| Summary: | Plants synthesize and store oil, mostly triacylglycerol (TAG), in various storage tissues that serves as a source of carbon and energy. The process is transcriptionally controlled by WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, that regulates most of the fatty acid biosynthesis genes. Among the four Arabidopsis WRI1 paralogs, only WRI2 is nonfunctional and failed to complement wri1-1 mutant seeds. The oleaginous Avocado (Persea americana) fruit mesocarp (60-70% DW oil) showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3. While the role of WRI1 as a master seed oil biosynthesis regulator is well-established, the function of WRI1 paralogs in non-seed tissues is poorly understood. We conducted structural analyses to elucidate distinct features of avocado WRI paralogs compared to their orthologs in seed tissues. Comprehensive comparative in silico analyses of WRI1 paralogs from Arabidopsis (dicot), maize (monocot), and avocado revealed distinct features associated with their function. Our analysis showed the presence of only one AP2 domain in all WRI2 orthologs, compared to two AP2 in others. The highly conserved N-terminal region and the less conserved C-terminal regions make up the primary structure of the proteins, with amino acid composition bias characteristic of intrinsically disordered proteins (IDPs). Additionally, the avocado WRI2 showed a high proportion of random coil secondary structure, although it lacks a C-terminal intrinsically disordered region (IDR). Also, both WRI1 and WRI2 have distinct predicted phosphorylation target sites compared to their orthologs, whereas WRI2 lacks a PEST motif. Finally, through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. Our study showed that avocado WRI2 is structurally different and is functional, unlike its ortholog in Arabidopsis. This study provides us with new targets to enhance oil biosynthesis in plants.
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