Functional characterization of flowering-associated PbMADS33 and PbMADS8 in Phoebe bournei

PbMADS8 and PbMADS33 from Phoebe bournei promote flowering when overexpressed in Arabidopsis and interact in the nucleus; their co-expression enhances PbGA20ox1 and PbFBX promoter activity in transient assays. Phoebe bournei has a prolonged juvenile phase that limits breeding, but the functions of its flowering-associated regulators remain poorly understood. Transcriptome analysis of leaves from trees of different ages and floral samples identified 5,921 differentially expressed genes. Weighted gene co-expression network analysis (WGCNA) identified a black module associated with leaves from flowering trees and containing three SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)-like genes, PbMADS8, PbMADS33, and PbMADS38. Comparative genomic analysis revealed expanded SOC1-like loci enriched in repetitive sequences. PbMADS8 and PbMADS33 were selected for functional analysis because of their higher transcript abundance relative to PbMADS38. Heterologous overexpression of either gene accelerated flowering in Arabidopsis thaliana under long-day conditions. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays supported an interaction between PbMADS33 and PbMADS8 in the nucleus. DNA affinity purification sequencing (DAP-seq) identified candidate PbMADS33-binding regions, and promoter assays supported regulation of PbGA20ox1 and the putative F-box gene PbFBX. Co-expression of PbMADS8 and PbMADS33 produced the highest reporter activity for both promoters in Nicotiana benthamiana. Together, these results nominate PbMADS8 and PbMADS33 as flowering-associated candidates and PbGA20ox1 and PbFBX as candidate promoter-associated genes in P. bournei. Their effects on endogenous gibberellin levels, protein turnover, and the juvenile phase of P. bournei remain to be established.