Antioxidative MEtabolism

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H 2 O 2 levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

During plantevirus interactions, defence responses are linked to the accumulation of reactive oxygen species (ROS). Importantly, ROS play a dual role by (1) eliciting pathogen restriction and often localized death of host plant cells at infection sites and (2) as a diffusible signal that induces antioxidant and pathogenesis-related defence responses in adjacent plant cells. The outcome of these defences largely depends on the speed of host responses including early ROS accumulation at virus infection sites. Rapid host reactions may result in early virus elimination without any oxidative stress (i.e. a symptomless, extreme resistance). A slower host response allows a certain degree of virus replication and movement resulting in oxidative stress and programmed death of affected plant cells before conferring pathogen arrest (hypersensitive response, HR). On the other hand, delayed host attempts to elicit virus resistance result in an imbalance of antioxidative metabolism and massively stressed systemic plant tissues (e.g. systemic chlorotic or necrotic symptoms). The final consequence of these processes is a partial or almost complete loss of control over virus invasion (compatible infections).

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H 2 O 2 levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Theobroma cacao gives higher yield when cultivated in full sun under irrigation system with fertilization, because is a species little conservative in relation the efficiency of water use. On the other hand, T. cacao is usually cultivated under shade conditions in 'Cabruca' and agroforestry systems but with low yield. It has been observed under field conditions that the genotypes of T. cacao with high concentration of anthocyanins in young leaves are more tolerant to high irradiance stress when grown in full sun. The accumulation of flavonoids or other UVabsorbing compounds in the leaf epidermis is one of the most important mechanisms to screening out UV-B radiation. The aim of this study was to evaluate the effects of different levels of light on three clonal cacao genotypes ('Catongo', SCA-6 and SJ-2), contrasting in relation to accumulation of anthocyanin levels in young leaves, by evaluations of photosynthesis, activity of guaiacol peroxidase (GPX), chloroplastid pigment contents and vacuolar (anthocyanins) flavonoids contents, anatomical characteristics and gene expression of the leaf. In summary, (i) the anthocyanins contents in leaf level did not provide protection against photoinhibition in T. cacao, (ii) the 'Catongo' and SJ-2 genotypes showed greater phenotypic plasticity to the morphology and the chloroplastidic pigment contents in the leaf, while the SCA-6 genotype allocated more in the flavonoids content and hsp70 gene expression; (iii) the relative expression of the genes psbA and psbO, did not vary between genotypes under irradiance stress.

Under abiotic stress conditions, plants suffer from oxidative damage caused by increased generation of reactive oxygen species (ROS). Although chloroplasts are the main source of ROS generation in plant cells, changes in ROS level in mitochondria affect plant stress response. In this study, we investigated whether overexpression of mitochondrial manganese superoxide dismutase (Mn-SOD) can enhance tolerance to photooxidative stress. We produced transgenic tobacco plants overexpressing rice Mn-SOD under the control of cauliflower mosaic virus 35S promoter, and assessed the stress tolerance of those plants. The transgenic lines showed higher total SOD activity by 2.8 to 5.2 fold than wild type plants. Chlorophyll fluorescence measurement revealed that the stress-induced inhibition of photosystem II was alleviated under high light, chilling and heat stresses in the transgenic lines. This result indicates that the Mn-SOD contributes to the defense against photooxidative damages in chloroplasts under these stress conditions.

