An R2R3‐MYB transcription factor represses the transformation of α‐ and β‐branch carotenoids by negatively regulating expression of <i>CrBCH2</i> and <i>CrNCED5</i> in flavedo of <i>Citrus reticulate</i>

<jats:title>Summary</jats:title><jats:p> <jats:list list-type="bullet"> <jats:list-item><jats:p>Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α ‐ and β‐branch carotenoid metabolism.</jats:p></jats:list-item> <jats:list-item><jats:p>In this study, an R2R3‐MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α‐ and β‐branch carotenoids was identified using Green Ougan (MT), a stay‐green mutant of <jats:italic>Citrus reticulata</jats:italic> cv <jats:italic>Suavissima</jats:italic>. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of <jats:italic>β‐carotene hydroxylases 2</jats:italic> (<jats:italic>CrBCH2</jats:italic>) and <jats:italic>9‐cis‐epoxycarotenoid dioxygenase 5</jats:italic> (<jats:italic>CrNCED5</jats:italic>) was responsible for the delay in the transformation of α‐ and β‐carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of <jats:italic>CrMYB68</jats:italic> in MT.</jats:p></jats:list-item> <jats:list-item><jats:p>Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate <jats:italic>CrBCH2</jats:italic> and <jats:italic>CrNCED5</jats:italic>. Moreover, transient overexpression experiments using leaves of <jats:italic>Nicotiana benthamiana</jats:italic> indicated that CrMYB68 can also negatively regulate <jats:italic>NbBCH2</jats:italic> and <jats:italic>NbNCED5</jats:italic>.</jats:p></jats:list-item> <jats:list-item><jats:p>To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.</jats:p></jats:list-item> </jats:list> </jats:p>