Table_5_BrTTG1 regulates seed coat proanthocyanidin formation through a direct interaction with structural gene promoters of flavonoid pathway and glutathione S-transferases in Brassica rapa L..docx

IntroductionSeed coat color is a significant agronomic trait in horticultural crops such as Brassica rapa which is characterized by brown or yellow seed coat coloration. Previous Brassica rapa studies have shown that BrTTG1 is responsible for seed coat proanthocyanidin formation, which is dependent on the MYB-bHLH-WD40 complex, whereas some studies have reported that TRANSPARENT TESTA GLABRA 1 (TTG1) directly interacts with the structural gene promoters of the flavonoid pathway.
MethodsHerein, the brown-seeded inbred B147 and ttg1 yellow-seeded inbred B80 mutants were used as plant materials for gene expression level analysis, gene promoter clone and transient overexpression.
ResultsThe analysis identified eleven structural genes involved in the flavonoid biosynthesis pathway, which are potentially responsible for BrTTG1- dependent seed coat proanthocyanidin formation. The promoters of these genes were cloned and cis-acting elements were identified. Yeast one-hybrid and dual-luciferase assays confirmed that BrTTG1 directly and independently interacted with proCHS-Bra008792, proDFR-Bra027457, proTT12-Bra003361, proTT19-Bra008570, proTT19-Bra023602 and proAHA10-Bra016610. A TTG1-binding motif (RTWWGTRGM) was also identified. Overexpression of TTG1 in the yellow-seed B. rapa inbred induced proanthocyanidin accumulation by increasing the expression levels of related genes.
DiscussionOur study unveiled, for the first time, the direct interaction between TTG1 and the promoters of the flavonoid biosynthesis pathway structural genes and glutathione S-transferases in Brassica rapa. Additionally, we have identified a novel TTG1-binding motif, providing a basis for further exploration into the function of TTG1 and the accumulation of proanthocyanidins in seed coats.

引言
种皮颜色是芸薹（Brassica rapa）等园艺作物的重要农艺性状，其种皮颜色以褐色或黄色为典型特征。过往针对芸薹的研究表明，BrTTG1负责调控种皮原花青素的合成，该过程依赖于MYB-bHLH-WD40复合体；而另有研究指出，透明种皮毛状体缺失蛋白1（TRANSPARENT TESTA GLABRA 1，TTG1）可直接结合黄酮类通路的结构基因启动子。
方法
本研究以褐籽自交系B147及ttg1突变型黄籽自交系B80为实验材料，开展基因表达水平分析、基因启动子克隆及瞬时过表达实验。
结果
本研究共鉴定出11个参与黄酮类生物合成通路的结构基因，这些基因可能参与BrTTG1依赖的种皮原花青素合成过程。研究人员克隆了上述基因的启动子并鉴定出其顺式作用元件。通过酵母单杂交及双荧光素酶实验证实，BrTTG1可直接且独立地结合proCHS-Bra008792、proDFR-Bra027457、proTT12-Bra003361、proTT19-Bra008570、proTT19-Bra023602及proAHA10-Bra016610的启动子。同时鉴定出一个TTG1结合基序（RTWWGTRGM）。在黄籽芸薹自交系中过表达TTG1，可通过上调相关基因的表达水平诱导原花青素的积累。
讨论
本研究首次揭示了芸薹中TTG1与黄酮类生物合成通路结构基因及谷胱甘肽S-转移酶启动子的直接结合作用。此外，本研究还鉴定出一个全新的TTG1结合基序，为后续探究TTG1的功能及种皮原花青素的积累机制提供了研究基础。