Table_6_Comparative Physiological and Transcriptomic Analyses Reveal Altered Fe-Deficiency Responses in Tomato Epimutant Colorless Non-ripening.xlsx

The mechanisms associated with the regulation of iron (Fe) homeostasis have been extensively examined, however, epigenetic regulation of these processes remains largely unknown. Here, we report that a naturally occurring epigenetic mutant, Colorless non-ripening (Cnr), displayed increased Fe-deficiency responses compared to its wild-type Ailsa Craig (AC). RNA-sequencing revealed that a total of 947 and 1,432 genes were up-regulated by Fe deficiency in AC and Cnr roots, respectively, while 923 and 1,432 genes were, respectively, down-regulated. Gene ontology analysis of differentially expressed genes showed that genes encoding enzymes, transporters, and transcription factors were preferentially affected by Fe deficiency. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed differential metabolic responses to Fe deficiency between AC and Cnr. Based on comparative transcriptomic analyses, 24 genes were identified as potential targets of Cnr epimutation, and many of them were found to be implicated in Fe homeostasis. By developing CRISPR/Cas9 genome editing SlSPL-CNR knockout (KO) lines, we found that some Cnr-mediated Fe-deficiency responsive genes showed similar expression patterns between SlSPL-CNR KO plants and the Cnr epimutant. Moreover, both two KO lines displayed Fe-deficiency-induced chlorosis more severe than AC plants. Additionally, the Cnr mutant displayed hypermethylation in the 286-bp epi-mutated region on the SlSPL-CNR promoter, which contributes to repressed expression of SlSPL-CNR when compared with AC plants. However, Fe-deficiency induced no change in DNA methylation both at the 286-bp epi-allele region and the entire region of SlSPL-CNR gene. Taken together, using RNA-sequencing and genetic approaches, we identified Fe-deficiency responsive genes in tomato roots, and demonstrated that SlSPL-CNR is a novel regulator of Fe-deficiency responses in tomato, thereby, paving the way for further functional characterization and regulatory network dissection.

有关铁（Fe）稳态调控的相关机制已得到广泛研究，但此类过程的表观遗传调控机制仍未得到充分解析。本研究发现，自然发生的表观遗传突变体无色非成熟（Colorless non-ripening, Cnr）相较于野生型艾尔莎·克雷格（Ailsa Craig, AC），表现出更强的缺铁响应。转录组测序（RNA-sequencing）结果显示，在AC与Cnr根系中，缺铁分别诱导947个和1432个基因上调，同时分别有923个和1432个基因下调。对差异表达基因进行基因本体（Gene Ontology, GO）富集分析发现，编码酶类、转运蛋白及转录因子的基因更易受缺铁胁迫影响。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析则显示，AC与Cnr对缺铁胁迫的代谢响应存在显著差异。通过比较转录组分析，本研究鉴定出24个可能为Cnr表观突变靶标的基因，其中多个基因参与铁稳态调控。本研究构建了SlSPL-CNR基因的CRISPR/Cas9基因组编辑敲除（knockout, KO）株系，发现部分由Cnr介导的缺铁响应基因在SlSPL-CNR KO植株与Cnr表观突变体中呈现相似的表达模式。此外，两株KO株系的缺铁诱导性黄化症状均较AC野生型更为严重。另外，相较于AC植株，Cnr突变体在SlSPL-CNR启动子的286 bp表观突变区域呈现高甲基化状态，这导致SlSPL-CNR的表达受到抑制。但缺铁胁迫并未改变286 bp表观等位基因区域及SlSPL-CNR基因全区域的DNA甲基化水平。综上，本研究通过转录组测序与遗传学手段，在番茄根系中鉴定出缺铁响应基因，并证实SlSPL-CNR是番茄缺铁响应的新型调控因子，为后续的功能验证及调控网络解析奠定了基础。