Galactono-1,4-lactone Dehydrogenase Research Articles

A New Perspective on the Effect of UV-B on l-Ascorbic Acid Metabolism in Cucumber Seedlings.

This study aimed to examine the effects of UV-B on AsA and gene expression in cucumber seedlings. Particular emphasis was placed on identifying genes that were responsive to UV-B to increase AsA levels and elucidate the key UV-B response pathway. We found that the activities of myo-inositol oxygenase (MIOX), galactono-1,4-lactone dehydrogenase (GLDH), ascorbate oxidase (AO), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and the transcript levels of CsMIOX1, CsGLDH, CsAO2, CsAO4, CsGR1, CsAPX5, and CsDHAR1 significantly increased with UV-B exposure. These observations indicate that UV-B induces the expression of genes involved in d-mannose/l-galactose and myo-inositol pathways and the ascorbate-glutathione system. Moreover, several genes related to the low and high UV-B fluence responses were considered. CsHY5 and CsMYB60 were involved with the low-fluence response and appeared to be responsive from 2 to 28 h. Together, these data show that these genes respond to UV-B to increase AsA levels through the low-fluence UV-B response pathway.

Cerium improves the vase life of Dianthus caryophyllus cut flower by regulating the ascorbate and glutathione metabolism

Abstract To study the role of cerium (Ce) in regulating the vase life of Dianthus caryophyllus cut flower, the effects of cerium nitrate (Ce(NO3)3) on the vase life, the activities of enzymes in the ascorbate and glutathione metabolism, the malondialdehyde (MDA) content and electrolyte leakage (EL), and the contents of reduced and total ascorbate and glutathione in the petals were investigated. The results showed that Ce(NO3)3 significantly enhanced the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), l -galactono-1,4-lactone dehydrogenase (GalLDH) and gamma-glutamylcysteine synthetase (γ-ECS), and the contents of reduced ascorbate (AsA), reduced glutathione (GSH), total ascorbate and total glutathione in the petals, compared with control. Meanwhile, Ce(NO3)3 significantly increased the vase life and decreased MDA content and EL. Our results suggested that Ce(NO3)3 increased the vase life of D. caryophyllus cut flower by enhancing the ascorbate and glutathione metabolism in the petals.

Interaction between antioxidants and reactive nitrogen species during pepper fruit ripening

Pepper fruit is of great interest for human nutrition due to its high content in vitamins A and, mainly, C. At ripening, decreases of the nitric oxide (NO) levels occur in peppers, accompanied by increases of nitrated and nitrosated proteins, two post-translational modifications favored by the NO-derived reactive nitrogen species (RNS). Catalase is the main enzyme which undergoes nitration at ripening and, in in vitro assays with RNS, it was found that this enzyme was inactivated. Besides, catalase from pepper fruits displays atypical molecular features comparing to other plant catalases. Recently, it was proved that NO delays ripening of peppers and that it interacts with a number of antioxidants thus involving changes in the nutritional properties of fruits. Treatment of pepper fruits with NO increased their content of ascorbate about 40%, and this was due to an induction of the galactono-lactone dehydrogenase (GalLDH) gene together a higher activity of GalLDH, the last enzyme of the ascorbate biosynthetic pathway in plants. At present, high throughput approaches based on RNA-Seq analysis have provided a set of genes affected by treatment of pepper fruits with NO, what could be used to gain added values through biotechnological approaches.

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.

P 014 - Rapid ascorbate response to bacterial elicitor treatment in Arabidopsis thaliana cells

An early event of the incompatible plant-pathogen interactions is an oxidative burst. The major water soluble, low molecular weight antioxidant, ascorbic acid plays a crucial role in Reactive Oxygen Species (ROS) balancing (scavenging). The regulation of ascorbate level therefore can be an important point of the fine tuning of ROS level during the early phase of plant-pathogen interaction. To evaluate how this interaction affects the biosynthesis, the recycling and the level of ascorbate, we challenged Arabidopsis thaliana cells with 2 different harpin proteins (HrpZpto and HrpWpto). HrpZpto and HrpWpto treatments caused a well-defined ROS peak. The expression of the alternative oxidase (AOX1a) and vtc5, one of the paralog genes that encode the rate limiting enzyme of ascorbate biosynthesis followed the elevation of ROS. Similarly the activity of ascorbate-peroxidase and galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate, mitochondria coupled step of ascorbate biosynthesis and the level of ascorbate and glutathione also followed the elevation of ROS due to harpin treatment. The enhanced expression of AOX1a, the elevated activity of GLDH and the increased level of ascorbate and glutathione all can contribute to the mitigation or absence of programmed cell death. Finally a new function, the fine tuning of redox balance during plant-pathogen interaction can be proposed to vtc5.

