Overproduction of ascorbic acid impairs pollen fertility in tomato

Abstract

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.

Improving oxidative stress resilience in plants

“…Although it is tempting to speculate that elevated AA levels improve stress tolerance predominantly because of an increase in the antioxidant capacity, the multifaceted role of AA makes the interpretation of these phenotypes challenging. Moreover, an increased antioxidant capacity might interfere with effective ROS signaling, ultimately leading to undesired consequences, such as impaired flower development and seedless fruits observed in AA-accumulating tomato mutants (Deslous et al, 2021).…”

Regulation of Vitamin C Accumulation for Improved Tomato Fruit Quality and Alleviation of Abiotic Stress

“…Indeed, VTC2 can be regulated by a non-canonical upstream open reading frame (uORF), which encodes for a peptide that acts as an inhibitor of translation when AsA is highly abundant, or a stimulator of GGP translation when AsA is at low levels [ 26 ]. Recent studies on a cis-acting uORF in GGP1 highlighted its role in regulating redox homeostasis, normal plant development, and pollen fertility in tomato [ 27 ]. On the other hand, the partial AsA deficiency in Arabidopsis vtc4 mutants, in combination with the fact that these mutants also contained low levels of myo -inositol reinforces the notion that this enzyme has probably a double activity on both l -galactose-1-P and d -myo-inositol 3-P [ 23 ].…”

“…Overexpression of GGP in a fruit-specific manner, resulted in a three-fold increase of AsA in ripe tomato fruit, whereas a similar approach for GPP did not result in a similar increase of fruit AsA (Kanellis et al, unpublished data). More recently, using combined forward genetics with mapping-by-sequencing approaches, the impaired pollen fertility that resulted in seedless tomato fruits has been related to the over-accumulation of AsA (up to 5-fold WT level) in AsA-enriched mutants [ 27 ]. In a less successful effort, Wang et al [ 130 ] achieved a nearly 50% decrease in leaf AsA content, by down-regulating a tomato gene coding GGP .…”

“…This efficient method of manipulating the translation of mRNA resulted in a 1.5-fold increase in AsA contents, and further improved stress tolerance to MV-induced oxidative stress in lettuce. More recently, AsA-enriched tomato mutants were generated by targeting the uORF of the SlGGP1 coding sequence, linking high AsA levels with impaired floral development and pollen fertility, as well as seedless fruits [ 27 ]. This intriguing finding that links AsA and GSH with plant reproduction has been previously reported in tomato lines overexpressing SlGGP1 [ 126 ].…”

“…This intriguing finding that links AsA and GSH with plant reproduction has been previously reported in tomato lines overexpressing SlGGP1 [ 126 ]. Additionally, RNA-seq analysis of these CRISP-mutated lines confirmed that this particular uORF acts as a regulator of AsA synthesis and redox state to enable normal plant/organ development [ 27 ].…”