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Plants synthesize and store oil mostly as triacylglycerols (TAG) in seeds that is transcriptionally controlled by WRINKLED 1 (WRI1), an APETALA2 (AP2) transcription factor. In contrast, avocado (Persea americana) mesocarp, which accumulates ~70% lipids by dry weight showed high expression of WRI2, along with WRI1 and WRI3. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Through in silico analysis, we identified that PaWRI2 has a single intact AP2 DNA-binding domain and lacks a C-terminal intrinsically disordered region (IDR3) and PEST motif, which likely results in a relatively stable protein, compared to its Arabidopsis ortholog. We further demonstrated that avocado WRI2 is functional, unlike Arabidopsis WRI2, and accumulates TAG when transiently expressed in Nicotiana benthamiana leaves. Additionally, co-infiltration of PaWRI2 with PaWRI1 and genes encoding for terminal step in TAG assembly, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and/or phospholipid:diacylglycerol acyltransferase 1 (PDAT1) also led to further increase in the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of > 40 fatty acid biosynthetic pathway genes revealed that several were distinctly up or down regulated by the expression of PaWRI2 and PaWRI1. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in nonseed 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 nonseed tissues.

Transcription factor Wrinkled (WRI) 1 is associated with triacylglycerol (TAG) biosynthesis and accumulation in plant tissues. In avocado (Persea americana), a basal angiosperm, four WRI orthologs (1-4) were identified by transcriptome studies and the gene expression of WRI1, 2 and 3 was associated with TAG accumulation in mesocarp tissue. Therefore, it is hypothesized that putative PaWRI1, 2 and 3 but not PaWRI4 are responsible for TAG synthesis in non-seed tissues. To this extent, various in silico analyses were performed to identify similarities and distinct features of putative WRI genes in basal angiosperm relative to maize and Arabidopsis, a monocot and dicot respectively. Predicted structural comparison of these orthologs is expected to reveal the distinct features of avocado WRI paralogs that are associated with the regulation of oil biosynthesis in non-seed tissues.

The biofuel market is dominated by ethanol and biodiesel derived from cellulosic and lipid-based biomass crops. This is largely due to the relatively low costs and reliability of production. At present, production of non-food plant-derived oils for biofuel production in the U.S. is minimal. A research team from the Commonwealth Scientific and Industrial Research Organization (CSIRO), an independent Australian federal government research institution, has developed an efficient transgenic system to engineer oil production in tobacco leaves. This novel system is comprised of multiple transgenes that direct the endogenous metabolic flux of oil precursors towards triacylglycerol (TAG) production. Additional genes were incorporated to store and protect the accumulated oil in vegetative tissues. Preliminary greenhouse tests by the CSIRO research group indicated an oil content of > 30% by dry weight (DW) in tobacco leaf lamina. Here we evaluated two transgenic lines against a non-transgenic control in 2017 and 2018 in greenhouse and field production systems. The 2017 pilot study showed that the high leaf-oil tobacco line was viable and will grow in the field in Kentucky. Chemical analyses revealed significantly higher oil content compared to the non-transgenic control despite several logistical setbacks. These promising discoveries prompted the deployment of additional transgenic line assessments and further data validation in 2018. Line evaluations in 2018 revealed that the LEC2:WRI1:DGAT:OLE transgenic line had the highest leaf oil content (≥ 19.3% DW-1) compared to both the WRI1:DGAT:OLE transgenic line (≤ 5.6% DW-1) and non-transgenic control (≤ 2.1% DW-1). The results of this research will contribute to the successful development of transgenic tobacco lines engineered to accumulate high concentrations of TAG in the leaves.