| The plant leaf senescence is induced and affected by internal and external environmental factors,and involves extensive reprogramming of gene expression.H2O2 as an important signal molecule in plants,it is an important exogenous factor affecting cell metabolism and plant senescence.Darkness treatments also accelerate plant senescence and cell death.However,the molecular mechanism of H2O2 and darkness affected plant senescence and cell death is still unclear.It is known that WRKY53 is an important regulator at early stage of plant senescence.H2O2 and darkness treatments promoted the WRKY53 gene expression leading to leaf senescence.The previous research in our lab has showed WHIRLY1 was an upstream regulator of WRKY53 and could bind to the WRKY53 promoter region to repress the expression level of WRKY53 gene to regulate the plant senescence and the cell death.Further CHIP-seq data have shown a series of downstream target genes of WHIRLY1.In this study,four downstream target genes of WHIRLY1:senescence related gene WRKY53,cell death related gene WRKY33,pathogen response related gene PR1 and topoisomerase gene SPO11-2 are selected to analyse the histone methylation and acetylation modification as well as the RNA polymerase Ⅱ(RNA Pol Ⅱ)recruitment at their promoter regions after H2O2 and darkness treatment,which might result in affecting the transcriptional activation or inhibition of these genes inducing leaves senescence in Arabidopsis.In other hand,by using the WHIRLY1 mutant(whirlyl)and overexpression WHIRLY1(oenwhirlyl)transgenic lines to address whether WHIRLY1 protein affected the RNA Pol Ⅱrecruitment at the transcription and promoter region of these four genes during plant aging.The main results as follows:(1)After H2O2 treatment for 2,4,6,8 hrs,Arabidopsis plant showed significantly accelerated senescence phenotype,the senescence associated genes WRKY53,WRKY33 and SAG12 was up-regulated by RT-qPCR analysis,and the expression level of WRKY53 was up to peak at 4 hrs after H2O2 treatment,which this time point as used to following experiment.The CHIP-qPCR analysis showed that after H2O2 treatment for 4hrs,the less enrichment of histone H4ac at the transcription region and histone H3K4me2,H4ac and H3K9ac at the promoter region of WRKY53 gene were related to the activation of WRKY53 transcription;however,there was no direct relationship between H3K27me2 modification and RNA Pol Ⅱ recruitment with WRKY53 transcription.The less enrichments of histone H3K4me2 and H4ac at the transcription region and the promoter region of WRKY33 gene were related to the activation of WRKY33 transcription;however,there was no direct relationship between H3K27me2,H3K9ac modification and RNA Pol Ⅱrecruitment with WRKY33 transcription;The less enrichments of H3K4me2,H4ac and H3K9ac at the promoter region of PR1 and SPO11-2 genes were related to the transcription of PR1 and SPO11-2 genes,but there was no direct relationship between H3K27me2 modification and RNA Pol Ⅱ recruitment with PR1 and SPO11-2 transcription.(2)After darkness treatment for 2,4,6,12,24 hrs,Arabidopsis plant showed no significantly accelerated senescence phenotype,the senescence associated genes WRKY53,WRKY33 and SAG12 was up-regulated by RT-qPCR analysis,and the expression level of WRKY53 was up to peak at 4 hrs after dark treatment,which time point as used to following experiment.The CHIP-qPCR analysis showed that after dark treatment for 4 hrs,the histone H3K4me2,H3K27me2,H4ac and H3K9ac modification at the transcription region and the promoter region of WRKY53 gene were not related to the activation of WRKY53 transcription;however,the increased RNA Pol II recruitment activated WRKY53 transcription.The increasing enrichments of histone H3K27me2 modification at the transcription region and the promoter region of WRKY33 gene were related to the activation of WRKY33 transcription;however,there was no direct relationship between the histone H3K4me2,H4ac,H3K9ac modification and the RNA Pol II recruitment with WRKY33 transcription;There was no direct correlation between the modification of H3K4me2,H4ac and H3K9ac and the transcription of PR1 and SPO11-2,but it was related to increasing enrichments of H3K27me2 modification.In addition,the transcription of SPO11-2 is associated with the activation of RNA Pol Ⅱ recruitment.(3)By using WT,whirlyl mutant and oenwhirlyl transgenic plants,we found that the RNA Pol Ⅱ recruitment at the transcription region and promoter regions of WRKY53 was increased in the 7th week,and the expression level of WRKY53 was up-regulated.WHIRLY1 could inhibit the expression of WRKY53 by inhibiting the RNA Pol Ⅱ recruitment.The RNA Pol Ⅱ recruitment at the transcriptional and promoter regions of WRKY33 and at the promoter regions of PR1,SPO11-2 were affected in the whirly1 mutant in the 7th and 8th week,but there was no positive correlation in the transcription level.WHIRLY1 affected the RNA Pol Ⅱrecruitment of WRKY53 not by influencing the expression of the Rpb1 gene or not by WHIRLY1 interaction with RNA Pol Ⅱ in the 7th week,WHIRLY1 may influence the RNA Pol Ⅱ recruitment of WRKY53 by the indirect effect. |
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