Genomic Identification and Expression Profiling of CoFAD3 Genes during Seed Development in Camellia oleifera

Camellia oil, a nutritionally rich edible oil extracted from seeds of oil Camellia , contains α-linolenic acid (ALA) as a key quality determinant. Although fatty acid desaturase 3 ( FAD3 ) has been characterized in ALA biosynthesis across various plant species, its characteristics, functions, and regulatory mechanisms in oil Camellia remain poorly understood. Here, we identified ten CoFAD3 genes from the tetraploid Camellia oleifera genome and analyzed their genomic architecture and protein properties, and revealed conserved structural features and evolutionary expansion through segmental duplications, with most duplicated gene pairs maintaining functional conservation. Multiple cis -elements associated with light responsiveness, growth regulators, phytohormones, and stress responses were identified in CoFAD3 promoters. Quantitative PCR showed that specific CoFAD3 genes exhibit expression patterns closely correlated with ALA accumulation during seed maturation. Among these, CoFAD3.6 emerged as a critical contributor to seed ALA biosynthesis, functioning within the endoplasmic reticulum and significantly enhancing seed ALA content in transgenic plants. Within the CoFAD3.6 promoter, three WRINKLED1 ( WRI1 )-binding AW-box motifs were identified. Yeast one-hybrid and dual-luciferase reporter assays confirmed that CoWRI1 binds to the CoFAD3.6 promoter and activates its expression. Arabidopsis overexpressing CoWRI1 showed significantly elevated seed ALA levels, demonstrating that CoWRI1 may enhance ALA accumulation by transcriptionally activating CoFAD3.6. Additionally, CoFAD3.6 increased tolerance to cold and ABA stress. Consistently, its expression was induced by cold stress, suggesting that phytohormones and environmental signals modulate ALA accumulation via CoFAD3.6 . Collectively, these findings provide fundamental insights into the genomic architecture, protein properties, expression patterns and stress responses of CoFAD3 genes, and further reveal the CoWRI1- CoFAD3.6 regulatory module during ALA biosynthesis in C. oleifera , offering valuable genetic targets for improving oil quality in oilseed crops through biotechnological approaches.