Functional Analysis Of Arabidopsis Glycosyltransferase Genes UGT76D1 And UGT73C7 In Plant Immunity

Innate immunity is one of the most efficient and common forms of plant immunities.There are two types of immune receptors in innate immunity.One is pattern recognition receptors(PRRs),which on the surface of cells recognize the conserved molecular features of pathogens and trigger PTI(PAMP-triggered immunity),and another is intracellular immune receptors R protein(Resistance protein)to recognize the presence of relatively diverse pathogen effectors and trigger ETI(effector-triggered immunity).The process of ETI activation usually leads to cell death at the site of pathogen infection,which is called hypersensitive response(HR).Plant secondary metabolites play an important role in plant immunity.Salicylic acid(SA),for example,is an important signal molecule in the plant immunity.Different concentrations of SA activate different downstream signaling pathways,which determine the fate of cells during pathogen infection.In addition to hormone,small molecular compounds in phenylpropanoid pathways have also been reported to be involved in plant defense against pathogens.Glycosylation can regulate the content,molecular characteristics and functions of small molecular compounds.The process of glycosylation is mainly performed by glycosyltransferases.Glycosyltransferases are believed to contribute an important part to plant immune responses.However,the mechanism of glycosyltransferase involvement in plant immunity is not well understood and needs more investigations.In this study,two Arabidopsis glycosyltransferase genes,UGT76D1 and UGT73C7,were analysed for their functions in plant immunity.It was revealed that the glycosylation of small moleculs catalyzed by the glycosyltransferases UGT76D1 and UGT73C7 plays a key role in plant immunity.The main contents are as follows.1.The glycosyltransferase gene UGT76D1 modulates salicylic acid-associated immunity by DHBA glycosylationIn this study,we characterized a unique UDP-glycosyltransferase gene,UGT76D1,which plays an important role in SA homeostasis and immune response in Arabidopsis thaliana.Firstly,it was found that UGT76D1 gene was significantly induced by pathogenic Pst DC3000 and SA.Overexpression of UGT76D1 resulted in the massive accumulation of SA,significant up-regulation of Pathogenesis-related genes(PR genes)and a hypersensitive response(HR)-like lesion mimic phenotype.When HR-inducible strain Pst DC3000(avrRpt2)was used to induce HR of wild-type and UGT76D1 mutants,it was found that HR was significantly delayed in UGT76D1 mutants compared with that of wild-type.Those results indicated that UGT76D1 participates in HR process.Since SA was accumulated in UGT76D1 overexpression lines,the transgenic plants of SA hydroxylase NahG and the SA synthase mutants(sid2-1)were crossed to UGT76D1 overexpression lines.It was found that the HR-like phenotype can be restored in the hybrid lines,suggesting the immune phenotype caused by UGT76D1 is SA dependent.To know the biochemical function of glycosyltransferase encoded by UGT76D1,we conducted extensive substrate screening with the purified UGT76D1 recombinant enzyme.Biochemical assays showed that UGT76D1 could glycosylate 2,3-dihydroxybenzoic acid(2,3-DHBA)and 2,5-DHBA,the major catabolic forms of SA,to their glucose and xylose conjugates in vitro and in vivo.Specific activity analysis revealed that UGT76D1 preferred glucose to xylose as sugar donor in vitro.S3H and S5H are two hydroxylases that catalyze the formation of 2,3-DHBA and 2,5DHBA from SA,which were the substrates of UGT76D1.s3h s5h block the formation of 2,3-DHBA and 2,5-DHBA.Under s3h s5h background,UGT76D1 overexpression did not cause the HR-like lesion mimic phenotype,suggesting the cruicial role of DHBA glycosylation for triggering immune response.Following infection with Pst DC3000,ugt76d1 mutants displayed a delayed immune response,with reduced levels of DHBA glycosides and SA,and down-regulated expression of SA synthase gene ICS1.By contrast,UGT76D1 overexpression lines showed an enhanced immune response and increased SA biosynthesis before and after pathogen infection.Thus,we propose that UGT76D1 plays an important role in SA homeostasis and plant immune responses by facilitating glycosylation of DHBA.2.The glycosyltransferase gene UGT73C7 functions in SNC1-mediated immunity by modulating phenylpropanoid metabolismSUPPRESSOR OF NPR1,CONSTITUTIVE 1(SNC1)is one of the most effective disease resistance proteins in Arabidopsis thaliana.However,the upstream regulatory factors of SNC1,especially at the transcription level,is laregly unkown.Here,our work revealed that Arabidopsis glycosyltransferase gene UGT73C7 could regulate SNC1 expression through modulating phenylpropanoid metabolism.We found that UGT73C7 could be induced by pathogens and SA.Overexpression of UGT73C7 promoted the transcript accumulation of SNC1 and other immunity-related genes and caused a constitutive defense response with the phenotype of dwarf and twisted leaves.Conversely,the loss of function of UGT73C7 substantially reduced SNC1 expression and partially restored the constitutive immunity phenotype conferred by gain-of-function mutant(sncl-1)of SNC1.These results indicated that UGT73C7 may be a positive regulator of SNC1.When UGT73C7 was overexpressed in snc1-11 mutant background(a loss-of-function mutant of SNC1),we found that the phenotype of dwarf and twisted leaves caused by UGT73C7 overexpression was totally suppressed,suggesting that SNC1 acts downstream of UGT73C7.SA is one of the important downstream signal molecules in the immunity activated by SNC1.SA synthase mutant sid2-1 can partially restore the plant immunity activated by UGT73C7,suggesting that the plant immunity activated by UGT73C7 is partially dependent on SA.Since UGT73C7 is a glycosyltransferase,finding the substrate of UGT73C7 will help us further study the mechanism that activates SNC1.We first used online software to predict the putative substrate of UGT73C7 as the phenolic compounds.Then,a widely-targeted metabolomics method was used to analyze more than 200 natual glycosides of ugt73c7 mutants and wild type.It was found that flavonoid glycosides and acylated flavone glycosides accumulated in large amounts in wild type while not detectable in the mutants.The majority of increased acylated flavone glycosides in wide-type were hydroxycinnamic acid derivetives.The connection between flavone and hydroxycinnamic acids is usually an glucosidic bands.In vitro enzymatic reaction results indicated that UGT73C7 can glycoside hydroxycinnamic acids and some flavonoids respectively.In vivo metabolic profiling further showed that the enhanced UGT73C7 expression in overexpression lines could increase the glucose conjugates of hydroxycinnamic acids and hydroxycinnamic acids itselves,especially p-coumaric acids.In the mutant,however,those metabolites decreased compared to wild type.In addition,the contents of cell wall-bound hydroxycinnamic acids were dramatically increased in UGT73C7 overexpression lines,but decreased in ugt73c7 knockout lines.These data suggested that the in vivo activity of UGT73C7 promotes the metabolic flux to hydroxycinnamic acids and their derivatives,and thus has a profound impact on the phenylpropanoid metabolism pathway.To further explore the effect of phenylpropanoid metabolites related to UGT73C7 on the the SNC1 expression,the substrates of UGT73C7,i.e.three hydroxycinnamic acids and flavonoids,were exogenously applied to wild type plants.We found that hydroxycinnamic acids,especially p-counaric acid,could clearly induce SNC1 upregulation,demonstrating the role of hydroxycinnamic acids and their derivatives in regulating SNC1 expression.C4H is the key enzyme responsible for the biosysthesis of(multiple hydroxycinnamic acids and flavonoids)in phenylpropanoid pathway.ref3-3 is an incomplete mutation of C4H.Our data showed that ref3-3 could eliminate the immune response phenotype of UGT73C7 overexpression lines in hybrid lines.Taken together,our findings suggested that the pathogen responsive glycosyltransferase UGT73C7 is involved in SNC1-mediated immunity probably through modulating the phenylpropanoid metabolism pathway.As for the mechanism remains to be answered.