Functional Divergence of the G-Box Regulatory Factor Genes CsGRF2 and CsGRF7 in Tea Plants

The G-box regulatory factor gene family (GRF, also known as 14-3-3) gene family, a highly conserved group of regulatory genes in eukaryotes, plays crucial roles in stress adaptation and growth and development regulation. However, the functional characterization of GRF genes in tea plants remains largely unexplored. In this study, we identified two key GRF members, CsGRF2 and CsGRF7, which exhibited pronounced differentiation in expression levels and functions across different tea plant cultivars. Sequence analysis showed that CsGRF2 was stably located on Chr04 among the eight tested tea cultivars, while CsGRF7 was only detected on Chr14 in four of them, indicating genetic differentiation between CsGRF2 and CsGRF7 in tea cultivars. Subcellular localization assays showed that CsGRF2 was localized in the cell membrane and cytoplasm, whereas CsGRF7 was specifically localized in the nucleus. Expression pattern analysis demonstrated that CsGRF2 was significantly upregulated under cold stress, while CsGRF7 was cold-insensitive. Transient silencing of CsGRF2 in tea shoots significantly reduced cold tolerance. The expression of CsGRF7 in the axillary/floral buds of floriferous cultivars (FDDB, ZH2) was significantly higher than that in oliganthous cultivars (PYTZ, HYFS) during the flower bud differentiation period. CsGRF7 expression showed a positive correlation with flowering abundance across different cultivars. Overexpression of CsGRF7 in Arabidopsis thaliana accelerated flowering, and CsGRF7 negatively regulated the expression of EARLY FLOWERING 3 (CsELF3), whose homolog is a negative regulator of flowering time in Arabidopsis. Our findings deepen the understanding of GRF gene specialization in perennial plants and provide valuable gene targets for molecular breeding of cold-tolerant and low-flowering tea cultivars.