Age-related changes in oxidative stress markers and abscisic acid levels in a drought-tolerant shrub, Cistus clusii grown under Mediterranean field conditions

Compared with our knowledge of senescence in annuals and biennials, little is known about age-related changes in perennials. To get new insights into the mechanisms underlying aging in perennials, we measured oxidative stress markers in leaves and organelles, together with abscisic acid levels in leaves of 2- and 7-year-old Cistus clusii dunal plants grown under Mediterranean field conditions. Recently emerged leaves, which either appeared during autumn or spring, were compared to evaluate the effects of environmental constraints on oxidative stress and abscisic acid accumulation as plants aged. Plant aging led to an enhanced oxidation of α-tocopherol and ascorbate, increased lipid peroxidation and reduced PSII efficiency in leaves during the more stressful conditions of spring and summer, but not during autumn. Analyses of lipid peroxidation in organelles isolated from the same leaves revealed that oxidative stress occurred both in chloroplasts and mitochondria. Although both plant groups showed similar leaf water and nitrogen contents throughout the study, abscisic acid levels were markedly higher (up to 75%) in 7-year-old plants compared to 2-year-old plants throughout the study. It is concluded that (a) meristematic tissues of C. clusii maintain the capacity to make new leaves with no symptoms of oxidative stress for several years, unless these leaves are exposed to environmental constraints, (b) leaves of oldest plants show higher oxidative stress than those of young plants when exposed to adverse climatic conditions, thus supporting the idea that the oxidative stress associated with aging is due at least partly to extrinsic factors, (c) at the subcellular level, age-induced oxidative stress occurs both in chloroplasts and mitochondria, and (d) even in the absence of environmental stress, newly emerged leaves accumulate higher amounts of ABA as plants age.     

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.     

Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus

In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13-15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

砂仁不同叶位叶片的光合作用和氧化胁迫

衰老时砂仁叶片Pmax降低,这与叶片Gs、Chi含量和可溶性蛋白质含量的降低有关.随着叶片的衰老,NPQ、AQY、F/Fm、φPsIl和qp均降低,热耗散减少,光抑制加剧,衰老后期出现光破坏.但这些参数下降的幅度均小于Pmax下降幅度.光暗反应失衡,活性氧生成增加.衰老初期(老化)叶片MDA含量没有升高,衰老中后期叶片MDA含量显著升高,表明老化叶片能有效地耗散或清除活性氧,衰老叶片则不能,尽管其sOD、APX和POD等抗氧化酶活力显著升高.上述结果表明砂仁叶片老化与氧化胁迫关系不大,衰老与氧化胁迫密切相关.     

Nitric oxide protects against cellular damage produced by methylviologen herbicides in potato plants.

Methylviologen compounds are normally used in agronomy as herbicides. They cause an overproduction of reactive oxygen species (ROS) within chloroplasts, subjecting the plant to a severe oxidative stress. Since nitric oxide (NO) is a bioactive ROS scavenger, we analyzed its effect over some toxic processes caused by the methylviologens diquat and paraquat in potato leaves (Solanum tuberosum L. cv. Pampeana). Three NO donors, (i) sodium nitroprusside (SNP), (ii) S-nitroso-N-acetylpenicillamine, and (iii) a mixed solution of ascorbic acid and NaNO2, were able to prevent chlorophyll loss. Residual products from NO generation and decomposition failed to prevent chlorophyll decline and a specific NO scavenger, carboxy-PTIO, arrested NO-mediated chlorophyll protection. Dichlorophenyldimethylurea, an inhibitor of chloroplastic electron transport, mimicked NO-mediated chlorophyll protection. During oxidative stress, cell ion leakage to intercellular compartments occurs as an early step, leading to a special kind of programmed cell death. NO proved to specifically decrease the extent of ion leakage originated by diquat, since the protection originated by 100 microM SNP was completely arrested by carboxy-PTIO. These results suggest that NO can strongly protect plants from methylviologen damage and strengthen the evidence in favor of NO as a potent antioxidant in some situations.     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.     

