Regulatory Mechanisms Of MaBZRs Transcription Factors In Controlling Banana Ripening

Banana(Musa acuminate)is a typical climacteric fruit,the ripening of which is controlled by plant hormone and gene expression.However,the ripe fruit are prone to mechanical injuries and pathogen infection,which severely shorten the storage periods.Therefore,studying the molecular mechanism of banana ripening is of both scientific and commercial significance for improving the postharvest quality and prolonging the preservation period of the fruit.Given that ethylene plays a dominant role in the ripening process of harvested banana fruit,whether other plant hormones such as brassinosteroid(BR)along with its signal perception component BZR are involved in this process is still largely unknown.In this context,exogenous brassinosteroid(BR)was used to treat the harvested banana fruit,and three MaBZRs genes namely MaBZR1-MaBZR3 were characterized on the basis of the banana transcriptomic database.Then,the protein sequence,expression pattern,subcellular localization and transcriptional activity of MaBZRs were analyzed.Furthermore,the transcriptional regulation of ethylene biosynthetic genes and cell wall modified genes by MaBZRs were investigated.Finally,the interacting proteins of MaBZRs were also studied.The main results are as follows:1.Exogenous BR treatments accelerate the ripening of harvested bananas.Treatments of 1,2 and 4 μM of BR were able to fastern the ripening of banana fruit,which is probably due to the elevated ethylene production caused by expression of ethylene biosynthetic genes.2.MaBZR1 to MaBZR3 act as transcriptional repressors in banana ripening.Three BZR genes which were named as MaBZR1-MaBZR3 were characterized according to banana transcriptomic database.Subcellular localization and transcriptional activity analyses indicated that MaBZR1 to MaBZR3 located in both nuclus and cytoplasm,and possessed transcriptional inhibitory capacities.The real-time quantitative PCR analysis showed that the expression of MaBZRs were gradually decreased along with the ripening procedure.Moreover,the promoter activities of MaBZR1-MaBZR3 were suppressed in response to exogenous ethylene.Together,these findings indicate that MaBZR1-MaBZR3 function as negative regulators of banana ripening.3.MaBZR1/2 bind directly to and repress the promoters of a subset of ripening-associated genes.Electrophoretic mobility shift assay(EMSA)illustrated that MaBZR1/2 bind specifically to the BRRE elements in the promoters of ethylene biosynthetic genes Ma ACS1,Ma ACO13 and Ma ACO14,as well as cell wall modified genes Ma EXP2,Ma PL2 and Ma XET5.Dual-luciferase reporter assay(DLR)indicated that MaBZR1/2 inhibit the promoter activities of these genes.4.The interacting candidates of MaBZRs were identified.Using the yeast twohybrid and bimolecular fluorescence complementation experiments,a mitogen-activated protein kinase Ma MPK14 was identified as an interacting partner of MaBZR1/2,and these protein-protein interactions were able to enhance MaBZR1/2-mediated transcriptional repression abilities.Additionally,MaBZR3 interacted with Mab ZIP4,which could bind to the promoter of aroma biosynthetic gene Ma AAT1.Collectively,our findings describe the regulatory network of MaBZR transcription factors in banana ripening,which not only expands the understanding of the molecular machinery of fruit ripening,but also provide essential theoretical basis for innovating the postharvest handling strategies in practice.