Epigenetic Regulation And Genomic Imprinting In Castor Bean Endosperms

The study of endosperm development in flowering plants has received special attentions and has been an important frontal filed in plant reproductive development, epigenetics and crop improvement. DNA methylation, one of the most important epigenetic marks, has been shown broad-ranging functions, such as involvement in chromatin organization, repression of transposable elements and regulation of gene expression and genomic imprinting. Increasing evidence demonstrates that DNA methylation plays crucial roles in the control of endosperm development and seed size. Investigations of genomic DNA methylation in the seeds of Arabidopsis, rice and maize revealed extensive hypomethylation in the endosperm genome. However, the conservation, functional roles and regulatory mechanism of genomic hypomethylation in endosperm are largely unclear. Specifically, in most eudicots including model plant Arabidopsis, their endosperms are ephemeral and gradually consumed by the embryo tissue during seed development. Thus, it appears difficult to dissect the potential mechanism of DNA methylation and genomic imprinting occurring in a eudicot's seeds. Castor bean (Ricinus communis), a member of Euphorbiaceae famliy, has relatively large and persistent endosperm throughout seed development. It has been considered as a model plant for studies on endosperm and seed development, and provides an excellent system for studies on regulatory mechanism underlying DNA methylation and genomic imprinting.In this study, we investigated the pattern and level of DNA methylation, regulatory mechanism of DNA methylation and effects of DNA methylation on gene expression in castor bean inbred ZB107 seeds by deep DNA methylation sequencing, small RNA sequencing and mRNA sequencing. Main results and conclusions were summarized as follows:1. Genomic DNA hypomethylation in castor bean endospermBased on DNA methylation sequencing (Bisulfite-treated DNA sequencing), we found that the genomic methylation percentage of CG and CHG sequence contexts was, respectively,30.3% and 18.3% in endosperm, and 40.7% and 24.0% in embryo, suggesting the obvious reduction of CG and CHG methylation level in endosperm relative to embryo. This is consistent with the reports in Arabidopsis, rice and maize. However, the CHH methylation percentage in endosperm did not exhibited obviously changes compared with the embryo. Moreover, castor bean showed rich CHH methylation (68%) in both endosperm and embryo. Most of CG and CHG sequence contexts were heavily methylated (above 90% methylation level), while the methylation level of CHH context apparently exhibited a more uniform distribution between 30% and 100% in castor bean seeds. Almost all DMRs (differentially methylated regions) were linked to the genomic hypomethylation of endosperm and resulted mainly from the reduction of CG and CHG methylation.2. DNA methylation profiles and effects of DNA methylation on gene expressionWhile inspecting the distribution of CG, CHG and CHH methylations in gene and transposable element (TE) regions, we observed that the CG methylation occurred in the genes and TE regions, whereas the CHG and CHH methylation sparsely distributed in the gene regions and mainly occurred in the TE regions. Unsurprisingly, the endosperm clearly exhibited the CG, CHG and CHH hypomethylation in both gene and TE regions. To determine whether the genomic DNA methylation level might affect global gene expression in castor bean seeds, we investigated the methylation changes of CG, CHG and CHH contexts for the genes at different expression level (high expression:RPKM> 100; middle expression:10< RPKM< 100; low expression:1< RPKM? 10 and no expression:RPKM? 1). The results showed that the relationship between DNA methylation and global gene expression was not strong and DNA methylation, in a way, mainly repressed gene expression in castor bean. For the genes with preferential expression in endosperm tissues, however, there was a significant decrease of CG and CHG methylation level in the promoter region of genes relative to embryo tissues. This indicated that the CG and CHG hypomethylation might be the potential mechanism for altering genes preferentially expressed in castor bean endosperm.3.24-siRNAs-mediated RdDM pathway and DNA methylationTo dissect the mechanism of rich CHH methylation in castor bean seeds, we investigated the relationship between small RNAs and DNA methylation. By high-throughput small RNA sequencing, we found that the 24-nucleotide (nt) small RNA was the most abundant in both endosperm and embryo. Moreover, the 24-nt siRNAs expression were strongly associated with CHG and CHH methylation patterns and levels. In addition, we found that the 24-nt siRNAs abundance in gene body regions was significant lower than that in TE regions. The abundance of 24-nt siRNAs in both gene and TE regions in endosperm was substantially lower than that in embryo. In particular, there were more 24-nt siRNAs enriched in CHH hypermethylated regions but fewer siRNAs in the hypomethylated regions in both endosperm and embryo tissues. These results clearly indicated that 24-nt siRNAs could significantly induce the increase of non-CG methylation level, especially for CHH methylation.4. The regulatory mechanism of DNA methylation in castor beanTo understand how DNA methylation is established and maintained in castor bean seeds, we assayed the expression profiles of DNA methylation-related genes in difficult tissues. The results showed that the repression of RcMETl and RcCMT expression and the activity of RcDME in endosperm resulted in genomic hypomethylation of CG and CHG at some discrete loci. The expression of key genes in RdDM pathway (including RcPoI ?, RcPo ?, RcRDR2, RcDLC3, RcAGO4 and RcDRM3) and the abundant 24-nt siRNAs might result in an effective maintenance of CHH methylation in both endosperm and embryo tissues.Genomic imprinting is a typical epigenetic phenomenon that mainly manifests itself in the endosperm of flowering plants, and plays import roles in endosperm and seed development. In present study, we performed deep mRNA sequencing in castor bean hybrid endosperm derived from reciprocal crosses between ZB107 and ZB306. We identified and characterized the imprinted genes in castor bean endosperm. The main results obtained in this study were summarized as follows:1. Identification and validation of imprinted genes in castor bean endospermTo discriminate the parental origin of allelic expression in hybrids, we identified the genomic variation between ZB107 and ZB306 and found a total of 1007066 SNPs and 100615 indels. Based on these high-confident SNPs and strict criterion, we identified 184 MEGs (maternally imprinted genes) and 9 PEGs (paternally imprinted genes) in reciprocal endosperm of castor bean. Of the 67 imprinted genes we selected, 57 were validated by RT-PCR sequencing and five genes exhibited a significant accession dependency. By tracking the imprinting status of several imprinted genes throughout the lifecycle, we found that most of them exhibited a dynamic imprinted expression pattern during endosperm development. In addition, we also identified 14 imprinted long non-coding RNAs and confirmed them by RT-PCR sequencing. Most of imprinted genes tested were expressed in different tissues, only 39% genes were confined to endosperm-specific or preferential expression.2. Characterization of imprinted genes in castor bean endospermComparisons of the genomic distance between imprinted genes showed that most of imprinted loci were not organized in co-localized clusters, and that only three regions in the genome contained the imprinted genes cluster. The analysis of GO function enrichment revealed that most imprinted genes were involved into specific endosperm development. Additionally, the specific LTR/gypsy TE family enriched around imprinted genes was identified in castor bean. Compared with imprinted genes in other plants, we found that the conservation of imprinted genes among different species was rather limited, suggesting the functional differentiation and rapid evolution of imprinted genes in different species.3. Regulation of DNA methylation on genomic imprintingBy DNA methylation sequencing in hybrid endosperm and embryo, we noted a significant enrichment of DMRs (differentially methylated regions) around the imprinted genes. Moreover, endosperm hypomethylated regions mainly occurred in LTR/gypsy TE family, suggesting TE could be a potential factor in driving imprinted gene expression. Besides, we found that six MEGs exhibited hypomethylation in maternal alleles and hypermethylation in paternal alleles, suggesting that allelic differential methylation is involved in the control of a small fraction of imprinted genes.