Tocopherol content and enzymatic antioxidant activities in chloroplasts from NaCl-stressed tomato plants

Changes in tocopherol, chlorophyll and TBARS levels and the activities of antioxidant enzymes i.e., GSH-Px, GST, and SOD in chloroplasts of tomato plants subjected to moderate (50 mM) and severe (150 mM) NaCl stress were determined. Increase in tocopherol content around the second day under both stresses did not correlate with the chlorophyll degradation while such correlation was observed from the fifth day of severe stress. The activities of GSH-Px and GST as well as TBARS content showed NaCl-induced enhancement which was dose- and time-dependent. However, chloroplastic SOD was rather not involved in the response of tomato plants to NaCl stress. The obtained results suggest that under the moderate stress similarly as in the early phase of severe stress tocopherol functions as a typical antioxidant, while in the late phase of the latter it may be involved in senescence signaling pathway and enables the recovery and recycling of the compounds significant for a plant organism.     

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second‐stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post‐inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down‐regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter R_fd, defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non‐photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine‐tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.     

Cadmium-induced changes in the growth and oxidative metabolism of pea plants

The effect of growing pea (Pisum sativum L.) plants with CdCl(2) (0-50 microM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 microM CdCl(2), showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl(2) can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.     

Regulation and function of ascorbate peroxidase isoenzymes

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

Exogenous nitric oxide promotes waterlogging tolerance as related to the activities of antioxidant enzymes in cucumber seedlings

We investigated the effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on growth of cucumber (Cucumis sativus L., cv. Jinyou No.1) seedlings and antioxidant enzyme activities in cucumber leaves under waterlogging stress. The growth of cucumber seedlings was significantly inhibited when plants were exposed to waterlogging, whereas shoot spraying with SNP significantly alleviated the inhibition of growth from this type of stress: height, fresh and dry weights of the flooded plants increased obviously. Waterlogging also caused the activation of the antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)), the reduction of the chlorophyll content, and the accumulation of MDA and protein in leaves. It was found that SNP treatment further potentiated the antioxidant enzyme activities and maintained the chlorophyll and protein content during the entire water-logging period; however, it reduced the MDA content. Thus, NO protects plants from oxidative damage and promotes growth by activation of antioxidant enzymes in leaves in an extent sufficient for the alleviation of membrane injury. However, exogenous NO had no significant effects on cucumber seedlings growth and antioxidant enzyme activities under nonstress conditions.     

PHYSIOLOGICAL EFFECTS OF MANGANESE DEFICIENCY RELATED TO AGE IN SOYBEANS (GLYCINE MAX)

Cooper , Eugene E., and Raymond E. Girton . (Purdue U., Lafayette, Ind.) Physiological effects of manganese deficiency related to age in soybeans (Glycine max). Amer. Jour. Bot. 50(2): 105–110. Illus. 1963.—Soybean plants when grown in manganese-deficient silica sand cultures developed typical manganese deficiency symptoms of interveinal chlorosis and necrosis. Physiological effects including depression of photosynthesis, respiration, growth, and relative chlorophyll contents were studied. The depression of photosynthesis was not always proportional to reduced chlorophyll content. This is taken to indicate the importance of manganese in reactions concerned in photosynthesis in addition to chlorophyll formation. Age of leaves related to position on the plant and actual aging of the plants with time sometimes produced different results when related to photosynthetic rates, which mainly decreased with age of plants. Chlorophyll content in young leaves increased with plant age, except for a consistent decrease after leaf maturity. Respiration rates generally decreased with age. For the most part, the effects of aging on photosynthesis, respiration, and chlorophyll contents were the same for soybeans as for other species reported in the literature.     

Physiological behaviour, oxidative damage and antioxidative protection of olive trees grown under different irrigation regimes

