Structural and functional insights into the LBD family involved in abiotic stress and flavonoid synthases in Camellia sinensis

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors that play a crucial role in growth and development, as well as metabolic processes. However, knowledge of the function of LBD proteins in Camellia sinensis is limited, and no systematic investigations of the LBD family have been reported. In this study, we identified 54 LBD genes in Camellia sinensis. The expression patterns of CsLBDs in different tissues and their transcription responses to exogenous hormones and abiotic stress were determined by RNA-seq, which showed that CsLBDs may have diverse functions. Analysis of the structural gene promoters revealed that the promoters of CsC4H, CsDFR and CsUGT84A, the structural genes involved in flavonoid biosynthesis, contained LBD recognition binding sites. The integrative analysis of CsLBD expression levels and metabolite accumulation also suggested that CsLBDs are involved in the regulation of flavonoid synthesis. Among them, CsLOB_3, CsLBD36_2 and CsLBD41_2, localized in the nucleus, were selected for functional characterization. Yeast two-hybrid assays revealed that CsLBD36_2 and CsLBD41_2 have self-activation activities, and CsLOB_3 and CsLBD36_2 can directly bind to the cis-element and significantly increase the activity of the CsC4H, CsDFR and CsUGT84A promoter. Our results present a comprehensive characterization of the 54 CsLBDs in Camellia sinensis and provide new insight into the important role that CsLBDs play in abiotic and flavonoid biosynthesis.