Histone Epigenetic Methylation Regulation Of Flowering Core Gene FT And FLC In Arabidopsis Exerted By ?-aminobutyric Acid

?-Aminobutyric acid(GABA)is an important signal molecule in regulating plant development,exogenous GABA can promote Arabidopsis thaliana flowering.In plants,FT(FLOWERING LOCUS T)and FLC(FLOWERING LOCUS C)are the core genes in controlling flowering,and their expression is modulated by epigenetic regulation of H3 histone methylation.However,the direct link of this regulating mechanism between GABA and H3 histone methylation largely remains unknown.In this study,five-week Arabidopsis thaliana leaves were used as the materials for genome chromatin extraction,the antibodies of H3K4me3,H3K27me3 and H3K36me3 were specifically used for Chromatin immunoprecipitation(CHIP).The purpose of this CHIP-PCR technology is trying to clarify the link between GABA and H3 histone methylation of FT and FLC around transcriptional starting site(TSS)and in gene body area.The results are as follows:1.GABA(1.0 mmol/L)was foliage spraying in another day from 2-week seedlings until flowering.The flowering time of plants treated by GABA was significantly earlier than that in the control,flowering time is 33 days in GABA treatment versus 28 days of the control,average 5 days is in advance than the control;The average height of GABA treated plants is 38.94 cm versus 26.27 cm in the control(P=0.003);2.FT encodes a key factor to promote flowering in the photoperiodic pathways.The results of CHIP-PCR showed that around the transcriptional start site(TSS,-63 bp-+64 bp)of FT,the level of H3K4me3 in the GABA treatment plant is 1.20 fold of that in the control,with no significant difference(P=0.12);However,the level of H3K36me3 in the GABA treatment proup is 2.02 fold of that in the control group,reaching the extremely significant difference level(P=0.002);H3K27me3 is a type of histone modifications which's function is to inhibit the expression of the genes.H3K27me3 level in the GABA treatmented plant is 0.67 fold of that in the control,with no significant difference(P=0.40);3.In the gene body area(+1477 bp-+1583 bp)of FT,GABA has different effect on H3 histone methylation.H3K4me3 level in the treatment plant is 1.50 fold that in the control group,having a significant difference(P=0.03);H3K36me3 level is 1.26 fold of that in the control group,with no significant difference(P=0.37);H3K27me3 level is 0.86 fold of that in the control,with no significant difference(P=0.56);4.In double mutant of GABA synthesis gad1-2 plants,and around the FT TSS(-63 bp-+64 bp),the level of H3K4me3 is 0.29 fold of that in the control group,reaching a very significant difference(P=0.003);H3K36me3 and H3K27me3 level is 0.31 fold and 0.34 fold of that in the control,respectively,both with the extremely significant difference(P value is 0.007 and 0.04,respectively);5.In the gene body area(1477 bp to 1583 bp)of FT,in the GABA synthesis double mutant of gad1-2,H3K4me3 levels is 0.3 fold of that of in the control,H3K36me3 is 0.18 fold and H3K27me3 fold is 0.48 fold of that in the control,all reaching the significantly difference(P value is 0.03,0.008 and 0.03,respectively);6.FLC is negative factor to control flowering.The results of CHIP-PCR showed that around the transcription starting site(TSS,-54 bp-+58 bp)of FLC,H3K4me3 level in the GABA treatmented plant is 1.84 fold of that in the control,with significant difference(P= 0.04);the level of H3K36me3 in the GABA treatmented group is 2.09 fold of that in the control,having significant difference(P=0.02),and H3K27me3 level is 1.24 fold of that in the control,with no significant difference(P =0.28);7.Around TSS(-54 bp-+58 bp)of FLC in the gad1-2 double mutant of GABA synthesis,H3K4me3 level is 0.29 fold of that in the control,reaching significant difference level(P= 0.03),and the level of H3K36me3 is 0.38 fold of that in the control,with significant difference(P=0.034).However,the level of H3K27me3 is 0.63 fold of that in the control,with no significant difference(P=0.33).Conclusion:1)Around TSS(-63 bp-+64 bp)of FT,as for the histone epigenetic regulation,exogenous GABA mainly regulates H3K36me3 level,and in gene body(1477 bp-1583 bp)of FT,external GABA mainly regulating H3K4me3 levels;2)Around TSS(-54 bp-+58 bp)of FLC,as for the histone epigenetic regulation,exogenous GABA significantly enhance both the level of H3K4me3 and H3K36me3;3)In the gad1-2 double mutant of GABA synthesis,the histone epigenetic modification of H3K4me3 and H3K36me3 are markedly decreased in the around TSS of FT,FLC and in the gene body of FT.The results aforementioned showed that the synthesis of endogenous GABA metabolism directly or indirectly affects the histone methylation epigenetic modifications,and exogenous GABA could stimulate Arabidopsis early flowering directly linked with the expression of FT,a key gene in the photoperiodic flowering control.Exogenous GABA participated in the FT gene expression,depending on modulating the histone methylation modifications of FT.This regulation increases the level of H3K36me3 around TSS and the level of H3K4me3 in the gene body area of FT.However,the complex epigenetic histone regulation of FLC still needs further study.