Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.     

Reactive oxygen intermediates modulate nitric oxide signaling in the plant hypersensitive disease-resistance response

The mechanisms involved in plant defense show several similar characteristics with the innate immune systems of vertebrates and invertebrates. In animals, nitric oxide (NO) cooperates with reactive oxygen intermediates (ROI) to kill tumor cells and is also required for macrophage killing of bacteria. Such cytotoxic events occur because unregulated levels of NO determine its diffusion-limited reaction with O₂^– generating peroxynitrite (ONOO^–), a mediator of cellular injury in many biological systems. In soybean suspension cells, unregulated NO production during the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate the hypersensitive cell death, which is triggered only by fine tuning the NO/ROI ratio. Furthermore, that hypersensitive cell death is activated following interaction of NO with H₂O₂, rather than O₂^–. Increasing O₂^– levels reduces NO-derived toxicity, and the addition of ONOO^– to soybean suspensions does not affect cell viability. Consistently with the fact that ONOO^– is not an essential mediator of NO/RO-induced cell death, during the HR superoxide dismutase (SOD) accelerates O₂^– dismutation to H₂O₂ and therefore minimizes the loss of NO by reaction with O₂^– and triggers hypersensitive cell death through the NO/H₂O₂ synergism. Consequently, the rates of production and dismutation of O₂^– generated during the oxidative burst play a crucial role in modulating NO signaling through the cell death pathway, which proceeds through mechanisms different from those commonly observed in animals.     

In planta measurements of oxidative bursts elicited by avirulent and virulent bacterial pathogens suggests that H2O2 is insufficient to elicit cell death in tobacco

A rapid and localized programmed cell death – the hypersensitive response (HR) – is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H₂O₂ generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H₂O₂ generation following challenge with avirulent strains of Pseudomonas syringae pv. (P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H₂O₂-responsive transgene AoPR10-GUS, the dually responsive (H₂O₂ and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10- and PR1a-GUS expression and cell death. Conversely, varying H₂O₂ generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci, which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H₂O₂ generation by an unknown mechanism.     

The role of jasmonic acid in root mitochondria disruption

Methyl jasmonate (MeJA) produces an important reduction in the accumulation of proteins related to energy metabolism. The treatment of hairy roots (HR) with MeJA increased the accumulation of H₂O₂ during the first 48 h and this H₂O₂ accumulation was also observed in isolated mitochondria. Peroxidase and catalase activities decreased in the presence of MeJA, and this decrease directly correlated with the increase of H₂O₂ in HR treated with MeJA. This suggests that the H₂O₂ burst due to MeJA is the initial response to mitochondria disruption in the roots.     

Characterization of the hypersensitive response‐like cell death phenomenon induced by targeting antiviral lectin griffithsin to the secretory pathway

Griffithsin (GRFT) is an antiviral lectin, originally derived from a red alga, which is currently being investigated as a topical microbicide to prevent transmission of human immunodeficiency virus (HIV). Targeting GRFT to the apoplast for production in Nicotiana benthamiana resulted in necrotic symptoms associated with a hypersensitive response (HR)‐like cell death, accompanied by H₂O₂ generation and increased PR1 expression. Mannose‐binding lectins surfactant protein D (SP‐D), cyanovirin‐N (CV‐N) and human mannose‐binding lectin (hMBL) also induce salicylic acid (SA)‐dependent HR‐like cell death in N. benthamiana, and this effect is mediated by the lectin's glycan binding activity. We found that secreted GRFT interacts with an endogenous glycoprotein, α‐xylosidase (XYL1), which is involved in cell wall organization. The necrotic effect could be mitigated by overexpression of Arabidopsis XYL1, and by co‐expression of SA‐degrading enzyme NahG, providing strategies for enhancing expression of oligomannose‐binding lectins in plants.     

