TY - JOUR
T1 - Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine
AU - Orduña, Luis
AU - Li, Miaomiao
AU - Navarro-Payá, David
AU - Zhang, Chen
AU - Santiago, Antonio
AU - Romero, Pablo
AU - Ramšak, Živa
AU - Magon, Gabriele
AU - Höll, Janine
AU - Merz, Patrick
AU - Gruden, Kristina
AU - Vannozzi, Alessandro
AU - Cantu, Dario
AU - Bogs, Jochen
AU - Wong, Darren C.J.
AU - Huang, Shao shan Carol
AU - Matus, José Tomás
N1 - Funding Information:
This work was supported by Grant PGC2018-099449-A-I00 and by the Ramón y Cajal program (grant RYC-2017-23 645), both awarded to JTM, and to the FPI scholarship (PRE2019-088044) granted to LO from the Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain), Agencia Estatal de Investigación (AEI, Spain), and Fondo Europeo de Desarrollo Regional (FEDER, European Union). CZ is supported by China Scholarship Council (CSC; no. 201906300087). KG and ZR were supported by the Slovenian Research Agency (grants P4-0165 and Z7-1888). SCH is partially supported by the National Science Foundation (grant PGRP IOS-1916804). This article is based upon work from COST Action CA 17111 INTEGRAPE, supported by COST (European Cooperation in Science and Technology). Data have been treated and uploaded in public repositories according to the FAIR principles, in accordance to the guidelines found at INTEGRAPE. The genomic data presented here will be re-analysed and associated to the PN40024.v4 assembly and its structural annotation upon its release. Grapevine cell cultures in solid media were kindly provided by Roque Bru (Universidad de Alicante). Special thanks to Pere Mestre and Philippe Hugueney (INRAE Colmar) for providing the 35S:STS48 construct and the 35S:ROMT1 positive control used for agroinfiltration, to Anne-Francoise Adam-Blondon and Nicolas Francillonne (URGI INRAE Versailles) for guidance in the adaptation of JBrowse during an INTEGRAPE Short-Term Scientific Mission of LO, and to Susana Selles-Marchart, for her help with stilbenoid quantification.
Funding Information:
This work was supported by Grant PGC2018‐099449‐A‐I00 and by the Ramón y Cajal program (grant RYC‐2017‐23 645), both awarded to JTM, and to the FPI scholarship (PRE2019‐088044) granted to LO from the Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain), Agencia Estatal de Investigación (AEI, Spain), and Fondo Europeo de Desarrollo Regional (FEDER, European Union). CZ is supported by China Scholarship Council (CSC; no. 201906300087). KG and ZR were supported by the Slovenian Research Agency (grants P4‐0165 and Z7‐1888). SCH is partially supported by the National Science Foundation (grant PGRP IOS‐1916804). This article is based upon work from COST Action CA 17111 INTEGRAPE, supported by COST (European Cooperation in Science and Technology). Data have been treated and uploaded in public repositories according to the FAIR principles, in accordance to the guidelines found at INTEGRAPE. The genomic data presented here will be re‐analysed and associated to the PN40024.v4 assembly and its structural annotation upon its release. Grapevine cell cultures in solid media were kindly provided by Roque Bru (Universidad de Alicante). Special thanks to Pere Mestre and Philippe Hugueney (INRAE Colmar) for providing the 35S: construct and the 35S: positive control used for agroinfiltration, to Anne‐Francoise Adam‐Blondon and Nicolas Francillonne (URGI INRAE Versailles) for guidance in the adaptation of JBrowse during an INTEGRAPE Short‐Term Scientific Mission of LO, and to Susana Selles‐Marchart, for her help with stilbenoid quantification. STS48 ROMT1
PY - 2022/4
Y1 - 2022/4
N2 - 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 −10 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.
AB - 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 −10 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.
KW - DNA affinity purification sequencing
KW - regulatory networks
KW - secondary metabolism
KW - transcription factors
KW - transcriptional regulation
KW - Gene Expression Regulation, Plant/genetics
KW - Plant Proteins/genetics
KW - Gene Regulatory Networks
KW - Stilbenes/metabolism
KW - Shikimic Acid
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U2 - 10.1111/tpj.15686
DO - 10.1111/tpj.15686
M3 - Article
C2 - 35092714
AN - SCOPUS:85125544198
SN - 0960-7412
VL - 110
SP - 529
EP - 547
JO - Plant Journal
JF - Plant Journal
IS - 2
ER -