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.

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.

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.