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International audience; 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.

Highlight The two tomato GDP-D-mannose epimerase isoforms play specific roles in cell wall biosynthesis and plant development but participate similarly in ascorbate biosynthesis., GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.

International audience; Besides being a model for fleshy fruits and Solanaceae species, tomato represents one of the main sources of ascorbate (vitamin C) in human diet in many parts of the world. Ascorbate fulfills various roles in plants due to its antioxidant potential and to its connection with other metabolic pathways e. g. cell wall biosynthesis. Among the functional genomic tools recently developed in tomato, EMS (ethyl methanesulfonate) mutant collections provide an opportunity for identifying allelic series of mutations in target genes by TILLING (Targeting Induced Local Lesions IN Genomes). We describe here the use of tomato EMS mutant collections in the miniature cv. Micro-Tom for the discovery of allelic variants in three ascorbate biosynthetic genes encoding the GDP-D-mannose pyrophosphorylase (GMP), the GDP-D-mannose epimerase (GME) and the GDP-L-galactose phosphorylase (GGP) respectively. We report on the discovery of several missense, truncation and splice junction mutations in these genes affecting plant ascorbate content to various levels, and show that several tomato mutant lines with strongly reduced ascorbate content undergo severe bleaching upon exposure to high light intensity.