How the Ethylene Biosynthesis Pathway of Semi-Halophytes Is Modified with Prolonged Salinity Stress Occurrence?

The mechanism of ethylene (ET)-regulated salinity stress response remains largely unexplained, especially for semi-halophytes and halophytes. Here, we present the results of the multifaceted analysis of the model semi-halophyte Mesembryanthemum crystallinum L. (common ice plant) ET biosynthesis pathway key components' response to prolonged (14 days) salinity stress. Transcriptomic analysis revealed that the expression of 3280 ice plant genes was altered during 14-day long salinity (0.4 M NaCl) stress. A thorough analysis of differentially expressed genes (DEGs) showed that the expression of genes involved in ET biosynthesis and perception (ET receptors), the abscisic acid (ABA) catabolic process, and photosynthetic apparatus was significantly modified with prolonged stressor presence. To some point this result was supported with the expression analysis of the transcript amount (qPCR) of key ET biosynthesis pathway genes, namely ACS6 (1-aminocyclopropane-1-carboxylate synthase) and ACO1 (1-aminocyclopropane-1-carboxylate oxidase) orthologs. However, the pronounced circadian rhythm observed in the expression of both genes in unaffected (control) plants was distorted and an evident downregulation of both orthologs' was induced with prolonged salinity stress. The UPLC-MS analysis of the ET biosynthesis pathway rate-limiting semi-product, namely of 1-aminocyclopropane-1-carboxylic acid (ACC) content, confirmed the results assessed with molecular tools. The circadian rhythm of the ACC production of NaCl-treated semi-halophytes remained largely unaffected by the prolonged salinity stress episode. We speculate that the obtained results represent an image of the steady state established over the past 14 days, while during the first hours of the salinity stress response, the view could be completely different.

Endophytic yeast protect plants against metal toxicity by inhibiting plant metal uptake through an ethylene-dependent mechanism.

Toxic metal pollution requires significant adjustments in plant metabolism. Here, we show that the plant microbiota plays an important role in this process. The endophytic Sporobolomyces ruberrimus isolated from a serpentine population of Arabidopsis arenosa protected plants against excess metals. Coculture with its native host and Arabidopsis thaliana inhibited Fe and Ni uptake. It had no effect on host Zn and Cd uptake. Fe uptake inhibition was confirmed in wheat and rape. Our investigations show that, for the metal inhibitory effect, the interference of microorganisms in plant ethylene homeostasis is necessary. Application of an ethylene synthesis inhibitor, as well as loss-of-function mutations in canonical ethylene signalling genes, prevented metal uptake inhibition by the fungus. Coculture with S. ruberrimus significantly changed the expression of Fe homeostasis genes: IRT1, OPT3, OPT6, bHLH38 and bHLH39 in wild-type (WT) A. thaliana. The expression pattern of these genes in WT plants and in the ethylene signalling defective mutants significantly differed and coincided with the plant accumulation phenotype. Most notably, down-regulation of the expression of IRT1 solely in WT was necessary for the inhibition of metal uptake in plants. This study shows that microorganisms optimize plant Fe and Ni uptake by fine-tuning plant metal homeostasis.

At the Edges of Photosynthetic Metabolic Plasticity-On the Rapidity and Extent of Changes Accompanying Salinity Stress-Induced CAM Photosynthesis Withdrawal.

