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.

Arabidopsis WRINKLED1 (AtWRI1) (At3g54320) is a pivotal transcription factor in the regulation of plant oil biosynthesis. Our previous study identified a WRI1 homolog highly expressed in oil palm mesocarp (EgWRI1). EgWRI1 protein is 93 amino acids shorter than AtWRI1 with low sequence conservation and all deletions occurring in the Cterminal region. We asked whether this divergent EgWRI1 protein, from a non-seed tissue with very high oil content, and from a phylogenetically distant species is able to complement the Arabidopsis loss-of-function mutant wri1-1. We show that expression of EgWRI1 is able to restore several phenotypes of wri1-1, including reduced seed fatty acid content, the “wrinkled" appearance of the seed coat, reduced seed germination, and impaired seedling establishment. Comparison of the protein sequence of WRI1 orthologs across many diverse plant species revealed the conservation of a 9 bp exon encoding “VYL". However, this small exon is missing in one of three predicted AtWRI1 splice forms. To understand expression of WRI1 splice forms we performed RNASeq analysis of Arabidopsis developing seeds and queried other Arabidopsis EST and RNASeq databases derived from a number of tissues and from a range of plant species. In all cases, only splice form 3 is expressed in Arabidopsis and VYL is observed in the cDNA of all WRI1 orthologs investigated. Site-directed mutagenesis showed that amino acid substitutions within the ‘VYL' exon of AtWRI1 results in failure to restore reduced oil content of wri1-1 seeds, providing direct evidence for the crucial role of this small exon in AtWRI1 function.

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants.