Ubiquitin-mediated degradation of the inhibitor FvMYB1 and the activator FvBBX20 by FvCSN5 balances anthocyanin biosynthesis in strawberry fruit

Abstract

Plant CSN5 is widely recognized as the subunit of the COP9 signalosome and CSN5 is mainly involved in plant growth and development, and tolerance to biotic and abiotic stresses. However, the molecular mechanism of CSN5 regulating anthocyanin biosynthesis in plants is still largely unknown. Here, we identified FvCSN5 from the woodland strawberry yeast two-hybrid library using the anthocyanin pathway inhibitor MYB1 as bait. We demonstrated the interaction of FvCSN5 and FvMYB1 by H2Y, Pull-down, LCI, and BiFC assays. FvCSN5 was expressed in all test tissues and localized in the nucleus and cytosol with self-activation activity. Stable overexpression of FvCSN5 in woodland strawberries reduced anthocyanin accumulation in fruits. The protein level of FvMYB1 greatly decreased in overexpressing FvCSN5 plants compared with wild-type plants. Protein degradation assay and MG-132 treatment (a proteasome inhibitor blocking 26S proteasome activity) revealed FvCSN5 degraded FvMYB1 through the ubiquitination pathway. In addition, FvCSN5 also interacted with the anthocyanin activator FvBBX20 and FvBBX20 could be degraded by FvCSN5. Moreover, transient expression analysis showed the expression of anthocyanin biosynthetic genes FvCHS and FvF3H was greatly increased and decreased when FvCSN5 was co-expressed with FvMYB1 and FvBBX20, respectively. These results indicate that FvMYB1-FvCSN5-FvBBX20 is a novel ternary complex that regulates anthocyanin biosynthesis by the ubiquitination pathway.