The common ice plant (Mesembryanthemum crystallinum L.) is a facultative crassulacean acid metabolism (CAM) plant, and its ability to recover from stress-induced CAM has been confirmed. We analysed the photosynthetic metabolism of this plant during the 72-h response period following salinity stress removal from three perspectives. In plants under salinity stress (CAM) we found a decline of the quantum efficiencies of PSII (Y(II)) and PSI (Y(I)) by 17% and 15%, respectively, and an increase in nonphotochemical quenching (NPQ) by almost 25% in comparison to untreated control. However, 48 h after salinity stress removal, the PSII and PSI efficiencies, specifically Y(II) and Y(I), elevated nonphotochemical quenching (NPQ) and donor side limitation of PSI (YND), were restored to the level observed in control (C3 plants). Swelling of the thylakoid membranes, as well as changes in starch grain quantity and size, have been found to be components of the salinity stress response in CAM plants. Salinity stress induced an over 3-fold increase in average starch area and over 50% decline of average seed number in comparison to untreated control. However, in plants withdrawn from salinity stress, during the first 24 h of recovery, we observed chloroplast ultrastructures closely resembling those found in intact (control) ice plants. Rapid changes in photosystem functionality and chloroplast ultrastructure were accompanied by the induction of the expression (within 24 h) of structural genes related to the PSI and PSII reaction centres, including PSAA, PSAB, PSBA (D1), PSBD (D2) and cp43. Our findings describe one of the most flexible photosynthetic metabolic pathways among facultative CAM plants and reveal the extent of the plasticity of the photosynthetic metabolism and related structures in the common ice plant.

Phytohormone based biostimulant combined with plant growth promoting endophytic fungus enhances Ni phytoextraction of Noccaea goesingensis.

To improve the efficiency of Ni phytoextraction, the metal hyperaccumulator N. goesingensis was subject to treatment with a combination of a Ni uptake stimulating microorganism and the commercially available, IAA- based biostimulating seaweed extract - Kelpak. Additionally, we compared the plant growth promoting and Ni uptake capabilities of the two biofertilizers. Treatment with the Kelpak alone had no significant effect on plant growth or Ni accumulation. Inoculation of N. goesingensis with Phomopsis columnaris significantly improved the biomass of the hyperaccumulating plant and Ni yield per plant and improved several plant biometric features such as fresh and dry weight and several others related to leaf and root size. However, the combination of the two treatments yielded the best results; plants treated with the two growth promoting agents yielded 85% more biomass compared to not treated plants and accumulated 48% more Ni per plant. To verify plant inoculation with the fungus we generated a GFP expressing strain of P. columnaris and visualized the fungus in both plant leaves and roots. To trace the development of the fungus in planta and to evaluate the effect of biostimulant treatment on mycelium development fungal translational elongation factor 1α (tef1α) DNA was quantified with qPCR. Upon biofertilizer the abundance P. columnaris in plant leaves increased nearly 5-fold. The utilization of plant growth stimulating microorganisms, endophytic fungi in particular, can significantly improve Ni phytoextraction in hyperaccumulator N. goesingensis.

The effect of endophytic fungi on growth and nickel accumulation in Noccaea hyperaccumulators.

The role of endophytic fungi isolated from different populations of European Ni hyperaccumulators was investigated in regard to the microorganisms' ability to enhance the hyperaccumulation of Ni in Noccaea caerulescens. Effects of particular species of endophytic fungi on adaptation of N. caerulescens to excess Ni were tested by co-cultivation with single strains of the fungi. Seven of these had a positive effect on plant biomass production, whereas two of the tested species inhibited plant growth; biomass production of inoculated plants was significantly different compared to non-inoculated control. Inoculation with six fungal strains: Embellisia thlaspis, Pyrenochaeta cava, Phomopsis columnaris, Plectosphaerella cucumerina, Cladosporium cladosporioides and Alternaria sp. stimulated the plant to uptake and accumulate more Ni in both roots and shoots, compared to non-inoculated control. P. columnaris was isolated from all plant species sampled. Strains isolated from Noccaea caerulescens and Noccaea goesingensis increased Ni root and shoot accumulation of their native hosts (compared to non-inoculated control). Inoculation of different populations of Noccaea with P. columnaris of foreign origin did not cause its host to accumulate more Ni, with the exception of the Ni-unadapted ecotype of N. goesingensis. Inoculation with P. columnaris from N. caerulescens significantly improved Ni uptake, but the effect of the fungus was not as prominent as in the case of N. caerulescens. By comparing the transcriptomes of N. caerulescens and N. goesingensis from Flatz inoculated with P. columnaris, we showed that enhanced uptake and accumulation of Ni in the plants is accompanied by an upregulation of several genes mainly involved in plant stress protection and metal uptake and compartmentation.

