Gene Regulation Analysis Of Response To Drought Stress By Acer Truncatum Bunge

With the strong adaptability to drought,Acer truncatum Bunge is widely distributed in China as an important economic and ecological tree species.As the key organ for water transportation,xylem is a unique carrier to study the growth of trees and the ability to adaptation to drought.This study focuses on the subject of A.truncatum branch,by monitoring the water physiology of branches dynamically,mapping the light response curves under drought stress to evaluate the photosynthetic capacity,performing transcriptome sequencing and analyzing gene expression to understand the response of A.truncatum to drought stress at the transcriptome level.And TTG1-encoded WD40 transcription factor is an important component of the MYB complex that regulates the flavonoid synthesis.We studied the regulatory methods and regulatory sites of genes related to flavonoid synthesis by TTG1-dependent MYB complex.The results are as follows.1.In the natural drought process of potted A.truncatum,the response rules of physiological and ecological indexes and photosynthetic indexes to soil moisture content are as follows:stems water potential and PLC decreased with the decrease of soil moisture content.Under the mild drought stress,stems water potential and PLC declined slowly,while the apparent quantum yield??_c?,intrinsic quantum efficiency??₀?and water use efficiency?WUE?risen,while the maximum net photosynthetic rate(P_nmax)declined.With the continuous decrease of soil moisture content,stems water potential,PLC declined rapidly,followed by LSP?P_nmax??_c??₀.The WUE had shown no significant difference under severe drought,which was far lower than both under the average and mild drought stress.2.A total of 70,442 Unigenes were obtained after the transcriptome sequencing results were assembled,including 41,089 annotated Unigenes.Among which,13,000 Unigenes were annotated in KEGG database,18,913 Unigenes were annotated in COG database,38,628 Unigenes were annotated in Nr database,and 32,734 Unigenes were annotated in Swissprot database.There were 9,682 Unigenes can be annotated in all the4 databases.On the contrary,29,353 Unigenes could not be annotated in any database.3.The analysis of differentially expressed genes?DEGs?showed that,under drought stress,387 Unigenes expressions were up-regulated and 335 were down-regulated in the transcriptome.While in the re-watering process,42 Unigenes expressions were up-regulated and 19 were down-regulated.Pathway significant enrichment analysis was performed on these differentially expressed genes,which showed that 10 pathways were significantly enriched under drought stress and 5 in the re-watering process.Among which,flavonoid bio-synthesis showed the most significant enrichment in both processes.4.There were 66 annotated Unigenes related to water transportation and response.In this study,the expression of these genes was relatively stable under both the drought stress and re-watering process with no significant differential expression.There were 8 significantly up-regulated genes relative to flavonoid bio-synthesis under drought stress,which were respectively annotated as chalcone synthase?CHS?,anthocyanin synthase?ANS?,anthocyanin reductase?ANR?and flavonoid-3'-hydroxylase?F3?H?.In the re-watering process,4 Unigenes were significantly up-regulated,which were respectively annotated as ANS,ANR,F3?H.5.We performed quantitative real-time PCR?q RT-PCR?to confirm the RNA-Sequencing results.The results showed that there were 1 regulatory gene and 10 structural genes related to flavonoid bio-synthesis,and 9 regulatory genes and 1 structural gene were related to trichome-inducing.From which,there were 4 regulatory genes involved in both trichome formation and flavonoid accumulation with a significant decline of gene expression.6.After DEX treatment of ttg1-13:35S:TTG1-GR,we estimated whether the TTG1-dependent MYB complex could regulate the flavonoid accumulation and trichome formation.It showed a positive result with a significant increase in the expression of TTG2,TT8,F3'H,DFR,ANS,UGT79B1,UGT75C1,5MAT,BLT,ANL2,PAL4,DMR6,FLS3,GSTF12,MYB5 and MYB23.This suggests that the above genes are regulated by TTG1.7.After DEX and CYC treatment of ttg1-13:35S:TTG1-GR,we obtained the direct target genes of TTG1-dependent MYB complex.The expression of TTG2,TT8,F3'H,DFR,ANS,UGT79B1,UGT75C1,5MAT and BLT were dramatically up-regulated.This indicates that these 9 genes are direct target genes of the TTG1-dependent MBW complex.At the same time,in the trichome formation,the direct target gene of the TTG1-dependent MBW complex is the regulatory gene BLT,the indirect regulatory genes are the regulatory gene SPL8,ETC1,HDG11,CPC,MYB106,MYB5,SIM, MYB23,and structural gene SVB.Meanwhile,in the the flavonoid synthesis,the direct target genes of the TTG1-dependent MBW complex are regulatory gene TT8 and structural gene F3?H,DFR,ANS,UGT79B1,UGT75C1,5MAT,and the indirect target genes are the regulatory gene ANL2 and structural gene PAL4,DMR6,FLS3,GSTF12.8.We performed Chromatin Immunoprecipitation?Ch IP?assay to obtain the binding site of TTG1-dependent MBW complex to the target gene.Results showed that the TTG1-dependent MBW complex binds directly to the loci of TTG2,TT8,F3?H,DFR,ANS,UGT79B1,UGT75C1,5MAT and BLT to promote their expression.In summary,under drought stress,flavonoids biosynthetic metabolism was the most active in A.truncatum branches,and its key genes were directly regulated by TTG1-dependent MBW complex transcription factor.Our research results can provide theoretical support for further scientific research on drought tolerance mechanism of A.truncatum,the effect of drought on the accumulation of secondary metabolites,and the practice of tree genetics and breeding,etc.