PrGRFZF1, as an interacting protein, is involved in regulating α-linolenic acid synthesis in tree peony seeds

Tree peony has emerged as a promising woody oil crop due to its abundant α-linolenic acid (ALA) content in its seeds. However, the molecular regulatory mechanisms governing ALA synthesis in tree peony seeds remain incompletely understood. Previous studies have demonstrated that the bZIP family transcription factor ABI5 in tree peony seeds regulates ALA synthesis by activating the expression of FAD3. Building upon these findings, this study utilized Paeonia rockii (a tree peony variety with the highest ALA accumulation) to construct a seed cDNA yeast library and screen for interacting proteins of PrABI5 using it as bait. The interacting protein PrGRFZF1, a C6-type Zinc Finger protein, was identified through yeast two-hybrid (Y2H) and Bimolecular fluorescence complementation (Bi-FC) assay. Yeast one-hybrid (Y1H) assays and dual-luciferase (dual-LUC) reporter assays further confirmed that PrGRFZF1 does not directly bind to the PrFAD3 promoter but instead enhances the transcriptional activation of PrFAD3 by PrABI5 through their physical interaction. Transient overexpression in Nicotiana benthamiana and virus-induced gene silencing (VIGS) experiments in P. rockii seeds demonstrated that the interaction between PrGRFZF1 and PrABI5 significantly promotes the accumulation of total fatty acids and ALA content in tree peony seeds. Collectively, this study establishes a transcriptional regulatory module of PrGRFZF1-PrABI5-PrFAD3 for ALA synthesis in tree peony, providing theoretical insights for molecular breeding of plants with elevated ALA content.