CsMIEL1-CsMYB113 regulatory module mediates temperature-dependent anthocyanin accumulation in blood orange

Abstract Background The accumulation and pigmentation of anthocyanins in blood oranges are highly temperature-dependent. However, the molecular mechanism by which high temperatures suppress anthocyanin biosynthesis and metabolism remains unclear. Results In this study, blood oranges at different developmental stages were subjected to gradient temperature treatments, followed by comprehensive metabolic profiling analysis. The results revealed that high temperature specifically blocks the synthesis and metabolism of cyanidin-3-O-glucoside and cyanidin-3-O-(6-O-malonyl-beta-D-glucoside), which are responsible for the pigmentation of blood oranges. Transcriptional analysis revealed that the expression levels of CsF3H , CsDFR , CsANS , and CsBZ1 , which are responsible for the biosynthesis of cyanidin-3-O-glucoside and cyanidin-3-O-(6-O-malonyl-beta-D-glucoside), were significantly and positively correlated with CsMYB113 and were negatively regulated by high temperature. The transient transformation assay of tobacco leaves and the expression profile analysis revealed that the ubiquitinated protein CsMIEL1, activated by high temperature, inhibited the anthocyanin accumulation induced by CsMYB113 in tobacco leaves. Protein-protein interaction assays further confirmed the interaction between CsMIEL1 and CsMYB113 , emphasizing the ubiquitination process of CsMYB113 mediated by CsMIEL1 . Conclusion In summary, this study identified the key anthocyanin subtypes that determine the pigmentation of blood oranges and the corresponding biosynthetic pathways. A hypothesis was constructed that CsMIEL1 induced by high temperature ubiquitinated and degraded CsMYB113, blocking anthocyanin biosynthesis and resulting in the loss of pigment in blood oranges. These findings provide novel insights into the molecular mechanism by which high temperature inhibits anthocyanin biosynthesis in blood oranges.