Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves

Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had Km values of 70 microM and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 micromol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.     

Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa

Dehydroascorbate reductase (DHAR) is a key enzyme in the ascorbate–glutathione cycle that maintains reduced pools of ascorbic acid and serves as an important antioxidant. In this study, to investigate functional divergence of plant DHAR family and catalytic characteristics of the glutathione binding site (G-site) residues of DHAR proteins, we cloned three DHAR genes (PtoDHAR1/2/3) from Populus tomentosa and predicted the G-site residues. PtoDHAR1 protein was localized in chloroplast, while PtoDHAR2/3 proteins showed cytosolic localizations. Three DHAR proteins showed different enzymatic activities, apparent kinetic characteristics, optimum T _m and pH profiles, indicating their functional divergence. Cys20, Lys8, Pro61, Asp72 and Ser73 of PtoDHAR2 were predicted as G-site residues based on their N-terminal amino acid sequence identity and the available crystal structures of glutathione S-transferases. The biochemical functions of these residues are examined in this study through site-directed mutagenesis. The aforementioned five residues are critical components of active sites that contribute to the enzyme’s catalytic activity. Cys20, Pro61 and Asp72 of PtoDHAR2 are also responsible for maintaining proper protein structure. This study provides new insights into the functional divergence of the plant DHAR family and biochemical properties of the G-site residues in plant DHAR proteins.     

Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes

Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic DHAR might play main important roles in improving the AsA contents of potato tubers.     

Effect of Selenium on Ascorbate�CGlutathione Metabolism During PEG-induced Water Deficit in Trifolium repens L.

To elucidate the effect of selenium (Se) on the ascorbate?Cglutathione (ASC?CGSH) cycle under drought stress, the activities of antioxidant enzymes and the levels of molecules involved in ASC?CGSH metabolism were studied in Trifolium repens seedlings subjected to polyethylene glycol (PEG)-induced water deficit alone or combined with 5???M Na₂SeO₄. Compared to the control, H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) contents increased, whereas a constant content of glutathione (GSH) and decreases in ASC/DHA and GSH/GSSG ratios were observed in the presence of PEG. The activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were upregulated, except for monodehydroascorbate reductase (MDHAR) activity during PEG-induced water deficit. Se application decreased the contents of H₂O₂, TBARS, DHA, and GSSG, increased the levels of GSH and ASC, and inhibited the decreases of ASC/DHA and GSH/GSSG ratios. Although it did not affect APX activity significantly, Se addition improved the activities of MDHAR, DHAR, and GR. Furthermore, GR activity showed the highest increase followed by that of DHAR and MDHAR in decreasing order. These data indicated that fluctuations in ASC?CGSH metabolism resulting from Se may have a positive effect on drought stress mitigation, and the regulation in the ASC?CGSH cycle can be attributed mainly to GR and DHAR in PEG?+?Se-treated T. repens seedlings.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. Three functional DHAR genes are encoded in the Arabidopsis genome. Ozone exposure increased the expression of the cytosolic DHAR (cytDHAR) gene alone. We characterized an Arabidopsis mutant with a deficient cytDHAR. The mutant completely lacked cytDHAR activity and was highly ozone sensitive. The amounts of total ascorbate and glutathione were similar in both lines, but the amount of apoplastic ascorbate in the mutant was 61.5% lower. These results indicate that the apoplastic ascorbate, which is generated through the reduction of DHA by cytDHAR, is important for ozone tolerance.     

Exogenous jasmonic acid modulates the physiology,antioxidant defense and glyoxalase systems in imparting drought stress tolerance in different Brassica species

This study examined the ability of jasmonic acid (JA) to enhance drought tolerance in different Brassica species in terms of physiological parameters, antioxidants defense, and glyoxalase system. Ten-day-old seedlings were exposed to drought (15 % polyethylene glycol, PEG-6000) either alone or in combination with 0.5 mM JA. Drought significantly increased lipoxygenase activity and oxidative stress, levels of malondialdehyde and H₂O₂. Drought reduced seedling biomass, chlorophyll (chl) content, and leaf relative water content (RWC). Drought increased proline, oxidized ascorbate (DHA) and glutathione disulfide (GSSG) levels. Drought affected different species differently: in B. napus, catalase (CAT) and glyoxalase II (Gly II) activities were decreased, while glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities were increased in drought-stressed compared to unstressed plants; in B. campestris, activities of glutathione reductase (GR), glyoxalase I (Gly I), GST, and GPX were increased, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), CAT and other enzymes were decreased; in B. juncea, activities of ascorbate peroxidase, GR, GPX, Gly I were increased; Gly II activity was decreased and other enzymes did not change. Spraying drought-stressed seedlings with JA increased GR and Gly I activities in B. napus; increased MDHAR activity in B. campestris; and increased DHAR, GR, GPX, Gly I and Gly II activities in B. juncea. JA improved fresh weight, chl, RWC in all species, dry weight increased only in B. juncea. Brassica juncea had the lowest oxidative stress under drought, indicating its natural drought tolerance capacity. The JA improved drought tolerance of B. juncea to the highest level among studied species.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

