Molecular Characterization Of MYB Transcription Factors That Are Involved In Starch Degradation During Banana Fruit Ripening

Banana fruit ripening is an intricate process with obvious physiological changes,and the harvested bananas exhibit rapid ripening,leading to a very short storage life.Starch degradation occurs during the ripening of banana fruit,which results in texture softening and sugars production.It is well-known that quite a lot of genes play important roles in regulating fruit ripening.Among these genes,transcription factors are one of the focus issues in fruit ripening research,which regulate the expression of downstream genes through transcriptional activation or inhibition of their activities.In the current work,RNA-Seq were performed using the banana fruit with green maturity,color turning and fully ripening stages,and the ripening-induced transcription factors LBDs and BSD were selected to varify the RNA-Seq results.Then MYB transcription factors were characterized from banana fruit and their roles in starch degradation during banana fruit ripening were also studied by qRT-PCR,electrophoretic mobility shift assay(EMSA),dual-luciferase reporter assay(DLR),protein-protein interation,DNA methylation detection,chromatin immunoprecipitaion(ChIP)approaches.Our results provide novel insights into the regulatory network of tanscription factors in regulating starch degradation during banana fruit ripening,and DNA methylation is also involved in the process.The main results and conclusion are as follows:1.The RNA-seq databases of banana fruit during ripening were available,and some ripening-induced transcription factors MaLBD1/2/3 and MaBSD1 were selected to confirm the results of RNA-Seq.A total of 12,774 differentially expressed genes were identified based on the RNA-Seq databases,which could be divided into eight expression trends.A large number of transcription factors related to the ripening of banana fruit were found,in which the ripening-induced transcription factors LBDs(MaLBD1/2/3)and BSD(MaBSD1)were selected to confirm the RNA-Seq results.qRT-PCR analyses indicated that the accumulation of MaLBD1/2/3 and MaBSD1 was increased along with the progression of fruit ripening.Moreover,MaLBD1/2/3 and MaBSD1 acted as transcriptional activators in banana ripening at least partially throuth activating the transcription of cell-wall modified genes MaEXP1/2.2.The possible mechanisms underlying MaMYBs in controlling starch breakdown during banana ripening were studied.Banana fruits under low temperature storage were ripened by ethylene treatment,with inhibition of peel color turning,the endogenous ethylene production,texture softening and starch degradation comared to the control fruits stored at room temperature.qRT-PCR assays showed that the negative regulators MaMYB3/4/32 were induced by low temperature treatment while the starch degradation-related genes MaAMY3,MaBAM2,MaGWD1 and MaPWD1 were inhibited by the treatment.Moreover,protein-protein interaction analyses indicated that MaMYB4,MaMYB3 and MaMYB32 interacted with each other.Transient expression assays showed that MaMYB3/4/32 which contained the Ethylene-responsive element binding factor-associated Amphiphilic Repression(EAR)domain possessed transcriptional inhibitory activities,and they could inhibit the promoter activities of some starch breakdown genes MaAMY3,MaBAM2,MaGWD1 and MaPWD1.More importantly,the interactions between MaMYB4,MaMYB3 and MaMYB32 could enhance the inhibition of these genes,possibly due to the forming protein complexes.It was found that the interactions between MaMYB4-mEAR,MaMYB3-mEAR,and MaMYB32-mEAR occurred when the EAR motif mutated,but their inhibitory abilities were disappeared.These results suggested that low temperature treatment could delay the banana fruit ripening,and that MaMYB3/4/32 could control the banana fruit ripening by transcriptional regulation of the starch degradation genes MaAMY3,MaBAM2,MaGWD1 and MaPWD1.3.The DNA methylation and transcriptome in three key periods of banana fruit ripening were sequenced and analyzed.Results showed that inverse patterns between DNA methylation level and transcripts were found in some genes,which are related to the plant hormone signal transduction pathway by using pathway enrichment analysis,and MaAMY3,MaBAM2,MaGWD1 and MaPWD1 genes were found in this pathway.Furthermore,the DNA methylation level in their promoter was negatively correlated with their transcripts level.These results suggest that the DNA methylation is an important machinary that affects the expression of these starch degradation genes.