Local and Systemic Changes in Photosynthetic Parameters and Antioxidant Activity in Cucumber Challenged with Pseudomonas syringae pv lachrymans.

We studied changes in gas exchange, photochemical activity and the antioxidant system in cucumber leaves locally infected with Pseudomonas syringae pv lachrymans and in uninfected systemic ones. Infection-induced declined net photosynthesis rate and the related changes in transpiration rate, the intracellular CO2 concentration, and prolonged reduction in maximal PSII quantum yield (Fv/Fm), accompanied by an increase in non-photochemical quenching (NPQ), were observed only in the infected leaves, along with full disease symptom development. Infection severely affected the ROS/redox homeostasis at the cellular level and in chloroplasts. Superoxide dismutase, ascorbate, and tocopherol were preferentially induced at the early stage of pathogenesis, whereas catalase, glutathione, and the ascorbate-glutathione cycle enzymes were activated later. Systemic leaves retained their net photosynthesis rate and the changes in the antioxidant system were partly like those in the infected leaves, although they occurred later and were less intense. Re-balancing of ascorbate and glutathione in systemic leaves generated a specific redox signature in chloroplasts. We suggest that it could be a regulatory element playing a role in integrating photosynthesis and redox regulation of stress, aimed at increasing the defense capacity and maintaining the growth of the infected plant.

The Common Ice Plant (Mesembryanthemum crystallinum L.)-Phytoremediation Potential for Cadmium and Chromate-Contaminated Soils.

The common ice plant (Mesembryanthemum crystallinum L.) is a widely studied model due to its tolerance to numerous biotic and abiotic stresses. In this study, carried out in model pots, the plants were treated with variant doses of Cd(II) and Cr(VI) and proved resistant to extreme levels of these heavy metals. Initial toxicity symptoms were observed upon final concentrations of 818 mg Cd kg-1 soil d.w., and 1699 mg Cr kg-1 applied as potassium chromate. Biometric analyses revealed that none of the Cr(VI) doses affected dry weight of the plant organs thus maintaining the shoot-to-root ratio. The Cd and Cr hypertolerance strategies were divergent and resulted in different accumulation patterns. For the case of Cd(II), an excluder-like mechanism was developed to prevent the plant from toxicity. For chromate, high accumulation potential together with Cr(VI) root-to-shoot translocation at sublethal concentrations was revealed (up to 6152 mg Cr kg-1 shoot at 4248 mg Cr kg-1 soil). It is concluded that M. crystallinum reveals considerable phytoremediation capabilities due to unique growth potential in contaminated substrates and is suitable for bioreclamation of degraded soils. The plant is especially applicable for efficient phytoextraction of chromate-contamination, whereas for Cd-affected areas it may have a phytostabilizing effect.

Expression of Genes Involved in Heavy Metal Trafficking in Plants Exposed to Salinity Stress and Elevated Cd Concentrations.

Many areas intended for crop production suffer from the concomitant occurrence of heavy metal pollution and elevated salinity; therefore, halophytes seem to represent a promising perspective for the bioremediation of contaminated soils. In this study, the influence of Cd treatment (0.01-10.0 mM) and salinity stress (0.4 M NaCl) on the expression of genes involved in heavy metal uptake (irt2-iron-regulated protein 2, zip4-zinc-induced protein 4), vacuolar sequestration (abcc2-ATP-binding cassette 2, cax4-cation exchanger 2 pcs1-phytochelatin synthase 1) and translocation into aerial organs (hma4-heavy metal ATPase 4) were analyzed in a soil-grown semi-halophyte Mesembryanthemum crystallinum. The upregulation of irt2 expression induced by salinity was additionally enhanced by Cd treatment. Such changes were not observed for zip4. Stressor-induced alterations in abcc2, cax4, hma4 and pcs1 expression were most pronounced in the root tissue, and the expression of cax4, hma4 and pcs1 was upregulated in response to salinity and Cd. However, the cumulative effect of both stressors, similar to the one described for irt2, was observed only in the case of pcs1. The importance of salt stress in the irt2 expression regulation mechanism is proposed. To the best of our knowledge, this study is the first to report the combined effect of salinity and heavy metal stress on genes involved in heavy metal trafficking.

