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      High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

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          Abstract

          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.

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          Most cited references38

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          Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid).

          A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.
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            The biosynthetic pathway of vitamin C in higher plants.

            Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.
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              Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase.

              L-Ascorbic acid (vitamin C) in fruits and vegetables is an essential component of human nutrition. Surprisingly, only limited information is available about the pathway(s) leading to its biosynthesis in plants. Here, we report the isolation and characterization of GalUR, a gene from strawberry that encodes an NADPH-dependent D-galacturonate reductase. We provide evidence that the biosynthesis of L-ascorbic acid in strawberry fruit occurs through D-galacturonic acid, a principal component of cell wall pectins. Expression of GalUR correlated with changing ascorbic acid content in strawberry fruit during ripening and with variations in ascorbic acid content in fruit of different species of the genus Fragaria. Reduced pectin solubilization in cell walls of transgenic strawberry fruit with decreased expression of an endogenous pectate lyase gene resulted in lower ascorbic acid content. Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content two- to threefold, demonstrating the feasibility of engineering increased vitamin C levels in plants using this gene.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                19 December 2013
                : 8
                : 12
                : e84474
                Affiliations
                [1 ]INRA, UR 1115 Plantes et Système de cultures Horticoles, Avignon, France
                [2 ]EA4279, Université d’Avignon, Avignon, France
                [3 ]INRA, UMR 1332 Biologie du Fruit et Pathologie, France Université de Bordeaux, Bordeaux, France
                [4 ]INRA, UR 1052, Génétique et Amélioration des Fruits et Légumes, Montfavet, France
                Universidade Federal do Rio Grande do Sul, Brazil
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CM RS HG. Performed the experiments: CM HG. Analyzed the data: CM HG. Contributed reagents/materials/analysis tools: CM DB FLL VT PB RS. Wrote the manuscript: CM DB RS HG.

                Article
                PONE-D-13-28250
                10.1371/journal.pone.0084474
                3868655
                24367665
                5b6c81a3-fccb-4df2-b0bc-ecb406e21e1e
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 July 2013
                : 21 November 2013
                Funding
                This work was supported by Plantinov'Ser fundings (Pays de Loire, France). INRA and Region Provence Alpes Cote d'Azur co-funded a PHD grant for Capucine Massot. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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