Supporting data for "Transcriptional regulation of flavone biosynthesis in rice"

Flavones, a major flavonoid subclass, are abundantly accumulated in monocots and they are commonly present in the forms of flavone C-glycosides and O-conjugated flavones. In rice, the biosynthetic pathways of flavone and flavone C-glycosides are well characterized, but the transcriptional regulation of key enzymes remained largely unknown. This study characterizes the MYB transcription factors involved in the regulation of flavone and flavone C-glycoside biosynthesis genes in rice.
In rice, chalcone synthase (OsCHS1) initiates flavonoid biosynthesis by producing naringenin chalcone. Whether OsCHS1 is continuously expressed spanning the rice life cycle was unclear. In this study, a systematic analysis of the expression of OsCHS1 was carried out using the non-invasive RUBY reporter system. The formation of red betalain indicates the expressionof OsCHS1, and the results presented in "Figures_chapter2.pptx" suggest the tissue-specific and stage-specific expression patterns of OsCHS1. Meanwhile, transient overexpression in rice protoplasts identified various culm-expressed R2R3-MYB proteins, functioning as higher hierarchical regulators for the expression of OsCHS1 and flavone biosynthesis genes, and their transcriptional activities are described in "Figures_chapter2.pptx" and "Dataset_chapter2".
This study also characterizes the role of OsMYB58/63 in the regulation of flavone biosynthesis. The files "Figures_chapter3.pptx" and "Dataset_chapter3.xlsx" include the results of gene expression analysis, metabolitc analysis, dual-luciferase reporter assays and in vitro protein-DNA binding assays which explain how OsMYB58/63 functions as transcriptional activator in regulating flavone biosynthesis. Moreover, the data in the file "Dataset_Supplementary.xlsx" suggest the multifunctional roles of OsMYB58/63 in regulating specialized metabolism in rice.
Lastly, this study characterizes a R2R3-MYB protein, OsP1L, as a transcriptional activator of the flavone C-glycoside biosynthesis in rice. The files "Figures_chapter4.pptx" and "Dataset_chapter4.xlsx" include the results of gene expression analysis, DAP-seq analysis, dual-luciferase reporter assays and EMSA which elucidate how OsP1L regulates the expression of OsCHI, OsCYP93G2 and OsCYP75B3. Moreover, these files include the expression patterns of flavone C-glycoside biosynthesis genes and the metabolic analysis of flavone C-glycosides in various tissues.

黄酮（flavones）是黄酮类化合物（flavonoid）的核心亚类之一，在单子叶植物中大量积累，且主要以黄酮C-糖苷（flavone C-glycosides）与O-缀合黄酮（O-conjugated flavones）两种形式存在。在水稻中，黄酮与黄酮C-糖苷的生物合成途径已得到充分解析，但相关关键酶的转录调控机制仍未被充分揭示。本研究针对水稻中参与黄酮及黄酮C-糖苷生物合成基因调控的MYB转录因子（MYB transcription factors）开展了系统表征与功能解析。

在水稻中，查尔酮合酶（chalcone synthase, OsCHS1）通过催化合成柚皮素查尔酮，启动黄酮类化合物的生物合成进程。此前学界尚不明确OsCHS1是否会在水稻的整个生命周期中持续表达。本研究采用无创RUBY报告基因系统（RUBY reporter system），对OsCHS1的表达模式展开了系统性分析：红色甜菜红素（betalain）的生成可直接指示OsCHS1的表达情况，《"Figures_chapter2.pptx"》中呈现的实验结果表明，OsCHS1的表达具有显著的组织特异性与发育阶段特异性。同时，本研究通过水稻原生质体瞬时过表达实验，鉴定出多种在茎秆中特异性表达的R2R3-MYB蛋白（R2R3-MYB proteins）；这类蛋白作为高阶调控因子，参与调控OsCHS1及黄酮生物合成基因的表达，其转录活性的相关细节详见《"Figures_chapter2.pptx"》与《"Dataset_chapter2"》。

本研究同时解析了OsMYB58/63在黄酮生物合成调控中的功能。《"Figures_chapter3.pptx"》与《"Dataset_chapter3.xlsx"》中收录了基因表达分析、代谢组分析、双荧光素酶报告基因检测以及体外蛋白质-DNA结合实验的相关结果，这些结果阐明了OsMYB58/63作为转录激活因子调控黄酮生物合成的分子机制。此外，《"Dataset_Supplementary.xlsx"》中的数据表明，OsMYB58/63在水稻次生代谢调控中兼具多重功能。

最后，本研究鉴定出一种R2R3-MYB蛋白OsP1L，其作为转录激活因子参与调控水稻黄酮C-糖苷的生物合成。《"Figures_chapter4.pptx"》与《"Dataset_chapter4.xlsx"》中收录了基因表达分析、DAP-seq分析、双荧光素酶报告基因检测以及电泳迁移率变动分析（EMSA）的相关结果，这些结果阐明了OsP1L调控OsCHI、OsCYP93G2及OsCYP75B3基因表达的分子机制。此外，上述文件还包含了黄酮C-糖苷生物合成基因的表达模式，以及不同组织中黄酮C-糖苷的代谢组分析结果。