Nicotiana tabacum transgenic plants overexpressing manganese (Mn) superoxide dismutase (SOD) targeted to plastids were studied upon 12.5 PM cadmium (Cd) application. No differences in tolerance were observed in comparison to wild type plants. Seed germination inhibition, seedling growth, and plant biomass were measured at different developmental stages. Cd analysis revealed the usual distribution of Cd between roots and shoots, 10-times higher in roots. Lipid peroxidation level, a characteristic parameter of oxidative stress, indicated that little protection was gained by overexpressing MnSOD. However, chlorophyll concentration was not affected upon Cd treatment in 35S:MnSOD transgenic plants, suggesting that a possible protection localised at the chloroplast could indeed occur. Three antioxidant enzymes sensitive to metal stress were also analysed after in gel activity staining: SOD, ascorbate peroxidase (APX) and glutathione reductase (GR). As expected, MnSOD activity was remarkably higher in transgenic N. tabacum plants. Interestingly, Cu/ZnSOD activity decreased upon exposure of plants to 12.5 PM Cd. GR and APX activities augmented in metal-treated plants. In particular, APX activity was slightly higher in MnSOD over-expressing plants, which was possibly related with an enhanced generation of H2O2 through higher SOD activity.

While authors have traditionally emphasized the deleterious effects of reactive oxygen species (ROS) on seed biology, their role as signaling molecules during seed dormancy alleviation and germination is now the focus of many studies around the world. Over the last few years, studies using ''-omics'' technologies together with physiological and biochemical approaches have revealed that seed germination is a very complex process that depends on multiple biochemical and molecular variables. The pivotal role of phytohormones in promoting germination now appears to be interdependent with ROS metabolism, involving mitogen-activated protein kinase cascade activation, gene expression and post-translational protein modifications. This review is, thus, an attempt to summarize the new discoveries involving ROS and seed germination. The study of these interactions may supply markers of seed quality that might eventually be used in breeding programs to improve crop yields. Keywords Antioxidative metabolism Á Germination Á Plant hormones Á ROS signaling Á Seed proteome Communicated by N. Stewart. P. Diaz-Vivancos and G. Barba-Espín contributed equally to this work.

The effect of over‐expression in tobacco plants of cytosolic Cu,Zn‐superoxide dismutase (cytsod) and ascorbate peroxidase (cytapx) alone, or in combination, against bacterial wildfire and crown gall diseases, caused by Pseudomonas syringae pv. tabaci and Agrobacterium tumefaciens, respectively, was investigated. Disease tolerance was observed in all the transgenic lines against the two causal agents, with various levels of resistance, with the double transformants (lines 35 and 39) the most resistant against bacterial wild fire. In the case of P. syringae pv. tabaci, disease tolerance and symptom decrease was associated with a lower bacterial population and a higher level of several antioxidant defence enzymes. Transgenic lines also exhibited an enhanced tolerance against A. tumefaciens, with the transgenic line harbouring cytapx (line 51) the most resistant to crown gall disease. However, this was only observed with strain C58 among the three pathogenic strains tested. These result...

SummaryTo fortify the antioxidant capacity of plum plants, genes encoding cytosolic antioxidants ascorbate peroxidase (cytapx) and Cu/Zn‐superoxide dismutase (cytsod) were genetically engineered in these plants. Transgenic plum plants expressing the cytsod and/or cytapx genes in cytosol have been generated under the control of the CaMV35S promoter. High levels of cytsod and cytapx gene transcripts suggested that the transgenes were constitutively and functionally expressed. We examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress (100 mm NaCl). Several transgenic plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5‐5 and J8‐1 (expressing several copies of sod and apx, respectively). Transformation as well as NaCl treatments influenced the antioxidative metabolism of plum plantlets, including enzymatic and nonenzymatic antioxidants. Transgenic plantlets exhibited higher contents of nonenzyma...

Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. Line J8-1 exhibited an e...

Salinity is one of the main constraints for agriculture productivity worldwide. This important abiotic stress has worsened in the last 20 years due to the increase in water demands in arid and semi-arid areas. In this context, increasing tolerance of crop plants to salt stress is needed to guarantee future food supply to a growing population. This review compiles knowledge on the use of phytoprotectants of microbial origin (arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria), osmoprotectants, melatonin, phytohormones and antioxidant metabolism-related compounds as alleviators of salt stress in numerous plant species. Phytoprotectants are discussed in detail, including their nature, applicability, and role in the plant in terms of physiological and phenotype effects. As a result, increased crop yield and crop quality can be achieved, which in turn positively impact food security. Herein, efforts from academic and industrial sectors should focus on defining the treatment conditions and plant-phytoprotectant associations providing higher benefits.

