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.     

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.     

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.     

星星草脱氢抗坏血酸还原酶（PtDHAR）cDNA的克隆及表达分析

脱氢抗坏血酸还原酶是抗坏血酸代谢循环中的关键酶,在多种植物中与抗胁迫相关。为了获得抗盐碱植物星星草中该基因序列,利用RACE技术,从星星草中克隆出脱氢抗坏血酸还原酶基因（PtDHAR）的cDNA全长序列,其GenBank登录号为HM125046。PtDHAR cDNA核苷酸序列长度为987bp,开放阅读框为639bp,编码213个氨基酸。该基因编码的氨基酸序列与水稻、小麦等禾本科作物具有很高的同源性。Northern杂交分析表明,该基因在盐碱胁迫下表达量显著升高。     

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.     

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.     

Differential Responses in Activity of Antioxidant Enzymes to Different Environmental Stresses in Arabidopsis thaliana

Arabidopsis thaliana . Three-week-old plants were exposed to a high temperature (30 C), an enhanced light intensity (200 μE/m²/sec), water deficiency (water deprivation for 2 days), a chilling temperature (5 C), or ultraviolet-B (UV-B) radiation (0.25 or 0.094 W/m²) for 1 week (except for water deficiency). The high temperature and enhanced light treatments increased only dehydroascorbate reductase (DHAR) activity. Water deficiency enhanced the activities of DHAR and guaiacol peroxidase (PER). Chilling temperature increased the activities of ascorbate peroxidase (APX) and glutathione reductase (GR), whereas it decreased catalase (CAT) activity. UV-B at an intensity of 0.25 W/m² elevated the activities of APX, monodehydroascorbate reductase (MDHAR), GR, PER and superoxide dismutase (SOD). It was suggested that the amounts of phenylpropanoid compounds increased during treatments of plants with enhanced light intensity, chilling temperature, and UV-B. These results suggest that some differences exist among the oxidative stress conditions caused by the different treatments, although all of these treatments seem to be related to active oxygen production. We propose that in A. thaliana, environmental stresses may be classified into those which induce DHAR activity and those which induce APX activity. Received 11 January 1999/ Accepted in revised form 22 April 1999     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.     

Purification and Characterization of Dehydroascorbate Reductase from Rice

Dehydroascorbate reductase (DHAR; EC 1.8.5.1 [EC] ) is an enzyme thatis critical for maintenance of an appropriate level of ascorbatein plant cells. This report describes the purification and characterizationof a GSH-dependent DHAR from rice (Oryza saliva) bran and isthe first, to our knowledge, of such an analysis of DHAR froma monocot. The enzyme was a monomeric thiol enzyme, resemblingDHARs purified from dicots, but it was different from them interms of heat stability and antigenicity. The amino-terminalamino acid sequence of the DHAR from rice did not show any obvioussimilarity to those of known proteins with DHAR activity, suchas, glutaredoxin (thioltrans-ferase), protein disulfide isomerase,and trypsin inhibitor. Immunoprecipitation analysis showed thatthis enzyme was a major DHAR in etiolated seedlings. Westernblot analysis indicated that this enzyme was distributed ubiquitouslyin rice tissues. A similar protein was found in barley but notin dicots. (Received July 18, 1996; Accepted December 4, 1996)     

Red 'Anjou' pear has a higher photoprotective capacity than green 'Anjou'

