Overproduction of ascorbic acid impairs pollen fertility in tomato.

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.