BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase,improves stress tolerance in transgenic tobacco

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the l-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T₁ seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H₂O₂ treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.     

Cytosolic monodehydroascorbate reductase gene affects stress adaptation and grain yield under paddy field conditions in <Emphasis Type="Italic">Oryza sativa L. japonica</Emphasis>

Monodehydroascorbate reductase (MDHAR), which is responsible for growth, development and stress response in plants, is a key enzyme in the maintenance of the ascorbate (AsA) pool through the AsA–glutathione (AsA–GSH) cycle and is induced by abiotic stresses. It has highly conserved regions containing FAD- and NAD(P)H-binding domains. In particular, NAD(P)H is a significant electron donor in the AsA–GSH pathway. In this context, we introduced RNA interference (RNAi) to determine the functional role of Oryza sativa L. japonica MDHAR isoform 3 (OsMDHAR3) and developed transgenic (mdhar3) rice plants in which the NAD(P)H domain was silenced. The mdhar3 rice plants were more sensitive to salt stress than the wild-type (WT) plants. In addition, the mdhar3 rice plants showed decreased ability for environmental adaptation because of an imbalance in the redox homeostasis and reduced AsA pool. These plants showed increased hydroperoxide levels and ion leakage, and decreased chlorophyll content and ascorbate/dehydroascorbate ratio under the paddy field conditions; they also exhibited a reduction in the total biomass and grain yield. Furthermore, the activity of a purified E196A mutant of the OsMDHAR protein decreased to approximately 70% of the activity of the WT protein. These results suggest that OsMDHAR3 plays a critical role in the intrinsic resistance, as well as in the sensitivity of seed maturation and productivity, of rice plants to environmental stresses, thereby indicating the functional importance of NADH in MDHAR activity, in vivo and in vitro.     

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.     

Effects of exogenous hydrogen sulfide on the redox states of ascorbate and glutathione in maize leaves under salt stress

This study investigated the effects of exogenous hydrogen sulfide (H₂S) on the redox states of ascorbate (AsA) and glutathione (GSH) in maize leaves under NaCl (100 mM) stress. Salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), Γ-glutamylcysteine synthetase (Γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), malondialdehyde content and electrolyte leakage, and reduced the ratios of reduced and oxidised forms of AsA (AsA/DHA) and GSH (GSH/GSSG) compared with control. Pretreatment with NaHS (H₂S donor) further enhanced the activities of the above enzymes except MDHAR and ameliorated the decrease in the ratios of AsA/DHA and GSH/GSSG compared with the salt stress alone. Pretreatment with NaHS significantly reduced the malondialdehyde content and electrolyte leakage induced by the salt stress. Pretreatment with NaHS alone did not affect any of the above mentioned parameters compared with the control. Our results suggest that exogenous H2S could maintain the redox states of ascorbate and glutathione by up-regulating the ascorbate and glutathione metabolism and thus play an important role for acquisition of salt stress tolerance in maize.     

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.     

Effects of lanthanum on the ascorbate and glutathione metabolism of Vigna radiata seedlings under salt stress

In order to elucidate the role of lanthanum (La) in response of Vigna radiata to a salt stress, we investigated the effects of La on the ascorbate and glutathione metabolism. The results show that in comparison with a control, the salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), γ-glutamylcysteine synthetase (γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), and the content of ascorbic acid (AsA) and glutathione (GSH). It also increased the malondialdehyde content (MDA) and electrolyte leakage. The salt stress significantly decreased the ratios of AsA/dehydroascorbate (DHA) and GSH/glutathione disulphide (GSSG) compared with the control. The pretreatment with La not only significantly increased the activities of the above enzymes, the content of AsA, GSH, and the ratios of AsA/DHA and GSH/GSSG, but also significantly reduced the MDA content and electrolyte leakage compared with the salt stress alone. Our results suggest that La could up-regulate the ascorbate and glutathione metabolisms and could have an important role for acquisition of salt stress tolerance in Vigna radiata.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.     

The expression of BrMDHAR gene in chloroplasts and mitochondria enhances tolerance to freezing stress in Arabidopsis thaliana

In chloroplasts and mitochondria, antioxidant mechanisms include the ascorbate-glutathione cycle, and monodehydroascorbate reductase (MDHAR) is important for regeneration of ascorbate (AsA) from monodehydroascorbate (MDHA). To improve detoxification of reactive oxygen species (ROS), we established a construct of the MDHAR gene from Brassica rapa fused to the targeting signal peptides of Pisum sativum glutathione reductase (GR), which was controlled by a stress-inducible SWPA2 promoter, and introduced this expression system into Arabidopsis thaliana. Transgenic (TG) plants overexpressing BrMDHAR targeted to chloroplasts and mitochondria through signal peptides showed an elevated MDHAR activity and an increased ratio of AsA to dehydroascorbate (DHA) when compared to wild-type (WT) plants under a freezing stress. These led to increased photosynthetic parameters, redox homeostasis, and biomass in TG plants when compared to the WT plants. Our results suggest that the overexpression of the BrMDHAR gene targeted to chloroplasts and mitochondria conferred an enhanced tolerance against the freezing stress, and a stress adaptation of dual-targeted BrMDHAR was better than that of single BrMDHAR.     

