Tomato is currently the model plant for fleshy fruit development and for Solanaceae species. Recent genomic approaches including transcriptome, proteome and metabolome analyses and genetic mapping have produced a wealth of candidate genes whose function needs to be assessed. The recent development in model and crop plants of TILLING (Targeting Induced Local Lesions IN Genomes), which reveals allelic series corresponding to several independent point mutations, and the current availability of deep sequencing tools further increase the interest of generating artificiallyinduced genetic diversity in tomato. We describe here the generation and use of EMS (ethyl methanesulfonate) tomato mutants in the miniature cultivar Micro-Tom and provide as example the identification of new fruit size and morphology mutants. We further propose new deep sequencing-based strategies for the discovery of mutations underlying phenotypic variations observed in mutant collections that will considerably increase the interest of exploiting Micro-Tom mutant collections for gene discovery in tomato.
HighlightThe two tomato GDP-D-mannose epimerase isoforms play specific roles in cell wall biosynthesis and plant development but participate similarly in ascorbate biosynthesis.
Ascorbate is a major antioxidant buffer in plants, so several approaches have been developed to increase the ascorbate contents of fruits and vegetables. In this study, we combined forward genetics with mapping-by-sequencing approaches using an EMSMicro-Tom population to identify putative regulators underlying a high ascorbate phenotype in fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5 line, up to 5 times the WT) strongly impaired flower development and produced seedless fruit. Without progeny, genetic characterization was performed by outcrossing the P17C5 line with S. Lycopersicum cv. M82. We successfully identified the mutation responsible for the high ascorbate trait in a cis-acting upstream open reading frame (uORF) that is involved in the downstream regulation of GDP-L-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enrichment trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5xM82 plants, and the CRISPR-mutated line. These studies revealed that a high ascorbate content is linked to impaired floral organ architecture, particularly anthers and pollen development, thus leading to male sterility. RNAseq analysis suggests that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.
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