ABSTRACT The biosynthesis of flavonols and anthocyanins is precisely regulated by different transcription factors in plants. WRKY11 promotes the biosynthesis of flavonoids in apple, but the molecular mechanism of WRKY11 regulating flavonols biosynthesis, and whether WRKY11 plays the same roles in other plants species remains to be further studied. Here, we cloned four NtWRKY11 genes from tobacco, which all contained the conserved WRKYGQK heptapeptide and a zinc-finger motif. The NtWRKY11b showed higher expression levels than the other NtWRKY11 genes in all the tobacco tissues examined, especially in tobacco leaves. Silencing of NtWRKY11b in tobacco leaves reduced the content of flavonols to 45.2%-69.8 % of that in the WT plants, but overexpression of NtWRKY11b increased the flavonols content by 37.8%-80.7%. Transcriptome analysis revealed 8 flavonoids related differentially expressed genes (DEGs) between NtWRKY11b-OE and WT plants, among which the transcription of NtMYB12, NtFLS, NtGT5, and NtUFGT was significantly induced by posttranslational activation of NtWRKY11b with the presence of protein synthesis inhibitor, indicating a putative direct promotion of NtWRKY11b on the transcription of these flavonoids related genes. Chromatin immunoprecipitation assays further demonstrated that NtWRKY11b could bind to the promoter regions of NtMYB12, NtFLS, NtGT5, and NtUFGT to activate the transcription of these genes. Moreover, ectopic expression of NtWRKY11b also promoted the expression levels of NtCML38, NtCTL1, NtWRKY44, and NtCML37 genes, which have been shown to enhance plant resistance to various stresses. Our findings revealed the molecular mechanism of NtWRKY11b regulating flavonols biosynthesis, and provided a promising target for increasing flavonols content in tobacco.

中文翻译：

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NtWRKY11b促进烟草黄酮醇生物合成的分子克隆及功能表征

摘要 植物体内不同转录因子精确调控黄酮醇和花青素的生物合成。WRKY11促进苹果中黄酮类化合物的生物合成，但WRKY11调控黄酮醇类生物合成的分子机制，以及WRKY11在其他植物物种中是否发挥同样的作用还有待进一步研究。在这里，我们从烟草中克隆了四个 NtWRKY11 基因，它们都包含保守的 WRKYGQK 七肽和一个锌指基序。NtWRKY11b 在所有检查的烟草组织中表现出比其他 NtWRKY11 基因更高的表达水平，尤其是在烟叶中。烟草叶片中 NtWRKY11b 的沉默将黄酮醇含量降低到 WT 植物中的 45.2%-69.8%，但 NtWRKY11b 的过表达使黄酮醇含量增加了 37.8%-80.7%。转录组分析揭示了 NtWRKY11b-OE 和 WT 植物之间的 8 个类黄酮相关差异表达基因 (DEGs)，其中 NtMYB12、NtFLS、NtGT5 和 NtUFGT 的转录受 NtWRKY11b 的翻译后激活和蛋白质合成抑制剂的存在显着诱导NtWRKY11b 对这些类黄酮相关基因转录的推定直接促进。染色质免疫沉淀分析进一步证明 NtWRKY11b 可以与 NtMYB12、NtFLS、NtGT5 和 NtUFGT 的启动子区域结合以激活这些基因的转录。此外，NtWRKY11b 的异位表达还促进了 NtCML38、NtCTL1、NtWRKY44 和 NtCML37 基因的表达水平，这些基因已被证明可以增强植物对各种胁迫的抗性。