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Plant Physiol. 2019 Feb;179(2):544-557. doi: 10.1104/pp.18.01187. Epub 2018 Nov 20.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Author information

1
School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK.
2
Department of Plant Science, Kulliyyah of Science, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia.
3
Institution of Vegetable Research, Shanxi Academy of Agricultural Sciences, Taiyuan City, China 030031.
4
Electron Microscopy Facility, Centre for Core Biotechnology Services, University of Leicester, Leicester LE1 7RH, UK.
5
Plant Sciences Department, University of California, Davis, California, 95616.
6
Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
7
School of Biological Sciences, Plant Molecular Sciences, University of London, Surrey TW20 0EX, UK.
8
School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK graham.seymour@nottingham.ac.uk.

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 remodeling 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 remodeling. 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 color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-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.

PMID:
30459263
PMCID:
PMC6426429
DOI:
10.1104/pp.18.01187
[Indexed for MEDLINE]
Free PMC Article

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