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Characterization of CRISPR mutants targeting genes modulating pectin degradation in ripening tomato

Wang, Duoduo; Samsulrizal, Nurul; Yan, Cheng; Allcock, Natalie S; Craigon, Jim; Blanco-Ulate, Barbara; Ortega-Salazar, Isabel; Marcus, Susan E; Bagheri, Hassan Moeiniyan; Perez-Fons, Laura; Fraser, Paul David; Foster, Timothy; Fray, Rupert George; Knox, J. Paul; Seymour, Graham B.

Authors

Duoduo Wang

Laura Perez-Fons

Paul David Fraser

Timothy Foster

Rupert George Fray

J. Paul Knox

Graham B. Seymour

Nurul Samsulrizal

Cheng Yan

Natalie S Allcock

Jim Craigon

Barbara Blanco-Ulate

Isabel Ortega-Salazar

Susan E Marcus

Hassan Moeiniyan Bagheri

Abstract

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodelling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin degrading enzymes are involved in cell wall remodelling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a) and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit colour and weight. Pectin localisation, distribution and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to de-esterified homogalacturonan (HG), INRA-RU1 to rhamnogalacturonan I, LM5 to β1-4-galactan and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

Journal Article Type Article
Publication Date Feb 1, 2019
Journal Plant Physiology
Print ISSN 0032-0889
Electronic ISSN 1532-2548
Publisher American Society of Plant Biologists
Peer Reviewed Peer Reviewed
Volume 179
Issue 2
Pages 544-557
DOI https://doi.org/10.1104/pp.18.01187
Keywords Plant Science; Genetics; Physiology
Publisher URL http://www.plantphysiol.org/content/early/2018/11/20/pp.18.01187

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