Orchestration of the stilbene synthase gene family and their regulators by subgroup 2 MYB genes

The control of plant specialised metabolism is exerted by transcription factors and co-regulators acting on cis -regulatory DNA sequences of pathway-structural genes, determining when, where, and how metabolites are accumulated. A particularly interesting case for studying the transcriptional control of metabolism is represented by stilbenoids, produced within the phenylpropanoid pathway, as their ability to inhibit infection by coronaviruses MERS-CoV and SARS-CoV has been recently demonstrated in vitro. Integrative omics studies in grapevine (Vitis vinifera L.), including gene co-expression networks, have previously highlighted several transcription factors (TFs) from different gene families as potential modulators of stilbenoid accumulation, conferring an ideal framework for gene function characterisation using genome-wide approaches. In the context of non-model plant species, DNA affinity purification sequencing (DAP-Seq) results a novel and potentially powerful tool for the analysis of novel uncharacterised regulators, however, it has not yet been applied in fruit crops. Accordingly, we tested as a proof-of-concept the binding of two previously characterized R2R3-MYB TFs to their known targets of the stilbene pathway, MYB14 and MYB15, obtaining 5,222 and 4,502 binding events assigned to 4,038 and 3,645 genes for each TF, respectively. Bound genes (putative targets) were overlapped with aggregated gene centred co-expression networks resulting in shared and exclusive High Confidence Targets (HCTs) suggesting a high, but not complete, redundancy. Our results show that in addition to the previously known but few STS targets, these regulators bind to almost half of the complete STS family in addition to other phenylpropanoid and stilbenoid-related genes. We also suggest they are potentially involved in other processes such as the circadian rhythm or the synthesis of biotin. We searched the activated transcriptomes of transiently MYB15 -overexpressing grapevine plants and observed a large activation of its high confidence targets, validating our methodological approach. Our results also show that MYB15 seems to play a role in regulating other stilbenoid-related TFs such as WRKY03.