Manipulation of the Rice L-Galactose Pathway: Evaluation of the Effects of Transgene Overexpression on Ascorbate Accumulation and Abiotic Stress Tolerance

Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3'',5''-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.     

Nitric oxide modulates antioxidant defense and the methylglyoxal detoxification system and reduces salinity-induced damage of wheat seedlings

The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal (MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the H₂O₂ and lipid peroxidation levels. Exogenous NO pre-treatment of the seedlings had little influence on the non-enzymatic and enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

The Xerophyta viscosa Aldose Reductase (ALDRXV4) Confers Enhanced Drought and Salinity Tolerance to Transgenic Tobacco Plants by Scavenging Methylglyoxal and Reducing the Membrane Damage

We report the efficacy of an aldose reductase (ALDRXV4) enzyme from Xerophyta viscosa Baker in enhancing the prospects of plant’s survival under abiotic stress. Transgenic tobacco plants overexpressing ALDRXV4 cDNA showed alleviation of NaCl and mannitol-induced abiotic stress. The transgenic plants survived longer periods of water deficiency and salinity stress and exhibited improved recovery after rehydration as compared to the wild type plants. The increased synthesis of aldose reductase in transgenic plants correlated with reduced methylglyoxal and malondialdehyde accumulation and an elevated level of sorbitol under stress conditions. In addition, the transgenic lines showed better photosynthetic efficiency, less electrolyte damage, greater water retention, higher proline accumulation, and favorable ionic balance under stress conditions. Together, these findings suggest the potential of engineering aldose reductase levels for better performance of crop plants growing under drought and salt stress conditions.     

Early Abscisic Acid Accumulation Regulates Ascorbate and Glutathione Metabolism in Soybean Leaves Under Progressive Water Stress

Ascorbate (AsA) and glutathione (GSH) play an important role in improving the tolerance of plants to water stress. The objective of this study was to investigate the effect of early abscisic acid (ABA) accumulation on AsA and GSH metabolism in soybean plants after 24 h of exposure to progressive water stress. The results showed that AsA, total AsA, GSH and total GSH content, and ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), GSH reductase (GR), GSH peroxidase (GPX), l-galactono-1,4-lactone dehydrogenase (GLDH), and γ-glutamylcysteine synthetase (γ-GCS) activities were increased by progressive water stress. The above increases, except for total GSH content and the activities of GLDH and γ-GCS, were blocked by pretreatment with tungstate, an ABA biosynthesis inhibitor, which significantly suppressed the early increase in ABA and reactive oxygen species (ROS) in stressed plants. Application of ABA reversed the effects of tungstate. Pretreatments with several ROS scavengers, such as Tiron and dimethylthiourea (DMTU), and an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), significantly arrested the early accumulation of ROS but not ABA in stressed plants. Furthermore, the above-mentioned pretreatments remarkably prevented any increase in APX, MDHAR, DHAR, GR, and GPX activities, as well as AsA, total AsA and GSH levels in stressed plants. Our results indicated that early ABA accumulation caused by progressive water stress triggers an early rise in ROS levels, which, in turn, leads to regulation of AsA and GSH metabolism.

     

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.

     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings

Salinity, a severe environmental factor, has limited the growth and productivity of crops. Many compounds have been applied to minimize the harmful effects of salt stress on plant growth. An experiment was conducted to investigate the interactive effects of exogenous ascorbic acid (AsA) and gibberellic acid (GA₃) on common bean (Phaseolus vulgaris L. cv. Naz) seedlings under salt stress. The changes of growth parameters, photosynthetic and non-photosynthetic pigments and potassium content showed that the addition of 1 mM AsA and/or 0.05 mM GA₃ considerably decreased the oxidative damage in common bean plants treated with 200 mM NaCl. The NaCl-stressed seedlings exposed to AsA or GA₃, specifically in their combination, exhibited an improvement in sodium accumulation in both roots and shoots, as compared to NaCl-treated plants. NaCl treatment increased hydrogen peroxide (H₂O₂) content and lipid peroxidation indicated by accumulation of malondialdehyde (MDA), whereas the interaction of AsA with GA₃ decreased the amounts of MDA and H₂O₂. In the meantime, interactive effect of these substances enhanced protein content and the activity of the antioxidant enzyme, guaiacol peroxidase, in common bean plants under salt stress. It was concluded that synergistic interaction between AsA and GA₃ could alleviate the adverse effects of salinity on P. vulgaris seedlings.