Functional analysis of grapevine phosphoenolpyruvate carboxylase kinases (VvPPCKs) reveals a new role as regulators of the defence response and induction of resveratrol production in grapevine cells under elicitation

Hurtado-Gaitán, Elías, Martínez-Márquez, Ascensión, Martínez-Esteso, María José, Morante-Carriel, Jaime Alfredo, Hartwell, James ORCID: 0000-0001-5000-223X and Bru-Martínez, Roque (2026) Functional analysis of grapevine phosphoenolpyruvate carboxylase kinases (VvPPCKs) reveals a new role as regulators of the defence response and induction of resveratrol production in grapevine cells under elicitation [Preprint]

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Abstract

<p>Phosphoenolpyruvate carboxylase (PEPC) and its regulatory protein kinase, phosphoenolpyruvate carboxylase kinase (PPCK), have been proposed to play key functions in the defence response leading to <em>trans</em>-resveratrol (<em>t</em>-R) biosynthesis in grapevine. This has led to the hypothesis that PEPC phosphorylation by PPCK governs the flow of carbon at a crucial crossroads between primary and secondary metabolism. Here, we used a grapevine suspension cell culture system subjected to stress elicitation with methyl-β-cyclodextrin (MBCD) to dissect the function of PPCK and PEPC phosphorylation in <em>t</em>-R production. Specifically, an <em>in vivo</em> functional analysis was carried out using both stable overexpression and hairpin RNA-mediated RNAi silencing of the two grapevine <em>VvPPCK</em> paralogues, <em>VvPPCK1</em> and <em>VvPPCK2, </em>in stable transgenic cell lines. The phenotypic consequences of overexpression and silencing of the two <em>VvPPCKs</em> was tested via: i) their influence on PEPC activity; ii) monitoring the level of <em>t</em>-R production in mildly elicited cell cultures, and, iii) their influence on total polyphenol accumulation. The results revealed that stable overexpression of <em>PPCK </em>diminished <em>t</em>-R biosynthesis and enhanced PEPC activity, while the knock-down line for <em>PPCK</em> had the opposite effect. It proved challenging to recover stable silenced cell lines targeting <em>VvPPCK</em>, suggesting PEPC regulation by PPCK plays a vital role in cell growth and development. Our functional data from stable transgenic grapevine cell cultures establish a novel role for PPCK, and consequently for PEPC phosphorylation, in the negative regulation of the <em>t</em>-R biosynthesis in response to elicitation stress conditions.</p>

Item Type:	Preprint
Uncontrolled Keywords:	3108 Plant Biology, 31 Biological Sciences, Biotechnology, Genetics, 1.1 Normal biological development and functioning
Divisions:	Faculty of Health & Life Sciences Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology > Inst. Systems, Molec & Integrative Biology (T&R Staff) Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology > Biochemistry, Cell and Systems Biology
Depositing User:	Symplectic Admin
Date Deposited:	10 Jul 2026 07:16
Last Modified:	10 Jul 2026 07:16
DOI:	10.21203/rs.3.rs-10120666/v1
Related Websites:	Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3199340
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