Zbigniew Miszalski

Lesion simulating disease 1 is required for acclimation to conditions that promote excess excitation energy

The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H2O2. A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H2O2 accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.

Arabidopsis Chloroplastic Glutathione Peroxidases Play a Role in Cross Talk between Photooxidative Stress and Immune Responses

Glutathione peroxidases (GPXs; EC 1.11.1.9) are key enzymes of the antioxidant network in plants and animals. In order to investigate the role of antioxidant systems in plant chloroplasts, we generated Arabidopsis (Arabidopsis thaliana) transgenic lines that are depleted specifically in chloroplastic (cp) forms of GPX1 and GPX7. We show that reduced cpGPX expression, either in transgenic lines with lower total cpGPX expression (GPX1 and GPX7) or in a gpx7 insertion mutant, leads to compromised photooxidative stress tolerance but increased basal resistance to virulent bacteria. Depletion of both GPX1 and GPX7 expression also caused alterations in leaf cell and chloroplast morphology. Leaf tissues were characterized by shorter and more rounded palisade cells, irregular spongy mesophyll cells, and larger intercellular air spaces compared with the wild type. Chloroplasts had larger and more abundant starch grains than in wild-type and gpx7 mutant plants. Constitutively reduced cpGPX expression also led to higher foliar ascorbic acid, glutathione, and salicylic acid levels in plants exposed to higher light intensities. Our results suggest partially overlapping functions of GPX1 and GPX7. The data further point to specific changes in the chloroplast ascorbate-glutathione cycle due to reduced cpGPX expression, initiating reactive oxygen species and salicylic acid pathways that affect leaf development, light acclimation, basal defense, and cell death programs. Thus, cpGPXs regulate cellular photooxidative tolerance and immune responses.

Redox control of oxidative stress responses in the C3-CAM intermediate plant Mesembryanthemum crystallinum

Abstract Crassulacean acid metabolism (CAM) is named after the Crassulaceae family of succulent plants, in which this type of metabolism was first discovered at the beginning of the 19th century. In recent years, Mesembryanthemum crystallinum, a facultative halophyte and C3–CAM intermediate plant, has become a favoured plant for studying stress response mechanisms during C3–CAM shifts. Recent studies in this and related areas can provide a new model of how such mechanisms could operate for acclimation to high salinity or excess excitation energy. These include roles for photosynthetic electron transport chain components and reactive oxygen species. The diurnal rhythms of catalase, superoxide dismutase and some CAM-related enzyme activities are discussed in relation to the protective role of photorespiration during C3–CAM transition. The role of excess excitation energy and redox events in the proximity of photosystem II (PSII) in regulation of ascorbate peroxidase (APX), superoxide dismutase (SOD): copper/zinc SOD (Cu/ZnSOD), iron SOD (FeSOD), and NAD(P)-malic enzyme gene expression are also discussed. We suggest a model in which the chloroplast plays a major role in regulation of acclimation to high salinity and/or excess exitation energy.

Spatial patterns of senescence and development-dependent distribution of reactive oxygen species in tobacco (Nicotiana tabacum) leaves

Senescence of tobacco leaves is distributed non-uniformly over a leaf blade. While photosynthetic competence and expression of photosynthesis-associated genes decline in interveinal areas of the leaf lamina with advancing age of the leaf, they are maintained at high levels in the tissue surrounding the veins. In contrast, expression of senescence-associated genes (SAG) was enhanced in both areas of the leaf blade. Accumulation of hydrogen peroxide was shown to precede the phase of senescence initiation in the veinal tissue. In the interveinal tissue, the level of hydrogen peroxide was increased with senescence progression and paralleled by an increase in the level of superoxide anions. It is hypothesized that the spatial differences in superoxide anions are important for the non-uniform down-regulation of photosynthesis-associated genes (PAG), while hydrogen peroxide is responsible for up-regulation of SAG.

Catalase Activity During C3-CAM Transition in Mesembryanthemum crystallinum L. Leaves

Treatment with 0.4 mol dm(-3) NaCl caused a C3-CAM shift in Mesembryanthemum crystallinum L. leaves. In parallel to the CAM induction the activity of CAT was significantly decreased. In C3 and in CAM plants CAT activity showed daily fluctuations, with the maximum at the end of the light period. The oscillations of CAT were more pronounced in CAM than in C3 plants. In M. crystallinum CAT activity seems to respond more to CAM induction than to salinity.

Mycorrhizal fungi modulate phytochemical production and antioxidant activity of Cichorium intybus L. (Asteraceae) under metal toxicity.

