Phosphorylation of CsLARb by mitogen-activated protein kinase cascade enhances catechins biosynthesis in tea plants ( <i>Camellia sinensis</i> )

Abstract Catechins, including monomeric forms and oligomeric proanthocyanidins (PAs), are the primary compounds responsible for tea astringency. Under stress conditions, changes in the biosynthetic regulation of catechins are part of the molecular mechanisms underlying the resistance of tea plants. Mitogen-activated protein kinase (MAPK) cascades play crucial regulatory roles in plant responses to biotic and abiotic stresses. However, the role of MAPK cascades in catechin biosynthesis in tea plants is still unclear. Through omics analyses, this study focused on the phosphorylation of leucoanthocyanidin reductase b (LARb), a key terminal enzyme in catechin biosynthesis, by multiple MAPKs at Ser-5 in its N-terminal sequence, including CsMAPK3–1, CsMAPK4–2, and CsMAPK6. Furthermore, by integrating omics data with protein interaction validation, we identified the ‘CsMEKK1–1–CsMKK1/6–CsMAPK3–1/4–2/6’ and ‘CsMEKK7–1-CsMKK5-CsMAPK3/4–2’ cascades. These cascades appear to play significant roles in the regulation of catechin and PA synthesis by modulating CsLARb phosphorylation. This regulation occurred in response to various environmental stimuli, including variations in exogenous sucrose levels and abiotic stresses, such as cold and UV radiation. Moreover, phosphorylation significantly enhanced CsLARb stability. Additional experiments demonstrated that the overexpression of CsLARb and CsLARbS5D in transgenic tobacco increased catechin levels. These findings not only elucidate the role of MAPK cascades in regulating stress-induced flavonoid metabolism in tea plants via phosphorylation but also identify novel targets for improving tea quality and stress resilience.