植物通过转录和转录后水平的调控响应非生物胁迫，该过程涉及到多种表观遗 传修饰的参与。然而，受限于尚未全面了解表观遗传修饰的所有可能类型，无法全 面认识表观修饰在植物非生物胁迫响应中的作用。N4-乙酰脱氧胞嘧啶修饰（N4- acetyldeoxycytosine，4acC）是近期在真核生物中发现的新型 DNA 乙酰化修饰，在拟 南芥基因组上其修饰丰度与基因表达水平呈正相关，在转录起始位点（Transcription Start Site，TSS）修饰程度最高，且与拟南芥冷胁迫响应有关，但其在水稻非生物胁 迫过程中的作用仍未可知。本研究应用 4acC-IP-seq（4acC-Immunoprecipitation sequencing）、RNA-seq（RNA-sequencing）等方法绘制了冷和盐胁迫下水稻基因组的 4acC 修饰图谱和基因表达图谱，为进一步揭示 4acC 修饰在水稻非生物胁迫中的调控 作用提供了理论基础。主要研究结果如下： 1. 为了明确 4acC 修饰在水稻基因组中的分布及其与基因表达的相关性，本研究 利用 4acC-IP-seq 绘制了水稻幼苗 4acC 修饰的全基因组图谱。结果发现 4acC 修饰分 布在基因间区和启动子区域，特异富集的 motif 序列为 CYTYCTCYTYYTCYYYYTC YY（Y=C，T）。计算全部基因和上下游 2 kb 的 4acC 修饰发现其在水稻基因的 TSS 丰度最高。结合 RNA-seq 数据分析发现高表达基因在 TSS 的 4acC 丰度显著高于低 表达基因，38%的表达基因中含有 4acC 修饰，而 4acC 修饰的基因中有 73%是表达 基因；TSS 存在 4acC 修饰的基因表达水平显著高于 TSS 无 4acC 修饰的基因。因此， 水稻的 4acC 修饰水平与基因表达呈正相关。 2. 为了研究 4acC 修饰的生物学功能，研究以 4acC-IP-seq 构建冷和盐胁迫下水 稻全基因组 4acC 修饰图谱。结果发现冷胁迫下，4acC 修饰在基因组上的分布模式与 对照一致。在对照中，共鉴定到 31718 个 4acC 修饰区间，冷胁迫下有 25485 个。差 异区间分析发现冷胁迫下共鉴定到 711 个差异区间，其中 681 个在冷胁迫后显著下 调，仅 30 个显著上调，说明冷胁迫后 4acC 修饰的分布不变，但其平均丰度显著下 降。盐胁迫下，4acC 修饰在基因组上分布模式不变。在对照中，共鉴定到 65825 个 4acC 修饰区间，而盐胁迫下有 58440 个。差异区间分析发现盐胁迫下共有 2541 个差 异区间，其中 2418 个在盐胁迫后显著上调，仅 123 个显著下调，但平均丰度显著上升。冷和盐胁迫下 4acC 修饰差异区间的重叠分析发现冷和盐共有的上调差异区间仅 有 4 个，共有的下调差异区间为 0。上述结果说明 4acC 修饰在水稻冷和盐胁迫中存 在明显差异。 3. 为了解析冷胁迫下 4acC 修饰对基因表达的影响，本研究利用 RNA-seq 技术绘 制冷和盐胁迫下的基因表达图谱，发现冷胁迫下，表达水平显著上调的基因有 10133 个，显著下调的基因有 9742 个；盐胁迫下显著上调的基因有 5736 个，显著下调的 有 4130 个，说明两种胁迫均可诱导大量的基因表达差异。对两种胁迫下的差异表达 基因进行聚类，结果发现金属离子转运、跨膜运输功能的基因在两种胁迫下表达均 下降；细胞分解代谢过程、信号调节过程功能的基因趋向于在冷胁迫下出现表达上 调，离子转运、跨膜运输功能的基因在盐胁迫下出现表达上调。冷胁迫诱导表达上 调基因 TSS 的 4acC 修饰趋向下调，而盐胁迫诱导表达上调基因 TSS 处的 4acC 修饰 趋向上调。进一步分析已报道的冷、盐胁迫调控关键基因，发现其 TSS 或基因上游 的 4acC 修饰水平和基因表达水平均发生了显著变化，这些结果暗示 4acC 修饰可通 过调控关键基因的表达来响应冷和盐胁迫。 4. 为了深入研究 4acC 的动态变化机制，本研究首先通过系统进化分析，鉴定了 水稻和拟南芥中的乙酰转移酶基因，以挑选可能的 4acC 修饰酶候选基因。其中拟南 芥全基因组的乙酰转移酶有 22 个，优先选择冷胁迫处理后表达下降的 12 个乙酰转 移酶基因为候选基因，获得其纯合突变体，并通过点杂和超高效液相色谱进行 4acC 修饰水平的鉴定。最终鉴定到 2 个 4acC 修饰明显下降的候选乙酰转移酶突变体 mut- 1 和 mut-2，为水稻乙酰转移酶的鉴定提供线索。 综上所述，水稻 4acC 修饰与基因表达呈正相关，胁迫后 4acC 修饰在基因组上 的分布模式不变，但冷胁迫诱导 4acC 修饰下调，盐胁迫诱导修饰上调。本研究为探 究新型 DNA 修饰 4acC 响应非生物胁迫的表观遗传调控机制提供了可靠线索。

Plants respond to abiotic stress through the regulation of transcription and posttranscriptional levels, involving various epigenetic modifications. However, due to the incomplete understanding of all possible types of epigenetic modifications, the role of epigenetic modifications in plant response to abiotic stress is not fully understood. N4- acetyldeoxycytosine(4acC) modificationis a newly discovered DNA acetylation modification in eukaryotes, and its abundance in the Arabidopsis genome is positively correlated with gene expression levels, with the highest modification degree at the Transcription Start Site(TSS). It has been found that 4acC modification is related to cold stress response in Arabidopsis, but its role in rice response to abiotic stress is still unknown. This study used 4acC-IP-seq, RNA-seq, and other methods to map the 4acC modification and gene expression patterns in the rice genome under cold and salt stress, providing a theoretical basis for further understanding the regulatory role of 4acC modification in rice response to abiotic stress. The main research results are as follows: 1. To clarify the distribution of 4acC modifications in the rice genome and their correlation with gene expression, this study used 4acC-IP-seq to map the whole-genome profile of 4acC modifications in rice seedlings. The results showed that 4acC modifications were distributed in intergenic regions and promoter regions, with a specifically enriched motif sequence of CYTYCTCYTYYTCYYYYTCYY (Y=C, T). By calculating the 4acC modifications for all genes and their 2 kb upstream and downstream regions, it was found that the abundance of 4acC was highest at the TSS of rice genes. Combining RNA-seq data analysis revealed that genes with high expression had significantly higher 4acC abundance at the TSS compared to low-expression genes. About 38% of expressed genes contained 4acC modifications, and among genes with 4acC modifications, 73% were expressed; genes with 4acC modifications at the TSS had significantly higher expression levels than those without. Therefore, the level of 4acC modification in rice is positively correlated wit gene expression. 