Cichorium intybus (common chicory), a perennial plant, common in anthropogenic sites, has been the object of a multitude of studies in recent years due to its high content of antioxidants utilized in pharmacy and food industry. Here, the role of arbuscular mycorrhizal fungi (AMF) in the biosynthesis of plant secondary metabolites and the activity of enzymatic antioxidants under toxic metal stress was studied. Plants inoculated with Rhizophagus irregularis and non-inoculated were grown on non-polluted and toxic metal enriched substrata. The results presented here indicate that AMF improves chicory fitness. Fresh and dry weight was found to be severely affected by the fungi and heavy metals. The concentration of hydroxycinnamates was increased in the shoots of mycorrhizal plants cultivated on non-polluted substrata, but no differences were found in plants cultivated on metal enriched substrata. The activity of SOD and H2O2 removing enzymes CAT and POX was elevated in the shoots of mycorrhizal plants regardless of the cultivation environment. Photochemical efficiency of inoculated chicory was significantly improved. Our results indicate that R. irregularis inoculation had a beneficial role in sustaining the plants ability to cope with the deleterious effects of metal toxicity.

Superoxide dismutase-like protein from roots of the intermediate C3-CAM plant Mesembryanthemum crystallinum L. in in vitro culture

The facultative halophyte Mesembryanthemum crystallinum can shift its mode of carbon assimilation from C3-pathway to CAM (crassulacean acid metabolism) in response to salinity. The effect of salt-induced stress on antioxidative enzyme superoxide dismutase (SOD) in leaves of M. crystallinum was studied previously in in vivo conditions ‘Plant Cell Environ. 21 (1998) 169’. We have demonstrated that NaCl-induced C3-CAM transition takes place in plants cultured in vitro. The same SOD forms were identified in leaves and roots of plants cultured in vitro. In M. crystallinum roots, additionally to ‘leaf form’ of MnSOD (MnSOD I) another MnSOD-like form (MnSOD II) was detected. The estimated native molecular mass of MnSOD II is in range of 186–201 kDa (native gradient electrophoresis) and 93 kDa (gel filtration). The subunit mass is estimated to be about 57 kDa. The new SOD-like form exhibits a high thermal stability and resistance to a reducing factor, mercaptoethanol. It is suggested that MnSOD II form can exist as active aggregates. In salt-induced CAM mode of photosynthesis the activity of MnSOD I and Cu/ZnSOD increases significantly, while the activity of the new MnSOD-like protein does not change. The potential physiological role of the MnSOD II is discussed.

The fungal endophyte Epichloë typhina improves photosynthesis efficiency of its host orchard grass (Dactylis glomerata)

Main conclusionAccording to the results presented in this paper the fungal endophyteEpichloë typhinasignificantly improves the growth, PSII photochemistry and C assimilation efficiency of its hostDactylis glomerata.In this paper, we present a comprehensive study of the impact of the endophytic fungi Epichloë typhina on its plant hosts’ photosynthesis apparatus. Chlorophyll a fluorescence, gas exchange, immuno-blotting and spectrophotometric measurements were employed to assess photosynthetic performance, changes in pigment content and mechanisms associated with light harvesting, carbon assimilation and energy distribution in Dactylis glomerata colonized with Epichloë typhina. According to the results presented in this study, colonization of D. glomerata results in improved photosynthesis efficiency. Additionally, we propose a new mechanism allowing plants to cope with the withdrawal of a significant fraction of its energy resources by the endophytic fungi. The abundance of LHCI, LHCII proteins as well as chlorophyll b was significantly higher in E+ plants. Malate export out of the chloroplast was shown to be increased in colonized plants. To our knowledge, we are the first to report this phenomenon. Epichloë colonization improved PSII photochemistry and C assimilation efficiency. Elevated energy demands of E+ D. glomerata plants are met by increasing the rate of carbon assimilation and PSII photochemistry.

Adaptation of the obligate CAM plant Clusia alata to light stress: Metabolic responses

In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO(2) and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200micromol m(-2)s(-1)) to high light (650-740micromol m(-2)s(-1)), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.

EPR spectroscopy as a tool for investigation of differences in radical status in wheat plants of various tolerances to osmotic stress induced by NaCl and PEG-treatment.

Two kinds of wheat genotypes with different tolerance to osmotic stress (NaCl and PEG-treatment) were investigated with biochemical analyses, including the measurements of total antioxidant capacity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power and starch content. The results were compared with electron paramagnetic resonance (EPR) data concerning the nature and amounts of stable long lived radicals present in the control and stressed plants. In addition, the changes in manganese content upon stress conditions were monitored. Different mechanisms of protection against PEG stress in sensitive and tolerant wheat genotypes were postulated. In sensitive genotypes, electrons were created in excess in stress conditions, and were stabilized by polysaccharide molecules, whereas in tolerant genotypes, protection by antioxidants dominated. Moreover, the quinone-semiquinone balance shifted towards semiquinone, which became the place of electron trapping. NaCl-treatment yielded significant effects mainly in sensitive genotypes and was connected with the changes of water structure, leading to inactivation of reactive oxygen species by water molecules.