Genome-Wide Identification,Characterization, and Stress-Responsive Expression Profiling of Genes Encoding LEA (Late Embryogenesis Abundant) Proteins in Moso Bamboo (Phyllostachys edulis)

Late embryogenesis abundant (LEA) proteins have been identified in a wide range of organisms and are believed to play a role in the adaptation of plants to stress conditions. In this study, we performed genome-wide identification of LEA proteins and their coding genes in Moso bamboo (Phyllostachys edulis) of Poaceae. A total of 23 genes encoding LEA proteins (PeLEAs) were found in P. edulis that could be classified to six groups based on Pfam protein family and homologous analysis. Further in silico analyses of the structures, gene amount, and biochemical characteristics were conducted and compared with those of O. sativa (OsLEAs), B. distachyon (BdLEAs), Z. mays (ZmLEAs), S. bicolor (SbLEAs), Arabidopsis, and Populus trichocarpa. The less number of PeLEAs was found. Evolutionary analysis revealed orthologous relationship and colinearity between P. edulis, O. sativa, B. distachyon, Z. mays, and S. bicolor. Analyses of the non-synonymous (Ka) and synonymous (Ks)substitution rates and their ratios indicated that the duplication of PeLEAs may have occurred around 18.8 million years ago (MYA), and divergence time of LEA family among the P. edulis-O. sativa and P. edulis–B. distachyon, P. edulis-S. bicolor, and P. edulis-Z. mays was approximately 30 MYA, 36 MYA, 48 MYA, and 53 MYA, respectively. Almost all PeLEAs contain ABA- and (or) stress-responsive regulatory elements. Further RNA-seq analysis revealed approximately 78% of PeLEAs could be up-regulated by dehydration and cold stresses. The present study makes insights into the LEA family in P. edulis and provides inventory of stress-responsive genes for further functional validation and transgenic research aiming to plant genetic improvement of abiotic stress tolerance.     

Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress

Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.     

Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD⁺-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family.     

Increased Vitamin C Content Accompanied by an Enhanced Recycling Pathway Confers Oxidative Stress Tolerance in Arabidopsis

Vitamin C (L-ascorbic acid, AsA) has important antioxidant and metabolic functions in both plants and animals. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR, EC 1.8.5.1). To analyze the physiological role of DHAR catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, we examined whether increasing the level of AsA through enhanced AsA recycling would limit the deleterious effects of oxidative stress. A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-dhar gene was introduced into Arabidopsis thaliana. Transgenic plants were biochemically characterized and tested for responses to oxidative stress. Western blot indicated that the dhar-transgene was successfully expressed. In homozygous T_4 transgenic seedlings, DHAR overexpression was increased up to 1.5 to 5.4 fold, which enhanced foliar ascorbic acid levels 2- to 4.25-fold and ratio of AsA/DHA about 3- to 16-fold relative to wild type. In addition, the level of glutathione, the reductant used by DHAR, also increased as did its redox state. When whole plants were treated with high light and high temperature stress or in vitro leaf discs were subjected to 10 μM paraquat, transgenic plants showed a larger AsA pool size, lower membrane damage, and a higher level of chlorophyll compared with controls. These data suggested that increasing the plant vitamin C content through enhanced ascorbate recycling could limit the deleterious effects of environmental oxidative stress.     

Molecular cloning and characterization of a rice dehydroascorbate reductase

Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet. Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1). A recombinant Dhar1p produced in Escherichia coli was functional. The expression sequence tag database suggests that Dhar1p homologs exist in various plants. Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one. The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature.     

Species-Specific Expansion and Molecular Evolution of the 3-hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR) Gene Family in Plants

The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.