Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress

Laboratoire de Biotechnologie et Physiologie Végétales, Centre de biotechnologie végétale et microbienne biodiversité et environnement, Faculté des Sciences, Université Mohammed V de Rabat, Rabat 1014, Morocco

GBF, Université de Toulouse, INRA, 31326 Castanet-Tolosan, France

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Genes 2020, 11(3), 272; https://doi.org/10.3390/genes11030272

Received: 23 December 2019 / Revised: 23 February 2020 / Accepted: 28 February 2020 / Published: 3 March 2020

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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Abstract

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies. View Full-Text

Keywords: ARF4; Auxin; CRISPR-Cas9; osmotic stress; salt; tolerance; tomato

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MDPI and ACS Style

Bouzroud, S.; Gasparini, K.; Hu, G.; Barbosa, M.A.M.; Rosa, B.L.; Fahr, M.; Bendaou, N.; Bouzayen, M.; Zsögön, A.; Smouni, A.; Zouine, M. Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress. Genes 2020, 11, 272. https://doi.org/10.3390/genes11030272

AMA Style

Bouzroud S, Gasparini K, Hu G, Barbosa MAM, Rosa BL, Fahr M, Bendaou N, Bouzayen M, Zsögön A, Smouni A, Zouine M. Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress. Genes. 2020; 11(3):272. https://doi.org/10.3390/genes11030272

Chicago/Turabian Style

Bouzroud, Sarah, Karla Gasparini, Guojian Hu, Maria Antonia Machado Barbosa, Bruno Luan Rosa, Mouna Fahr, Najib Bendaou, Mondher Bouzayen, Agustin Zsögön, Abdelaziz Smouni, and Mohamed Zouine. 2020. "Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress" Genes 11, no. 3: 272. https://doi.org/10.3390/genes11030272

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Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress

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