Salicylic Acid Hydroxylases Coordinate The Growth And Disease Resistance In Rice

Salicylic acid(SA)as an important class of plant hormones is involved in plant immune response and growth.Rice has higher SA content than Arabidopsis and other plants,but its SA content is not induced by pathogens.The mechanism of SA in rice disease resistance and growth of rice remain to be investigated.In this study,gene expression of rice hydroxylase genes and the enzyme activities were studied.CRIPSR/Cas9 technology was used to knock out the salicylic acid hydroxylase of NIP and Yandao 8 to obtain mutants with a certain increase in salicylic acid content.At the same time,transgenic plants with reduced salicylic acid content were obtained by overexpressing salicylate hydroxylase.The above materials were used to further study the role of SA hydroxylase and SA in rice growth and disease resistance.The main findings are as follows:(1)The profilings of SA and hydroxylated SA of Arabidopsis and riceat flowering stage showed that the SA content in rice is 2.3 times higher than that of Arabidopsis.SA in Arabidopsis thaliana can be hydroxylated to form two products,2,3-DHBA and 2,5-DHBA,but only 2,5-DHBA can be detected in rice.(2)Four SA candidate hydroxylase gene expressions all can be induced by exogenous SA.In vitro enzyme activity assays revealed that OsS5H1 and OsS5H2 have SA hydroxylase activity but OsS5H3 and OsS5H4 only have slightly enzyme activities.(3)The s5h1s5h2 double mutant materials in different breed backgrounds were created through hybridization or CRISPR/Cas9 methods.(4)The SA metabolism of s5h1s5h2 material and OsS5H1/OsS5H2 overexpressed plants changed significantly.Through metabolic analysis of OsS5H1 and OsS5H2 mutants and overexpressed materials in different periods,it was found that the SA content in the overexpressed material decreased compared with the control,the metabolite content increased,and the mutant showed the opposite trend.It is proved that OsS5H1 and OsS5H2 have SA hydroxylation activity in rice,and the catalytic activity of OsS5H1 hydroxylase is stronger than OsS5H2 in vivo.(5)s5h1s5h2 mutant has enhanced disease resistance.After inoculation with Xanthomonas oryzae pv.oryzae P6,s5h1s5h2 in the background of NIP and Yandao 8 both displayed enhanced pathogen resistance.Overexpression of OsS5H1 or OsS5H2 in the NIP background increased the sensitivity of rice to Xanthomonas oryzae pv.oryzae P6.(6)OsS5H1 or OsS5H2 coordinate the relationship between rice disease resistance and yield.By counting the agronomic traits of s5h1s5h2,we found that the number of secondary branches,the grain number per panicle and the grain number per plant of the double mutants under the background of NIP decreased compared with the control,redulting grain weight loss per plant.Interestingly,the double mutants in the background of Yandao 8 background decreased the grain number per panicle,but the effective tiller number increased,thus the grain weight per plant showed no difference to the control.This shows that the s5h1s5h2 mutations can increase the pathogen resistance without the penalty to the yield in some Yandao 8 variety.OsS5H1 or OsS5H2 overexpression lines did not significantly increase the yield,which proved that reducing the SA content of rice could not increase its yield.(7)oss5h1 and oss5h2 mutation resulted in shorter plant height and rice rachis internode length.We found that oss5h1 and oss5h2 mutation can affect the rachis internode length under the background of NIP and Yandao 8,indicating that SA accumulation negatively regulate the plant height and rachis internode length.In summary,the SA content and hydroxylation metabolism in rice and Arabidopsis are inconsistent.OsS5H1 and OsS5H2 are both SA 5-hydroxylases which can catalyze the formation of 2,5-DHBA in vitro and in vivo.OsS5H1 and OsS5H2 coordinated rice growth and disease resistance by adjusting SA accumulation.In the future,rice salicylate hydroxylase is expected to be used as a key marker in molecular breeding of rice varieties with strong disease resistance and high-yield.