The plastidic 2-cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage

The chloroplastic drought-induced stress protein of 32 kD (CDSP32) is composed of two thioredoxin modules and is induced by environmental and oxidative stress conditions. We investigated whether the plastidic protein BAS1, which is related to eubacterial 2-Cys peroxiredoxin, is a target for CDSP32. Using a CDSP32 active-site mutant, we showed that the BAS1 and CDSP32 proteins form a mixed disulfide complex in vitro. Moreover, affinity chromatography indicated that BAS1 is a major target for CDSP32 in chloroplasts. CDSP32 was able to reduce BAS1 in vitro, and BAS1 displayed CDSP32-dependent peroxidase activity. The function of CDSP32 was investigated in transgenic potato lines without detectable levels of the protein as a result of cosuppression. Under conditions of photooxidative stress induced by incubation with either methyl viologen or t-butyl hydroperoxide or by exposure to low temperature under high light, plants lacking CDSP32 exhibited decreased maximal photosystem II photochemical efficiencies compared with the wild type and transgenic controls. In addition, plants without CDSP32 retained much less chlorophyll than controls under stress, indicating increased damage to photosynthetic membranes. We conclude that CDSP32 is a thioredoxin with a critical role in plastid defense against oxidative damage and that this role is related to its function as a physiological electron donor to the BAS1 peroxiredoxin.     

Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses

The chloroplastic drought-induced stress protein of 32 kDa (CDSP32) is a thioredoxin induced by environmental stress conditions. To gain insight into the function of CDSP32, we applied two strategies to analyze its targets. First, using affinity chromatography with an immobilized CDSP32 active site mutant, we identified six plastidic targets of CDSP32. Three of them are involved in photosynthetic processes: ATP-ase gamma-subunit, Rubisco and aldolase. The three others participate in the protection against oxidative damage: two peroxiredoxins, PrxQ and the BAS1 2-Cys peroxiredoxin, and a B-type methionine sulfoxide reductase. Then, we developed a novel strategy to trap targets directly in leaf extracts. The method, based on co-immunoprecipitation using extracts from plants overexpressing Wt CDSP32 or CDSP32 active site mutant, confirmed the interaction in vivo between CDSP32 and the PrxQ and BAS1 peroxiredoxins. We showed that CDSP32 is able to form heterodimeric complexes with PrxQ and that the peroxiredoxin displays CDSP32-dependent peroxidase activity. Under photooxidative stress induced by methyl viologen, plants overexpressing CDSP32 active site mutant exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared with Wt plants and with plants overexpressing Wt CDSP32. We propose that the increased sensitivity results from trapping in planta of the targets involved in the protection against oxidative damage. We conclude that CDSP32, compared with other plant thioredoxins, is a thioredoxin more specifically involved in plastidic responses against oxidative stress.     

Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.     

Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.     

Accumulation of plastid lipid-associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species.

Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Enhanced sensitivity to oxidative stress in an Arabidopsis nitric oxide synthase mutant

The possible involvement of nitric oxide (NO) in oxidative stress tolerance was studied using Arabidopsis thaliana wild type (WT) and Atnos1 mutant plants, in which endogenous NO production is greatly diminished because 80% of nitric oxide synthase (NOS) activity is eliminated due to T-DNA insertion in the first exon of the NOS1 gene. Compared with WT, Atnos1 mutant plants showed increased hypersensitivity to salt stress and methyl viologen (MV) treatment. The maximal photochemical efficiency of photosystem II (F(v)/F(m)) and membrane integrity decreased in WT and Atnos1 mutant plants under stresses, but the extent was higher in the mutant. Treatment with sodium nitroprusside (SNP) (a NO donor) to Atnos1 mutant plants alleviated the damage. Instead, inhibition of nitric oxide accumulation in the WT plants produced opposite effects. Hydrogen peroxide and lipid peroxidation increased and the extent was higher in Atnos1 mutant plants than that in WT plants under MV stress. These results indicated that nitric oxide could protect the damage against NaCl and MV treatments.     

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.     

过表达番茄GDP-L-半乳糖磷酸酶基因提高烟草抗MV诱导的氧化胁迫能力

为了探讨番茄GDP—L-半乳糖磷酸酶对烟草抗坏血酸（AsA）含量及抗氧化能力的影响,从番茄叶片中分离了GDP-L-半乳糖磷酸酶基因（LeGGP）,并转入到烟草中。以野生型（WT）和转正义LeGGP烟草株系T1-3和T1-15为试材,测定了甲基紫精（MV）处理下AsA、脱氢抗坏血酸（DHA）、H2O2、O2-和叶绿素含量、抗坏血酸过氧化物酶（APX）活性、光合速率和叶绿素荧光参数等。Northem杂交分析表明LeGGP的表达受MV的诱导,在MV处理下,野生型烟草的离体叶圆片发生比转基因烟草更严重的光漂白,转基因烟草的AsA含量及清除H2O2和O2-的能力明显强于野生型,过表达LePGG胀高了烟草的生长量。并且转基因烟草比野生型具有更高的净光合效率（Pn）和光系统Ⅱ（PSII）最大光化学效率（眠）。结果表明,LeGGP的过表达有助于提高烟草AsA含量及抗氧化胁迫能力。     

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.     

Overexpression of a peroxiredoxin gene from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, <Emphasis Type="Italic">ThPrx1</Emphasis>, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H₂O₂) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H₂O₂ and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H₂O₂, superoxide (O₂^??) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.     

Overexpression of the pepper antimicrobial protein <Emphasis Type="Italic">CaAMP1</Emphasis> gene regulates the oxidative stress- and disease-related proteome in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Proteomics facilitates our understanding of cellular processes and network functions in the plant defense response during abiotic and biotic stresses. Here, we demonstrate that the ectopic expression of the Capsicum annuum antimicrobial protein CaAMP1 gene in Arabidopsis thaliana confers enhanced tolerance to methyl viologen (MV)-induced oxidative stress, which is accompanied by lower levels of lipid peroxidation. Quantitative comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified some of the oxidative stress- and disease-related proteins that are differentially regulated by CaAMP1 overexpression in Arabidopsis leaves. Antioxidant- and defense-related proteins, such as 2-cys peroxiredoxin, l-ascorbate peroxidase, peroxiredoxin, glutathione S-transferase and copper homeostasis factor, were up-regulated in the CaAMP1 transgenic leaf tissues. In contrast, GSH-dependent dehydroascorbate reductase and WD-40 repeat family protein were down-regulated by CaAMP1 overexpression. In addition, CaAMP1 overexpression enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and also H₂O₂ accumulation in Arabidopsis. The identified antioxidant- and defense-related genes were differentially expressed during MV-induced oxidative stress and Pst DC3000 infection. Taken together, we conclude that CaAMP1 overexpression can regulate the differential expression of defense-related proteins in response to environmental stresses to maintain reactive oxygen species (ROS) homeostasis.     

Suppression of tomato <Emphasis Type="Italic">SlGGP</Emphasis> aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.     

Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants

Two chloroplastic proteins, named CDSP 32 and CDSP 34 for chloroplastic drought-induced stress protein of 32 and 34 kDa, were previously shown to be substantially synthesized in Solanum tuberosum plants subjected to water deficit. We investigated the localization of CDSPs in leaf chloroplasts from control and wilted potato plants using immunocytochemistry. Observation of electron micrographs did not reveal any important change in plastid structures of drought-stressed plants, except an increased number and a larger size of plastoglobuli. In well-watered plants, very little labeling corresponding to CDSP 32 was detected. Consecutively to water stress, a higher abundance of CDSP 32 was revealed, the protein being exclusively localized in the stroma. Immunocytochemical data indicated the presence of some CDSP 34 protein in well-watered plants and confirmed its accumulation upon water deficit. CDSP 34 was found to be preferentially associated with stromal lamellae thylakoids, but some protein was revealed in the stroma. No association of CDSP 34 with grana and plastoglobuli was noticed in chloroplasts from control and stressed plants.     

Specific roles of alpha- and gamma-tocopherol in abiotic stress responses of transgenic tobacco

Tocopherols are lipophilic antioxidants that are synthesized exclusively in photosynthetic organisms. In most higher plants, alpha- and gamma-tocopherol are predominant with their ratio being under spatial and temporal control. While alpha-tocopherol accumulates predominantly in photosynthetic tissue, seeds are rich in gamma-tocopherol. To date, little is known about the specific roles of alpha- and gamma-tocopherol in different plant tissues. To study the impact of tocopherol composition and content on stress tolerance, transgenic tobacco (Nicotiana tabacum) plants constitutively silenced for homogentisate phytyltransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) activity were created. Silencing of HPT lead to an up to 98% reduction of total tocopherol accumulation compared to wild type. Knockdown of gamma-TMT resulted in an up to 95% reduction of alpha-tocopherol in leaves of the transgenics, which was almost quantitatively compensated for by an increase in gamma-tocopherol. The response of HPT and gamma-TMT transgenics to salt and sorbitol stress and methyl viologen treatments in comparison to wild type was studied. Each stress condition imposes oxidative stress along with additional challenges like perturbing ion homeostasis, desiccation, or disturbing photochemistry, respectively. Decreased total tocopherol content increased the sensitivity of HPT:RNAi transgenics toward all tested stress conditions, whereas gamma-TMT-silenced plants showed an improved performance when challenged with sorbitol or methyl viologen. However, salt tolerance of gamma-TMT transgenics was strongly decreased. Membrane damage in gamma-TMT transgenic plants was reduced after sorbitol and methyl viologen-mediated stress, as evident by less lipid peroxidation and/or electrolyte leakage. Therefore, our results suggest specific roles for alpha- and gamma-tocopherol in vivo.     

A novel cis-element that is responsive to oxidative stress regulates three antioxidant defense genes in rice

All organisms have defense systems against oxidative stress that include multiple genes of antioxidant defense. These genes are induced by reactive oxygen species under condition of oxidative stress. In this study, we found that a 28-bp motif is conserved on the promoter regions of three antioxidant defense genes in rice (Oryza sativa): cytosolic superoxide dismutase (sodCc1), cytosolic thioredoxin (trxh), and glutaredoxin (grx). We demonstrated that the 28-bp sequence acts as a cis-element responsive to oxidative stress by transient expression assay and designated it as CORE (coordinate regulatory element for antioxidant defense). The CORE was activated by methyl viologen treatment and induced a 3.1-fold increase in expression of the reporter gene, but it did not respond to hydrogen peroxide. The expressions of the sodCc1, trxh, and grx genes were coordinately induced by methyl viologen, suggesting that multiple genes involved in antioxidant defense are controlled by a common regulatory mechanism via CORE. Application of the mitogen-activated protein kinase kinase inhibitor caused the constitutive induction of the sodCc1, trxh, and grx genes and the activation of CORE without methyl viologen treatment. These results indicate that a mitogen-activated protein kinase cascade is involved in the gene regulation mediated by CORE.