Environmental dependence of visible anthocyanin accumulation mediated by the CmMYB6–CmbHLH2 complex

The anthocyanin biosynthesis pathway has been investigated as a candidate for visual reporting in plants, due to its pigment-producing capacity. Activation of this pathway is predominantly regulated by MYB-bHLH regulatory complexes. In a previous study, two chrysanthemum transcription factors, CmMYB6 and CmbHLH2 , were shown to induce anthocyanin accumulation when co-expressed. However, the stability and reliability of the visible pigmentation mediated by this complex under varying environmental conditions remain unclear. In this study, the visible anthocyanin phenotype was assessed under different environmental conditions, including darkness, high temperature, and drought stress. Our results show that anthocyanin accumulation mediated by the 35S::CmMYB6-CmbHLH2 complex is highly dependent on light and temperature. High temperature reduced anthocyanin accumulation mainly by decreasing transgene expression efficiency, whereas darkness suppressed pigmentation primarily at the post-transcriptional level, while drought stress moderately enhanced anthocyanin accumulation. Collectively, these findings indicate that environmental factors regulate anthocyanin accumulation mediated by the CmMYB6-CmbHLH2 complex through distinct regulatory mechanisms, thereby suggesting that stable visible pigmentation mediated by the MYB-bHLH complex requires sufficient light intensity and a moderate temperature condition.