Salinity is one of the main constraints for agriculture productivity worldwide. This important abiotic stress has worsened in the last 20 years due to the increase in water demands in arid and semi-arid areas. In this context, increasing tolerance of crop plants to salt stress is needed to guarantee future food supply to a growing population. This review compiles knowledge on the use of phytoprotectants of microbial origin (arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria), osmoprotectants, melatonin, phytohormones and antioxidant metabolism-related compounds as alleviators of salt stress in numerous plant species. Phytoprotectants are discussed in detail, including their nature, applicability, and role in the plant in terms of physiological and phenotype effects. As a result, increased crop yield and crop quality can be achieved, which in turn positively impact food security. Herein, efforts from academic and industrial sectors should focus on defining the trea...

Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. Line J8-1 exhibited an e...

To fortify the antioxidant capacity of plum plants, genes encoding cytosolic antioxidants ascorbate peroxidase (cytapx) and Cu/Zn-superoxide dismutase (cytsod) were genetically engineered in these plants. Transgenic plum plants expressing the cytsod and/or cytapx genes in cytosol have been generated under the control of the CaMV35S promoter. High levels of cytsod and cytapx gene transcripts suggested that the transgenes were constitutively and functionally expressed. We examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress (100 mM NaCl). Several transgenic plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and J8-1 (expressing several copies of sod and apx, respectively). Transformation as well as NaCl treatments influenced the antioxidative metabolism of plum plantlets, including enzymatic and nonenzymatic antioxidants. Transgenic plantlets exhibited higher contents of nonenzymatic antioxidants glutathione and ascorbate than nontransformed control, which correlated with lower accumulation of hydrogen peroxide. Overall, our results suggest that transformation of plum plants with genes encoding antioxidant enzymes enhances the tolerance to salinity.

In this work we used two different pea cultivars, JI281 is a semidomesticated land race of pea from Ethiopia whereas JI399 is a typical domesticated pea variety. Exposure of pea leaves to excess light (EL) for 1 h caused a reversible photoinhibition of photosynthesis as showed by changes in Fv / Fm. Although little difference existed between the two pea genotypes with respect to photoinhibition, after 60 min of EL the decline in Fv / Fm was higher in JI281 than in JI399 leaves. As a consequence of EL, H2O2 increased in both pea cultivars, whereas lipid peroxidation and protein oxidation slightly increased, although differences between cultivars were minimal. The redox state of ascorbate shifted towards its oxidized form under EL stress in both cultivars. Transcript levels of genes coding antioxidant enzymes varied with EL in both cultivars, but the response was more pronounced in JI399. The induction observed during EL was maintained or increased after the stress period, as occurred...

The biosafety of chemically synthesized graphene nanosheets on leaves ultrastructure of pepper and eggplants, especially chloroplast. Improvement of plants growth and yield attributes in response to graphene nanosheets spraying. No signs of oxidative burst or membrane damage, activation of health promoting components and antioxidants were coined in plants received graphene nanosheets.

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a Sadenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and ROS accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidised ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated Spd and Spm, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

Background Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions significantly accumulate during biotic and abiotic stress and cause oxidative damage and eventually cell death. There is accumulating evidence that ROS are also involved in regulating beneficial plant–microbe interactions, signal transduction and plant growth and development. Due to the relevance of ROS throughout the life cycle and for interaction with the multifactorial environment, the physiological phenotyping of the mechanisms controlling ROS homeostasis is of general importance. Results In this study, we have developed a robust and resource-efficient experimental platform that allows the determination of the activities of the nine key ROS scavenging enzymes from a single extraction that integrates posttranscriptional and posttranslational regulations. The assays were optimized and adapted for a semi-high throughput 96-well assay format. In a case study, we have analyzed tobacco leaves chal...

