Cold Induced Modification Of DNA Methylation In Hevea Brasiliensis

Hevea brasiliensis is native to the rainforests of tropical regions of the Amazon basin.In the late 1970s,rubber plantations spread to sub-optimal environments in high-latitude non-traditional planting areas.Cold stress becomes one of the most important factors limiting rubber planting and production.The successful extension of Hevea brasiliensis cultivation to high-latitude areas has long been believed to benefit from the breeding of cold-tolerant cultivars.However,the germplasm resources of rubber tree are very narrow,the growth cycle of rubber tree is very long,whereas the big size of rubber seeds and low planting density,which strongly limit the selections of rubber hybrid breeding.However,cold resistant cultivars of rubber tree were successfully developed within such a short period of time,which raised a question,what kind of molecular mechanism was adopted during the breeding?In order to explain the puzzling incongruity,the epigenetics related mechanism involved in cold tolerance of rubber tree was investigated in this work and following results were obtained:(1)In order to study whether the cold responsive genes and the methylation-related genes are induced by low temperature,seedlings of rubber tree were treated with cold,the expression profiles of HbICEI,HbCBF2,and other cold stress responsive genes(COR),as well as DNA methylation related were analysed.qRT-PCR results showed that these genes were regulated by cold treatment at different degrees.(2)To further investigate whether the cold treatment caused alteration of epigenetic modification,seedlings of Reyan7-33-97 were treated with long-term cold treatment.Obvious phenotypic changes such as leaves chlorosis,decreased chlorophyll content and retarded growth were found after the long-term cold treatment.Methylated CpG Island Amplification(MCA)showed that the methylation modification of CpG island was changed by long-term cold treatment at genome-wide.In addition,long-term cold treatment not only increased the transcriptional levels of HbICE1,HbCBF2 and COR genes,but also induced DNA demethylation of HbICE1,HbCBF2 and HbMET gene promoters.The expression of DNA demethylation-related genes such as HbDMEs,HbROSs and HbDMLs was up-regulated by long-term cold treatment,but the demethylation of gene promoters did not occur.In other ways,short-term,multi-species and tissue-specific expression analysis of DNA demethylation-related gene families.It is further proved that the expression mechanism of DNA methylation family genes is more obviously changed due to prolonged accumulation of low temperature.(3)In order to reveal whether cold induced DNA demethylation occurred under natural conditions,leaves samples of rubber tree were collected from the Sanya and Wenchang City of Hainan province,and Xishuangbanna and Puer City of Yunnan province for expression analysis and methylation analysis in April and October,respectively.Four collection sites represent different latitudes of rubber planting locations.Bisulphite sequencing results showed that general demethylation was observed in the HbICE1 and HbMET gene promoters in the samples collected from in Wenchang,Xishuangbanna and Puer in April,but not in the sample collected in Sanya.The HbICE1 and HbMET gene promoters switched to hypermethylation status in almost all the samples collected in October.DNA methylation modification showed a significant correlation with low temperature but not with the genetic background of rubber clones,suggesting that the environmental temperature may alter the epigenetic modification such as DNA methylation modification,so as to affect the expression of the cold related genes and cold tolerance of the rubber tree.(4)Additionally,possible correlation between the methylation status and gene expression in natural conditions.Gene expression analysis and thermal map show a high correlation between cold-induced DNA demethylation and the expression of COR gene.Given that little SNPs existing in gene promoters of the HbICEl and HbMET in 12 rubber cultivars tested,cold tolerance of the rubber tree was more closely correlated with epigenetics,but not with the genetic variation among of different cultivars of rubber tree.In another word,cold-induced epigenetic modification plays a much more important role than the genetic variation in cold tolerance in rubber tree.(5)Furthermore,high-throughput Digital Gene Expression(DGE)analysis was used to globally screen the cold responsive genes in rubber tree.DGE result showed that the 3693 genes were up-regulated and 2043 gene were down regulated after low-temperature treatment.Differentially expressed genes were mainly clustered as Metabolism,Environmental Information Processing,Genetic Information Processing,Organismal Systems,Cellular Processes,and Metabolic pathway in the KEGG metabolic pathway.Total 122 pathways involved in cold response in rubber tree.(6)Finally,the correlation between cold induced differentially expressed genes and DNA methylation status were conducted by Reduced Representation Bisulfite Sequencing(RRBS).Combined analysis of DNA methylome and trasncriptome reveal that CpG islands were widely demethylated and distributed in the coding region and functional regulation region of the gene.Through analyzing the distribution of methylation regions(DMR)of differentially expressed genes,it was found that the longer distance from the coding region,the higher degree of DNA methylation appeared in the untreated sample,DNA demethylation was significantly increased after cold treatment,in which CHG and CHH were the main type of CpG island.Interestingly,methylation degree of the transposon increased after after cold treatment,in which CG was main CpG type.Combined analysis of DNA methylome and trasncriptome reveal that more than 400 genes showed significant alteration of DNA methylation after coldtreatment,including methyltransferas,zinc finger proteins,chloroplastid related genes and stress-related gene family,which were not only regulated at methylation level,but also at transcriptional level by cold treatment.Taken together,cold-related genes and DNA-methylation related genes were induced by cold treatment.Long-term cold treatment not only elevated the transcriptional activities but also induced DNA demethylation of the genes promoters.Cold treatment increased the transcriptional activities of demethylation-related genes such as the HbDME、HbROS and HbDML genes,but did not alter the promoter methylation status.Furthermore,the HbICEl and HbMET promoters were hypomethylated in samples collected at the end of winter from 12 different cultivars grown in four geographical locations,but their methylation status switched to hypermethylation at the end of summer.The DNA demethylation induced by cold was highly correlated with the expression level of COR genes and low temperature,but not with the genetic backgrounds of cultivars.Little genetic diversity was observed in the HbICE1 and HbMET promoters in the 12 cultivars,suggesting that cold-induced epigenetic modification is more important than genetic variation in cold tolerance of H.brasiliensis.Our findings may provide valuable evidence to clarify the molecular mechanism of rubber tree under cold stress,which might help us to breed the cold resistant rubber cultivar in the future.