Distinct roles of avocado transcription factors PaRAP2.4-2 and PaERF082-1 in fatty acid synthesis regulation in transgenic Arabidopsis thaliana

• Identified PaRAP2.4-2 and PaERF082-1 as key regulators of avocado FA synthesis. • Generated PaRAP2.4-2 mutants in Arabidopsis using Agrobacterium and CRISPR/Cas9. • Overexpression of PaRAP2.4-2 upregulated 16 genes linked to FA synthesis and assembly. • PaRAP2.4-2 significantly affects FA accumulation, while PaERF082-1 has a minor role. Avocado is renowned for its high content of fatty acids (FAs), which are essential for human nutrition. Understanding the specific functions of unknown transcription factors (TFs) in FA synthesis in avocado mesocarp is crucial for comprehending the regulation of FA accumulation. PaRAP2.4-2 and PaERF082-1 may play a role in regulating FA synthesis and increasing accumulation. To understand their specific function in FA synthesis, PaRAP2.4-2 and PaERF082-1 overexpressing and atrap2.4–2 knockout mutants of Arabidopsis thaliana were generated using Agrobacterium -mediated transformation and the CRISPR/Cas9 system. Transcriptome, FA accumulation, and lipid droplet histological observations were then performed. Furthermore, functional characterization of these TFs was conducted to investigate their roles in FA accumulation. This study determined that PaRAP2.4-2 and PaERF082-1 are in the nucleus. Transcriptomic, biochemical, and histological analyses of PaRAP2.4-2 overexpression in transgenic A. thaliana plants revealed 16 up-regulated genes related to FA synthesis, triacylglycerol assembly, and lipid droplet formation, as well as increased FA contents and lipid droplet area compared to the wild type, implying that PaRAP2.4-2 contributes to enhancing FA accumulation. Moreover, the overexpression of PaERF082-1 in transgenic A. thaliana plants led to up-regulation of the expression of only 7 genes linked to FA synthesis and TAG (triacylglycerol) assembly, exerting a negligible effect on FA accumulation. However, the FA contents and lipid droplet area in the atrap2.4–2 knockout mutant were not significantly changed compared with the wild type. These results highlight the distinct roles of PaRAP2.4-2 and PaERF082-1 in modulating FA metabolism in avocado, providing a foundation for further studies on the relationship between AP2/ERF transcription factors and FA accumulation.