2. To investigate the biological function of 4acC modifications, the study constructed whole-genome 4acC modification maps under cold and salt stress using 4acC-IP-seq. The results showed that, under cold stress, the distribution pattern of 4acC modifications in the genome remained consistent with the control. In the control, 31718 4acC modification intervals were identified, while under cold stress there were 25485. Differential interval analysis identified 711 differential intervals under cold stress, with 681 significantly downregulated and only 30 significantly upregulated, indicating that the distribution of 4acC modifications remained unchanged but their average abundance significantly decreased under cold stress. Under salt stress, the distribution pattern of 4acC modifications remained unchanged. In the control, 65825 4acC modification intervals were identified, whereas under salt stress there were 58440. Differential interval analysis identified 2541 differential intervals under salt stress, with 2418 significantly upregulated and only 123 significantly downregulated, although the average abundance significantly increased. Overlapping analysis of differential intervals under cold and salt stress revealed only 4 common upregulated intervals and 0 common downregulated intervals. These results indicate significant differences in 4acC modifications under cold and salt stress in rice. 3. To analyze the impact of 4acC modifications on gene expression under cold stress, the study used RNA-seq technology to map gene expression profiles under cold and salt stress. It was found that under cold stress, 10133 genes were significantly upregulated and 9742 were significantly downregulated; under salt stress, 5736 genes were significantly upregulated and 4130 were significantly downregulated, indicating that both stresses induced large-scale gene expression changes. Clustering of differentially expressed genes under both stresses revealed that genes related to metal ion transport and transmembrane transport functions were downregulated under both stresses; genes involved in cellular catabolic processes and signaling regulation tended to be upregulated under cold stress, while genes related to ion transport and transmembrane transport functions were upregulated under salt stress. The 4acC modification at the TSS of genes upregulated by cold stress tended to be downregulated, whereas those upregulated by salt stress tended to have increased 4acC modification. Further analysis of reported key genes regulated by cold and salt stress revealed significant changes in their 4acC modification levels at the TSS or upstream regions, suggesting that 4acC modifications can respond to cold and salt stress by regulating the expression of key genes.In summary, 4acC modification in rice is positivel correlated with gene expression, and its distribution pattern changes in response to abiotic stress. This research provides valuable insights for exploring the role of the novel modification 4acC in response to abiotic stress, which is of great significance for deciphering the epigenetic mechanisms of rice response to abiotic stress. 4. To further study the dynamic mechanisms of 4acC, the study first performed a phylogenetic analysis to identify acetyltransferase genes in rice and Arabidopsis, selecting potential candidate 4acC modifying enzymes. Among them, 22 acetyltransferases were identified in the Arabidopsis genome, and 12 acetyltransferase genes downregulated after cold stress were selected as candidates, obtaining their homozygous mutants, and 4acC modification levels were identified through dot blot and ultra-high-performance liquid chromatography. Finally, two candidate acetyltransferase mutants, mut-1 and mut-2, with significantly reduced 4acC modifications were identified, providing clues for identifying rice acetyltransferases. In summary, 4acC modifications in rice are positively correlated with gene expression. After stress, the genomic distribution pattern of 4acC modifications remains unchanged, but cold stress induces a decrease, while salt stress induces an increase in these modifications. This study provides reliable clues for exploring the epigenetic regulatory mechanisms of novel DNA modification 4acC in response to abiotic stres。

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