1-C-2,OsS5H-1-C-3,OsS5H-2-C-1,OsS5H-2-C-2 and all overexpression lines displayed decreased content of SA and increased content of 2,5-DHBA;SA in icsl-1,icsl-2 and icsl-3 decreased 67%at 15day after germination;there is no significant difference between mutants and Nip at 40day after germination;SA in s5h-1-1,s5h-1-2,s5h-1-3 increased obviously and 2,5-DHBA in mutants decreased sinificantly;SA in s5h-2-1 and s5h-2-2 have no obvious difference with Nip.In summary,our results demonstrated that OsICS1 is the key gene which regulates the SA in rice seedling;OsS5H-1 and OsS5H-2 could catalytze SA into 2,5-DHBA;disease resistance in rice is related to SA.