Overproduction of ascorbic acid impairs pollen fertility in tomato.

Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) 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-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

The effect of low ascorbic acid content on tomato fruit ripening.

MAIN CONCLUSION: The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

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.

Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato.

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.