Role of hydrogen peroxide and antioxidant enzymes in the interaction between a hemibiotrophic fungal pathogen, Leptosphaeria maculans, and oilseed rape

Reactive oxygen species play a dual role in host-pathogen interaction. They impede the spread of biotrophic pathogens via stimulating cell death and hypersensitive response (HR), and, on the other hand, they provide access to nutrients for necrotrophic pathogens feeding on dead tissues and facilitate their colonizing the host. The participation of ROS in defending plants from pathogens with a combined lifestyle (hemibiotrophs) is not yet understood, and it varies in its dependence on the particular host-pathogen combination. In the present study, we inoculated rapeseed plants (Brassica napus) with a hemibiotrophic fungus, Leptosphaeria maculans, and manipulated the H₂O₂ content in cotyledons by infiltrating catalase and/or H₂O₂ into tissues. The action of catalase resulted in a significant decrease in lesions development, but when H₂O₂ was applied instead, lesion formation was only moderately stimulated compared to the untreated control. When H₂O₂ toxicity to L. maculans was tested in vitro, concentrations above 5 mM and 10 mM H₂O₂ were lethal for germinating conidia and growing mycelia of L. maculans, respectively. We can assume that L. maculans behaves as a necrotroph during this early stage of infection even though its resistance to H₂O₂ does not exceed standard concentrations. To investigate antioxidant mechanisms implicated in the response of B. napus to L. maculans, the cotyledons were both inoculated with conidia and treated with L. maculans elicitor. Increased activities of guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and superoxide dismutase were recorded both in L. maculans-infected and elicitor-treated cotyledons. The results indicate the importance of these enzymes for ROS scavenging in B. napus-L. maculans interaction.     

Chloroplasts and mitochondria have multiple heat tolerant isozymes of SOD and APX in leaf and inflorescence in Chenopodium album

Thermal stability of antioxidant defense enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) was studied in chloroplasts and mitochondria of leaf and inflorescence in heat adaptive weed Chenopodium album. Leaf samples were taken in March (31 °C/14 °C) and young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). Leaf and INF chloroplast and mitochondrial fractions were subjected to elevated temperatures in vitro (5–100 °C) for 30′. SOD and APX showed activity even after boiling treatment in both chloroplast and mitochondria of leaf and INF. SOD was more heat stable than APX in both chloroplasts and mitochondria in both the tissues. Chloroplast contained more heat stable SOD and APX isozymes than mitochondria in both leaf and INF. To the best of our knowledge this is the first report showing presence of thermostable APX isozymes (100 °C for 30′) in chloroplasts and mitochondria in C. album. Heat stable isozymes of SOD and APX in chloroplasts and mitochondria in leaves and inflorescence may contribute to heat tolerance in C. album.     

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.     

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

Leishmania donovani: dyskinetoplastid cells survive and proliferate in the presence of pyruvate and uridine but do not undergo apoptosis after treatment with camptothecin

We have shown that treatment with luteolin in leishmanial cells causes loss of mt-DNA and induces apoptosis through mitochondria dependent pathway [Sen, N., Das, B.B., Ganguly, A., Banerjee, B., Sen, T., Majumder, H.K., 2006. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology, in press]. Here, we report that mitochondrial DNA depleted leishmanial cells require exogenous sources of pyruvate and uridine to survive and proliferate. The presence of pyruvate and uridine in a growing media help them to produce sufficient amount of glycolytic ATP to maintain the mitochondrial membrane potential in the absence of their functional ETC. Treatment of wild type cells with CPT causes generation of ROS that leads to apoptosis. But unlike the normal cells ROS was not generated in these mt-DNA depleted cells after treatment with CPT. Taken together we have shown for the first time that dyskinetoplastid cells are auxotrophic for pyruvate and uridine and apoptosis cannot be induced in these cells in the presence of CPT. Therefore, the presence of mitochondrial DNA is absolutely necessary for the cytotoxicity of CPT in kinetoplastid parasites.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Warriors at the gate that never sleep: non-host resistance in plants

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.     

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression

Hydrogen sulfide (H₂S) is considered as a cellular signaling intermediate in higher plants, but corresponding molecular mechanisms and signal transduction pathways in plant biology are still limited. In the present study, a combination of pharmacological and biochemical approaches was used to study the effect of H₂S on the alleviation of GA-induced programmed cell death (PCD) in wheat aleurone cells. The results showed that in contrast with the responses of ABA, GA brought about a gradual decrease of l-cysteine desulfhydrase (LCD) activity and H₂S production, and thereafter PCD occurred. Exogenous H₂S donor sodium hydrosulfide (NaHS) not only effectively blocked the decrease of endogenous H₂S release, but also alleviated GA-triggered PCD in wheat aleurone cells. These responses were sensitive to hypotaurine (HT), a H₂S scavenger, suggesting that this effect of NaHS was in an H₂S-dependent fashion. Further experiment confirmed that H₂S, rather than other sodium- or sulphur-containing compounds derived from the decomposing of NaHS, was attributed to the rescuing response. Importantly, the reversing effect was associated with glutathione (GSH) because the NaHS triggered increases of endogenous GSH content and the ratio of GSH/oxidized GSH (GSSG) in GA-treated layers, and the NaHS-mediated alleviation of PCD was markedly eliminated by l-buthionine-sulfoximine (BSO, a selective inhibitor of GSH biosynthesis). The inducible effect of NaHS was also ascribed to the modulation of heme oxygenase-1 (HO-1), because the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPP) significantly suppressed the NaHS-related responses. By contrast, the above inhibitory effects were reversed partially when carbon monoxide (CO) aqueous solution or bilirubin (BR), two of the by-products of HO-1, was added, respectively. NaHS-triggered HO-1 gene expression in GA-treated layers was also confirmed. Together, the above results clearly suggested that the H₂S-delayed PCD in GA-treated wheat aleurone cells was associated with the modulation of GSH homeostasis and HO-1 gene expression.     

Pharmacological induction of ischemic tolerance in hippocampal slices by sarcosine preconditioning

Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD.