Functional Study Of Histone Deacetylase Genes SlHDA1 And SlHDT3 In Tomato

Histone acetylation and histone deacetylation on lysine residues is a reversible biological process which depends on the action of histone acetyltransferases and histone deacetylases.The reduced ionic interactions between the positively charged histone and the negatively charged DNA backbone and the reduced internucleosomal interactions make histone hyperacetylation to be a more advantageous chromatin structure correlating with gene transcription.In general,histone acetylation is associated with increased gene activity,whereas histone deacetylation is related to transcriptional repression.The level of histone acetylation is regulated by the opposing activities of histone acetyltransferases(HATs)and histone deacetylases(HDACs).Studies performed in different plant species have shown that histone acetylation is associated with several aspects of development.Different plant HDACs have been characterized in detail,especially in Arabidopsis and rice.However,histone deacetylase genes have not been functionally analyzed in tomato to date.Based on the sequence similarity and the dependency on co-factors,HDACs in all eukaryotes are divided into three subfamilies: RPD3/HDA1 subfamily,HD2 subfamily and SIR2 subfamily.There are 18 HDAC genes in Arabidopsis,18 HDAC genes in rice,5 HDAC genes in maize and 14 HDAC genes in tomato.In this study,14 SlHDAC genes encoding putative SlHDACs were isolated and characterized.Nine of which belong to RPD3/HDA1 subfamily(SlHDA1-SlHDA9),three of which belong to HD2 subfamily(SlHDT1-SlHDT3),and two of which belong to SIR2 subfamily(SIR1 and SIR2).Alignment of the amino acid sequences of these nine tomato histone deacetylases suggested that they contain a typical deacetylase catalytic domain.An analysis of tissue-specific expression of these SlHDAC genes was performed using quantitative RT-PCR.The results suggesting that they may have diverse and crucial roles during tomato growth and development.In addition,we further investigated their expression profiles under multiple abiotic stresses(NaCl,dehydration,and high/low temperature).Varying degrees of induction were detected in the expression of these Sl HDAC genes.Our results provide valuable information for further investigation of these SlHDAC genes in tomato growth,development and in response to abiotic stress,and one or more of them may hold promise for improving stress tolerance in tomato via genetic engineering.Then we picked out the SlHDA1 gene by gene expression pattern analysis whose transcripts show high accumulation in ripening fruits,indicating that SlHDA1 may be involved in the regulation of fruit ripening.In our study,an RNA interference(RNAi)expression vector targeting SlHDA1 was constructed and transformed into tomato plants.Shorter fruit ripening time and decreased storability were observed in Sl HDA1 RNAi lines.The accumulation of carotenoid was increased through an alteration of the carotenoid pathway flux.Ethylene content,ethylene biosynthesis genes(ACS2,ACS4 and ACO1,ACO3)and ripening-associated genes(RIN,E4,E8,Cnr,TAGL1,PG,Pti4 and LOXB)were significantly up-regulated in SlHDA1 RNAi lines.In addition,the expression of fruit cell wall metabolism genes(HEX,MAN,TBG4,XTH5 and XYL)was enhanced compared with wild type.Furthermore,SlHDA1 RNAi seedlings displayed shorter hypocotyls and were more sensitive to ACC(1-aminocyclopropane-1-carboxylate)than the wild type.The results of our study indicate that SlHDA1 functions as a negative regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation.Crop growth and productivity were often limited by the abiotic stresses,especially salinity and drought.The genes of histone deacetylase play important roles in various stress responses.However,to date only little information regarding stress-related histone deacetylase genes is available in tomato.Sl HDA1-SlHDA9 involved in response of various abiotic stresses have been reported in our previously study.The expression of SlHDA1 was also induced significantly by NaCl and drought stress.To gain further insight into the function of the SlHDA1 gene in response to abiotic stress,we investigated the effects of salt and drought stress on wild-type and SlHDA1-RNAi transgenic tomato plants.The results indicated that the growth of roots and hypocotyls in RNAi plants were more inhibited by ABA and salt stress compared with that of wild-type at post-germination stage,and transgenic plants became less tolerant to salt and drought stress in soil,which were demonstrated by higher water loss rate,lower levels of chlorophyll and water contents in their leaves compared with wild-type plants under stressed conditions.In addition,multiple stress-related genes were down-regulated in SlHDA1-RNAi plants under both control and salt-stressed conditions.Together,these results highlighted the important role of SlHDA1 in the positive modulation of salt-stress tolerance,possibly through an ABA-dependent signaling network,and may have promising applications in the engineering of salt-and drought-tolerant tomato.The HD2 proteins are plant-specific HDACs and are involved in plant responses to abiotic stresses in different plant species.Phylogenetic and amino acid homology analysis showed that SlHD2 s was homologous to At HD2 s and possessed the same active sites as other plant HDACs.The results of tissue-specific expression analysis of SlHDT1 and SlHDT3 genes suggesting that they are tissue-specific expression profiles in fruits and they may have crucial roles during tomato development and ripening.In addition,we further investigated their expression profiles under multiple abiotic stresses(NaCl,dehydration,and high/low temperature).Varying degrees of induction were detected in the expression of SlHDT1 and Sl HDT3 genes.With the aim of characterizing the tomato HD2 family of histone deacetylases,an RNA interference(RNAi)expression vector of SlHDT3 was constructed and transformed into tomato plants.The time of fruit ripening was delayed and the shelf life of the fruit was prolonged in SlHDT3 RNAi lines.The accumulation of carotenoid was decreased by altering of the carotenoid pathway flux.Ethylene content was also reduced and expression of ethylene biosynthetic genes(ACS2,ACS4 and ACO1,ACO3)and ripening-associated genes(RIN,E4,E8,PG,Pti4 and LOXB)was significantly down-regulated in SlHDT3 RNAi lines.The expression of genes involved in fruit cell wall metabolism(HEX,MAN,TBG4,XTH5 and XYL)was inhibited compared with wild type.These results indicate that SlHDT3 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation and that SlHDT3 lies upstream of SlMADS-RIN in the fruit ripening regulatory network.In summary,nine histone deacetylase RPD3/HDA1 subfamily genes were identified in this study,and we focus on analyzing the potential functions of SlHDA1 in fruit ripening and abiotic stress response and the HD2 s subfamily gene SlHDT3 in fruit ripening,which provided a set of significant data and important clues for elucidation of the functions of SlHDA1 and SlHDT3 in tomato and laid a foundation for potential applications in genetic engineering.