Functional and Predictive Structural Characterization of WRINKLED2, A Unique Oil Biosynthesis Regulator in Avocado

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants

In Silico Structural Analyses of Avocado WRINKLED Orthologs

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

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. 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

Comparative in silico analysis of WRINKLED 1 paralogs in angiosperms

WRINKLED 1(WRI1), a member of AP2/EREBP class of transcription factors regulates carbon allocation between glycolytic and fatty acid biosynthetic pathway. Additionally, among the four WRI1 paralogs in arabidopsis, WRI3 and 4 but not WRI2, are also able to increase fatty acid content in different tissues. While the role of WRI1 is well established in seeds, the potential or WRI1 or its paralogs as master regulators in oil-rich nonseed tissues is poorly understood. Recent transcriptome studies of avocado (Persea americana) mesocarp revealed that the ortholog of WRI2, along with WRI1 and WRI3 was highly expressed during oil accumulation. Through transient expression assays, we further demonstrated that both PaWRI1 and PaWRI2 can accumulate oil in tobacco leaves. We conducted a comprehensive and comparative in silico analysis of WRI paralogs from a dicot, monocot and a basal angiosperm to identify distinct features associated with function. These data provide insights into the possible evolutionary changes in WRI1 homologs and allow for identification of new targets to enhance oil biosynthesis in diverse tissues

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

C-Terminal Region of WRI1 Paralogs in Avocado Is a Potential Target for Oil Enhancement in Nonseed Tissues

WRINKLED1 (WRI1), a member of AP2/EREBP class of transcription factors regulates carbon allocation between glycolytic and fatty acid biosynthetic pathway in plants. Additionally, among the four WRI1 paralogs in arabidopsis, WRI3 and 4 but not WRI2, are also able to increase fatty acid content in seed tissue. While the role of WRI1 is well established in seeds, the potential role of WRI1 or its paralogs as master regulators in oil-rich nonseed tissues is poorly understood. One of the basal angiosperm avocado (Persea americana) accumulates high oil content in its mesocarp throughout its fruit development. Recent transcriptome studies of avocado mesocarp revealed that the ortholog of WRI2, along with WRI1 and WRI3 were highly expressed throughout the developmental stages. Through transient expression assays, we further demonstrated that both PaWRI1 and PaWRI2 can accumulate oil in tobacco leaves. We conducted a comprehensive and comparative in silico analysis of WRI1, 2 & 3 orthologs from A. thaliana (dicot), Z. mays (monocot) and P. americana (basal angiosperm) to identify distinct features associated with function. Our data shows a difference in C-terminal intrinsically disordered region (IDR) and potential phosphorylation sites in PamWRI1 & 2, which might suggest their possible role in high oil biosynthesis in mesocarp tissue. Also, enrichment with hydrophobic amino acid and depletion of hydrophilic amino acid leads to high random coil secondary structure with PamWRI1 &2 showing the highest percentage among all. Absence of C- terminal PEST motif in PamWRI1 & 2 might result in their stability in nonseed tissue and thus leads to high oil biosynthesis and accumulation. These data suggest that variable C-terminal region among the WRI1 orthologs is a potential target to enhance oil biosynthesis in nonseed tissues