Tomato being sensitive to high temperature experiences mild to high temperature stresses under climate change conditions. To understand the response of tomato genotypes to mild temperature stress, a study was conducted in temperature gradient tunnel facility. The results revealed that across the genotypes studied, specific activity of antioxidant enzymes viz., superoxide dismutase (SOD), peroxidase (POX) and glutathione reductase (GR) increased significantly. Among the genotypes, increase in SOD activity was highest in cv. Arka Vikas, followed by IIHR 2195 and least in Abhinava. The GR activity was highest in Abhinava, followed by IIHR 2195 and least in cv. Arka Vikas. The mild temperature stress caused reduction in catalase (CAT) activity. The decrease in CAT activity and concomitant increase in POX activity was observed in cv. Arka Vikas. Low leaf water potential (W leaf) and higher electrolyte leakage indicated that the membrane integrity was affected across the tomato genotypes even under mild temperature stress. Among the genotypes studied, cv. Arka Vikas showed greater activity of SOD and POX, higher membrane stability and least reduction in water potential under mild temperature stress.

Various abiotic stresses like drought, salinity, high temperature, and chilling adversely affect plant growth and productivity. Terminal drought stress is one of the major concerns which limits the growth and yield of chickpea. CAMTA (Calmodulin binding transcription activator) plays a vital role in stress tolerance in plants. In this study, we have selected a CAMTA1 gene to explore its role against salinity and drought stress in an economically important crop, chickpea (Cicer arietinum L.). CAMTA1 gene was then over-expressed in chickpea and was exposed to drought and salinity. The over-expression of CAMTA1 enhanced the activities of various antioxidant enzymes (ascorbate peroxidase; APX, catalase: CAT, glutathione S-transferase; GST, superoxide dismutase; SOD, monodehydroascorbate reductase; MDHAR). The reduced stress markers TBARS and H2O2 enhanced the survival of plants against both stresses. The physiological parameters (net photosynthesis; PN, transpiration; E, stomatal conduc...

The oxidative stress response of rice cytosolic ascorbate peroxidase (APX) was examined. The transcript level of cytosolic APX was significantly increased when suspension cultures of germinating rice embryos were treated with paraquat (7.9-fold) or H 2 O : (6.1-fold). Induction by paraquat reached a maximum at 8 h. Induction by H 2 O 2 peaked earlier at 4 h of treatment. This result suggests that the induction by paraquat might be caused by the H 2 O 2 generated from superoxide. Treatment with a superoxide dismutase inhibitor, diethyldithiocarbamate, which is supposed to decrease the cellular H 2 O 2 level, reduced the paraquat induction of cytosolic APX. In contrast, when APX and catalase were inhibited by hydroxyurea or aminotriazole, cellular H 2 O 2 content was elevated and cytosolic APX mRNA was markedly increased without paraquat or H 2 O 2 treatment. This suggests that cytosolic APX is regulated by the H 2 O 2 level within cells. An increase in H 2 O 2 content was observed in the paraquat treated embryos, suggesting that paraquat induction of cytosolic APX was caused by H 2 O 2 generated through superoxide dismutation. These results indicate that H 2 O 2 is involved in oxidative stress signalling, leading to the induction of cytosolic APX.

Agrobacterium tumefaciens and Agrobacterium vitis significantly reduced the entire criteria (galls, embedded stages, final population, Pf/Pi and egg production) of the root-knot nematode, Meloidogyne incognita, when they were in concomitance with grape (Vitis vinifera var) superior roots. Greater suppression in such criteria was observed with high inoculum levels (5X10 7 cfu/pot) than with lower (2.5X10 7 cfu/pot) ones. All treatments resulted in the increase of leaves contents of H 2 O 2 and lipid peroxidation (TBARS) dramatically, which were considered the most damaging stresses in plant cells. The contents of AsA, GSH, TPH and PAL specific activity increased as a strongly antioxidant defense compound against induced oxidative damage. In addition, the increase in the activity of various antioxidant defense specific enzymes (SOD, APX, CAT and GST) represented the protective activity used to counteract the oxidative injury promoted by nematode and nematode-bacteria infections. The r...

