Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Significance of antioxidants and electron sinks for the cold-hardening-induced resistance of winter rye leaves to photo-oxidative stress

The contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold-hardened leaves (CHL) by incubating non-hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L -galactonic acid-γ-lactone and 2-oxothiazolidine-4-carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D , L -buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo-inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquat-induced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat-induced photo-inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat-induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature-induced photo-inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature-induced photo-inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non-photochemical quenching of excitation energy.     

Apoplastic antioxidative system responses to ozone stress in strawberry leaves

Cell wall polysaccharides, pectin composition, as well as apoplastic superoxide dismutase and peroxidase activities were investigated in strawberry (Fragaria × ananassa) cultivars (cvs) Korona and Elsanta differing in their ozone sensitivity. Plants were exposed to 140–170 μg m⁻³ ozone either short-term for 7 days or long-term for 2 months in order to investigate whether differences in ozone sensitivity were due to differences in the apoplastic antioxidative systems. Cell wall polysaccharides were increased after 7 days and 2 months of ozone stress. While water-soluble pectins, low methoxy pectinates and NaOH-soluble pectinates were elevated after 7 days, their contents were unaffected (water-soluble pectins) or lower (low methoxy pectinates, NaOH-soluble pectinates) after 2 months. In cv. Elsanta, ozone treatment resulted in a significant reduction of superoxide dismutase activity after 7 days and 2 months, while it remained similar in cv. Korona. After 7 days, peroxidase activity was significantly higher in ozone-exposed leaves of cv. Korona, whereas after 2 months it was similar to or higher than in controls. Superoxide dismutase in cv. Korona detoxified ozone and its products in the apoplast, and the resulting elevated levels of H₂O₂ were balanced within 7 days by an increase in peroxidase activity. Long-term peroxidase activity may not play a comparably significant role in ozone defence, but the increase in cell wall polysaccharides and cell wall thickness measured after 2 months, resulting in a decrease in specific leaf area, reflected structural modifications that limited activities of reactive oxygen species efficiently. In contrast, the reduction of superoxide dismutase activity in cv. Elsanta indicated a less efficient apoplastic radical scavenging system, at least during the first 7 days of ozone stress, which was accompanied by membrane leakage and contributed to accelerated leaf senescence. Long-term, the reduction of intercellular air space volume in leaves contributed to ozone tolerance of cv. Elsanta as in cv. Korona.     

Photosynthetic and anatomic responses of peanut leaves to cadmium stress

Cadmium (Cd) treatments caused an inhibition in the net photosynthetic rate (P _N) of peanut (Arachis hypogaea) plants, due to the reduction of stomatal conductance (g _s) and photosynthetic pigment contents, as well as the alteration in leaf structure. The decrease of the transpiration rate and g _s might result from the Cd-induced xerophyte anatomic features of leaves (i.e. thick lamina, upper epidermis, palisade mesophyll, high palisade to spongy thickness ratio, as well as abundant and small stomata). The decline of P _N was independent of the impairment in photosystem 2.     

Photosynthetic and stomatal responses of spinach leaves to salt stress

The gas exchange of spinach plants, salt-stressed by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar, was studied 3 weeks after starting NaCl treatment. Photosynthesis became light saturated at 1100 to 1400 micromoles per square meter per second in salt-treated plants and at approximately 2000 micromoles per square meter per second in control plants. Photosynthetic capacity of the mesophyll measured as a function of intercellular partial pressure of CO₂ at the light intensity prevailing during growth and at light saturation were both decreased in the salttreated plants. The CO₂ compensation points and relative enhancements of photosynthesis at low O₂ were not affected by salinity. The lower photosynthetic rates in salt-treated leaves at 450 micromoles per square meter per second were associated with a 70% reduction in stomatal conductance and low intercellular CO₂ (219 microbars; cf. 285 microbars for controls). Increasing photon flux density to light saturation extended the linear portions of the CO₂ response curves, increased stomatal conductances, increased intercellular CO₂ in the salt-treated plants, but lowered it in controls, and accentuated differences in photosynthetic rate (area basis) between the treatments.

Leaves from salt-treated plants were thicker but contained about 73% of the chlorophyll per unit area of control plants. When photosynthetic rates were expressed on a chlorophyll basis there was no difference in initial slope of assimilation versus intercellular CO₂ between treatments. Photosynthetic rates (chlorophyll basis) at light saturation differed only by 20% which was also observed earlier with isolated, intact chloroplasts (Robinson et al. 1983 Plant Physiol 73: 238-242).

Measurement of carbon isotope ratio revealed less discrimination against ¹³C with salt treatment and confirmed the persistence of low intercellular partial pressures of CO₂ during plant growth. The development of a thicker leaf with less chlorophyll per unit area during salt treatment permitted stomatal conductance and intercellular partial pressure of CO₂ to decline without restricting photosynthesis and had the benefit of greatly increasing water use efficiency.

