DNA Methylation Mediated Regulation Of Betulin Biosynthesis In Birch (Betula Platyphylla Suk.)

Birch(Betula platyphylla Suk.)is one of the important precious broad-leaved timber species in China.It is widely distributed in the alpine forests of Northeast,North China,Northwest and Southwest China.There were great differences in environmental conditions among different geographical regions,and significant genetic differences among populations and individuals within populations.Abiotic stress is one of the key obstacles to the growth and development of birch.In order to cope with abiotic stress caused by climate change,secondary metabolites produced by birch play a crucial role.Betulin,a secondary metabolite of pentacycltriterpenoid,mainly accumulates in the outer epidermis of birch and plays an important role in plant resistance to cold environment in northern winter.Its content is affected by climatic factors such as winter temperature and summer precipitation.At present,the key enzymes involved in betulin biosynthesis pathway have been fully elucidated.However,the mechanism by which betulin responds to climate change in its habitat is still unknown,especially whether epigenetics are involved in climate change and affect betulin biosynthesis is rarely reported.In this study,the chemical agent Zebularine(ZEB),which can affect DNA methylation,was used as mutagenesis agent.Birch seeds were treated with different concentrations of ZEB(0-400?M)to obtain the mutagenesis population of birch.Physiological experiments and genome DNA methylation sequencing were carried out.Combining molecular biology,epigenetics and bioinformatics techniques,this study attempted to reveal the regulatory mechanism of DNA methylation modification on betulin biosynthesis.In order to provide theoretical basis and technical support for elucidate the epigenetic mechanism of forest environmental adaptation and forest epigenetic breeding,we further analyzed the correlation among climate factors,DNA methylation level and betulin accumulation in the natural population of birch with obvious climate and environmental differences,providing theoretical basis and technical support for forest tree epigenetic breeding.The results as follow:(1)Altered the germination and betulin contents of birch with ZEB treatment.To investiagate the effects of DNA methylation on growth and development of birch,different ZEB concentrations were used to treat birch seeds,and the germination rate was calculated.The results showed that when ZEB concentration was lower than 200 ?M,the seed germination of birch was not significantly affected.When the concentration was higher than 200 ?M,the germination rate decreased significantly.ZEB treatment had no significant effect on biomass related phenotypes,such as plant height,DBH and lateral branch development,in the growth and development of birch.Moreover,HPLC analysis showed that betulin content in 50 ?M ZEB treatment was significantly higher than that in control group.There was no significant difference in betulincontent between ZEB 100?300 ?M and control group.The betulin content of 400 ?M ZEB treatment was significantly lower than that of the control group.The concentration of betulin in ZEB treatment groups showed hormesis effect pattern.(2)Altered the genome-wide CHH methylation of TEs in birch with ZEB treatment.Genome-wide DNA methylation sequencing analysis of ZEB treated birch showed that there were no significant differences in mCG,mCHG and mCHH methylation in gene regions,while there were significant differences in mCHH methylation in transposon(TE)regions.The DNA methylation level of 50 ?M ZEB treatment group was significantly lower than the control group,200 ?M and 400 ?M treatment group was higher than the control group,revealing the remarkable hormesis patterns.In transposon regions,there was no significant change in the number of DMR in mCG and mCHG,while the number of mCHH-DMR was significantly increased.GO enrichment of mCHH-DMR linked genes showed that betulin biosynthesis related genes were significantly enriched.(3)Altered the DNA methylation and gene expression levels of the key genes in betulin biosynthetic pathway in birch with ZEB treatment.Real-time fluorescence quantitative PCR and McrBC-qRT-PCR were used to analyze the expression levels and DNA methylation levels of betulin biosynthesis related transcription factor bHLH9 and the key structure genes FPS,SS,SE,and LUS in the biosynthetic pathway of betulin.The results showed that:All the key structure genes were expressed at high levels in the low-concentration ZEB treatment group,but decreased in the high-concentration treatment group,but the DNA methylation levels of these genes were not consistent with the gene expression pattern.While,we found only DNA methylation level of bHLH9 shows the reverse pattern of its expression.The results showed that ZEB treatment affected the expression of betulin biosynthesis gene by influencing the DNA methylation level in the promoter region of transcription factor bHLH9,and thus affected the expression levels of structure genes and the contents of betulin.(4)Association of winter temperature and summer precipitation with DNA methylation as well as biosynthesis of betulin.Correlation analysis of betulin content,genome-wide DNA methylation levels,and climate factor data from WorldClim showed that only DNA methylation levels in bHLH9 promoter region were significantly negatively correlated with betulin contents in birch samples growing under different natural environment conditions.Furthermore,DNA methylation level of bHLH9 promoter region was negatively correlated with winter temperature and positively correlated with summer precipitation.High temperature in winter and drought in summer reduced the DNA methylation level of bHLH9 promoter and increased its expression level and active the structure genes,thus promoting the synthesis of betulin.In summary.ZEB mutagens,winter temperature and summer precipitation in climate factors can affect betulin content and DNA methylation level in Betula platyphylla.Whichregulate the expression of key genes in betulin biosynthesis by influencing DNA methy lation level of bHLH9 promoter region,and control betulin biosynthesis.This study highlights the role of environmental signals to induce epigenetic changes that result in betulin production in a tree species,possibly helping to develop resilient plants to combat ongoing climate change and enhance secondary metabolite production.