Reducing AsA Leads to Leaf Lesion and Defence Response in Knock-Down of the AsA Biosynthetic Enzyme GDP-D-Mannose Pyrophosphorylase Gene in Tomato Plant

As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.     

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.     

Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.     

An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.     

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2 weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24 h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.     

Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.     

Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene

GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type.     

外源α-萘乙酸对花期长期干旱大豆叶片抗氧化系统的影响

以不同耐旱型品种‘南农99-6’和‘科丰1号’大豆为材料,2012年在南京农业大学牌楼试验站进行为期110 d的盆栽试验,研究大豆花期叶面喷施α-萘乙酸(NAA)对长期干旱条件下大豆植株抗氧化系统的影响.结果表明: 干旱胁迫显著降低了大豆地上部干物质量,叶片中丙二醛(MDA)含量及活性氧(ROS)水平显著升高,同时,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、单脱氢抗坏血酸还原酶（MDHAR）、谷胱甘肽还原酶(GR)和谷胱甘肽过氧化物酶(GPX)活性,还原型抗坏血酸(AsA)、还原型谷胱甘肽(GSH)含量及AsA/DHA(双脱氢抗坏血酸)和GSH/GSSG(氧化型谷胱甘肽)比值显著升高,其中‘科丰1号’大豆的抗氧化能力更高,从而维持较低的ROS水平和MDA含量.NAA可显著提高叶片中的APX、POD、CAT、MDHAR活性及AsA/DHA、GSH/GSSG比值,其中‘科丰1号’大豆叶片的脱氢抗坏血栓还原酶（DHAR）活性和AsA含量极显著增加.     

Overexpression of SlGGP-LIKE gene enhanced the resistance of tomato to salt stress

Ascorbic acid (AsA) plays an important role in scavenging reactive oxygen species (ROS) and reducing photoinhibition in plants, especially under stress. The function of SlGGP which encodes the key enzyme GDP-L-galactose phosphorylase in AsA synthetic pathway is relatively clear. However, there is another gene SlGGP-LIKE that encodes this enzyme in tomato, and there are few studies on it, especially under salt stress. In this study, we explored the function of this gene in tomato salt stress response using transgenic lines overexpressing SlGGP-LIKE (OE). Under normal conditions, overexpressing SlGGP-LIKE can increase the content of reduced AsA and the ratio of AsA/ DHA (dehydroascorbic acid), as well as the level of xanthophyll cycle. Under salt stress, compared with the wild-type plants (WT), the OE lines can maintain higher levels of reduced AsA. In addition, OE lines also have higher levels of reduced GSH (glutathione) and total GSH, higher ratios of AsA/DHA and GSH/oxidative GSH (GSSR), and higher level of xanthophyll cycle. Therefore, the OE lines are more tolerant to salt stress, with higher photosynthetic activity, higher antioxidative enzyme activities, higher content of D1 protein, lower production rate of ROS, and lighter membrane damage. These results indicate that overexpressing SlGGP-LIKE can enhance tomato resistance to salt stress through promoting the synthesis of AsA.

     

Metal/metalloid stress tolerance in plants: role of ascorbate,its redox couple,and associated enzymes

The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water-soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.     

Senescence-induced increases in intracellular oxidative stress and enhancement of the need for ascorbic acid in human fibroblasts

Many studies have suggested that there is a close correlation among declines in internal ascorbic acid (AsA) levels, various disorders, and senescence. To clarify the relationships between age-associated changes in intracellular AsA levels and the effects of AsA administration on intracellular reactive oxygen species (ROS) levels, we investigated aging-related changes in AsA uptake, ROS levels, and the effects of AsA administration on intracellular ROS levels in young and old (senescent) human fibroblasts. Our results demonstrated that AsA uptake was increased in old cells compared with young cells, although mRNA and protein expression of sodium-dependent vitamin C transporter 2 was barely altered between the young and old cells. We also demonstrated that the intracellular superoxide anion level was higher in young cells, whereas the level of intracellular peroxides was significantly increased in old cells under both normal and oxidative stress conditions. Moreover, AsA administration markedly decreased the augmentation of intracellular peroxides in old cells, whereas there was no effect of AsA treatment in young cells under both normal and oxidative stress conditions. Therefore, our results also indicate that AsA could play an important role in regulating the intracellular ROS levels in senescent cells and that the need for AsA is enhanced by cellular senescence.     

Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat

Although ascorbic acid (AsA) is one of the most important and abundantly occurring water soluble antioxidants in plants, relatively little is known about its role in counteracting the adverse effects of salt stress on plant growth. To address this issue that whether exogenous application of ascorbic acid (AsA) through rooting medium could alleviate the adverse effects of salt stress on wheat plants, a hydroponic experiment was conducted under glasshouse conditions using two wheat cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive). Plants of both cultivars were subjected to 0 or 150 mM NaCl solution supplemented with 0, 50, or 150 mg L⁻¹ AsA for 58 days. Imposition of salt stress reduced the growth of both wheat cultivars by causing reduction in photosynthesis, and endogenous AsA level, and enhancing accumulation of Na⁺ and Cl⁻ coupled with a decrease in K⁺ and Ca²⁺ in the leaves and roots of both cultivars thereby decreasing tissue K⁺/Na⁺ ratio. However, root applied AsA counteracted the adverse effects of salt stress on the growth of cv. S-24 only, particularly at 100 mg L⁻¹ AsA level. AsA-induced enhancement in growth of salt-stressed plants of S-24 was associated with enhanced endogenous AsA level and CAT activity, and higher photosynthetic capacity, and accumulation of K⁺ and Ca²⁺ in the leaves. Although root applied AsA did not improve the growth of salt-stressed plants of MH-97, it enhanced endogenous level of AsA, CAT activity, photosynthetic capacity, and leaf K⁺ and Ca²⁺. These findings led us to conclude that root applied AsA counteracts the adverse effects of salt stress on growth of wheat by improving photosynthetic capacity of wheat plants against salt-induced oxidative stress and maintaining ion homeostasis, however, these effects were cultivar specific.     

Alpha-momorcharin, a RIP produced by bitter melon, enhances defense response in tobacco plants against diverse plant viruses and shows antifungal activity in vitro

Alpha-momorcharin (α-MMC) is type-1 ribosome inactivating proteins (RIPs) with molecular weight of 29 kDa and has lots of biological activity. Our recent study indicated that the α-MMC purified from seeds of Momordica charantia exhibited distinct antiviral and antifungal activity. Tobacco plants pre-treated with 0.5 mg/mL α-MMC 3 days before inoculation with various viruses showed less-severe symptom and less reactive oxygen species (ROS) accumulation compared to that inoculated with viruses only. Quantitative real-time PCR analysis revealed that the replication levels of viruses were lower in the plants treated with the α-MMC than control plants at 15 days post inoculation. Moreover, the coat protein expression of viruses was almost completely inhibited in plants which were treated with the α-MMC compared with control plants. Furthermore, the SA-responsive defense-related genes including non-expressor of pathogenesis-related genes 1 (NPR1), PR1, PR2 were up-regulated and activities of some antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were increased after the α-MMC treatment. In addition, the α-MMC (500 μg/mL) revealed remarkable antifungal effect against phytopathogenic fungi, in the growth inhibition range 50.35–67.21 %, along with their MIC values ranging from 100 to 500 μg/mL. The α-MMC had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of Sclerotinia sclerotiorum. The α-MMC showed a remarkable antiviral and antifungal effect and hence could possibly be exploited in crop protection for controlling certain important plant diseases.     

