Ireneusz Ślesak

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.

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.

Isochorismate synthase 1 is required for thylakoid organization, optimal plastoquinone redox status, and state transitions in Arabidopsis thaliana

Isochorismate synthase 1 (ICS1) is a crucial enzyme in the salicylic acid (SA) synthesis pathway, and thus it is important for immune defences. The ics1 mutant is used in experiments on plant–pathogen interactions, and ICS1 is required for the appropriate hypersensitive disease defence response. However, ICS1 also takes part in the synthesis of phylloquinone, which is incorporated into photosystem I and is an important component of photosynthetic electron transport in plants. Therefore, photosynthetic and molecular analysis of the ics1 mutant in comparison with wild-type and SA-degrading transgenic NahG Arabidopsis thaliana plants was performed. Photosynthetic parameters in the ics1 mutant, when compared with the wild type, were changed in a manner observed previously for state transition-impaired plants (STN7 kinase recessive mutant, stn7). In contrast to stn7, deregulation of the redox status of the plastoquinone pool (measured as 1–q p) in ics1 showed significant variation depending on the leaf age. SA-degrading transgenic NahG plants targeted to the cytoplasm or chloroplasts displayed normal (wild-type-like) state transition. However, ics1 plants treated with a phylloquinone precursor displayed symptoms of phenotypic reversion towards the wild type. ics1 also showed altered thylakoid structure with an increased number of stacked thylakoids per granum which indicates the role of ICS1 in regulation of state transition. The results presented here suggest the role of ICS1 in integration of the chloroplast ultrastructure, the redox status of the plastoquinone pool, and organization of the photosystems, which all are important for optimal immune defence and light acclimatory responses.

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.

Oxygen and Hydrogen Peroxide in the Early Evolution of Life on Earth: In silico Comparative Analysis of Biochemical Pathways

In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O(2), reactive oxygen species (ROS), among them hydrogen peroxide (H(2)O(2)), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O(2) appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O(2) and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O(2)-and H(2)O(2)-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O(2)-and H(2)O(2)-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H(2)O(2) and O(2) should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O(2)/H(2)O(2) was a key adaptation of LUCA to already existing weakly oxic zones in Earths primordial environment.

Role of phytochromes A and B in the regulation of cell death and acclimatory responses to UV stress in Arabidopsis thaliana

Highlight Phytochromes A and B are complex regulators of photosynthesis, reactive oxygen species and salicylic acid homeostasis, and UV-C-induced programmed cell death in Arabidopsis thaliana.

Superoxide dismutase activity in C3 and C3/CAM intermediate species of Clusia

The C3-CAM intermediate Clusia minor L. and the C3 obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 µmol m−2 s−1 for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.

Malate accumulation in different organs of Mesembryanthemum crystallinum L. following age-dependent or salinity-triggered CAM metabolism

Different organs of Mesembryanthemum crystallinum exhibit differing levels of CAM (Crassulacean acid metabolism), identifiable by quantification of nocturnal malate accumulation. Shoots and also basal parts of young leaves were observed to accumulate high concentrations of malate. It was typically found in mature leaves and especially prominent in plants subjected to salt stress. Small amount of nocturnal malate accumulation was found in roots of M. crystallinum plants following age-dependent or salinity-triggered CAM. This is an indication that malate can be also stored in non-photosynthetic tissue. Measurements of catalase activity did not produce evidence of the correlation between activity of this enzyme and the level of malate accumulation in different organs of M. crystallinum although catalase activity also appeared to be dependent on the photoperiod. In all material collected at dusk catalase activity was greater than it was observed in the organs harvested at dawn.

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b6f complex is active in plastoquinone reduction: Implications for cyclic electron transport

In this study, we have compared three isolation methods of cytochrome b₆f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b₆f-associated ferredoxin:NADP+ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH-PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b₆f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2-n-nonyl-4-hydroxyquinoline N-oxide, an inhibitor of the cytochrome b₆ f complex at the Q(i) site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c(i) . The obtained data indicate that FNR associated with the cytochrome b₆ f complex can participate in the cyclic electron transport as PSI-PQ or NADPH-PQ oxidoreductase. Moreover, we have shown that PQ can be non-enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non-photochemical reduction pathways of the PQ-pool in chloroplasts.

Ozone fumigation results in accelerated growth and persistent changes in the antioxidant system of Brassica oleracea L. var. capitata f. alba.

The growth response and antioxidant capacity of Brassica oleracea var. capitata f. alba plants treated with 70ppb of ozone was examined. Four week old cabbage seedlings were fumigated with O3 for 3 days before being transplanted into the growing field. The effect of O3 treatment was determined directly after fumigation and over the course of field cultivation. Plants subjected to O3 treatment had an increased diameter of rosettes and number of leaves after 3 and 7 weeks in agriculture, respectively. In addition, the vast majority of fumigated plants reached marketable quality faster than control plants, indicating a positive role of episodes of increased O3 concentrations during vegetation on growth and yielding. Our analysis revealed that by fumigating juvenile white cabbage plants with moderate doses of O3 the activity of catalases (CAT) and peroxidases was elevated. The activity of the examined enzymes was not affected directly after fumigation, but it increased after several weeks in the experimental field. Increased CAT activity was accompanied by changes in 2 out of the 3 CAT genes CAT1 and CAT2, where CAT2 seemed to be responsible for the induced CAT activity. The biosynthesis of low-molecular stress protectants - tocopherols and the glucosinolate (GLS) sinigrin was transiently affected by ozone. γ-Tocopherol (γ-toc) content significantly increased directly after fumigation, but after 3 weeks of vegetation in the field its concentration reached values similar to control. The biosynthesis of α-tocopherol (α-toc) and sinigrin seemed to be upregulated in fumigated plants. However, the response was delayed; no differences were registered directly after treatment, but 3 weeks after transplanting the concentration of sinigrin and α-toc was elevated.