OP20 - The evolution of vitamin C biosynthetic pathways in plants and algae

Ascorbic acid (vitamin C) is present in high concentrations in leaves and, in some species, in fruit. In leaves it has a major role in photoprotection of photosynthesis both as scavenger of reactive oxygen species but also as a cofactor for violaxanthin de-epoxidase, an enzyme required for the synthesis of the photoprotective xanthophyll pigment zeaxanthin. Animals use an ER-localised gulonolactone oxidase (GULO) for ascorbate biosynthesis but this also generates hydrogen peroxide. The biosynthetic pathway in plants and photosynthetic protists is distinct from animals in using a mitochondrial galactonolactone dehydrogenase (GLDH) in the last step that does not generate hydrogen peroxide. Genome sequence analysis and biochemical data suggest that GULO has been lost repeatedly throughout eukaryote evolution (resulting in inability to synthesise ascorbate in primates and some other animal groups) and has been functionally replaced by GLDH in all photosynthetic eukaryotes. Green algae and plants derived from primary endosymbiosis with a cyanobacterium synthesise ascorbate by a pathway distinct from animals while photosynthetic protists with chloroplasts derived by secondary endosymbiosis appear to have a “hybrid” pathway combining an animal-like pathway with plant-like GLDH in the final step. It is proposed that unlike the animal pathway, the plant pathway and the “hybrid” pathway provide a high capacity for ascorbate biosynthesis without hydrogen peroxide formation that provides sufficient ascorbate for photoprotection of photosynthesis.

L-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Responses to drought stress of the biosynthetic and recycling metabolism of glutathione and ascorbate in <I>Agropyron cristatum</I> leaves on the Loess Plateau of China

Aims Our objective was to clarify the mechanism of drought tolerance for Agropyron cristatum on the Loess Plateau of China through investigation of drought stress on the biosynthetic and recycling metabolism of glutathione and ascorbate in leaves. Methods We investigated the levels and redox status of glutathione and ascorbate and the activities of galactonolactone dehydrogenase (GalLDH), gamma glutamyl cysteine synthetase (γ-ECS), ascorbate peroxidase glutathione reductase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) in the leaves of A. cristatum under different levels of artificial drought stress using pot experiments. Important findings Levels of glutathione and ascorbate and their redox status were maintained under drought stress by increasing activities of GalLDH, γ-ECS, APX, GR, DHAR and MDHAR involved in the metabolism of glutathione and ascorbate. This protected A. cristatum from oxidative damage induced by drought stress. However, there were differences in the responses of the metabolism of glutathione and ascorbate related to different drought stress levels with prolonged stress. With 24 or fewer days of stress treatment, the metabolism of glutathione and ascorbate was enhanced under severe drought stress. After 24 days of stress treatment, the activities of GalLDH, γ-ECS, APX, GR, DHAR and MDHAR decreased significantly under severe drought stress, because A. cristatum suffered more severe oxidative damage. However, the activities of these enzymes were maintained throughout the whole period of stress under moderate drought stress. This suggested that A. cristatum could effectively protect itself from oxidative damage induced by moderate drought stress for long time periods. However, A. cristatum could only effectively protect itself from oxidative damage induced by severe drought stress for limited time, and its antioxidant capacity decreased significantly with prolonged stress. Our results also suggested that ascorbate and glutathione had important roles in protecting against oxidative damage under drought stress.

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO(2) assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO(2) assimilation/intercellular CO(2) curves at low CO(2), and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.

Ascorbate content of wheat leaves is not determined by maximal l‐galactono‐1,4‐lactone dehydrogenase (GalLDH) activity under drought stress

ABSTRACTAlthough ascorbic acid (AA) is a high‐abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l‐galactono‐1,4‐lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well‐watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.