The Functional Studies Of Histone Modifying Enzymes GmFLD And HDA6

The histone epigenetic modifications(methylation,acetylization et al)are one of the important part in epigenetic studies,and they are very significant for the expression regulation of genes.In plant growth and development,and during the process of plant resistance to biotic and abiotic stresses,the histone epigenetic modification genes play important roles.In this study,we studied two histone epigenetic modification factors,the lysine-specific histone demethylase GmFLD in soybean and the histone deacetylase HDA6 in Arabidopsis,both of which function in repression of the expression of genes,and characterized their functions in plant growth and development(flowering)and in biotic stress(pathogen infection)to explore the contributing molecular mechanisms of histone epigenetic modification factors in life.1.The soybean gene GmFLD can promote flowering in Arabidopsis thalianaFlowering at an appropriate time is crucial for seed maturity and reproductive success in all flowering plants.Soybean(Glycine max)is a typical short day plant,and both photoperiod and autonomous pathway genes exist in soybean genome.Since the whole genome sequence of soybean was released,many photoperiod pathway genes have been cloned and studied.However,there were few reports about the research on soybean autonomous pathway genes.The Arabidopsis FLD(Flowering Locus D)gene is one of the important autonomous pathway genes.FLD encodes a plant ortholog of the human Lys-Specific Demethylase 1(LSD1)protein.FLD functions in histone H3K4 demethylation and H3/H4 deacetylation to repress the expression of FLC.In A.thaliana genome,two other FLD homolog,LSD1-LIKE1(LDL1)and LSD1-LIKE2(LDL2),act in partial redundancy with FLD to repress FLC expression.However,LDL1 and LDL2 act independently of FLD in the silencing of FWA(FLOWERING WAGENINGEN),a homeodomain-containing transcription factor.The sequence homology analysis by BLAST(basic local alignment search tool)showed that,in soybean genome,there are four FLD homologs(E value=0.0):LOC100786453(Glyma02g18610),LOC100810687(Glyma09g31770),LOC100783933(Glyma07g09980)and LOC100809901(Glyma06g38600)with identity of 73%,57%,53%and 52%,respectively.Through the reverse BLAST,the conserved domain assay and the phylogenetic tree assay,we determined that LOC100786453(Glyma02g18610)has the highest homology to the Arabidopsis FLD and is designated as GmFLD,LOC100783933(Glyma07g09980)and LOC100810687(Glyma09g31770)are designated as GmLDL1A and GmLDLIB,and LOC100809901(Glyma06g38600)is designated as GmLDL2,respectively.In this study,we characterized the functions of GmFLD and GmLDL2 in the regulation of plant flowering through genetic and molecular methods.In soybean,GmFLD and GmLDL2 have similar tissue expression patterns:both of them was expressed highly in cotyledons,roots and pods,moderately in seedlings,hypocotyls,epicotyls and flowers,and very lowly in true leaves,including unifoliate and trifoliate leaves.This expression pattern was not absolutely consistent with that of Arabidopsis homogeneous genes FLD and LDL2.Both of the Arabidopsis genes were mainly expressed in root tip and stem tip.The subcellular localization analysis showed that both of GmFLD and GmLDL2 localized in the plant cell nuclei.When was over-expressed in Arabidopsis,GmFLD could promote plant flowering and it could complement the late flowering phenotype of Arabidopsis fld mutant.Our results demonstrated that in GmFLD transgenic plants(Col or fld background),the FLC(FLOWERING LOCUS C)transcript levels decreased whereas the floral integrators,FT and SOC1,were up-regulated when compared with the corresponding non-transgenic genotypes.However,the transgenic Arabidopsis plants of over-expressed GmLDL2 did not show any flowering associated phenotype.Furthermore,chromatin immuno-precipitation(ChIP)analysis showed that in the transgenic rescued lines(fld background),the levels of both tri-methylation of histone H3 Lys-4 and acetylation of H4 decreased significantly around the transcriptional start site of FLC.This is consistent with the function of GmFLD as a histone demethylase.Taken together,our results suggest that GmFLD is a functional ortholog of the Arabidopsis FLD and may play an important role in the regulation of chromatin state in soybean.The present data provides the first evidence for the evolutionary conservation of the components in the autonomous pathway in soybean.2.HDA6 could suppress the expression of the pathogenic reaction associated genesThe histone deacetylases(HDAC),which are exist extensively in procaryotic and eukaryotic organisms,are a class of enzymes that remove acetyl groups from a lysine amino acid on a histone,allowing the histone to wrap the DNA more tightly.They play important roles in the histone modifications and the expression regulation of genes.The HDA6 is one of the histone deacetylases,which plays important roles in plant growth and development and response to biotic and abiotic stresses.However,in-depth studies are also needed to elucidate its comprehensive functions and the contributing molecular mechanism.In the previous studies,through the EMS mutagenesis and the map-based cloning,our lab obtained a new Arabidopsis HDA6 mutant 1502.In this study,we found that the 1502 mutant plants show pleiotropic phenotype:plants are short and small and most of the organs,such as leaves and pods,accordingly become small;the flowering time is delayed;the flowers develop abnormally;the stamen become short,the anthers are far away from stigma and the dehiscence is blocked that resulted in lower percentage of filled grain;the color of seed coat turn into yellow and the thousand seed weight become light.In the 1502 mutant,the expression of the exogenous genes(hygromycin resistance gene and luciferase marker gene)are activated.The ChIP experiment demonstrated that,compared to wild type 9-1,the histone acetylization modification level of the exogenous genes was obliviously up-regulated in the 1502 plants.The more interesting is that the cotyledons of the seedlings and the early rosette leaves of 1502 mutants show the phenotype similar to pathogenic hypersensitivity response,such as etiolation and dying gradually,which was not reported in other HDA6 mutants identified by previous researchers.In order to find the downstream genes regulated by HDA6 to characterize the HDA6 functions and explain the phenotype of the 1502 mutants,we performed transcriptome sequencing assay.The transcriptome sequencing results showed that,compared to wild type 9-1,the expression of 329 genes were up-regulated in 1502 mutant plants.Among these genes,the annotation functions of 66 genes are associated with biotic stress stimulus.These genes include 17 hypersensitivity response associated genes and 5 WRKY family genes.Through RT-PCR,we further confirmed that the expression of these genes were up-regulated in 1502 mutants and recovered to the level of wild type in complementing plants.Then,we randomly selected 8 hypersensitivity response associated genes to perform ChIP assay.The ChIP results showed that the acylation levels around the transcriptional start sites of these gene were obviously up-regulated suggesting that they are the target genes directly regulated by HDA6 histone deacetylase activity.The pathogen infection experiment showed that,compared to wild type 9-1,the ability of resistance to the pathogen(Pst DC3000)was enhanced in 1502 mutants suggesting that HDA6 negatively regulates plant resistant response and represses the expression of pathogenic response associated genes in noninfectious conditions.