花青素是具有既定健康益处的主要类黄酮化合物。尽管MYB-碱性螺旋-环-螺旋(bHLH)-WD-重复蛋白(MBW)复合物中的MYB转录因子(TFs)在花色苷生物合成中的分子机制已经被揭示,在此过程中无法形成MBW复合体的其他MYBTFs的功能仍不清楚。在这项研究中,我们发现并广泛表征了洋葱(洋葱)中的R2R3-MYBTF,命名为AcMYB96,被鉴定为潜在的花色苷激活剂。AcMYB96被分类为R2R3-MYBTF家族的第1亚组,缺乏与bHLHIIIfTF相互作用所需的保守序列。始终如一,酵母双杂交试验表明,AcMYB96不与任何bHLHIIIfTFs相互作用,包括AcB2和AtTT8。AcMYB96的转录模式与花青素积累水平相关,通过在洋葱鳞茎和拟南芥的上皮细胞中过表达证实了其在激活花青素生物合成中的作用。酵母单杂种,电泳迁移率偏移,和启动子反式激活实验进一步证明,AcMYB96通过与查尔酮合酶(AcCHS1)的启动子结合来促进花青素的生物合成,花青素合成酶(AcANS),和UDP-葡萄糖-类黄酮3-O-葡萄糖基转移酶(AcUFGT)基因,从而独立于bHLHIIIfTF激活它们的表达。这些结果表明,AcMYB96激活花青素生物合成而不形成MBW复合物,为进一步丰富花色苷积累和选育高花色苷红葱的基因资源提供理论基础。
Anthocyanins are major flavonoid compounds with established health benefits. Although the molecular mechanisms of MYB transcription factors (TFs) within the MYB-basic helix-loop-helix (bHLH)-WD-repeat protein (MBW) complex in anthocyanin biosynthesis have been revealed, the functions of other MYB TFs that are unable to form the MBW complex in this process remain unclear. In this study, we uncovered and extensively characterized an R2R3-MYB TF in onion (Allium cepa L.), named AcMYB96, which was identified as a potential anthocyanin activator. AcMYB96 was classified into subgroup 1 of the R2R3-MYB TF family and lacked the conserved sequences required for interactions with bHLH IIIf TFs. Consistently, yeast two-hybrid assays showed that AcMYB96 did not interact with any bHLH IIIf TFs examined, including AcB2 and AtTT8. The transcription pattern of AcMYB96 correlated with the level of anthocyanin accumulation, and its role in activating anthocyanin biosynthesis was confirmed through overexpression in the epithelial cells of onion bulbs and Arabidopsis. Yeast one-hybrid, electrophoretic mobility shift, and promoter transactivation assays further demonstrated that AcMYB96 promoted anthocyanin biosynthesis by binding to the promoters of the chalcone synthase (AcCHS1), anthocyanidin synthase (AcANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (AcUFGT) genes, thereby activating their expression independent of bHLH IIIf TFs. These results demonstrate that AcMYB96 activates anthocyanin biosynthesis without forming the MBW complex, providing a theoretical foundation to further enrich the gene resources for promoting anthocyanin accumulation and breeding red onions with high anthocyanin content.