Background: Phytoalexins are the secondary metabolites produced in crop plants after the attack of necrotizing pathogens. They are aimed to support homeostasis in stressed plants. Proteins are also part of this regime that is manipulated according to the type of infection or environmental stress. Chemical elicitors substitute the traditional fungicides and safe to use. They also modify protein levels due to their direct or indirect influence on the defense pathways. Methodology: An in vitro study was conducted on tomato seedlings inoculated with cucumber mosaic virus (CMV). Plants were treated with salicylic acid and BTH at different intervals to find out the alterations in the total soluble proteins. Purpose of study was to investigate the variation in total proteins during the viral attack and role of elicitors in this case. Results: Results indicated that protein level was initially reduced (133.3 mg) in the inoculated plants prior to the application of elicitors. However, BTH al...

Sharka, a disease caused by plum pox virus (PPV), has a significant economic impact on fruit tree production. In this work, we analysed the effect of (2,1,3)-benzothiadiazole (BTH) and L-2-oxo-4-thiazolidine-carboxylic acid (OTC) on plant growth and virus content. OTC reduced sharka symptom, stimulated plant growth and alleviated PPV-induced oxidative stress, indicated by a lack of changes in some oxidative stress parameters. PPV infection reduced chloroplast electron transport efficiency. However, in the presence of BTH or OTC, no changes in the chlorophyll fluorescence parameters were observed. PPV produced an alteration in chloroplast ultrastructure, giving rise to a decrease in starch contents that was less dramatic in OTC-treated plants. Furthermore, PPV reduced the abundance of proteins associated with photosynthesis, carbohydrate and amino acid metabolism and photorespiration. These changes did not take place in OTC-treated plants, and increases in the expression of proteins related with the aforementioned processes, including ADP-glucose pyro-phosphorylase, were produced, which correlated with the lower decrease in starch contents observed in PPV-infected plants treated with OTC. The results suggested that OTC treatment provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV-infected peaches. Thus, OTC could have practical implications in agriculture in improving the vigour of different plant species as well as in immunizing plants against pathogens.

We studied the effect of salicylic acid treatments on the response of pea plants to salinity. The NaCl-induced damage in leaves increased by salicylic acid: this response correlated with a reduction in plant growth.
The contents of reduced ascorbate and glutathione in leaves of salt-treated plants increased by effect of salicylic acid, although an accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxyde also occurred in the leaves of salt-exposed plants which were treated by salicylic acid.
In the absence of NaCl, salicylic acid increased the ascorbate peroxidase (100 µM) and glutathione-S transferase (50 µM) activities and increased catalase activity in a concentration-dependent manner. Salinity decreased glutathione reductase activity, but increased glutathione-S transferase and catalase activities. In salt-stressed plants, salicylic acid also produced changes in antioxidative enzymes: 100 µM decreased ascorbate peroxidase but increased glutathione-S transferase. Finally, a concentration-dependent increase in superoxide dismutase activity was induced by salicylic acid treatments in salt-stressed plants.
An induction of PR-1b was observed in NaCl-stressed plants treated with salicylic acid. The treatment with salicylic acid, as well as the interaction between the salinity and salicylic acid treatments, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mM NaCl, but it was statistically higher in the absence than in the presence of salicylic acid. Globally, the results show that salicylic acid treatments affected negatively the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidative metabolism. The data also show that salicylic acid treatment could enhance the resistance of salt-stressed plants to a possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.

