Transcriptional Regulation Of Postharvest Kiwifruit Starch Degradation By AdDof3

Ripening and softening are the main internal factors that determine postharvest shelf-life of fruit.'Hayward'(Actinidia deliciosa[A.Chev.]C.F.Liang et A.R.Ferguson var.deliciosa cv.Hayward)were used to investigate kiwifruit ripening transcriptional regulatory mechanism.Research methods such as RNA-seq,real-time PCR,promoter motif analysis,dual-luciferase assays,EMSA,as well as transient and stable over-expression analysis were used to screen and identify the function of genes involving in kiwifruit ripening.It provides a new basis mechanism for kiwifruit postharvest softening.The main results are as follows:1.Many physiological index were measured in different treated kiwifruit,including ethylene production,total soluble solid content,starch content,firmness and cell wall material content.A transcriptomics-based approach was used to identify transcriptional regulatory components associated with texture,ethylene and starch degradation in ripening kiwifruit.Twelve differentially expressed structural genes including seven involved in cell wall metabolism,four in ethylene biosynthesis and one in starch degradation and 14 transcription factors(TFs)induced by exogenous ethylene treatment and inhibited by the ethylene perception signalling inhibitor 1-methylcyclopropene were identified as changing intranscript levels during ripening.Moreover,analysis of the regulatory effects of differentially expressed genes(DEGs)identified a zinc finger TF,DNA BINDING WITH ONE FINGER(AdDof3),which showed significant trans-activation on the AdBAM3L(?-amylase)promoter.AdDof3 physically interacted with the AdBAM3L promoter and stable overexpression of AdBAM3L resulted in lower starch content in transgenic kiwifruit leaves,suggesting that AdBAM3L is a key gene for starch degradation.Moreover,transient overexpression analysis showed that AdDof3 upregulated AdBAM3L expression in kiwifruit.Thus,transcriptomics analysis facilitated characterization of a TF,AdDof3,and a key structural gene,AdBAM3L,in starch degradation.2.AdERF family members were isolated and obtained from the kiwifruit genome database(http://bioinfo.bti.cornell.edu).Phylogenetic and MEME analyses indicated that kiwifruit ERF genes are distributed in different subfamilies and all have single AP2/ERF domain.Moreover,some ERF members were putative repressors,as indicated by the presence of EAR motifs.The relationship between the ERF family and kiwifruit ripening we investigated with C2H4 treatment and CA storage.Gene expression analyses pointed out eight ripening related ERF genes,including four putative activators(AdERF10/14/15/75)and four putative repressors(AdERF7/8/19/21)of ripening.In conclusion,an ethylene responsive C2H2-type zinc finger transcription factor,AdDof3,regulates starch degradation in kiwifruit via trans-activationof the AdBAM3L promoter.Moreover,a few of other transcription factors were also characterized as the potential regulators for kiwifruit ripeing.