Exogenous 5-aminolevulinic acid pretreatment ameliorates oxidative stress triggered by low-temperature stress of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Low temperature is an important limiting factor in tomato production in early spring and winter. 5-Aminolevulinic acid (ALA) protects crops against varied abiotic stresses. However, the methodology to precisely use ALA to increase the cold tolerance in tomatoes is still not fully known. We therefore explored the effects of ALA concentration, application period, and dose on membrane lipid peroxidation, antioxidation, photosynthesis, and plant growth in different tomato cultivars (Zhongza No. 9, ZZ and Jinpeng No. 1, JP) at low-temperature stress. Results revealed that low temperature caused plants oxidative damage and growth inhibition in both ZZ and JP plants. The ROS (hydrogen peroxide and superoxide anion) accumulation and membrane lipid peroxidation (malondialdehyde content and the relative electrical conductivity) were more remarkable in JP plants than ZZ plants under low temperature. The catalase (CAT) and ascorbate–glutathione cycle (AsA–GSH) induced by ALA reliably eliminated excessive ROS to maintain the redox balance in both tomato cultivars under low-temperature stress. In AsA–GSH cycle, AsA regeneration was mainly catalyzed by dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR), from dehydroascorbate (DHA) to AsA and monodehydroascorbate (MDA) to AsA in ZZ plants, while AsA regeneration in JP plants was mostly catalyzed by DHAR, from DHA to AsA. The ALA optimum concentration was 25 mg L^?1. The tomato plants with five true leaves pretreated with 6 mL ALA were more effective than spraying after cold occurred. In conclusion, the two tomato varieties illustrated different capacities to bear low-temperature stress. And ZZ plants were more tolerant to low temperature than JP plants. Precise ALA pretreatment observably alleviated low temperature induced-damage via CAT and AsA–GSH cycle in both cultivars. The regeneration of AsA in AsA–GSH cycle may be more comprehensive in ZZ plants than JP plants, to better tolerate low-temperature stress.     

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3′,5′-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.     

Salmonella Typhimurium-specific bacteriophage ΦSH19 and the origins of species specificity in the Vi01-like phage family

Background

PHYVV and PepGMV are plant viruses reported in Mexico and Southern US as causal agents of an important pepper disease known as "rizado amarillo". Mixed infections with PHYVV and PepGMV have been reported in several hosts over a wide geographic area. Previous work suggested that these viruses might interact at the replication and/or movement level in a complex manner. The aim of present report was to study some aspects of a synergistic interaction between PHYVV and PepGMV in pepper plants. These include analyses of symptom severity, viral DNA concentration and tissue localization of both viruses in single and mixed infections.

Results

Mixed infections with PepGMV and PHYVV induced symptoms more severe than those observed in single viral infections. Whereas plants infected with either virus (single infection) presented a remission stage with a corresponding decrease in viral DNA levels, double-infected plants did not present symptom remission and both viral DNA concentrations dramatically increased. In situ hybridization experiments revealed that both viruses are restricted to the vascular tissue. Interestingly, the amount of viral DNA detected was higher in plants inoculated with PepGMV than that observed in PHYVV-infected plants. During mixed infections, the location of both viruses remained similar to the one observed in single infections, although the number of infected cells increases. Infections with the tripartite mixture PHYVV (A+B) + PepGMV A produced a similar synergistic infection to the one observed after inoculation with both full viruses. On the contrary, tripartite mixture PepGMV (A+B) + PHYVV A did not produce a synergistic interaction. In an attempt to study the contribution of individual genes to the synergism, several mutants of PHYVV or PepGMV were inoculated in combination with the corresponding wild type, second virus (wt PepGMV or wt PHYVV). All combinations tested resulted in synergistic infections, with exception of the TrAP mutant of PepGMV (PepGMV TrAP-) + PHYVV.

Conclusion

In this report, we have demonstrated that synergistic interaction between PHYVV and PepGMV during a mixed infection is mainly due to an increased DNA concentration of both viruses, without any noticeable effect on the localization of either virus on infected plant tissue. Our results have shown that the viral component A from PepGMV is important for synergism during PHYVV-PepGMV mixed infections.     

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.