The response of a model C3/CAM intermediate semi-halophyte Mesembryanthemum crystallinum L. to elevated cadmium concentrations.

Many areas exhibiting increased concentrations of soluble salts are simultaneously polluted with heavy metals (HM), and halophytes with extended tolerance to heavy metal toxicity seem to represent a promising tool for their phytoremediation. In this study, the response of the soil-grown C3-CAM (Crassulacean acid metabolism) intermediate halophyte Mesembryanthemum crystallinum (common ice plant) to increased concentrations of Cd (0.01-1 mM) was investigated. None of the tested Cd treatments affected growth parameters or tissue water content of either C3 or CAM-performing plants. Chlorophyll a fluorescence confirmed high tolerance of the photosynthetic apparatus of both metabolic states towards Cd. Plants performing both photosynthesis types accumulated significant Cd amounts only under the highest (1 mM) treatment, and the metal was primarily deposited in the roots, which are features typical of an excluding strategy. Upon the application of 1 mM Cd solution CAM-performing plants, due to the NaCl pre-treatment applied for CAM induction, were exposed to significantly higher amounts of bioavailable Cd in comparison with those of C3-performing plants. As a result, roots of CAM plants accumulated over 4-fold higher Cd amounts when compared with C3 plants. In our opinion, enhanced Cd-accumulating potential observed in CAM-performing plants was the effect of osmotic stress episode and resulting modifications e.g. in the detoxifying capacity of the antioxidative system. Increased antioxidative potential of NaCl pre-treated plants was pronounced with significantly higher activity of CuZnSOD (copper-zinc superoxide dismutase), not achievable in C3 plants subjected to high Cd concentrations. Moreover, the applied Cd doses induced SOD activity in a compartment-dependent manner only in C3 plants. We confirmed that none of the applied Cd concentrations initiated the metabolic shift from C3 to CAM.

Acclimation of the photosynthetic apparatus and alterations in sugar metabolism in response to inoculation with endophytic fungi.

The role of an endophytic Zygomycete Mucor sp. in growth promotion and adaptation of the photosynthetic apparatus to increased energy demands of its hosts Arabidopsis arenosa and Arabidopsis thaliana was evaluated. Inoculation with the fungus improved the water use efficiency of the plants and allowed for them to utilize incident light for photochemistry more effectively by upregulating the expression of several photosystem I- and II-related genes and their respective proteins, proteins involved in light harvesting in PSII and PSI and carbon assimilation. This effect was independent of the ability of the plants to acquire nutrients from the soil. We hypothesize that the accelerated growth of the symbiotic plants resulted from an increase in their demand for carbohydrates and carbohydrate turnover (sink strength) that triggered a simultaneous upregulation of carbon assimilation. Arabidopsis plants inoculated with Mucor sp. exhibited upregulated expression in several genes encoding proteins involved in carbohydrate catabolism, sugar transport, and smaller starch grains that indicate a significant upregulation of carbohydrate metabolism.

Withdrawal from functional Crassulacean acid metabolism (CAM) is accompanied by changes in both gene expression and activity of antioxidative enzymes.

In Mesembryanthemum crystallinum, crassulacean acid metabolism (CAM) is seemingly reversible, but unequivocal evidence for functional CAM withdrawal has yet to be shown. In this study, we confirmed the rapid downregulation of PEPC1 expression just 1 h after the removal of NaCl from the plant growth media. At the same time, the Δ malate values in desalted plants rapidly (1 d) re-established to values typical for C3 plants. This phenomenon allowed us to confirm functional CAM withdrawal in the desalted plants. Desalting altered the expression of the genes of the main antioxidative enzymes and/or the activity of their respective proteins; for catalase (CAT), both gene expression and protein activity were restored to levels observed in C3 plants in response to desalting, while for cooper-zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX), only protein activity was restored. Therefore, we conclude that during the C3→CAM transition the CAM-specific antioxidative enzyme activity profile constitutes a transient and fully reversible response to abiotic stress.

