Epigenetic Regulation Mechanism Of MaMYB4 Transcription Factor In Low-temperature-affected Ripening Of Harvested Banana Fruit

Banana,as a crucial global cash crop,is also the four major fruits in the world.Temperature affects the ripening and quality formation of postharvest banana fruit.Chilling injury is an urgent problem needed to be solved in the banana industry.Transcription factors play an important role in the ripening and quality formation of postharvest banana fruit,and the network and mechanisms of transcriptional regulatory involvement in fruit ripening have been reported,in recent years,much more attention has been made about the protein modification and epigenetic regulation which are involved in fruit ripening.Previous studies in our laboratory found that Ma ERF11 delayed fruit ripening through recruiting Ma HDA1 to affect the acetylation level of ripening-related genes and further delayed fruit ripening,while,Ma MYB3 couldn’t influence fruit ripening through recruiting or regulating Ma HDAs.MYB is one of the largest transcription factor families in plants,which is involved in plant growth and development,phenylalanine metabolism,adversity stress and hormone response,little is known about the roles of MYB transcription factors in fruit ripening with relation to epigenetic modification.Therefore,clarify more MYB transcription factors affecting fruit ripening and its mechanisms,especially with relation to epigenetic modification has great significance in expanding the regulatory network of fruit ripening.In this study,based on low temperature storage affecting banana fruit ripening process,the MYB transcription factors were selected to study the mechanism of involvement in fruit riepning through transcriptome sequencing(RNA-seq)and bioinformatics analysis.Of the fruit ripening-related MYB transcription factors,Ma MYB4 was chosen to study the potential binding elements and the target genes,using the co-analysis of DNA affinity purification sequencing(DAP-seq)and RNA-seq.and its transcriptional regulation to the target genes was further confirmed by electrophoresis mobility shift assay(EMSA),chromatin immunoprecipitation-quantitative PCR(Ch IP-q PCR)and tobacco transient expression system.Moreover,the ripening characteristics of tomato fruit overexpressed Ma MYB4 was also analyzed and clarified.The main results are listed as follows:(1)MYBs involved in low temperature-affecting fruit quality were obtained and MYB4 was chosen for further study.The appearance of banana fruit storage at 7℃ for 3 days turned browning and fruit ripened abnormally when fruit were returned to room temperature for further ripening,in addition,the ethylene peak,fruit softening and peel yellowing were delayed.The fruit samples at 0 d and 3 d of RT3 E group(Fruit stored at22℃ for 3 d were ripened by ethylene and then storage at 22℃)and LT3 E group(Fruit stored at 7℃ for 3 d were ripened with ethylene and then storage at 22℃)were selected for RNA-seq analysis to construct the expression profile of the effect of low-temperature on fruit ripening.Differentially expressed genes were selected based on FDR<0.05 and|log2FC|>1 analysis,and 55 types of differentially expressed transcription factors(TFs)were filtrated.The top ten differentially expressed TFs family included b HLH,MYB,C2H2,b ZIP,NAC,WRKY,HD-ZIP,G2-like,Dof and MYB-Related,and 130 MYB TFs exhibited in different expression patterens.In addition,986 differentially expressed genes were selected from the four comparison groups of RT3E-0 d vs RT3E-3 d,LT3E-0 d vs LT3E-3 d,RT3E-0 d vs LT3E-0 d and RT3E-3 d vs LT3E-3 d,and the gene expression trends were analyzed according to the order of RT3E-0 d,LT3E-0 d,RT3E-3 d and LT3E-3d and exhibited 20 kinds of gene expression trends.The 8th trend,whose accumulations were induced by low temperature and inhibited by ripening,was the most significant,428 differentially expressed genes were enriched.By intersecting these 428 differentially expressed genes with 130 MYB transcription factors,we obtained five MYB transcription factors whose accumulations were induced by low temperature and inhibited by ripening,and Ma MYB4(Ma01＿g19610)was chosen for further study.(2)Ma MYB4 was involved in the fruit ripening by inhibiting Ma ACS1,Ma XTH5,Ma PG3 and Ma EXPA15.Gene expression and protein accumulation of Ma MYB4 decreased during fruit ripening,while significantly induced in the fruit storage at low temperature.2490 Ma MYB4 binding target genes and two binding elements(motif1 and motif2)were obtained through Ma MYB4-DAP-seq analysis.Of 2490 Ma MYB4 binding target genes,188 were induced by ripening and inhibited significantly by low temperature through co-analysis with RNA-seq.According to the information of banana genome annotation,we selected Ma ACS1 related to ethylene synthesis and Ma XTH5,Ma PG3,Ma EXPA15 related to fruit softening for further study from 188 target genes,and q RT-PCR showed that the expression of Ma ACS1,Ma XTH5,Ma PG3 and Ma EXPA15 were closely associated with fruit ripening.Moreover,EMSA,Ch IP-q PCR and DLR assays showed that Ma MYB4 could bind the promoters of Ma ACS1,Ma XTH5,Ma PG3 and Ma EXPA15 and repressed their expressions.These results determined that Ma MYB4 regulated fruit ripening by inhibiting ethylene synthesis and cell wall metabolism.(3)The ripening process of tomato fruit overexpressed Ma MYB4 was delayed.The stable overexpression of Ma MYB4 in tomato delayed the ripening for 29 days comparision with the WT tomato fruits,and the transgenic tomato fruit was larger than that of wild type.Moreover,the expression patterns of Sl ACS2,Sl XTH5,Sl PG2 and Sl EXP1 were consistent with the changes of ethylene production,which increased at first and decreased during fruit ripening,further suggesting that Ma MYB4 delayed fruit ripening through inhibiting the expression of Sl ACS2 and Sl XTH5,Sl PG2 and Sl EXP1.(4)Ma MYB4 delayed fruit ripening through recruiting Ma HDA2 to affect the acetylation level of ripening-related genes and further delayed fruit ripening.The interaction of Ma HDA2 with Ma MYB4 was obtained,further confirmed by yeast two hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC)assays.In addition,DLR assays showed that Ma HDA2 enhanced the repression ability of Ma MYB4 to effect the expressions of target genes Ma ACS1,Ma XTH5,Ma PG3 and Ma EXPA15.Moreover,the histone acetylation level in the promoters of Ma ACS1,Ma XTH5,Ma PG3 and Ma EXPA15 increased during banana fruit ripening,but decreased in the fruits storage at low temperature,suggesting that Ma MYB4 and Ma HDA2 can form a complex to negatively regulated fruit ripening by inhibiting the expression of ripening-related genes,including Ma ACS1 and Ma XTH5,Ma PG3,Ma EXPA15.In addition,the histone acetylation H3 and H4 level of Sl ACS2,Sl XTH5,Sl PG2 and Sl EXP1 increased during fruit ripening in Ma MYB4 overexpressing tomato and was lower as compared to that of the wild type.These results further indicated that the acetylation levels in the promoters of ripening-related genes increased with fruit ripening,and Ma MYB4 regulated fruit ripening through affecting the acetylation level in the promoters of fruit ripening related genes.