A Systems Biology Study in Tomato Fruit Reveals Correlations between the Ascorbate Pool and Genes Involved in Ribosome Biogenesis, Translation, and the Heat-Shock Response.

Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes-ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.

Is monodehydroascorbate reductase activity in leaf tissue critical for the maintenance of yield in tomato?

Ascorbate redox metabolism and growth have been shown to be linked and related to the activity of enzymes that produce or remove the radical monodehydroascorbate, the semi-oxidized form of ascorbate (ascorbate oxidase or peroxidase and monodehydroascorbate reductase respectively). Previous work in cherry tomato has revealed correlations between monodehydroascorbate reductase and ascorbate oxidase activity and fruit yield: decreased whole plant MDHAR activity decreases yield while decreased whole plant ascorbate oxidase activity increases yield under unfavourable environmental conditions. We aimed to investigate if similar effects on yield are obtained in a large-fruited variety of tomato, Moneymaker. Furthermore we wished to establish whether previously observed effects on yield in cherry tomato following changes in whole plant enzyme activity could be reproduced by reducing MDHAR activity in fruit only by using a fruit-specific promoter in cherry tomato (West Virginia 106). In Moneymaker, RNAi lines for monodehydroascorbate reductase did not show significant yield decrease compared to control lines when plants were grown under optimal or non-optimal conditions of carbon stress generated by mature leaf removal. In addition, we show that a decrease in monodehydroascorbate reductase activity in fruit of cherry tomato had no effect on yield compared to a reduction in whole-plant monodehydroascorbate reductase activity: we therefore show that whole plant MDHAR activity is necessary to maintain yield in cherry tomato, suggesting that the carbon source in autotrophic tissue is more important than fruit sink activity. The present data also revealed differences between cherry and large fruited tomato that could be linked to a source of genetic variability in the response to monodehydroascorbate metabolism in tomato: maybe the domestication of tomato towards large-fruited lines could have affected the importance of MDHAR in yield maintenance.

Reduction of MDHAR activity in cherry tomato suppresses growth and yield and MDHAR activity is correlated with sugar levels under high light.

Ascorbate is oxidized into the radical monodehydroascorbate (MDHA) through ascorbate oxidase or peroxidase activity or non-enzymatically by reactive oxygen species. Regeneration of ascorbate from MDHA is ensured by the enzyme MDHA reductase (MDHAR). Previous work has shown that growth processes and yield can be altered by modifying the activity of enzymes that recycle ascorbate; therefore, we have studied similar processes in cherry tomato (Solanum lycopersium L.) under- or overexpressing MDHAR. Physiological and metabolic characterization of these lines was carried out under different light conditions or by manipulating the source-sink ratio. Independently of the light regime, slower early growth of all organs was observed in MDHAR silenced lines, decreasing final fruit yield. Photosynthesis was altered as was the accumulation of hexoses and sucrose in a light-dependent manner in plantlets. Sucrose accumulation was also repressed in young fruits and final yield of MDHAR silenced lines showed a stronger decrease under carbon limitation, and the phenotype was partially restored by reducing fruit load. Ascorbate and MDHA appear to be involved in control of growth and sugar metabolism in cherry tomato and the associated enzymes could be potential targets for yield improvement.

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-dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol.

Ascorbate is a powerful antioxidant in plants, and its levels are an important quality criteria in commercial species. Factors influencing these levels include environmental variations, particularly light, and the genetic control of its biosynthesis, recycling and degradation. One of the genes involved in the recycling pathway encodes a monodehydroascorbate reductase (MDHAR), an enzyme catalysing reduction of the oxidized radical of ascorbate, monodehydroascorbate, to ascorbate. In plants, MDHAR belongs to a multigene family. Here, we report the presence of an MDHAR isoform in both the cytosol and peroxisomes and show that this enzyme negatively regulates ascorbate levels in Solanum lycopersicum (tomato). Transgenic lines overexpressing MDHAR show a decrease in ascorbate levels in leaves, whereas lines where MDHAR is silenced show an increase in these levels in both fruits and leaves. Furthermore, the intensity of these differences is light dependent. The unexpected effect of this MDHAR on ascorbate levels cannot be explained by changes in the expression of Smirnoff-Wheeler pathway genes, or the activity of enzymes involved in degradation (ascorbate peroxidase) or recycling of ascorbate (dehydroascorbate reductase and glutathione reductase), suggesting a previously unidentified mechanism regulating ascorbate levels.

Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations.

Fresh fruit and vegetables are a major source of ascorbic acid (vitamin C), an important antioxidant for the human diet and also for plants. Ascorbic acid content in fruit exhibits a quantitative inheritance. Quantitative trait loci (QTL) for ascorbic acid content have been mapped in three tomato populations derived from crosses between cultivated tomato varieties (Solanum lycopersicum accessions) and three related wild species or subspecies. The first population consists of a set of introgression lines derived from Solanum pennellii, each containing a unique fragment of the wild species genome. The second population is an advanced backcross population derived from a cross between a cultivated tomato and a Solanum habrochaites (formerly Lycopersicum hirsutum) accession. The third population is a recombinant inbred line population derived from the cross between a cherry tomato line and a large fruited line. Common regions controlling ascorbic acid content have been identified on chromosomes 2, 8, 9, 10, and 12. In general, the wild alleles increased ascorbic acid content, but some improvement could also be provided by S. lycopersicum. Most QTLs appeared relatively stable over years and in different environments. Mapping of candidate genes involved in the metabolism of ascorbic acid has revealed a few colocations between genes and QTLs, notably in the case of a monodehydroascorbate reductase gene and a QTL present in two of the populations on chromosome 9 (bin 9-D), and a previously mapped GDP-mannose epimerase and a QTL on chromosome 9 (bin 9-J).

Technique for rapid, small-scale analysis of vitamin C levels in fruit and application to a tomato mutant collection.

We present a technique for easy, rapid analysis of both total and reduced forms of vitamin C in fruits using microplates and a plate reader. This technique has been compared with a spectrofluorometric technique classically used for assaying vitamin C in fresh tomato. We have applied these methods to a population of 118 tomato mutant lines and controls in search of variability for this trait. Six lines, identified as having high vitamin C levels, and four lines having low vitamin C levels have been chosen for further study. The vitamin C levels have been compared with sugar concentration, dry matter content, fruit weight, titratable acidity, and firmness. The correlations that often exist in tomato varieties between sugar and vitamin C content (positive correlation) or fruit weight and vitamin C content (negative correlation) can be uncoupled in the lines selected for further analysis.