Table_2_MaMYB4, an R2R3-MYB Repressor Transcription Factor, Negatively Regulates the Biosynthesis of Anthocyanin in Banana.XLSX

Anthocyanins spatiotemporally accumulate in certain tissues of particular species in the banana plant, and MYB transcription factors (TFs) serve as their primary regulators. However, the precise regulatory mechanism in banana remains to be determined. Here, we report the identification and characterization of MaMYB4, an R2R3-MYB repressor TF, characterized by the presence of EAR (ethylene-responsive element binding factor–associated amphiphilic repression) and TLLLFR motifs. MaMYB4 expression was induced by the accumulation of anthocyanins. Transgenic banana plants overexpressing MaMYB4 displayed a significant reduction in anthocyanin compared to wild type. Consistent with the above results, metabolome results showed that there was a decrease in all three identified cyanidins and one delphinidin, the main anthocyanins that determine the color of banana leaves, whereas both transcriptome and reverse transcription–quantitative polymerase chain reaction analysis showed that many key anthocyanin synthesis structural genes and TF regulators were downregulated in MaMYB4 overexpressors. Furthermore, dual-luciferase assays showed that MaMYB4 was able to bind to the CHS, ANS, DFR, and bHLH promoters, leading to inhibition of their expression. Yeast two-hybrid analysis verified that MaMYB4 did not interact with bHLH, which ruled out the possibility that MaMYB4 could be incorporated into the MYB-bHLH-WD40 complex. Our results indicated that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, likely due to a two-level repression mechanism that consists of reduced expression of anthocyanin synthesis structural genes and the parallel downregulation of bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that was identified by genetic methods in bananas, which will be helpful for manipulating anthocyanin coloration in banana programs in the future.

花青素在香蕉属特定物种的特定组织中呈时空特异性积累，MYB转录因子（MYB transcription factors, TFs）是其主要调控因子。然而，香蕉中调控花青素合成的确切分子机制仍有待阐明。本研究鉴定并表征了一个R2R3-MYB类转录抑制因子MaMYB4，其含有EAR（ethylene-responsive element binding factor–associated amphiphilic repression）和TLLLFR两个保守基序。MaMYB4的表达可被花青素积累所诱导。过表达MaMYB4的转基因香蕉植株，其花青素积累量较野生型显著下降。与上述结果一致，代谢组学分析显示，在决定香蕉叶片颜色的主要花青素组分中，所鉴定出的3种矢车菊素和1种飞燕草素的含量均出现下降；而转录组学与逆转录定量聚合酶链反应（RT-qPCR）分析均表明，在MaMYB4过表达植株中，多个花青素合成关键结构基因及转录调控因子的表达量均被下调。此外，双荧光素酶报告基因实验结果显示，MaMYB4可结合CHS、ANS、DFR及bHLH的启动子区域，进而抑制这些基因的表达。酵母双杂交实验证实，MaMYB4无法与bHLH发生相互作用，这排除了MaMYB4参与组装MYB-bHLH-WD40激活复合体的可能性。本研究结果表明，MaMYB4作为香蕉花青素生物合成的负调控因子发挥功能，其调控机制可能包含两个层面：一是直接抑制花青素合成结构基因的表达，二是通过平行下调bHLH的表达，干扰MYB-bHLH-WD40激活复合体的正常组装。据我们所知，本研究是首次通过遗传学方法在香蕉中鉴定得到调控花青素合成的MYB类转录因子，该成果可为未来香蕉育种中调控花青素色泽提供理论依据。