Functional Validation of Wrinkled Orthologs in Avocado Oil Biosynthesis
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.
Johnson City, TN
Bhatia, Shina; Rahman, Mahbubur Md.; and Kilaru, Aruna. (false). 2017. Functional Validation of Wrinkled Orthologs in Avocado Oil Biosynthesis. Appalachian Student Research Forum, Johnson City, TN. https://www.etsu.edu/studentresearch/2017/documents/2017_asrf_programbook.pdf