In this work we used two different pea cultivars: JI281 is a semidomesticated land race of pea from Ethiopia whereas JI399 is a typical domesticated pea variety. Exposure of pea leaves to excess light (EL) for 1 h caused a reversible photoinhibition of photosynthesis as showed by changes in Fv/Fm. Although little difference existed between the two pea genotypes with respect to photoinhibition, after 60 min of EL the decline in Fv/Fm was higher in JI281 than in JI399 leaves. As a consequence of EL, H2O2 increased in both pea cultivars, whereas lipid peroxidation and protein oxidation slightly increased, althought differenced between cultivars were minimal. The redox state of ascorbate shifted towards its oxidized form under EL stress in both cultivars. Transcript levels of genes coding antioxidant enzymes varied upon EL in both cultivars, but the response was more pronounced in JI399. The induction observed during EL was maintained or increased after the stress period, as occurred for cytGR and chlMDHAR. GR protein accumulation and activity correlated to the transcript accumulation in JI399, but not in JI288. In this work, a possible role for H2O2 and redox status of ascorbate in the photoxidative stress signalling is discussed.

In the phytotron experiment, the effect of elevated atmospheric CO 2 (EC, 750 μmol mol -1 ) on the drought tolerance was studied in two winter varieties (Mv Mambo, tolerant; Mv Regiment, moderately tolerant) and in one spring variety of wheat (Lona, sensitive to drought). Changes in net photosynthetic rate (P N ), stomatal conductance, transpiration, wateruse efficiency, effective quantum yield of photosystem II, and activities of glutathione reductase (GR), glutathione-Stransferase (GST), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were monitored during water withdrawal. Drought caused a faster decline of P N at EC, leading to the lower assimilation rates under severe drought compared with ambient CO 2 (NC). In the sensitive variety, P N remained high for a longer period at EC. The growth at EC resulted in a more relaxed activation level of the antioxidant enzyme system in all three varieties, with very low activities of GR, GST, APX, and POD. The similar, low values were due to decreases in the varieties which had higher ambient values. A parallel increase of CAT was, however, recorded in two varieties. As the decline in P N was faster at EC under drought but there was no change in the rate of electron transport compared to NC values, a higher level of oxidative stress was induced. This triggered a more pronounced, general response in the antioxidant enzyme system at EC, leading to very high activities of APX, CAT, and GST in all three varieties. The results indicated that EC had generally favourable effects on the development and stress tolerance of plants, although bigger foliage made the plants more prone to the water loss. The relaxation of the defence mechanisms increased potentially the risk of damage due to the higher level of oxidative stress at EC under severe drought compared with NC.

Nicotiana tabacum transgenic plants overexpressing manganese (Mn) superoxide dismutase (SOD) targeted to plastids were studied upon 12.5 PM cadmium (Cd) application. No differences in tolerance were observed in comparison to wild type plants. Seed germination inhibition, seedling growth, and plant biomass were measured at different developmental stages. Cd analysis revealed the usual distribution of Cd between roots and shoots, 10-times higher in roots. Lipid peroxidation level, a characteristic parameter of oxidative stress, indicated that little protection was gained by overexpressing MnSOD. However, chlorophyll concentration was not affected upon Cd treatment in 35S:MnSOD transgenic plants, suggesting that a possible protection localised at the chloroplast could indeed occur. Three antioxidant enzymes sensitive to metal stress were also analysed after in gel activity staining: SOD, ascorbate peroxidase (APX) and glutathione reductase (GR). As expected, MnSOD activity was remarkably higher in transgenic N. tabacum plants. Interestingly, Cu/ZnSOD activity decreased upon exposure of plants to 12.5 PM Cd. GR and APX activities augmented in metal-treated plants. In particular, APX activity was slightly higher in MnSOD over-expressing plants, which was possibly related with an enhanced generation of H 2 O 2 through higher SOD activity.

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco
(Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1)
or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance
against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least
apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water
stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general,
oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H2O2 levels, were higher in
non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects
tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in
transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also
observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the
positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the
regulation network of plant antioxidant defences during drought stress.
Key words: Cytosolic ascorbate peroxidase, cytosolic superoxide dismutase, tobacco, water stress.