Elżbieta Kuźniak

Ascorbate, glutathione and related enzymes in chloroplasts of tomato leaves infected by Botrytis cinerea

The effect of Botrytis cinerea infection on the total pool sizes of ascorbate, glutathione and their redox status, the activities of ascorbate-glutathione cycle related enzymes, APX, DHAR, GR as well as GST and GSH-Px activities in chloroplasts isolated from tomato leaves were studied. An accelerated chlorophyll loss in chloroplasts from infected leaves was observed. The AA content was not changed considerably but a burst in DHA formation, up to 319% of control, and a significant prooxidative shift in the ascorbate redox status were found in chloroplasts from infected leaves. A massive progressive decrease in GSH and total glutathione contents as well as GR activity increase were found after infection. However, similar but less-pronounced glutathione-related changes were observed in control suggesting that they could be at least partly the result of B. cinerea induced promotion of senescence and not a specific response to pathogen infection. As shown by the analysis of ascorbate-glutathione cycle related enzyme activities as well as correlations between the two antioxidants and enzymes responsible for keeping them in the reduced state GSH is the limiting factor for the whole cycle operation in chloroplasts. This is in accordance with our previous results for the whole-cell extracts.

The effect of Botrytis cinerea infection on ascorbate-glutathione cycle in tomato leaves

Changes in the total pool sizes of ascorbate and glutathione and their degree of oxidation as well as the activities of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase and glutathione transferase in tomato leaves after Botrytis cinerea infection have been studied. In infected leaves the concentration of reduced ascorbate was similar to that in control. A transient increase in dehydroascorbate content (55% above the control) and a decrease in the ascorbate redox status were observed only 3 days after inoculation. In the diseased leaves we found a significant progressive decrease, up to ∼50% 5 days after inoculation, in the reduced glutathione content while the oxidized glutathione concentration remained unaffected. Although a decline in reduced glutathione content was detected the ascorbate and glutathione redox ratios were maintained high up to the 5th day after inoculation. B. cinerea infection enhanced ascorbate peroxidase and glutathione reductase activities. Dehydroascorbate reductase activity was significantly decreased when compared with control, but its activity level in the inoculated leaves stayed rather constant. Total glutathione transferase activity remained unchanged. In the diseased tissues statistically significant (P<0.05) linear correlations between glutathione reductase and ascorbate peroxidase activities were found to occur 4 and 5 days after inoculation and ascorbate peroxidase activity was correlated with the total glutathione level and reduced glutathione content on the 4th day. The decreased reduced glutathione content found in the infected leaves could be a limiting factor for operation of the ascorbate-glutathione cycle related enzymes at the advanced stages of infection development.

Determination of cysteine and glutathione in cucumber leaves by HPLC with UV detection

Cysteine (Cys) and glutathione (GSH) have been identified as key players in redox-based defense and signaling under stress in plants. Here, we describe a new high-performance liquid chromatography assay for the determination of four different forms of Cys and GSH, based on reduction of disulfide bonds with tris(2-carboxyethyl)phosphine followed by derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate and UV detection. The presented method allows facile determination of total, reduced, free and protein bound aminothiols in cucumber leaves. Via this simple yet efficient method very good precision as well as accuracy were obtained. Linearity in detector response was observed over the range of 5–100 μmol L−1 for GSH and 1–20 μmol L−1 for Cys. The LOQs for GSH and Cys were 5 μmol L−1 and 1 μmol L−1 homogenate of cucumber leaf, respectively.

Involvement of salicylic acid, glutathione and protein S-thiolation in plant cell death-mediated defence response of Mesembryanthemum crystallinum against Botrytis cinerea

The response of Mesembryanthemum crystallinum plants performing C3 photosynthesis and crassulacean acid metabolism (CAM) to the non-host necrotrophic pathogen Botrytis cinerea was analyzed at the local and systemic levels. The induction of programmed cell death, lignin and callose deposition, changes in salicylic acid, glutathione and cysteinylglycine pools as well as the content of thiolated proteins were studied. The infected C3 and CAM plants exhibited hypersensitive-like defence response, however fluorescence staining with acridine orange and ethidium bromide revealed programmed cell death events in C3 plants only. The local immune response was not related to callose and lignin deposition. In the infected plants, salicylic acid, glutathione and cysteinylglycine, the first product of glutathione catabolism, as well as protein S-thiolation, predominantly S-glutathionylation, contributed to local defence at sites of inoculation. They (except protein thiolation) were also active in the establishment of systemic acclimation response monitored in the non-treated upper leaves. The extent to which they were involved in the local and systemic responses induced by B. cinerea differed in C3 and CAM plants. The accumulation of free salicylic acid, both in treated and upper leaves of the infected plants, was much more pronounced in CAM plants. The results have been discussed with respect to redox regulations in defence against necrotrophic pathogens and to stress acclimation.

