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.     

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.     

Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress

Quantitative trait loci (QTL) mapping is a step towards the identification of factors regulating traits such as fruit ascorbic acid content. A previously identified QTL controlling variations in tomato fruit ascorbic acid has been fine mapped and reveals that the QTL has a polygenic and epistatic architecture. A monodehydroascorbate reductase (MDHAR) allele is a candidate for a proportion of the increase in fruit ascorbic acid content. The MDHAR enzyme is active in different stages of fruit ripening, shows increased activity in the introgression lines containing the wild-type ( Solanum pennellii ) allele, and responds to chilling injury in tomato along with the reduced/oxidized ascorbate ratio. Low temperature storage of different tomato introgression lines with all or part of the QTL for ascorbic acid and with or without the wild MDHAR allele shows that enzyme activity explains 84% of the variation in the reduced ascorbic acid levels of tomato fruit following storage at 4 °C, compared with 38% at harvest under non-stress conditions. A role is indicated for MDHAR in the maintenance of ascorbate levels in fruit under stress conditions. Furthermore, an increased fruit MDHAR activity and a lower oxidation level of the fruit ascorbate pool are correlated with decreased loss of firmness because of chilling injury.     

Ectopic expression of Brassica rapa L. MDHAR increased tolerance to freezing stress by enhancing antioxidant systems of host plants

Ascorbate (vitamin C) plays an important role in detoxification of reactive oxygen species (ROS) in most living organisms. Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is crucial to regeneration of the oxidized form of ascorbate (monodehydroascorbate) so that it can be recycled to maintain ROS scavenging ability. The MDHAR gene from Brassica rapa L. was cloned and introduced into Arabidopsis thaliana (L.) Heynh. to test the hypothesis that enhanced ROS scavenging activity of BrMDHAR alleviates freezing stress. BrMDHAR was expressed under the control of either the CaMV 35S promoter or stress inducible SWPA2 promoter. Ectopic expression of BrMDHAR led to the up-regulation of many antioxidant genes, including APX, DHAR, GR, SOD, GPX, and PRX Q, which are involved in ascorbate–glutathione cycle. And, transgenic plants showed improved stress tolerance against freezing with exhibiting higher levels of chlorophyll content and antioxidant molecules such as ascorbate and glutathione as well as alleviated redox status and malondialdehyde contents. These results suggested that ectopic expression of BrMDHAR conferred improved tolerance to freezing stress not only by simply recycling ascorbate, but also by inducing co-regulation of the ascorbate–glutathione cycle, which in turn enhances the antioxidant capacity of the host plants.     

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.     

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.     

Physiological and biochemical responses of fruit exocarp of tomato (Lycopersicon esculentum Mill.) mutants to natural photo-oxidative conditions

Photo-oxidative stress was imposed under natural solar radiation on exposed and shaded sections of detached fruit of immature green tomato (Lycopersicon esculentum Miller = Solanum lycopersicum L.) mutants (anthocyanin absent, beta-carotene, Delta, and high pigment-1) and their nearly isogenic parents ('Ailsa Craig' and 'Rutgers'). After 5 h exposure to high solar irradiance, either with or without ultraviolet (UV) radiation, surface colour changes, pigment composition, photosynthetic efficiency, antioxidant metabolites and enzyme activities, and selected flavonoids and antioxidant proteins in exocarp tissue were evaluated. The imposed photo-oxidative stress reproduced the symptoms observed on attached fruit. Both high temperature and solar irradiance caused fruit surface discoloration with faster degradation of chlorophyll (Chl) than carotenoids (Car), leading to an increase in the Car/Chl ratio. Surface bleaching was mostly caused by visible light, whereas elevated temperatures were mostly responsible for the inactivation of photosynthesis, measured as decreased F(v)/F(m). Ascorbate, glutathione, and total soluble protein concentrations decreased in the exocarp as the duration of exposure increased. Specific activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), and catalase increased with exposure, suggesting that these proteins were conserved during the imposed stress. GR protein expression remained stable during the imposed stress, whereas, MDHAR protein expression increased. Quercetin and kaempferol concentrations increased rapidly upon exposure, but not to UV radiation, suggesting rapid photo-protection in response to visible light; however, naringenin synthesis was not induced. The apparent increased tolerance of hp-1 fruit is discussed.     

Ascorbate and ascorbate-dependent enzymes in detached tomato leaves under conditions modulating the ascorbate pool

The relationship between AA-metabolising enzymes and the AA pool was studied by subjecting tomato leaves to treatments up- or down-regulating the AA content. GalL feeding through leaf petioles increased the level of cellular ascorbate up to two-fold. This effect accompanied by a AA/total ascorbate ratio increase was observed after a 2-h incubation and remained constant during the following 4 days. Dark-incubated leaves showed a 56% decline in AA content concomitantly with DHA accumulation and significant ascorbate redox ratio decrease. Opposite changes were induced when the darkened leaves were transferred to light, as AA biosynthesis depends on light. The light-induced AA biosynthesis was partially inhibited by lycorine. Regardless of the pattern of AA pool changes the AA-related enzymes were similarly affected. Both the GalL/light-dependent AA increase and dark-induced AA depletion were accompanied by APX and MDHAR activity induction. No significant AA pool-dependent DHAR activity changes were found. The possible relevance of the AA system changes induced is discussed.     

Ascorbate degradation in tomato leads to accumulation of oxalate,threonate and oxalyl threonate

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non‐reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [¹⁴C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall‐rich) leaf material. No [¹⁴C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under‐expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation.     

Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii

The activities of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and SOD were studied in cell organelles of the cultivated tomato Lycopersicon esculentum (M82) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa). All four enzymes of the ascorbate-glutathione cycle were present in chloroplasts/plastids, mitochondria and peroxisomes of leaf and root cells of both tomato species. In all leaf and root organelles of both species, the activity of MDHAR was similar to, or higher than, that of APX, while the activity of DHAR was one order of magnitude lower than that of MDHAR. Based on these results, it is suggested that in the organelles of both tomato species, ascorbate is regenerated mainly by MDHAR. In both tomato species, GR activity, and to a lesser extent DHAR activity, was found to reside in the soluble fraction of all leaf and root cell organelles, while APX and MDHAR activities were distributed between the membrane and soluble fractions. A higher SOD to APX activity ratio in all Lpa organelles was the major difference between the two tomato species. It is possible that this higher ratio contributes to the inherently better protection of Lpa from salt stress, as was previously reported.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

樱桃蕃茄不同栽培方式比较研究

以7个樱桃蕃茄品种为试材,进行不同栽培方式试验,对每个品种在不同栽培方式下的生长势、单果重、可溶性固形物含量、单株前期产量和总产量、农药残留量、亚硝酸盐含量、灌溉排出液的硝酸盐含量及经济效益进行比较分析。结果表明,基质栽培方式是比较理想的栽培模式。从农业综合性状分析,圣女、3688F₁、韩3号为闽南地区适合推广种植的品种。     

Changes in the antioxidative systems in mitochondria during ripening of pepper fruits

The presence of enzymes of the ascorbate–glutathione cycle was studied in mitochondria purified from green and red pepper (Capsicum annuum L.) fruits. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were present in the isolated mitochondria of both fruit ripening stages. The activity of the reductive ascorbate–glutathione cycle enzymes (MDHAR, GR and DHAR) was higher in mitochondria isolated from green than from red fruits, while APX and the antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) were higher in the red fruits. The levels of ascorbate and L-galactono-γ-lactone dehydrogenase (GLDH; EC 1.3.2.3) activity were found to be similar in the mitochondria of both fruits. The higher APX and Mn-SOD specific activities in mitochondria from red fruits might play a role in avoiding the accumulation of any activated oxygen species generated in these mitochondria, and suggests an active role for these enzymes during ripening.     

Subcellular distribution of ascorbate in plants

The compartment specific distribution of ascorbate in plants is of great importance for plant development, growth and defense as this multifunctional metabolite plays important roles in the detoxification of reactive oxygen species (ROS), redox signaling, modulation of gene expression and is important for the regulation of enzymatic activities. Even though changes in ascorbate contents during plant growth and various stress conditions are well documented and the roles of ascorbate in plant defense during abiotic stress conditions are well established, still too little is known about its compartment specific roles during plant development and defense. This mini-review focuses on the subcellular distribution of ascorbate in plants and describes different methods that are currently used to study its compartment specific distribution. Finally, it will also briefly discuss data available on compartment specific changes of ascorbate during some abiotic stress conditions such as high light conditions and exposure to ozone.Key words: ascorbate, mitochondria, chloroplasts, electron microscopy, ozone, high light stress, reactive oxygen speciesAscorbate is one of the most important antioxidants in plants and animals. It detoxifies reactive oxygen species (ROS) either directly or through the glutathione-ascorbate cycle (Fig. 1) and is involved in redox signaling, modulation of gene expression and the regulation of enzymatic activities (extensively reviewed in ref. ¹ and ²). Ascorbate occurs in a reduced form (ascorbic acid) and two oxidized forms (mono- and dehydroascorbic acid). The ratio between reduced and oxidized ascorbate is essential for the ability of the plant to fight oxidative stress. During environmental stress situations when ROS are formed inside the cell, large amounts of dehydroascorbic acid can be formed by oxidation of ascorbic acid which shifts the ascorbate pool more towards the oxidative state and diminishes the antioxidative capacity of the plant. Additionally, environmental stress situations can change total ascorbate contents in plants which makes ascorbate an important stress marker during abiotic and biotic stress situations.^3–11 Ascorbate contents are typically measured biochemically in individual plant organs or tissues and the obtained values represent a combination of the ascorbate status of all individual organelles. As many environmental stress conditions induce highly compartment specific stress responses changes of ascorbate contents in individual organelles might not be detected when ascorbate is measured in whole organs or tissues. This is crucial as data obtained about the antioxidative status from individual organs are often used to interpret the stress response of the whole plant to the exposed stress conditions. Thus, in order to gain a deeper insight into the defense response of plants it is essential to measure changes in the subcellular distribution of these components during environmental stress situations.Open in a separate windowFigure 1Ascorbate-glutathione cycle in plants. Hydrogen peroxide (H₂O₂) within the plant cell can be detoxified by ascorbate peroxidase (APX). In this reaction the reduced form of ascorbate (Asc) is oxidized to monodehydroascorbate (MDHA). MDHA is then either reduced by monodehydroascorbate reductase (MDHAR) to Asc or, since very unstable, reacts to dehydroascorbate (DHA). DHA is reduced by dehydroascorbate reductase (DHAR) to Asc. In this reaction the reduced form of glutathione (GSH) is oxidized to glutathione disulfide (GSSG). GSSG is then reduced by glutathione reductase (GR) to GSH. The electron acceptor NADP is regenerated during the reduction of MDHA and GSSG by the respective enzymes. Asc and GSH are additional able to detoxify reactive oxygen species by direct chemical interaction. Thus, besides the total ascorbate level their redox state (reduced vs. oxidized state) which depends on the activity of the described enzymes (grey boxes) is also very important for successful plant protection.     

Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, <Emphasis Type="Italic">Physcomitrella patens</Emphasis> and characterization of the expressed enzymes

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K _m and k _cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s⁻¹, respectively, using NADH as the electron donor. The K _m values were at least an order of magnitude higher for NADPH. The K _m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.