Arabidopsis Histone H3K4 Methylases ATX4 And ATX5 Function In Abscisic Acid And Dehydration Stress Responses

Epigenetic mechanisms play essential roles in maintaining developmental stability and improving adaption of creatures in response to environmental conditions.Trithorax-group proteins(TrxGs)are one of most important epigenetic components which play essential regulatory roles in chromatin modification.TrxGs have been shown to be evolutionarily conserved and extensively involved in the activation of developmental genes,however,how the specific TrxGs function in the dehydration and abscisic acid(ABA)-mediated modulation of downstream gene expression remains unknown.In this study,we use the model plant Arabidopsis thaliana to screen ABA-hypersensitive ARABIDOPSIS TRITHORAX4(ATX4)and ARABIDOPSIS TRITHORAX5(ATX5)loss-of-function mutants via reverse genetics.Subsequently,we use multidisciplinary techniques and methods such as molecular biology,genetics,cell biology,plant physiology and bioinformatics to investigate the underlying functions and molecular mechanisms of ATX4 and ATX5 in the drought stress and ABA response.The main results are as follows:(1)The sequences of ATX4 and ATX5 are extremely similar,which share 85% homology and harbor highly conserved domains such as the SET and PHD domains.?-Glucuronidase(GUS)staining experiment demonstrates that the spatial and temporal expression patterns of ATX4 and ATX5 are very similar.In addition,subcellular localizations of ATX4 and ATX5 are observed at the nucleus by co-transfecting the Arabidopsis thaliana protoplasts with NLS-RFP.(2)During the vegetative and later reproductive stage,atx4 and atx5 grow abnormally under normal conditions.Intriguingly,these abnormal phenotypes become exaggerated along with decreased light intensity.Besides,ATX5 specifically involves in regulating the shape of curling cauline leaves and the architecture of inflorescence.(3)atx4 and atx5 display ABA-hypersensitive phenotypes during seed germination and seedling development.Besides,they show reduced water-loss rates and drought stress-tolerant phenotypes.(4)ATX4 and ATX5 are proved to be specific H3K4me2 and H3K4me3 methyltransferases via isolating nuclei and western blotting assay.(5)Genome-wide RNA-sequencing analyses show that the landscape of transcriptional regulation by ATX4 and ATX5 are really similar,the majority of downstream transcriptional targets are overlapped,however,there are a few targets genes are specifically regulated by ATX4 or ATX5.The overlapped genes are stratified into response to ABA stimulus and dehydration stress.(6)atx4-1 atx5-1 double mutant displays further exacerbation of the phenotypes during the developmental and reproductive stage or under ABA treatment.Thus,ATX4 and ATX5 play partially redundant roles in plant growth and ABA responses.(7)Using Gene Ontology analysis(GO)and functional annotation,AHG3 acting as an essential negative regulator of ABA signaling is focused.Subsequently,crossed atx4-1 atx5-1 ahg3-1 triple mutant shows more ABA-hypersensitive phenotypes than single mutant,which demonstrates AHG3 acts genetically downstream of ATX4 and ATX5 in response to ABA.(8)Chromatin Immunoprecipitation with qPCR(ChIP-qPCR)and Co-Immunoprecipitation(Co-IP)experiments show that ATX4 and ATX5 directly bind to RNA polymerase II,facilitating Pre-Initiation-Complex(PIC)assembly,and then,ATX4 and ATX5 directly bind to the gene body of AHG3 and trimethylate histone H3 of Lys 4(H3K4me3),and active gene expression in response to ABA.Our findings reveal novel molecular functions of A.thaliana TrxGs in dehydration stress and ABA responses,which provides new evidence of the regulation mechanism of histion transmethylase and the theoretical basis for creating drought-tolerence modified crops.