Effects of Botrytis cinerea and Pseudomonas syringae infection on the antioxidant profile of Mesembryanthemum crystallinum C3/CAM intermediate plant

Mesembryathemum crystallinum plants performing C(3) or CAM (crassulacean acid metabolism) appear to be highly resistant to Botrytis cinerea as well as to Pseudomonas syringae. Fungal hyphae growth was restricted to 48h post-inoculation (hpi) in both metabolic types and morphology of hyphae differed between those growing in C(3) and CAM plants. Growth of bacteria was inhibited significantly 24 hpi in both C(3) and CAM plants. B. cinerea and P. syringae infection led to an increase in the concentration of H(2)O(2) in C(3) plants 3 hpi, while a decrease in H(2)O(2) content was observed in CAM performing plants. The concentration of H(2)O(2) returned to the control level 24 and 48 hpi. Changes in H(2)O(2) content corresponded with the activity of guaiacol peroxidase (POD), mostly 3 hpi. We noted that its activity decreased significantly in C(3) plants and increased in CAM plants in response to inoculation with both pathogens. On the contrary, changes in the activity of CAT did not correlate with H(2)O(2) level. It increased significantly after interaction of C(3) plants with B. cinerea or P. syringae, but in CAM performing plants, the activity of this enzyme was unchanged. Inoculation with B. cinerea or P. syringae led to an increase in the total SOD activity in C(3) plants while CAM plants did not exhibit changes in the total SOD activity after interaction with both pathogens. In conclusion, the pathogen-induced changes in H(2)O(2) content and in SOD, POD and CAT activities in M. crystallinum leaves, were related to the photosynthetic metabolism type of the stressed plants rather than to the lifestyle of the invading pathogen.

Plastoquinone redox state modifies plant response to pathogen

The role of PQ (plastoquinione) redox state in establishment of response to pathogen infection (Botrytis cinerea) was tested along the regulation of main antioxidative enzymes (superoxide dismutase - SOD, catalase - CAT) and photochemistry of PSII (photosystem II) in Mesembryanthemum crystallinum plants performing C3 and CAM (Crassulacean acid metabolism) carbon metabolism. The redox state of PQ was modified by two inhibitors of photosynthetic electron transport resulting in a more oxidised (3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCMU) or reduced (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DBMIB) PQ redox state simulating darkness and high light conditions, respectively. Irrespective of the type of treatment (mock inoculation or pathogen inoculation) SOD activity depended on the PQ pool. Our results suggest that regarding changes in infection-induced CAT activity, plants developed response that is vital for hypersensitive-like (HR-like) response establishment only when PQ pool generated signal was similar to that in light presence (DBMIB pre-treatment). When PQ pool generated signal was similar to darkness, CAT activity response remained stress-independent, similarly to SOD. Fluorescence parameters of PSII, Qp (photochemical quenching coefficient) and NPQ (non-photochemical quenching) were affected only in the tissues treated with DCMU in stress-independent manner. We suggest that in case of abiotic and biotic stresses signals emerging from PQ pool indirectly orchestrate plant response and carbon metabolism affects this regulatory pathway.

Redox signals as a language of interorganellar communication in plant cells

Plants are redox systems and redox-active compounds control and regulate all aspects of their life. Recent studies have shown that changes in reactive oxygen species (ROS) concentration mediated by enzymatic and non-enzymatic antioxidants are transferred into redox signals used by plants to activate various physiological responses. An overview of the main antioxidants and redox signaling in plant cells is presented. In this review, the biological effects of ROS and related redox signals are discussed in the context of acclimation to changing environmental conditions. Special attention is paid to the role of thiol/disulfide exchange via thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs) in the redox regulatory network. In plants, chloroplasts and mitochondria occupying a chloroplasts and mitochondria play key roles in cellular metabolism as well as in redox regulation and signaling. The integrated redox functions of these organelles are discussed with emphasis on the importance of the chloroplast and mitochondrion to the nucleus retrograde signaling in acclimatory and stress response.

Automated image analysis for quantification of reactive oxygen species in plant leaves

The paper presents an image processing method for the quantitative assessment of ROS accumulation areas in leaves stained with DAB or NBT for H2O2 and O2- detection, respectively. Three types of images determined by the combination of staining method and background color are considered. The method is based on the principle of supervised machine learning with manually labeled image patterns used for training. The methods algorithm is developed as a JavaScript macro in the public domain Fiji (ImageJ) environment. It allows to select the stained regions of ROS-mediated histochemical reactions, subsequently fractionated according to the weak, medium and intense staining intensity and thus ROS accumulation. It also evaluates total leaf blade area. The precision of ROS accumulation area detection is validated by the Dice Similarity Coefficient in the case of manual patterns. The proposed framework reduces the computation complexity, once prepared, requires less image processing expertise than the competitive methods and represents a routine quantitative imaging assay for a general histochemical image classification.

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3–CAM intermediate plant Mesembryanthemum crystallinum

BACKGROUND AND AIMS Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant. METHODS The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. KEY RESULTS Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. CONCLUSIONS The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling.

Veinal-mesophyll interaction under biotic stress

According to microscopic observations, germinating hyphae of Botrytis cinerea, though easily penetrating Mesembryanthemum crystallinum mesophyll tissue, are limited in growth in mid-ribs and only occasionally reach vascular bundles. In mid-ribs of C3 and CAM leaves, we found significantly lower rbcL (large RubisCO subunit) abundance. Moreover, in CAM leaves, minute transcript contents for pepc1 (phosphoenolpyruvate carboxylase) and nadpme1 (malic enzyme) genes found in the mid-ribs suggest that they perform β-carboxylation at a low rate. The gene of the main H2O2-scavenging enzyme, catL (catalase), showed lower expression in C3 mid-rib parts in comparison to mesophyll. This allows maintenance of higher H2O2 quantities in mid-rib parts. In C3 leaves, pathogen infection does not impact photosynthesis. However, in CAM plants, the expression profiles of rbcL and nadpme1 were similar under biotic stress, with transcript down-regulation in mid-ribs and up-regulation in mesophyll (however, in case of rbcL not significant). After B. cinerea infection in C3 plants, transcripts for both antioxidative proteins strongly increased in mid-ribs, but not in mesophyll. In infected CAM plants, a significant transcript increase in the mesophyll was parallel to its decrease in the mid-rib region (however, in the case of catL this was not significant). Pathogen infection modified the expression of carbon and ROS metabolism genes in mid-ribs and mesophyll, resulting in the establishment of successful leaf defense.