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VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine

Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identi...

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Autores principales: Clemens, Molly, Faralli, Michele, Lagreze, Jorge, Bontempo, Luana, Piazza, Stefano, Varotto, Claudio, Malnoy, Mickael, Oechel, Walter, Rizzoli, Annapaola, Dalla Costa, Lorenza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9152544/
https://www.ncbi.nlm.nih.gov/pubmed/35656017
http://dx.doi.org/10.3389/fpls.2022.878001
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author Clemens, Molly
Faralli, Michele
Lagreze, Jorge
Bontempo, Luana
Piazza, Stefano
Varotto, Claudio
Malnoy, Mickael
Oechel, Walter
Rizzoli, Annapaola
Dalla Costa, Lorenza
author_facet Clemens, Molly
Faralli, Michele
Lagreze, Jorge
Bontempo, Luana
Piazza, Stefano
Varotto, Claudio
Malnoy, Mickael
Oechel, Walter
Rizzoli, Annapaola
Dalla Costa, Lorenza
author_sort Clemens, Molly
collection PubMed
description Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in ‘Sugraone’, a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.
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spelling pubmed-91525442022-06-01 VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine Clemens, Molly Faralli, Michele Lagreze, Jorge Bontempo, Luana Piazza, Stefano Varotto, Claudio Malnoy, Mickael Oechel, Walter Rizzoli, Annapaola Dalla Costa, Lorenza Front Plant Sci Plant Science Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in ‘Sugraone’, a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions. Frontiers Media S.A. 2022-05-17 /pmc/articles/PMC9152544/ /pubmed/35656017 http://dx.doi.org/10.3389/fpls.2022.878001 Text en Copyright © 2022 Clemens, Faralli, Lagreze, Bontempo, Piazza, Varotto, Malnoy, Oechel, Rizzoli and Dalla Costa. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Clemens, Molly
Faralli, Michele
Lagreze, Jorge
Bontempo, Luana
Piazza, Stefano
Varotto, Claudio
Malnoy, Mickael
Oechel, Walter
Rizzoli, Annapaola
Dalla Costa, Lorenza
VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title_full VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title_fullStr VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title_full_unstemmed VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title_short VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine
title_sort vvepfl9-1 knock-out via crispr/cas9 reduces stomatal density in grapevine
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9152544/
https://www.ncbi.nlm.nih.gov/pubmed/35656017
http://dx.doi.org/10.3389/fpls.2022.878001
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