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The study is aimed to determine the role of avocado DGAT1 and PDAT1 in seed oil synthesis. Triacylglycerol (TAG) has a nutritional and industrial value and is essential for plant growth. DGAT1 and PDAT1 catalyze the final step of TAG Assembly. We hypothesized that both PaPDAT1 and PaDGAT1, although predominantly expressed in non-seed tissues, could contribute to oil accumulation in seeds. Agrobacterium transformants with PaPDAT1 and PaDGAT1 cloned in pCAMBIA were generated to test this. Subsequently, the Agrobacterium-mediated transformation of Arabidopsis mutant lines and camelina was carried out by floral dipping. The T1 camelina seeds expressing the genes of interest were selected using fluorescence screening. Homozygous T3 lines were generated. The transgenic camelina seeds were evaluated for TAG content and fatty acid composition relative to wild-type seeds. Line D1 3-3-2 expressing PaDGAT1 and line P1 7-8 expressing PaPDAT1 showed a significant increase in C18:1 compared to the wild type.

Plants synthesize and store oil, mostly as triacylglycerols (TAG), in seeds that is transcriptionally controlled by WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Additionally, two rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid diacylglycerol acyltransferase (PDAT) catalyze the terminal step in TAG assembly and contribute to oil accumulation. Avocado (Persea americana) mesocarp, a non-seed tissue, accumulates significant amounts of TAG (~70% by dry weight) enriched with heart-healthy oleic acid. The oil accumulation stages in mesocarp coincide with the high expression of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional implication of these genes is not explored. Using structural analyses, we showed that PaWRI2 is a relatively stable protein, has a single intact AP2 DNA-binding domain, and has different C-terminal properties compared to its ortholog in Arabidopsis. Through transient expression, we demonstrated that PaWRI2 is functional and drives TAG accumulation in Nicotiana benthamiana leaves, unlike Arabidopsis WRI2. Additionally, co-infiltration of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1 further increased the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of >46 fatty acid biosynthetic pathway genes revealed that several were distinctly up- or down-regulated by the expression of PaWRI2 and PaWRI1. Further yeast-one-hybrid assay showed a unique characteristic of PaWRI2 being autoregulated and by PaWRI1. Also, both the proteins could bind to AW-box promoter elements in specific avocado genes. Deletion of the C-terminally-located ordered region in both the proteins further improved the lipid content with an altered composition in the leaf tissue. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in non-seed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and non-seed tissues.