Function And Molecular Mechanisms Of Glycosyltransferase Genes UGT76F1 And UGT71C3 In Arabidopsis Thaliana

During the long evolution process of plants,the surrounding environmental factors provides motivation for the adaptive growth of plants under natural selection,forming a bilateral influence between plants and environmental signals.When sensing external environments such as changing light signals,temperature signals,soil osmotic pressure and metal ions,plants not only make changes in the growth and development in order to adapt to these changes(such as changing plant height,leaf shape,and branches,etc.),but also stimulates the synthesis of secondary metabolites of plants.Secondary metabolism is a result of the interaction of plants with biotic and abiotic factors during long-term evolution.Glycosylation of secondary metabolites is a common modification of small molecule compounds,which is widely present in plant kingdom.It is a class of UDP-glycosyltransferases(UGTs)that catalyze this reaction.This type of glycosyltransferases has a wide range of biological functions in plants.Here,we focused on two glycosyltransferase genes,UGT76F1 and UGT71C.3,and clarified their role in regulating light and temperature-mediated adaptive growth and systemic acquired resistance in plants.The revealed molecular mechanism not only lays an important theoretical foundation for a deep understanding of the interaction between plants and biotic or abiotic environments,but also provides a reference for genetic breeding and gene editing of agricultural and forestry crops.The main contents and results of this research are as follows:1.UGT76F1 gene is involved in light-temperature mediated plant hypocotyl growth through IPyA glycosylation modification.By screening members in the Arabidopsis UGTs family that can respond to light signals,it was found that the UGT76F1 gene was significantly up-regulated by light induction(https://genevestigator.com).Therefore,the gene UGT76F1 was cloned,and its overexpression vector was constructed.A number of independent and over-expressing transgenic homozygous strains were obtained through genetic transformation.At the same time,CRISPR-Cas9 technology was used to obtain multiple independent homozygous mutant lines with base deletion or base insertion Under white light,red light,and blue light conditions,it was found that the ugt76fl mutant exhibited longer hypocotyls than the wild type,while the overexpression lines(OE lines)showed opposite changes.What's more,the UGT76F1 OE lines grown under dark conditions were more prone to cotyledon opening than WT.In order to confirm whether UGT76F1 participates in the light signal pathway,the expression of UGT76F1 in the photoreceptor mutant phyA,phyB,crylcry2 was investigated and it was demonstrated that UGT76F1 is regulated by multiple photoreceptors.On the other hand,the UGT76F1 gene was up-regulated in the pif4 mutant and down-regulated in the 35S:PIF4 plants,suggesting that this gene was negatively regulated by PIF4.Yeast one-hybrid experiments,ChIP-qPCR experiments,and Dual-luciferase reporter gene experiments further confirmed that PIF4 directly binds to the UGT76F1 promoter and negatively regulates UGT76F1 expression.Because PIF4 responds to both the light signal and the high temperature in the environment,it is speculated that UGT76F1 is also regulated by high temperature.Therefore,the expression level of UGT76F1 gene was detected in pif4 mutants and WT at 22? and 28?,respectively.It was found that the transcription of UGT76F1 was inhibited at 28? and was dependent on PIF4.Our genetic experiments have also confirmed that the pif4ugt76f1 double mutant can restore the short hypocotyl phenotype of the pif4 mutant,while 35S:PIF4 35S:UGT76F1 plants exhibited inhibition of the long hypocotyl phenotype caused by PIF4 overexpression.In order to understand the molecular mechanism of the UGT76F1 gene,biochemical experiments and metabolic analysis in vivo and in vitro were conducted and revealed that UGT76F1 can specifically glycosylate the main auxin precursor Indole-3-pyruvic acid(IPyA)to form IPyA glucoside(IPyA-Glc),but has no activity on active auxin(IAA)itself.Analysis of IPyA-Glc in the wild-type,ugt76f1 mutants and OE lines revealed that the biochemical function was consistent with that in vitro.The mutants produced less IPyA-Glc,while the OE lines accumulated more IPyA-Glc.At the same time,the level of IPyA-Glc in the pif4 mutant was also analyzed,and it was found that the pif4 mutant had a significantly increased IPyA-Glc level than wild type.This result is consistent with the conclusion that PIF4 negatively regulates UGT76F1.In order to analyze whether UGT76F1 regulates the