The Epigenetic Modifications Of Chorispora Bungeana And The Function Of ADH1 In Cold Response

Low temperature limits plant growth and development as well as plant distribution and crop productivity.At present,studies on alpine plants mainly focus on physiological and biochemical mechanism,it is not yet clear whether and how epigenetic modification mediates the adaptation to natural environment in alpine regions.C.bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia(-4~16?),where hailstone,snowfall,as well as chilling and freezing frequently fluctuate during the growing season.C.bungeana has evolved unique adaptation mechanisms over the course of millions of years,so it is an ideal material for the research of cold tolerance.In this research,changes of histone methylation and acetylation as well as DNA methylation caused by temperature fluctuation in nature were analyzed.At the same time,the changes of DNA methylation at chilling(4?)and freezing(-4?)treatments were investigated,and the differentially expressed genes that regulated by DNA methylation were identified.The5-methylcytosine level of Cb ADH1 changed significantly during the chilling and freezing treatments.We cloned and analyzed the function and epigenetic regulations of Cb ADH1 under cold stress,elucidating the special anti-freeze mechanism of CbADH1 in C.bungeana.Below are the main results:1.Cold stress response of C.bungeana was associated with epigenetic regulation.The semi lethal temperature was increased and cold resistance was recuced when C.bungeana treated with HDAC inhibitors TSA.Western blot assay showed that the histone H3,H3K4me3,H3K9 ac,H35Kac,H3K27me3 modifications of C.bungeana were changed once the temperature was fluctuant in the nature growth condition.2.DNA methylation potentially serve as a rapid and flexible mechanism for C.bungeana to adapt to the intricate cold stresses in the alpine areas.Methylation-dependent restriction-site associated DNA sequencing(MethylRAD-seq)and Methylation-sensitive amplified fragment-length polymorphism(MSAP)were used to analyze changes in genomic DNA methylation in response to chilling and freezing temperatures.The results showed that chilling and freezing induced distinct DNA methylation pattern.The results indicated that DNA methylation was involved in the cold response of C.bungeana,and C.bungeana rapidly through thealterations of DNA methylation to regulate the expression of cold responsive genes.3.Series of genes were regulated by DNA methylation under cold stress.Forty-three cold-induced polymorphic fragments were randomly selected.BLAST X analysis showed that 34 of the profiles represent active genic sequences.The remaining cloned fragments were related to functional proteins,transposons,gypsy-like retrotransposons and unknown proteins.Three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase(ADH1),UDP-glucosyltransferase(UGT)and polygalacturonase inhibiting protein(PGIP).The three genes were selected to verify the existence of different expressive patterns between chilling and freezing by chromatin immunoprecipitation(Ch IP)and quantitative real time polymerase chain reaction(qPCR).Results showed that DNA methylation of these genes decreased under cold stress,and the expression of Cb ADH1 significantly increased in the chilling treatment.4.CbADH1 was involved in plant growth and development and responded to various stresses.Using rapid amplification of cDNA ends(RACE),we cloned and characterized a cold-induced alcohol dehydrogenase 1 gene from C.bungeana,CbADH1.Multiple alignment result showed that the N-terminal domain of CbADH1 exists 43 unique amino acids,and the subcellular localization was affected.Except for plasma membrane,CbADH1 also expressed in the chloroplast.We analyzed the tissue localization and relative expression level of Cb ADH1 under multiple stresses.Gus staining experiments showed that CbADH1 expression was ubiquitous and higher in young leaves and pollen grains.CbADH1 was involved in plant growth and development.Quantitative assay for transcripts found that CbADH1 responded to various stresses.Compared to AtADH1 transgenic seedlings,overexpression of CbADH1 in Arabidopsis and tobacoo significantly improved cold shock tolerance.Combined co-localization,bimolecular fluorescence complementation(BiFC)and yeast two hybrid(Y2H)to analyze the functional mechanism of CbADH1.The results showed that CbADH1 interacted with ethylene response factor ERF75,histone deacetylase CbHDA6,pyruvate decarboxylase Cb PDC1,and UDP-glucosyltransferase CbUGT73.These results indicated that CbADH1 interacts with some proteins to enhance cold tolerance of plants.5.The expression of CbADH1 was regulated by epigenetic modifications.ChIP-qPCR results showed that the chromatin modifications of Cb ADH1 changed under chilling stress,concomitant with nucleosome loss and histone modifications.ChIP assays showed histone H3 was removed from the promoter region and middle-coding region.The H3K9 acetylation signal and H3K4 trimethylation signal increased at all regions and at the proximal promoter region,respectively.Moreover,different distribution of the Ser2 phosphorylation(Ser2P)and Ser5 phosphorylation(Ser5P)on the C-terminal heptad repeat domain(CTD)of RNA polymerase II indicated that RNApolymerase II is involved in the expression and extension of CbADH1 under cold stress.Bisulfite sequencing indicated that DNA methylation of CbADH1 occurred mainly at CG site.6.ADH1 affected the metabolites that are intermediates of glycolysis and tricarboxylic acid cycle under cold shock.The real-time quantitative PCR revealed a significant increase in ADH1 expression during low temperature treatment(4?).The semi lethal temperature of ADH1 deletion mutants(adh1)was higher compared to that in wild type(WT)plants.Metabolite analyses in the mutant adh1 showed that the soluble sugars(e.g.,sucrose)and amino acids(e.g.,asparagine)of Arabidopsis thaliana were changed under shock freezing treatment.Results indicated that ADH1 affected the metabolites in response to cold stress.In summary,CbADH1 improves cold tolerance,and this is the first study to address the role of epigenetic modifications in the expression of ADH1 in response to cold stress.Research on anti-freeze mechanism of C.bungeana provides a new viewpoint and theoretical basis on the tolerance mechanisms induced by adverse environment of alpine subnival plants,and expands our understanding of the evolutionary adaptability to environmental stress in alpine subnival plants.