Photoprotective function of anthocyanins along with xanthophyll cycle and antioxidant system in fruit peel was investigated in red 'Anjou' vs green 'Anjou' pear (Pyrus communis) during fruit development and in response to short-term exposure to high light. The sun-exposed peel of red 'Anjou' had higher maximum quantum yield of photosystem II (F(V)/F(M)) than that of green 'Anjou' and both the sun-exposed peel and the shaded peel of red 'Anjou' had smaller decreases in F(V)/F(M) after 2-h high light (photon flux density of 1500 mumol m(-2) s(-1)) treatment than those of green 'Anjou'. At the middle and late developmental stages, the xanthophyll cycle pool size on a chlorophyll basis, the activity of superoxide dismutase, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and the level of reduced ascorbate and total ascorbate pool in the sun-exposed peel were either the same or lower in red 'Anjou' than in green 'Anjou', whereas the xanthophyll cycle pool size on a chlorophyll basis and the activity of APX, catalase, MDAR, DHAR and GR in the shaded peel were higher in red 'Anjou' than in green 'Anjou'. It is concluded that red 'Anjou' has a higher photoprotective capacity in both the sun-exposed peel and the shaded peel than green 'Anjou'. While the higher anthocyanin concentration along with the larger xanthophyll cycle pool size and the higher activity of some antioxidant enzymes may collectively contribute to the higher photoprotective capacity in the shaded peel of red 'Anjou', the higher photoprotective capacity in the sun-exposed peel of red 'Anjou' is mainly attributed to its higher anthocyanin concentration.     

The Tropical Fig Ficus microcarpa L. f. cv. Golden Leaves Lacks Heat-Stable Dehydroascorbate Reductase Activity

Dehydroascorbate reductase (DHAR) is an enzyme which mediatesa regeneration of ascorbate from dehydroascorbate using GSHas the electron donor. Here we report a tropical fig lackinga heat-stable DHAR activity in leaves. The leaves of Ficus microcarpaL. f. cv. Golden Leaves are normally green under dim light butare yellow under full sunlight in the field. We compared DHARactivity from the leaves of Golden Leaves to that of the wildtype using two distinct assay methods. Total leaf DHAR activityof Golden Leaves, determined in the presence of 2-mercaptoethanoland EDTA, was 0.3 µmol min^–1 (mg protein)^–1which was comparable to that of the wild type or spinach leaves.This activity was completely eliminated by heat treatment at80°C, whereas 40% of the activity was resistant to thistreatment in the wild type leaves. Similar results were obtainedwith another assay method employing dithiothreitol and gel filtration.We conclude that the absence of heat-stable DHAR activity isa significant biochemical marker that distinguishes Golden Leavesfrom the wild type. A physiological role for DHAR in adaptationto high light stress is proposed. (Received December 4, 1998; Accepted April 15, 1999)     

低温胁迫对水稻幼苗抗坏血酸含量的影响

随着低温时间的延长,水稻幼苗抗坏血酸和GSH的含量均平行地下降,抗坏血酸含量与电解质渗出率之间呈负相关。增强低温胁迫程度,随着抗坏血酸含量逐渐减少,MDA含量则逐渐增加。经低温处理后在不同光强下恢复时,抗坏血酸和MDA含量的变化不同。抗氧化剂(GSH、半胱氨酸和巯基乙醇)的预处理可抑制低温引起的抗坏血酸含量的下降。     

Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.     

Modified responses of root growth and reactive oxygen species-scavenging system to combined salt and heat stress in transgenic rice

Root growth and modifications of ROS-scavenging systems were investigated in transgenic rice (Oryza sativa L., cv. Zhonghua no. 11) co-expressing glutathione-S-transferase (GST, EC. 2.5.1.18) and catalase 1 (CAT1, EC 1.11.1.6) and nontransgenic rice exposed to just salt or heat and their combination. The higher number of adventitious roots but the lower root to shoot ratio were observed in the stressed transgenics as compared with nontransgenics. Most antioxidant enzymes, such as CAT, GST, ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC.1.6.4.2), dehydroascorbate reductase (DHAR, EC.1.8.5.1), and the redox state of glutathione and ascorbate, measured in the transformant roots, were significantly different from those in nontransformant roots following the three types of stress. The variations of root growth and antioxidant systems in the stressed transgenic rice may be attributed to not only the GST and CAT1 transgenes but also the coordination of the ascorbate-glutathione cycle.     

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.