A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit.

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose:sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.

Light affects ascorbate content and ascorbate-related gene expression in tomato leaves more than in fruits.

Little is known about the light regulation of vitamin C synthesis in fruits. In contrast, previous studies in leaves revealed that VTC2 (coding for GDP-L: -galactose phosphorylase) was one of the key genes up-regulated by light in leaves. Our objective was to determine how the expression of ascorbate (AsA) synthesis genes in tomato (Solanum lycopersicum) was modified according to light irradiance in both leaves and fruits. Seven days of shading strongly decreased total ascorbate (reduced and oxidized form) content in leaves (50%) and to a lesser extent in fruits (10%). Among the last six steps of AsA biosynthesis, only two genes, VTC2 and GPP1 (one of the two unigenes coding for L: -galactose-1-P phosphatase in tomato), were down-regulated by long-term shading in red ripe fruits, compared to seven genes regulated in leaves. This underlines that light affects AsA-related gene expression more in leaves than in ripening fruits. Moreover, this study reveals strong daily changes in transcript levels of enzymes of the AsA biosynthetic pathway in leaves (11 of the 12 studied genes showed significant changes in their expression pattern). Among those genes, we found that diurnal variation in transcript levels of VTC2 and GME1 correlated to leaf AsA content measured 8 h later. This study provides a new hypothesis on the role of GME1 in addition to VTC2 in light-regulated AsA biosynthesis.

Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance.

BACKGROUND AND AIMS: The mechanisms involving light control of vitamin C content in fruits are not yet fully understood. The present study aimed to evaluate the impact of fruit and leaf shading on ascorbate (AsA) accumulation in tomato fruit and to determine how fruit sugar content (as an AsA precursor) affected AsA content. METHODS: Cherry tomato plants were grown in a glasshouse. The control treatment (normally irradiated fruits and irradiated leaves) was compared with the whole-plant shading treatment and with leaf or fruit shading treatments in fruits harvested at breaker stage. In a second experiment, the correlation between sugars and AsA was studied during ripening. KEY RESULTS: Fruit shading was the most effective treatment in reducing fruit AsA content. Under normal conditions, AsA and sugar content were correlated and increased with the ripening stage. Reducing fruit irradiance strongly decreased the reduced AsA content (-74 %), without affecting sugars, so that sugar and reduced AsA were no longer correlated. Leaf shading delayed fruit ripening: it increased the accumulation of oxidized AsA in green fruits (+98 %), whereas it decreased the reduced AsA content in orange fruits (-19 %), suggesting that fruit AsA metabolism also depends on leaf irradiance. CONCLUSIONS: Under fruit shading only, the absence of a correlation between sugars and reduced AsA content indicated that fruit AsA content was not limited by leaf photosynthesis or sugar substrate, but strongly depended on fruit irradiance. Leaf shading most probably affected fruit AsA content by delaying fruit ripening, and suggested a complex regulation of AsA metabolism which depends on both fruit and leaf irradiance and fruit ripening stage.

High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

Fluctuations in sugar content are not determinant in explaining variations in vitamin C in tomato fruit.

The present study aimed to clarify the relationship between sugars and vitamin C in fruit. The objective was to determine whether vitamin C content was regulated by sugar content due to the role of sugar as a precursor for vitamin C. During summer, maximal content in sugar and vitamin C were found in both genotypes tested Solanum lycopersicon 'Cervil' and 'Levovil'. During autumn, fruit pruning increased fruit size and hexose content but fruit vitamin C content did not increase. Therefore sugar substrate was not limiting for vitamin C synthesis during autumn. We demonstrated for two cultivars, 'Cervil' and 'Levovil', with different sugar accumulation profiles during ripening, that sugar content was not determinant in the regulation of vitamin C content. The strong correlation observed between sugars and vitamin C in 'Cervil' was due to their concomitant increase during fruit ripening.

An integrative genomics approach for deciphering the complex interactions between ascorbate metabolism and fruit growth and composition in tomato.

Very few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and other metabolic pathways in the fruit, we undertook a fruit systems biology approach. To this end, we have produced tomato transgenic lines altered in ascorbate content and redox ratio by RNAi-targeting several key enzymes involved in ascorbate biosynthesis (2 enzymes) and recycling (2 enzymes). In the VTC (ViTamin C) Fruit project, we then generated phenotypic and genomic (transcriptome, proteome, metabolome) data from wild type and mutant tomato fruit at two stages of fruit development, and developed or implemented statistical and bioinformatic tools as a web application (named VTC Tool box) necessary to store, analyse and integrate experimental data in tomato. By using Kohonen's self-organizing maps (SOMs) to cluster the biological data, pair-wise Pearson correlation analyses and simultaneous visualization of transcript/protein and metabolites (MapMan), this approach allowed us to uncover major relationships between ascorbate and other metabolic pathways.