     

Photosynthetic and anatomic responses of peanut leaves to zinc stress

In this study, photosynthetic performance, pigment content, chlorophyll a fluorescence, and leaf anatomy in peanut (Arachis hypogaea) subjected to zinc (Zn) stress were investigated. Zn stress resulted in reduction of photosynthetic and transpiration rates, pigment contents and root biomass. Zn-induced xerophyte structure in peanut leaves (i.e. thick lamina, upper epidermis, and palisade mesophyll, as well as abundant and small stomata) also contributed to decreased transpiration rate and stomatal conductance. This in turn, partially contributed to the limitation of photosynthesis.     

Singlet oxygen and photo-oxidative stress management in plants and algae

Photosynthetic organisms constantly face the threat of photo-oxidative stress from fluctuating light conditions and environmental stress. Plants and algae have developed an array of defences to protect the chloroplast from reactive oxygen species. Genetic and physiological studies have shown that antioxidant responses are important to high-light acclimation, both by directly scavenging or quenching reactive oxygen intermediates and by contributing reducing power for alternative electron transport pathways and excess energy dissipation. At present, the signalling events leading to up-regulation of antioxidant defences in high light remain a mystery. Recent advances toward understanding acclimation to oxidative stress in both photosynthetic and non-photosynthetic model organisms may illuminate how plants and algae respond to high-light stress. Although the role of hydrogen peroxide in high-light acclimation has been investigated, less is known about responses to singlet oxygen, a form of reactive oxygen that poses a significant threat specifically to photosynthetic organisms. This review will discuss some intriguing new findings in that area, focusing on recent findings regarding the nature of singlet-oxygen responses in the chloroplast.     

Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens‐mediated transformation. Catalase activity in progeny from transgenic plants was approximately three‐fold higher than that in wild‐type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild‐type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild‐type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H₂O₂, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild‐type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m^?2 s^?1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild‐type plants. The catalase activity and the photosynthesis activity (indicated by the F_v/F_m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO₂ fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m^?2 s^?1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo‐oxidative stress invoked by paraquat treatment, drought stress and chilling stress.     

Cinnamoyl-based Nrf2-activators targeting human skin cell photo-oxidative stress

Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress.     

Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress

The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO₂ exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700 absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

Imaging,screening and remote sensing of photosynthetic activity and stress responses

Journal of Plant Research -     

Nitric oxide interferes with plant photo-oxidative stress by detoxifying reactive oxygen species

Oxidative stress within chloroplasts is originated due to light‐dependent O₂ reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H₂O₂ and then, by the Fenton reaction, H₂O₂ may be reduced to the hydroxyl radical (OH^?). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm ) and S‐nitroso‐N‐acetylpenicillamine (200 µm ), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.     

Antioxidant system and photosynthetic characteristics responses to short-term PEG-induced drought stress in cucumber seedling leaves

The effect of short-term drought stress on the water content, antioxidant system and photosynthetic characteristics was investigated using cucumber (Cucumis sativus L.) seedlings. The results indicated that polyethylene glycol induced water stress reduced water content in shoots of cucumber seedling after treatment of 36 hours, and caused obvious reductions in net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration of leaves. In addition, water stress significantly reduced the photosynthetic pigment content and inhibited photochemical activity, including actual photochemical efficiency, maximal quantum yield of photosystem II photochemistry and coefficient for photochemical quenching. Meanwhile non-photochemical quenching increased. As responses to drought stress, significant increases in electrolyte leakage, malondialdehyde, superoxide anion and hydrogen peroxide levels were detected in leaves. The superoxide dismutases, catalase, glutathione reductase and dehydroascorbate reductase activities, protein, ascorbate and glutathione content, all decreased and peroxidases activity increased, while ascorbate peroxidase and monodehydroascorbate reductase activities exhibited different trend under different degree of water stress. Therefore, it can be concluded that water stress strongly disrupted the normal metabolism of leaves and restrained water absorption.     

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO₄ (10, 100, 200 μm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 μm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

Imaging photosynthesis in wounded leaves of Arabidopsis thaliana

Chlorophyll fluorescence imaging provides a non-invasive and non-destructive means with which to measure photosynthesis. This technique has been used, in combination with 14CO2 feeding, to study the spatial and temporal changes in source-sink relationships which occur in mechanically wounded leaves of Arabidopsis thaliana. Twenty-four hours after wounding, cells proximal to the wound margin showed a rapid induction of PhiII upon illumination (a measure of the efficiency of photosystem II photochemistry) whilst cells more distal to the wound margin exhibited a much slower induction of PhiII and a large, transient increase in NPQ (a measure of the rate constant for non-photochemical energy dissipation within the light-harvesting antenna). These results are indicative of an increase in sink strength in the vicinity of the wound and this was confirmed by the retention of 14C photosynthate in this region. It has been hypothesized that wound-induced cell wall (apoplastic) invertase (cwINV) activity plays a central role in generating localized increases in sink strength in stressed plant tissue and that hexose sugars generated by the sucrolytic activity of cwINV may act as a signal regulating gene expression. Enzyme activity measurements, quantitative RT-PCR, and T-DNA insertional mutagenesis have been used to determine that expression of AtcwINV1 is responsible for all induced cwINV activity in mechanically wounded leaves. Whilst inactivation of this gene abolished wound-induced cwINV activity, it did not affect localized alterations in source-sink relationships of wounded leaves or wound-regulated gene expression. The signals that may regulate source-sink relationships and signalling in wounded leaves are discussed.