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RNA-seq transcriptomes are poised to provide a key genetic resource for gene discovery, even in nonmodel system lacking substantial genomic resources. By considering a few key steps when designing experiments (see guidelines in Section 6.2 and examples in Section 6.3), workflows for PV gene discovery in any plant system can be tailored to the available resources, capabilities, and a priori background knowledge (Figure 6.2). In the most generalized scenario, such as the case of a nonmodel plant producing unique volatile(s) of interest but with little or no background information available, volatiles and transcriptomes of select tissues or organs, and at various developmental stages, can first be profiled. Genes that encode enzymes or TFs that match the qualitative or quantitative profile of the target metabolite(s), and maybe homologous to proteins of known function, can provide excellent starting candidates for further functional validation. Below, several other promising approaches for enhancing gene discovery are also emerging.

Harnessing TF Overexpression or Knockdown Coupled with Transcriptome (or Multi-omics) Analysis

In plants, structural gene and metabolic networks of the general phenylpropanoid pathways that generate an enormous array of specialized metabolites (flavonoid, anthocyanin, phenylpropanoid/ benzenoid volatiles inclusive) are often regulated by select TFs that belong to the R2R3-MYB and bFILH families (Allan and Espley 2018; Davies et al. 2012). Some studies have used overexpression of these TFs (with known roles) as a tool to discover novel genes. TF overexpression may accelerate the discovery of volatile pathway genes by activating multiple candidate genes simultaneously and bypassing the need for more complex analysis that takes into account the spatial and temporal variation in volatile gene expression. The opposite effect may also occur when target TFs are knocked down. For example, silencing of PH4, a petunia R2R3-MYB TF known for its role in activating vacuolar acidification, revealed a new role of the gene in floral volatile emission in petunia (Cna’ani et al. 2015).

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