Triacylglycerol (TAG) is a class of lipid molecules composed of three fatty acyl chains esterified to a glycerol backbone. In plants, TAG is synthesized in various tissues and serves as a carbon and energy source. Oil biosynthesis is well understood in oilseeds however how plants store oil in non-seed tissue is yet to be determined. In Avocado (Persea americana), a basal angiosperm, TAG is exclusively accumulated in mesocarp tissue and therefore is emerging as a model system to uncover underlying mechanisms of TAG biosynthesis in tissues other than seed. The mesocarp of Avocado fruit contains ~60-70% of oil by dry weight. Recent transcriptome studies revealed that the TAG biosynthesis is transcriptionally regulated in non-seed tissues. In seed tissues, TAG biosynthesis is regulated by many seed maturation factors directly or indirectly through downstream transcription factor WRINKLED1 (WRI1). Transcriptome studies revealed that in addition to ortholog of WRI1, orthologs for WRI2 and WRI3 were also highly expressed in avocado mesocarp during the period of oil accumulation. Based on the transcriptome data, I hypothesize that putative WRI genes (WRI1, 2, 3) of avocado enhance oil content in nonseed tissues. Currently, cloning of Putative PaWRI 1, 2 and 3 genes into a binary vector, followed by agrobacterium-mediated transformation to generate transient and stable transient lines, is underway. Full-length cDNA for PaWRI genes (1 & 2) were amplified and cloned into pK34 entry vector followed by sequence confirmation. PaWRI genes (1 & 2) were subcloned into pB110 destination vector and will be transformed into agrobacterium for their integration into the plants. Cloning of WRI3 is still ongoing. Transient expression of putative PaWRI 1, 2 and 3 genes, will be validated using tobacco leaf assay, are expected to enhance oil accumulation in leaf tissues. Agrobacterium bearing PaWRI genes and a viral silencing protein (p19) will be co-infiltrated on to the underside of Nicotiana benthamiana leaves. Infiltrated plants will be placed in growth room with 16:8 light/dark cycle. Four days post infiltration, infected leaf areas will be harvested and TAG content and composition will be determined by gas chromatography coupled with flame ionization detector. Functional validation of these orthologs is expected to reveal the preferred WRI isoform that likely participates in regulation of oil biosynthesis in avocado mesocarp. Additionally, this work may also elucidate the differences between regulation of TAG accumulation in seed and non-seed tissues and identify new targets to enhance TAG biosynthesis in plants.

Triacylglycerol (TAG) is a class of lipid molecules composed of three fatty acyl chains esterified to a glycerol backbone. In plants, TAG is synthesized in various tissues and serves as a carbon and energy source. Oil biosynthesis is well understood in oilseeds however how plants store oil in non-seed tissue is yet to be determined. In Avocado (Persea americana), a basal angiosperm, TAG is exclusively accumulated in mesocarp tissue and therefore is emerging as a model system to uncover underlying mechanisms of TAG biosynthesis in tissues other than seed. The mesocarp of Avocado fruit contains ~60-70% of oil by dry weight. Recent transcriptome studies revealed that the TAG biosynthesis is transcriptionally regulated in non-seed tissues. In seed tissues, TAG biosynthesis is regulated by many seed maturation factors directly or indirectly through downstream transcription factor WRINKLED1 (WRI1). Transcriptome studies revealed that in addition to ortholog of WRI1, orthologs for WRI2 and WRI3 were also highly expressed in avocado mesocarp during the period of oil accumulation. Currently, cloning of Putative PaWRI 1, 2 and 3 genes into a binary vector, followed by agrobacterium-mediated transformation to generate transient and stable transient lines, is underway. Transient expression of putative PaWRI 1, 2 and 3 genes, using tobacco leaf assay, are expected to enhance oil accumulation in leaf tissues. Stable expression of PaWRI 1, 2, and 3 in Atwri-/- is expected to restore oil accumulation in seeds. TAG content and composition will be determined by gas chromatography coupled with flame ionization detector. Functional validation of these orthologs is expected to reveal the preferred WRI isoform that likely participates in regulation of oil biosynthesis in avocado mesocarp. Additionally, this work may also elucidate the differences between regulation of TAG accumulation in seed and non-seed tissues and identify new targets to enhance TAG biosynthesis in plants.

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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.