Silencing cell wall remodeling enzymes in tomato and analysis of the effects on fruit softeningTools Samsulrizal, Nurul Hidayah (2017) Silencing cell wall remodeling enzymes in tomato and analysis of the effects on fruit softening. PhD thesis, University of Nottingham.
AbstractFruits are the seed dispersal units of flowering plants and fleshy fruits form an important part of the human diet. Ripening is a complex developmental process and involves many events such as textural and constitutional changes. The texture of fleshy fruits is one of the major criteria for consumer choice. However, the molecular determinants of ripening- associated changes in texture or “softening” are relatively poorly understood and seem to involve a large number of cell wall remodelling factors. The recent completion of the tomato genome sequence has revealed more than 50 cell wall structure-related genes that are expressed during fruit development and ripening and may impact texture changes in this fruit (Tomato Genome Consortium, 2012). The aim of the project was to compare, on a genome-wide scale, ripening-related gene expression in a range of fleshy fruits and especially those linked with cell wall remodelling. Then by identifying orthologous genes in different fruit species, to make predictions about those genes important for the softening process in all fleshy fruits. We hypothesise that there are a core set of genes that is always associated with fruit softening. Once this core set was identified we planned to use transgenic approaches to test the role of these cell wall genes in texture changes using tomato (Solanum lycopersicum) as a model system. Comparative genomics analysis of tomato (S. lycopersicum), banana (Musa acuminata), melon (Cucumis melo) and grape (Vitis vinifera), was undertaken using Inparanoid software. This analysis showed that a total of 8,982 (25.86%) gene models could be identified in common between all four genomes based on comparison of amino acid sequences. However, comparison of the expression patterns of cell wall remodelling genes across the various species revealed that most were expressed in tissues other than ripening fruits and of the fruit expressed genes only a small number were common between different species. We selected a CELLULOSE SYNTHASE (CESA) gene that was up-regulated in all fruit species for further analysis. The PHYTOENE SYNTHASE 1 (PSY1) was selected as a control, and PECTATE LYASE (PL) and POLYGALACTURONASE (PG) as genes that were highly expressed in many fruits including tomato. We then used the recently developed CRISPR/ Cas9 DNA editing technology to generate mutations in the target PSY1, CESA, PL and PG genes. A range of either single base insertions or deletions of more than ten bases were identified at Cas9 cleavage site in T0 plants in all cases except for the CESA lines where no mutant plants were recovered; suggesting that knocking out this gene gives a lethal phenotype. The efficacy of CRISPR/Cas9 constructs in tomato fruits was readily apparent when observing the phenotype in PSY1 line where yellow or orange fruits occurred in T0 individuals. In the PL lines, substantially firmer tomato fruits were produced at the red ripe stage. There were some much smaller impacts on texture in the PG CRISPR lines. The CRISPR approaches described in this thesis provide methods to enhance texture and extend shelf life in tomato without compromising fruit quality.
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