RNA polymerase IV is essential for tomato embryo development through mediating non-CG methylation [Bisulfite_seq]

Background: Plant-unique RNA polymerase IV (Pol IV) governs the establishment of small RNA-directed DNA methylation (RdDM) and contributes to the maintenance of transposable element (TE) silencing. Pol IV-dependent RdDM is highly active during embryonic development, and its absence occasionally reduces seed sets in species with high TE content. However, the mechanism underlying embryonic development failure due to the loss of Pol IV-RdDM remains largely unclear. Results: Here, we report that the loss-of-function of SlNRPD1, encoding the largest subunit of the Pol IV complex leads to aberrant embryogenesis in tomato (Solanum lycopersicum). The slnrpd1 mutants show non-CG hypomethylation and a burst of 21/22-nt sRNA from the distal chromatin region. We found that non-CG hypomethylation at the gene body region is generally associated with the activation of transcription, and knocking out DEMETER-like DNA demethylases cannot reverse slnrpd1 embryo lethality. mCHH hypomethylation in slnrpd1 disrupts gene expression related to auxin and WUSCHEL-related homeobox (WOX) signaling. TE reactivation in slnrpd1 mainly affects the expression of a limited number of nearby genes rather than contributing to excessive sRNA. Conclusion: We conclude that Pol IV-mediated RdDM is essential for tomato embryogenesis via non-CG methylation. mCHH hypomethylation in the slnrpd1 embryo leads to abnormal auxin/WOX signaling and TE reactivation which probably disrupts the tissue and organ initiation. This study provides new insights into the epigenetic regulation of gene and TE expression during embryogenesis. Keywords: DNA methylation, RdDM, NRPD1, Embryogenesis, Tomato Overall design: To investigate the function of NRPD1 in tomato embryogenesis, we collected 21 days after flowering (DAF) embryos of wild type and slnrpd1 to perform RNA-sequencing.

背景：植物特有RNA聚合酶IV（RNA polymerase IV, Pol IV）可调控小RNA指导的DNA甲基化（small RNA-directed DNA methylation, RdDM）通路的建立，并参与维持转座因子（transposable element, TE）的沉默。依赖Pol IV的RdDM在胚胎发育过程中活性极高，当该通路缺失时，高转座因子含量的物种偶尔会出现结籽量减少的情况。然而，Pol IV-RdDM缺失引发胚胎发育失败的潜在机制目前仍不甚明晰。
结果：本研究发现，编码Pol IV复合物最大亚基的SlNRPD1基因功能缺失会导致番茄（Solanum lycopersicum）胚胎发育异常。slnrpd1突变体呈现非CG低甲基化现象，且远端染色质区域出现21/22 nt小RNA（small RNA, sRNA）的爆发式积累。我们发现，基因体区域的非CG低甲基化通常与转录激活相关，而敲除DEMETER样DNA去甲基化酶（DEMETER-like DNA demethylases）无法挽救slnrpd1胚胎的致死表型。slnrpd1中的mCHH低甲基化会干扰与生长素（auxin）及WUSCHEL同源盒（WUSCHEL-related homeobox, WOX）信号通路相关的基因表达。slnrpd1中的转座因子激活主要影响少量邻近基因的表达，而非导致过量小RNA产生。
结论：本研究证实，Pol IV介导的RdDM通过非CG甲基化调控对番茄胚胎发育至关重要。slnrpd1胚胎中的mCHH低甲基化会引发异常的生长素/WOX信号通路及转座因子激活，这可能干扰组织与器官的起始过程。本研究为胚胎发育过程中基因及转座因子表达的表观遗传调控提供了新的见解。
关键词：DNA甲基化，RdDM，NRPD1，胚胎发生，番茄
整体实验设计：为探究NRPD1在番茄胚胎发生中的功能，本研究收集了开花后21天（days after flowering, DAF）的野生型与slnrpd1番茄胚胎，开展RNA测序（RNA-sequencing）。