auxin homeostasis,quantitative analysis of IAA in plants was performed by LC/MS.Loss function of UGT76F1 significantly increased the accumulation of IAA,while over-expressing UGT76F1 reduced endogenous IAA level.Consistent with this,DR5:GUS staining showed an increased auxin signal in the ugt76fl mutants and a decreased signal in overexpression lines.This results indicates that UGT76F1 can control the metabolic flow of IAA biosynthesis through IPyA glycosylation and participate in the auxin homeostasis.Previous studies have reported that PIF4 is an integrative factor of the light-temperature signaling pathway and PIF4 positively regulate the key enzyme YUCs for auxin synthesis.Because UGT76F1 is negatively regulated by PIF4,we investigated the different effects of UGT76F1 and YUCs on plant growth,confirming that UGT76F1 has an antagonistic effect on YUCs in controlling hypocotyl growthIn summary,the genetic and biochemical data in this part indicate that UGT76F1 is involved in the regulation of auxin level and light-temperature mediated plant hypocotyl elongation through IPyA glycosylation.IPyA glycosylation working as a key "watershed" in regulating auxin synthesis plays an important role in the plant responses to environmental changes and the maintenance of auxin dynamic balance2.UGT71C3 gene is involved in plant system acquired resistance through MeSA glycosylation modificationIn the previous work of this laboratory,Master Xiafei Meng proved that Arabidopsis glycosyltransferase UGT7I C3 can glycosylate MeSA in vitro and in vivo.It is inactive against the structural analogues of MeSA,SA,benzoic acid and methyl benzoate and initially showed that the enzyme may be involved in the SAR of plants However,research data were not systematic and complete.In this study,based on previous research,the molecular mechanism of UGT71C3 participating in SAR was explored in depth,some data were supplemented and improved,and also others were re-verified.Firstly,the expression pattern of UGT71C3 gene in Arabidopsis was investigated,and it was found that UGT71C3 gene can be strongly induced by SA and Pseudomonas syringae pv.tomato DC3000(Pst DC3000)/avrRpt2.And this gene showed an inducible expression pattern in the true leaves of young seedlings and adult seedlings,and a constitutive expression pattern in the roots of plants,indicating that the gene may be involved in the response to pathogenic bacteria in leaf tissues.MeSA glycoside standard was used to compare with enzymatic reaction product of UGT71C3 towards MeSA through LC/MS and both results are consistent.It is speculated that MeSA may be a natural substrate of glycosyltransferase UGT71C3 in vitro.In order to further study the biological function of UGT71C3 in vivo,this study obtained a new mutant using CRISPR-Cas9 technology in addition to the previous T-DNA insertion mutant.UGT71C3 mutants and overexpressions were used to detect their SAR response.After inoculation with the pathogen Pst DC3000/avrRpt2 or Pseudomonas syringae pv.maculicola ES4326(Psm ES4326)/avrRpt2,the SAR response of the ugt71c3 mutants were enhanced,while the SAR response of the OE lines were weakened compared to wild type plants.These data indicate that UGT71C3 negatively regulates the SAR immune response in plants.Based on this result,we further analyzed the content of MeSA in mutants and OE lines by gas chromatography.It was found that OE lines significantly reduced MeSA accumulation compared to wild type,while the ugt71c3 mutants accumulated much more.Since the initiation of the SAR response is ultimately caused by SA,we tested the SA content in the systemic leaves after inoculating pathogenic bacteria towards the local leaves.Experiment results showed that the ugt71c3 mutants accumulated much more free and total SA than wild type,while UGT71C3 OE lines accumulated much less free and total SA.This result indicates that UGT71C3 affects the SA level through MeSA glycosylation.Consistent with this,SAR-related disease resistance genes were found to be significantly up-regulated in the ugt71c3 mutants,but significantly down-regulated in OE lines.The results in this part indicate that UGT71C3 could enhance the glycosylation of the signal molecule MeSA,decrease the SA level and negatively regulate the SAR response after pathogenic bacteria infection.This negative regulation role of UGT71C3 may help to ensure plant systemic acquired resistance at an appropriate level,and may be a fine regulatory mechanism for plant SAR responses.Therefore,this study enriches the theory of SAR regulation,and also provides inspiration for the study of potential negative regulations through glycosylation of other immune signaling molecules in plants.