Oil trait and multi-omic analyses reveal the regulatory network of triacylglycerol and fatty acid accumulation in hexaploid Camellia oleifera across different harvesting stages.

Background: The regulatory network governing triacylglycerol (TAG) and fatty acid (FA) accumulation in hexaploid Camellia oleifera kernels remains unclear due to the lack of an appropriate reference genome. In this study, we combined oil trait measurements with multi-omics analyses (global transcriptomics, miRNA sequencing, proteomics, and metabolomics) across 3 harvest stages, using the hexaploid C. oleifera 'Changlin 40' genome as reference.

Results: Integrated analysis of oil traits and multi-omics data demonstrated that premature harvesting (1 week before the recommended date) decreased oil production and enhanced saturated FAs composition, while postponed harvesting (1 week after the recommended date) preserved oil yield and increased oleic and linolenic acid contents. Comprehensive pathway analysis identified 11 key metabolic pathways involved in TAG and FA biosynthesis and catabolism, revealing their coordination with terpene and flavonoid synthesis under the influence of carbohydrate metabolism. Within the 11 metabolic pathways, key regulatory enzymes comprised alcohol dehydrogenase and galactosidase that negatively regulated TAG biosynthesis, palmitoyl-protein thioesterase that mediated short-chain saturated FA formation, and acyl-CoA thioesterase that enhanced long-chain unsaturated FA production. Additionally, we detected novel associations between TAG/FA accumulation and non-canonical factors, including noncoding RNAs (circR007, lncR114741, miR167h, miR166, miR398a-3p, miR398d, and miR396a-3p), proteins (seed storage protein, caffeic acid O-methyltransferase, galactose oxidase, No Pollen Germination 1, Coronatine Insensitive 1), and metabolites (L-carnitine and L-glutamate). The reliability of our findings was confirmed through qRT-PCR (RNA validation) and Parallel Reaction Monitoring (protein validation).

Conclusion: This study established the regulatory network TAG and FA accumulation in hexaploid C. oleifera kernels, and provided foundational molecular framework for breeding high-yield, high-quality C. oleifera cultivars through marker-assisted selection.