DkHB4 Regulates The Postharvest Softening Of ’Mopan’ Persimmon Fruit

Persimmon(Diospyros kaki)fruit occurs rapid softening after harvest,which limited the fruit postharvest storage and shelf-life.In the present research,’Mopanshi’ was selected as experimental material through ethylene(ETH)treatment.Using the data from the RNA-Seq,through gene cloning,real-time PCR,dual-luciferase,subcellular localization,yeast two-hybrid assays.as well as BIFC,we identified a few ethylene responsive transcription factors and cell wall degradation related genes.Meanwhile,the regulatory roles of these transcription factors on target genes were also investigated.The main results are as follows:1.Isolation and expression analysis of genes related to persimmon fruit softening.According to RNA-seq results,transcription factors and cell wall related genes,which were up-regulated by ethylene treatment,were screened and obtained.Among them,the full-length CDS of 30 transcription factors and 13 cell wall related genes were obtained by RACE technology.Real-time PCR(Q-PCR)was used to verify that the expressions of these genes in postharvest persimmon fruit,and the results indicated that 27 transcription factors and 10 cell wall related genes were significantly induced by ethylene treatment.2.In vivo regulatory effects of selected transcription factors on cell wall related genes.DkHB4,DkGATA3,DkWRKY12 and DkNAC22 were identified to significantly activate the promoters of DkXTH9 and DkPE9 by using the dual-luciferase assay.Cis-element mutation experiments showed that DkHB4,DkGATA3,DkWRKY12 can directly bind DkXTH9 promoter,and DkNAC22 directly binds DkPE9 promoter.3.Analysis of synergistic regulation effect between DkHB4 and DkERFs.DkHB4 was further selected as the target to anaylsis its correlations with the previously characterized softening related DkERF transcription factors.Using dual-luciferase assay,it was found that combination of DkHB4 with individual of DkERF8,DkERF16 and DkERF19 could generate significantly higher activations on DkXTH9 promoter.Furthermore,the results of yeast two-hybridization and BiFC showed the protein-protein interactions between DkERF16 and DkHB4,which such interactions were absent between DkERF8/DkERF19 and DkHB4.In conclusion,this research identified some persimmon softening related transcription factors and cell wall genes.Among them,a few of transcription factors were shown with trans-activation on cell wall genes,such as DkHB4,DkGATA3,DkWRKY12 can regulate DkXTH9 promoter and DkNAC22 can regulate DkPE9 promoter.With specific analysis,DkHB4 not only directly binds to DkXTH9 promoter,but also interacts with DkERF16 and subsequently shows higher activation.These preliminary results indicated the novel transcriptional regulatory mechanism of persimmon fruit postharvest softening.