Mechanism Of MYB8 Regulating Ripening Of Banana Fruits During Storage At Low Temperature

Bananas is mainly distributed in tropical and subtropical areas and banana fruits are sensitive to low temperature.Storage at low temperature is prone to cold damage,which seriously affects their commerciality and causes economic losses.Ethylene,as an endogenous hormone in plants,is closely related to fruit ripening and quality formation,in adition,ethylene is also involved in plant stress and initiates a defense response by inducing gene expression to initiate a defense response.MYB transcriptors,widely present in higher plants,participate in the control of cell morphogenesis and the metabolism of plant secondary products,and play an important role in various abiotic stresses,plant growth and development.BAM(β-amylase)is involved in starch hydrolysis,and EXP(expansion protein)is involved in cell wall relaxation during fruit ripening,both of which play an important role in fruit softening.The ripening of banana fruit is delayed when stored at low temperature.Exogenous ethylene treatment before storage at low temperature can reduce the chilling injury of banana fruit storage,however,there are few reports about whether MYB transcription factor is involved and how to regulate the ripening of banana fruit stored at low temperature.In this paper,the two transcription factors of MaMYB8/9 were isolated from‘Brazilian’ banana.The expression characteristics of these two genes during the ripening process of fruits stored at low temperature were detected,and the effect of exogenous ethylene treatment on their expression were also examined,in addition,MaMYB8/9 were localized nucleus and exhibted transcription activation activity,more importantly,the mechanisms of MaMYB8 regulating ripening of banana fruit storage at low temperature by modifying starch degradation and cell wann metabolism were determined.The major results are as follows:1.The basic characteristics of MaMYB8/9 and with relationship to the ripening of fruits storage at low temperature are clarified: Phylogenetic tree analysis,amino acid sequence alignment,subcellular localization,and transcriptional activity analysis in yeast and tobacco showed that MaMYB8/9 is an R2R3-type transcription repressor,located in sub22 subfamily and located in the nucleus.Moreover,the accumulations of MaMYB8/9decreased during the ripening process of banana fruits stored at low temperature.Exogenous ethylene treatment before low temperature storage can significantly inhibit the expression of these two genes in the subsequent ripening process.2.Two E3 ubiquitinated ligase proteins interacting with MaMYB8 were obtained:MaMYB8 interacts with itself and MaMYB9 through yeast two-hybrid(Y2H)screening,meanwhile,two E3 ubiquitin ligase proteins Ma EHA2 A and Ma RMA1 interacting with MaMYB8 were obtained,and expressions of Ma EHA2 A and Ma RMA1 with relation to fruit ripening was studied.In addition,the MaMYB8 interacting with Ma EHA2 A and Ma RMA1 were confirmed by bimolecular fluorescence complementary experiment(Bi FC).3.The molecular mechanisms of MaMYB8 regulating banana fruit ripening were determined: The expressions of β-amylase gene MaBAM2/4/7/8 and the expansion protein gene Ma EXP1/7/8 were up-regulated with fruit ripening and are induced by exogenous ethylene,and validation analysis of gel retardation migration experiment(EMSA)and dual luciferase reporter gene experiment(DLR)showed that MaMYB8 binds and inhibits MaBAM2/7,Ma EXP1/8 promoters,meanwhile,the interacting of E3 ubiquitin ligases Ma EHA2 A and Ma RMA1 interact with MaMYB8 could reduce the inhibitory effect of MaMYB8 on the β-amylase gene MaBAM2.4.The binding motifs of MaMYB8 were identified: using DAP-seq,the motif of MaMYB8 binding target was obtained:(T/C)AAC(T/C/G)(G/A),and this motif is highly consistent with reported results,with high reference value.At the same time,we also obtained a new motif of MaMYB8:(A/T/G)C(A/C/G)GGTA,which needs to be further confirmed.