Cloning And Functional Analysis Of SlHDA1/SlHDA3 In Tomato During Fruit Development And Ripening

Histone acetylation and deacetylation are integral components of the epigenetic network in many eukaryotes and play crucial roles in the regulation of eukaryotic gene activity through histone acetylation and deacetylation at the N-terminus of histone tails.A growing number of studies have demonstrated the importance of histone acetylation/deacetylation on genome stability,transcriptional regulation,development and response to stress in Arabidopsis.However,less known about the regulatory mechanism of histone acetylation and deacetylation in fruit development and ripening.Tomato(Solanum lycopersicum)is an ideal model plant for fruit development and ripening research,which has many specific characteristics and advantages.SlHDA1 and SlHDA3 were reported expressed highly in tomato Break and ripening stage,and both may participate in fruit ripening process.Therefore,tomato was utilized as raw material for the study of SlHDA1 and SlHDA3 functions in tomato fruit development and ripening.These results will provide basis of experimental research to illuminating the molecular mechanism of epigenetic regulation in fruit development and ripening mediated by SlHDA1 or SlHDA3.Also,the study will make foundation for improving fruit quality by epigenetic regulation.In this study,Sl HDA1 and SlHDA3 were cloned from tamato genome,and the gene structure and protein conserved domain were analyzed.We analyzed the phylogenetic relationship of HDACs family proteins in tamato and Arabidopsis.We also analyzed the subcellular of SlHDA1 and SlHDA3 protein as well as the temporal and spatial expression patterns of the two genes.In addition,we analyzed the promoter information of the two genes and investigated the gene expression patterns under different treatments.To investigate the biological functions of the two genes in tomato fruit development and ripening stage,we constructed the overexpression vectors and RNAi suppression expression vectors for tomato transformation.More,we utilized the yeast two-hybrid method to screen the transcription factors which could interact with SlHDA1 or SlHDA3 for the biological functions analysis in the progress of fruit development and ripening.The main results are as follows:1.Gene structure analysis found that the length of SlHDA1 is 1497 bp,which contained 8 extrons,7 introns and encoded a polypeptide of 498 amino acids;the length of SlHDA3 was 1416 bp,which contained 6 extrons,5 introns and encoded a polypeptide of 471 amino acids.Based on the analysis of protein conserved domain,SlHDA1 and SlHDA3 contained the typical Arginases domain and HDACs domain as well as some metal binding sites.2.To address the subcellular localization of SlHDA1 and SlHDA3 proteins,we generated SlHDA1/ Sl HDA3: GFP fusion protein constructs and transiently expressed in BY2 tobacco cells.Laser scanning confocal microscopy analysis showed that the GFP signal for SlHDA1: GFP and SlHDA3: GFP fusion protein were exclusively localized to the nucleus.3.Based on online analysis and Real-time PCR,we investigated the temporal and spatial expression patterns of SlHDA1 and SlHDA3.These results indicated that SlHDA1 and SlHDA3 mainly expressed in floral ogans and fruits,and during the developmental stage of ovary or fruit,two genes highly expressed at Break and ripening period.4.Promoter function components analysis found that there were methyl jasmonic acid and gibberellin response components as well as some adversity related response components.These different treatments results showed that both SlHDA1 and SlHDA3 regulated by GA3 and SlHDA3 regulated by MeJA.In addition,both genes may participate in the progress of plant response to temperature and salt stress.5.Through the phenotype analysis in the overexpression and RNAi plants of these two genes,we found that overexpression of SlHDA1 and SlHDA3 induced smaller fruit and less seeds than wild type.And the SlHDA3-RNAi plants displayed pigment distribution asymmetrically on the surface of fruits,and the over ripening fruits displayed orange red rather than deep red.6.Yeast two-hybrid results showed that both SlHDA1 and SlHDA3 proteins could interact with two fruit development and ripening related transcription factors SlGRAS9 and SlGRAS40,moreover a zinc finger protein SlZF1 could interact with SlHDA1 too.The interactions among these proteins may take functions by regulating downstream genes expression to influence the progress of fruit development and ripening.