Evolution of the PEBP gene family in Juglandaceae and their regulation of flowering pathway under the synergistic effect of JrCO and JrNF-Y proteins.

Phosphatidyl ethanolamine-binding protein (PEBP) has a conserved PEBP domain and plays an important role in regulating the flowering time and growth of angiosperms. To understand the evolution of PEBP family genes in walnut family and the mechanism of regulating flowering in photoperiod pathway, 53 genes with PEBP domain were identified from 5 Juglandaceae plants. The PEBP gene family of Juglandaceae can be divided into four subgroups, FT-like, TFL-like, MFT-like and PEBP-like subgroups. These genes all show very high homology for motifs and gene structure in Juglandaceae. In addition, the results of gene replication and collinearity analysis showed that the evolution of PEBP genes was mainly purified and selected, and segmental repetition was the main driving force for the evolution of PEBP gene family in walnut family. We found that PEBP gene family played an important role in female flower bud differentiation, and most JrPEBP genes were highly expressed in leaf bud and female flower bud by qRT-PCR. In Arabidopsis, AtCO can not only directly bind to CORE2, but also interact with NF-Y complex to positively regulate the expression of AtFT gene. In this study, we proved that JrCO (the lineal homologue of AtCO) could not directly regulate the expression of JrFT gene, but could enhance the binding of JrNF-YB4/6 protein to the promoter of JrFT gene by forming a heteropolymer with NF-YB4/NF-YB6. We also confirmed that JrNF-YC1/3/7, JrNF-YB4/6 and JrCO can form a trimer structure similar to AtNF-YB-YC-CO of Arabidopsis, and then bind to the promoter of JrFT gene to promote the transcription of JrFT gene. In a word, through identification and analysis of PEBP gene family in Juglandaceae and study on the mechanism of photoperiod pathway regulating flowering in walnut, we have found that nuclear transcription factor NF-YB/YC plays a more important role in the trimer structure of NF-YB-YC-CO in walnut species. Our study has further perfected the flowering regulatory network of walnut species.