The Role of Strigolactone in the Cross-Talk Between Arabidopsis thaliana and the Endophytic Fungus Mucor sp.

Over the last years the role of fungal endophytes in plant biology has been extensively studied. A number of species were shown to positively affect plant growth and fitness, thus attempts have been made to utilize these microorganisms in agriculture and phytoremediation. Plant-fungi symbiosis requires multiple metabolic adjustments of both of the interacting organisms. The mechanisms of these adaptations are mostly unknown, however, plant hormones seem to play a central role in this process. The plant hormone strigolactone (SL) was previously shown to activate hyphae branching of mycorrhizal fungi and to negatively affect pathogenic fungi growth. Its role in the plant-endophytic fungi interaction is unknown. The effect of the synthetic SL analog GR24 on the endophytic fungi Mucor sp. growth, respiration, H2O2 production and the activity of antioxidant enzymes was evaluated. We found fungi colony growth rate was decreased in a GR24 concentration dependent manner. Additionally, the fungi accumulated more H2O2 what was accompanied by an altered activity of antioxidant enzymes. Symbiosis with Mucor sp. positively affected Arabidopsis thaliana growth, but SL was necessary for the establishment of the beneficial interaction. A. thaliana biosynthesis mutants max1 and max4, but not the SL signaling mutant max2 did not develop the beneficial phenotype. The negative growth response was correlated with alterations in SA homeostasis and a significant upregulation of genes encoding selected plant defensins. The fungi were also shown to be able to decompose SL in planta and to downregulate the expression of SL biosynthesis genes. Additionally, we have shown that GR24 treatment with a dose of 1 µM activates the production of SA in A. thaliana. The results presented here provide evidence for a role of SL in the plant-endophyte cross-talk during the mutualistic interaction between Arabidopsis thaliana and Mucor sp.

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.

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.

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 ß-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.

Ozone fumigation increases the abundance of nutrients in Brassica vegetables: broccoli (Brassica oleracea var. italica) and Chinese cabbage (Brassica pekinensis).

Brassicaceae vegetables, among them broccoli and Chinese cabbage, are well recognized due to the nutritional properties. Four-week-old Chinese cabbage and broccoli seedlings were fumigated with O3 for 3 days before being transplanted into the field. The effect of O3 treatment was determined after reaching marketable quality (ca. 10 weeks). The inflorescences of O3-treated broccoli were enriched in vitamin E (α-tocopherol and γ-tocopherol), whereas Chinese cabbage heads had an increased content of anthocyanins and ß-carotene. Ozone treatment did not significantly affect the productivity of both examined vegetables.

Differences in the activity and concentration of elements of the antioxidant system in different layers of Brassica pekinensis head.

Differences in the activity of superoxide dismutase, catalase (CAT) and ascorbate peroxidase (APX) as well as in the concentration of ascorbate, tocopherol and hydrogen peroxide (H2O2) were found in leaves from different layers of the Chinese cabbage (Brassica pekinensis (Lour.) Rupr.) head. The youngest chlorophyll-deficient leaves from the most inner layers of the cabbage head were characterized by a high concentration of ascorbate, high activity of iron superoxide dismutase (FeSOD), cooper/zinc superoxide dismutase (Cu/ZnSOD) and a low content of H2O2. On the other hand, activity of CAT, manganese superoxide dismutase (MnSOD) and APX and tocopherol content were highest in chlorophyll-rich leaves from outer parts. The results of this work are interesting from the human nutrition standpoint, as the measured antioxidants have beneficial effects on human health. They can also be utilized to improve storage conditions due to an unequivocal function of antioxidant molecules in maintaining postharvest quality of vegetables.

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.