Irrigation effects were investigated on an 8-year-old olive (Olea europaea L., cv. Cobrançosa) commercial orchard located in northeast Portugal. Trees were subjected to a rainfed control (T0) and three treatments (T1, T2, T3) that received a seasonal water amount equivalent to 30%, 60% and 100% of the estimated local evaporative demand by a drip irrigation system. Irrigation increases the photosynthetic activity of olive trees, in association with increases in water status, and reduces the midday and afternoon depression in gas exchange. The closely association between photosynthetic rate (A) and stomatal conductance (g _s) revealed that the decline in net photosynthesis over the course of the day was largely a consequence of stomatal limitation. However, the ratio of intercellular to atmospheric CO₂ concentration increased markedly from morning to midday in non-irrigated plants, in spite of lower g _s, suggesting that non-stomatal limitations of photosynthesis also occur when environmental conditions become more stressful. The occurrence of perturbations at chloroplastic level in rainfed plants was demonstrated by a lower maximum photochemical efficiency of photosystem II during the afternoon. Chlorophyll fluorescence measurements also revealed the occurrence of a dynamic photoinhibition in irrigated trees, mainly in T2 and T3, which seemed to be effective in protecting the photosynthetic apparatus from photodamage. Irrigation enhances antioxidant protection and decreases the oxidative damage at leaf level. Leaves grown under rainfed conditions revealed symptoms of oxidative stress, like the reduction (14%) in chlorophyll concentration and the increased levels (57%) of lipid peroxidation. We also found that the scavenging function of superoxide dismutase was impaired in rainfed plants. In contrast, the low thiobarbituric acid reactive substances concentration in T3 indicates that irrigation enhances the repairing mechanisms and decreases the oxidative damage by lipid peroxidation. Accordingly, leaves in T3 treatment had high levels of –SH compounds and the highest antioxidant potential. Meanwhile, the finding that guaiacol peroxidase activity increased in rainfed plants, associated with the appearance of oxidative damage, suggests that this enzyme has no major antioxidative function in olive.     

Antioxidant enzymes and physiological characteristics in two Jerusalem artichoke cultivars under salt stress

The effects of NaCl stress on the activity of antioxidant enzymes, lipid peroxidation, cell membrane stability, net photosynthetic rate, gas-exchange, and chlorophyll content were investigated in two Jerusalem artichoke cultivars, Dafeng (salt-tolerant) and Wuxi (salt-sensitive), grown under control (nutrient solution) or salt stress (nutrient solution containing 75, 150, and 225 mM NaCl) conditions for 7 days. In leaves of salt-tolerant cv. Dafeng, superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), and catalase (EC 1.11.1.6) activities significantly increased as compared to the controls, whereas no significant change was observed in cv. Wuxi. Lipid peroxidation and cell membrane injury were enhanced in both cultivars. Net photosynthesis and stomatal conductance decreased in response to salt stress, but cv. Dafeng showed a smaller reduction in photosynthesis than cv. Wuxi. The results indicated that stomatal aperture limited leaf photosynthetic capacity in the NaCl-treated plants of both cultivars. However, significant reduction in the leaf chlorophyll content due to NaCl stress was observed only in cv. Wuxi. These results suggested that salt-tolerant Jerusalem artichoke varieties may have a better protection against reactive oxygen species, at least in part, by increasing the activity of antioxidant enzymes under salt stress.     

Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.     

Salt-induced oxidative stress in rosemary plants: Damage or protection?

Mechanisms of photoprotection and antioxidant protection, including changes in chlorophylls, xanthophyll cycle components and levels of low-molecular-weight chloroplastic antioxidants (lutein, β-carotene and α-tocopherol) were studied together with levels of malondialdehyde, a product of lipid peroxidation, in the response of rosemary (Rosmarinus officinalis L.) plants to salt stress. Plants were exposed to increasing NaCl concentrations (50, 100 and 150 mM) for 6 weeks, and two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl₃, were used together with 100 mM NaCl to explore the extent to which these salts can alter the mechanisms of photoprotection, antioxidant protection and malondialdehyde accumulation in leaves. Increasing concentrations of NaCl decreased leaf water contents and photosynthetic pigment levels, while the contents of α-tocopherol and malondialdehyde increased, but with completely different kinetics. α-Tocopherol levels increased in a dose-dependent manner as stress progressed, while malondialdehyde levels increased at the highest dose (150 mM NaCl) but only during early phases of stress. Furthermore, although the addition of chloride salts to NaCl-treated plants apparently improved leaf physiological status, in terms of water and chlorophyll contents, plants showed an increased photoprotective demand and increased oxidative stress, particularly in FeCl₃-treated plants. It is concluded that (i) rosemary plants can withstand moderate doses of NaCl in the medium (at least 150 mM NaCl for 6 weeks), (ii) oxidative stress may be a mechanism for protecting plants from moderate doses of salt stress rather than causing damage to plants, and (iii) the addition of chloride salts to NaCl-treated plants may dramatically increase the photoprotective demand and oxidative stress of leaves, while plant growth is not negatively affected.