Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by Subgroup 2 R2R3‐MYBs in grapevine

Abstract

The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP-Seq) allows for the genome-wide TF-binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within À10 kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H, and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates among which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes were induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.

A COMPASS for VESPUCCI: A FAIR Way to Explore the Grapevine Transcriptomic Landscape

“…The second use case aims to characterize a grapevine gene family and identify those members that show tissue-specific expression and modulation upon biotic stresses. The last use case explores a high number of target genes modulated by the MYB14 transcription factor obtained in Orduña et al (2022) . This set of MYB14-bound genes was filtered according to the TF-binding position in: (i) upstream-bound (up to −3 kb from each transcription start site) and (ii) gene body and downstream-bound (coding sequence and untranslated regions, up to 2 kb from the end of each gene feature).…”

Dissecting the effect of soil on berry transcriptional plasticity in two Italian grapevine varieties ( V. vinifera L.).

“…Analysis of the cistrome in the promoter sequences of these genes shown that they are potential targets of the transcription factors MYB13, MYB14 and MYB15. The control of STS and other biosynthetic genes of the shikimate pathway is now a known case (Höll et al , 2013;Wong et al , 2016;Vannozzi et al , 2018;Orduña et al , 2022). More intriguing is the apparent involvement of the aforementioned TFs in the regulation of gene clusters linked to the biosynthesis of terpenes and other flavonoids, as well as their involvement in the regulation of genes that show divergent expression patterns.…”

“…The analysis highlighted thecis element GGTAGGTG (P-val 5.4E-14, FDR 9.1E-12) as the best ranked enriched motif. This element, in addition to representing a target of the TF VvMYB03 based on PlantDBTF, was found to be the binding site of VvMYB13, VvMYB14 and VvMYB15 based on a recent study in which the cistroma of these TFs was studied through a DNA Affinity Purification Assay (DAPseq) (Orduña et al , 2022). To shed light on the regulation of these soil-responsive genes we therefore screened the DAPseq data to verify how many of the 740 genes were effective targets of MYB13-15.…”

Transcriptomic and metabolomic integration as a resource in grapevine to study fruit metabolite quality traits

“…(2021) . The use of metabolomics (stilbenoid-oriented) and transcriptomics, coupled to the genome-wide exploration of transcription factor binding sites through DAP-seq, allowed Orduña et al. (2022) to identify new candidate genes coding for resveratrol modifying enzymes including laccasses, glycosyltransferases and O -methyl-transferases, potentially producing viniferin, piceid and pterostilbene, respectively.…”