Histone Deacetylases Are Involved In ERF-Mediated Transcriptional Regulation Of Banana Fruit Ripening

Banana is a climacteric fruit, and easy to become soft when ripe, creating great difficulty for storage and transportation. A lot of studies showed that ERF transcription factor and histone deacetylase(HDAC) played important roles in plant growth and development. Our previous study showed that Ma ERFs regulate banana fruit ripening via transcriptional regulation of ethylene biosynthesis genes Ma ACS1 and Ma ACO1, or interaction with Ma ACO1 protein. However, it is still unclear whether and how HDAC are involved in ERF regulating fruit ripening. In this study, based on Ma ERF11 involved in banana ripening, transcriptional regulation of ERF and HDAC in banana ripening was further studied through electrophoresis mobility shift assay(EMSA), dual-luciferase reporter assay(DLR), yeast two-hybrid(Y2H), bi-molecular fluorescence complementation(Bi FC), co-immunoprecipitation(Co IP), chromatin immunoprecipitation(Ch IP) and so on, which expands the knowledge of transcriptional regulatory network in fruit ripening, especially, HDAC involved in banana fruit ripening. The main results and conclusions are as follows:1. Ma ERF11 has transcriptional repression activity, might regulate ethylene biosynthesis and fruit soften through suppressing Ma ACO1 and Ma EXP2/7/8, further regulate banana fruit ripening. EMSA and Ch IP analyses demonstrated that Ma ERF11 binds to promoters of ethylene biosynthetic gene Ma ACO1 and three soften-related Ma EXP2/7/8. In addition, DLR showed that Ma ERF11 has transcriptional repression activity, and represses Ma ACO1 and Ma EXP2/7/8 promoters activity. The results indicated that Ma ERF11 acts as a transcriptional repressor, and regulates ethylene biosynthesis and fruit soften through suppressing Ma ACO1 and Ma EXP2/7/8, and further regulate ethylene biosynthesis and fruit softening in banana fruit ripening.2. Expression and histone acethylation levels of ripening-related Ma ACO1 and Ma EXP2/7/8 were involved in banana fruit ripening. Real-time quantitative polymerase chain reaction(q RT-PCR) displayed that transcripts of Ma ACO1 and Ma EXP2/7/8 were all increased dramatically in fruit ripening stage. Ch IP showed that the histone H3 and H4 acetylation level of Ma ACO1 and Ma EXP2/7/8 chromatin also increased during fruit ripening, which are correlated with the changes of expression of Ma ACO1 and Ma EXP2/7/8. The results showed that expression and histone acethylation levels of Ma ACO1 and Ma EXP2/7/8 are involved in fruit ripening.3. Ma ERF11 recruits Ma HDA1 to form a transcription repression complex, and both regulated Ma ACO1 and Ma EXP2/7/8. Y2 H, Bi FC and Co-IP assays all proved interaction of Ma ERF11 and Ma HDA1. Furthermore, Ma HDA1 contains a HDAC conserved domain and has histone deacetylase activity, might enhance the transcriptional repression ability of Ma ERF11 to downstream genes Ma ACO1 and Ma EXP2/7/8. The results declared that Ma ERF11 and Ma HDA1 formed a transcription repression complex, and both regulated Ma ACO1 and Ma EXP2/7/8, further regulated banana fruit ripening.4. Seventeen Ma HDACs from banana genome were grouped into RPD3/HDA1, HD2 and SIR2 subfamilies, and displayed different expression patterns during fruit ripening. Seventeen putative Ma HDAC genes were identified from the banana genome, and a phylogenetic tree analysis showed that Ma HDA1-12 all contained HDAC domain, and belonged to RPD3/HDA1 subfamily; Ma HDT1-3 all had MEFWG domain in N terminal, and belonged to HD2 subfamily; Ma SRT1/2 both contained SIR2 domain, and belonged to SIR2 subfamily. q RT-PCR analyses revealed that 17 Ma HDACs exhibited different expression patterns during fruit ripening, especially, Ma HDA6 was the most ripening-induced in three different treatments.5. Ma HDA6 is involved in ethylene signal transduction pathway and fruit ripening. Ch IP exhibited that Ma HDA6 could bind to the promoters of Ma ERF11/15, and acetylation levels of histone H3 and H4 were decreased in Ma ERF11/15 chromatinin ripe banana, which correlated with expression of Ma ERF11/15. These results indicated that Ma HDA6 might regulate expression of Ma ERF11/15 through reducing histone acetylation, further involved in ethylene signal transduction pathway and banana fruit ripening.