Genome-Wide Analysis Of DNA Methylation And Its Regulation In Citrus

Epigenetics refers to “any potentially stable and heritable change in gene expression that occurs without a change in DNA sequence”. In eukaryotes, chromatin structure and gene expression are affected by several epigenetic mechanisms to regulate plant development, which mainly include DNA methylation, histone modification, and certain aspects of small-interfering RNA(siRNA). Recently, studies have widely revealed that epigenetic mechanisms play important roles in regulating plant development and environment interactions. However, the regulations of DNA methylation during citrus development have not been characterized well. In order to inverstigate the involvements of DNA methylation regulations in citrus, we characterized the DNA methylation variations during citrus fruit development. Also, we utilized citrus callus which initially accumulated high level of carotenoids to study the regulations of DNA methylation in carotenoids metabolism. Moreover, we investigated the epigenetic mechanisms involved in citrus “polyembryony” from the epigenome perspective. Furthermore, citrus histone modification genes were identified based on the sequenced sweet orange genome. The main results are as follows:1. DNA methylation variations during fruit developmentAnalysis of genomic DNA methylation levels in different fruit tissues and six developmental stages indicated that methylation levels were not static during fruit development and ripening. To explore the possible contributions of methylation-related genes in fruit development and ripening process, the full length coding sequences corresponding to three types of DNA methyltransferase families(CsMET1, CsCMT3 and CsDRM1), a chromatin-remodeling gene(CsDDM1), and a demethylation gene(CsDME1) were isolated. The expression profiles of these methylation-related genes were analyzed in vegetative tissues(seedlings and leaves) and reproductive tissues(three stages of flowering and six stages of fruit development). The results showed that CsDRM1 was highly expressed in seedlings, leaves and flowers compared with other genes. During fruit development and ripening, CsMET1, CsCMT1, CsCMT2, CsDRM1 and CsDMEs showed higher expression in the peel than in the flesh. However, global DNA methylation level changes could not be explained by the expression of a single gene, suggesting that the methylation and demethylation system may cooperatively contribute to the final global DNA methylation pattern.2. Effects of 5-azacytidine treatment induced carotenoids degradation in citrus cell culturesTo investigate the involvements of DNA methylation in carotenoid metabolism, we utilized methyltransferase inhibitor 5-azacytidine(5azaC) to treat Citrus callus which initially accumulated high level of carotenoids. The treatment results in the carotenoids pigmentations were gradually degraded along with the increase of 5azaC concentrations. Moreover, the degradation of up-stream carotenoids resulted in the reduction of down-stream ABA content. The expression analysis of genes involved in carotenoid pathway showed that CpCCD1 was highly induced. Functional complement experiment in bacterial(Escherichia coli) verified that CpCCD1 exhibited the strong catalytic activities to zeaxanthin, β-carotene and lycopene. Moreover, we found that 5azaC induced the expression abundance of DNA methylation related genes which could be responsible for the hypermethylated loci caused by 5azaC. In order to inverstigate the gobal expression changes in response to 5azaC treatment, Digital Gene Expression Profiling was performed. A total of 3047 differentially expressed genes were identified, with 2047 up-regulated and 1333 down-regulated genes, indicating that lots of genes were activated by the 5azaC treatment. Also, a numbers of transcriptional factors(TFs) were activated by 5azaC treatment, such as MYBs, WRKYs, NACs and so on. Further methylome analysis revealed that the DNA methylation variations altered by 5azaC treatment mainly occurred in intergenic regions and up-stream 2kb and down-stream 2kb of gene elements. Totally, 3082 differentially methylated genes(682 hypermethylated; 2400 hypomethylated) including gene body and promoter regions were identified, indicating that the demethylation occurred among the whole genome. Functional category analysis showed that the differentially methylated genes were mainly involved in proteolysis, transcriptional factors, signaling, stress response, hormone metabolism and so on.3. Genome-wide DNA hypermethylation occurred during citrus ovules developmentThis study is the first methylome analysis of citrus developing ovules during nucellar embryo initiation based on the MeDIP-seq technology(Methylated DNA Immunoprecipitation Sequencing). Two developmental stages of Citrus sinensis ovules(10 days prior to anthesis-before nucellar embryo initiation; 15 days after anthesis-after nucellar embryo initiation) were applied to methylome analysis. A total of 2349 differentially methylated genes were identified, including 2245 hypermethylated genes and 104 hypomethylated genes. Global genes were hypermethylated indicated that DNA methylation was modified at the whole genome level during the ovule development. Together, the methylome analysis of Citrus clementina ovules across the fitilization process also comfirmed that genome-wide DNA methylation were increased during the early stages of seed development. Studies have revealed that DNA methylation patterns were reprogramming during the reproduction process. It is implied that the ovules in this study were in the stages of global DNA methylation re-establishment after undergoing the demethylation in gametogenesis phase. Moreover, transcriptome analysis was preformed to investigate the global expression changes during the ovule development. A total of 2487 differentially expressed genes(DEGs) were identified, including 1361 up-regulated genes and 1126 down-regulated genes. Functional category analysis showed that DEGs were mainly involved in hormone signal transduction, proteasome, microtubule-based process and so on. Furthermore, based on the polyembryony-associated regions identified by the previous studies, the changes of methylation status were analyzed in this region and several candidate genes were possibly involved in polyembryony.4. Identification of histone modification gene families and their expression analysisBased on the two recent sequenced sweet orange genome databases, a total of 136 CsHMs(Citrus sinensis histone modification genes), including 47 CsHMTs(histone methyltransferase genes), 23 CsHDMs(histone demethylase genes), 50 CsHATs(histone acetyltransferase genes), and 16 CsHDACs(histone deacetylase genes) were identified. These genes were categorized to 11 gene families. A comprehensive analysis of these 11 gene families was performed with chromosome locations, phylogenetic comparison, gene structures and conserved domain compositions of proteins. In order to gain an insight into the potential roles of these genes in citrus fruit development, 42 CsHMs with high mRNA abundance in fruit tissues were selected to further analyze their expression profiles at six stages of fruit development. Interestingly, a numbers of genes were expressed highly in flesh of ripening fruit and some of them showed the increasing expression levels along with the fruit development. Furthermore, we analyzed the expression patterns of all 136 CsHMs response to the infection of blue mold(Penicillium digitatum), which is the most devastating pathogen in citrus postharvest process. The results indicated that 20 of them showed the strong alterations of their expression levels during the fruit-pathogen infection. Based on the results, a candidate gene CsSDG7 were selected to further functional analysis. The down-regulated CsSDG7 Hongkong kumquats lines(Fortunella hindsii) showed the early flowering during the seedling stage.