Hydrogen peroxide is not involved in HrpN from Erwinia amylovora-induced hypersensitive cell death in maize leaves

Harpin elicits rapid and localized programmed cell death in plants, also known as the hypersensitive response (HR). Here we report that HrpN from Erwinia amylovora led to rapid cell death in maize leaves within 24 h and also induced the expression of systemic acquired resistance genes, such as ZmPR1 and ZmPR5. Surprisingly, the results of DAB staining showed that there was no H₂O₂ accumulation in maize leaves during the HR process, and semi-quantitative RT-PCR revealed that there was also no difference in the expression of the ZmRboh genes. These results suggest that HrpN-induced cell death may be independent of H₂O₂ accumulation in maize leaves.     

H2O2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species([F])

In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H₂O₂), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H₂O₂‐induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H₂O₂ levels induced the generation of nitric oxide (NO) and higher S‐nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H₂O₂‐induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H₂O₂‐induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [ Chengcai Chu (Corresponding author)]     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: An hypothesis

Production of reactive oxygen intermediates (ROI) has been thought for a long time to adversely affect the physiology and survival of a cell. There is now a growing body of evidence to suggest that ROI such as superoxide anion (O^·-₂) and hydrogen peroxide (H₂O₂) can influence the growth, as well as death, of animal cells in vitro. The observation that cells release O^·-₂ or its dismutation product H₂O₂, either constitutively in the case of tumor cells or following cytokine stimulation, has led to the speculation that they might possibly serve as intercellular messengers to stimulate proliferation via mechanisms common to natural growth factors. However, as the balance between cell populations in an organism is tightly controlled by the rate of proliferation and death of constituent cells, an increase in cell numbers could reciprocally be viewed as deregulation of cell death. Hence, it is equally important to decipher how ROI influence the response of cells to signals that activate cell death pathway(s). We propose that ROI not only regulate proliferation but also affect cell sensitivity to triggers which activate the cellular suicide program (apoptosis) versus those that cause accidental (necrotic) cell death.     

Superoxide Generation in Chemically Activated Resistance of Barley in Response to Inoculation with the Powdery Mildew Fungus

In a previous work, a phenotype-specific accumulation of superoxide radical anions (O^??₂) after attack of the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) in near-isogenic barley (Hordeum vulgare L.) lines bearing different Mlx genes for resistance was described (Hückelhoven and Kogel, 1998). We have now a histochemical study of the pathogenesis-related O^??₂ generation in the systemic activated resistance (SAR) response induced in barley cv Pallas by the plant activator 2,6-dichloroisonicotinic acid (DCINA). SAR-specific defence was conducted prevalently characterized by penetration resistance. Fungal arrest was observed before haustorium formation by a highly localized cell wall reinforcement (effective papillae) and, in most cases, by a subsequent hypersensitive cell death (HR). No O^??₂ generation was found in association with these plant defence responses. However, a strong O^??₂ burst in the attacked epidermal cells was detected in the control plants which were not activated by DCINA. This burst coincided with cell wall penetration and subsequent contact of the pathogen with the host plasma membrane. A strong SAR-related O^??₂ burst was induced in the mesophyll tissue beneath the attacked and hypersensitively reacting epidermal cells in plants treated with DCINA. The accumulation of O^??₂ was confined to chloroplasts. The remarkable burst in mesophyll tissue was not followed by mesophyll-HR indicating that chloroplastic O^??₂ generation is not sufficient for the hypersensitive cell death. Since the same pattern of pathogenesis-related O^??₂ accumulation was identified for race-specific response mediated by the Mlg gene for powdery mildew resistance, the present data are consistent with the hypothesis that the SAR phenotype is a phenocopy of the Mlg-type resistance (Kogel et al., 1994).     

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpinpss-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS)

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin_pss, an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin_pss-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coliwas able to significantly increase active oxygen species (AOS) generation when mixed with harpin_pss in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin_pss in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflpgene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin_pss-mediated hypersensitive cell death in vivo. In response to harpin_pss, suspension cells derived from Pflptransgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl₃) were used to study the involvement of AOS in harpin_pss-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin_pss inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.     

Different responses of two tobacco cultivars and their cell suspension cultures to quercinin,a novel elicitin from Phytophthora quercina

In this study we describe the response of two tobacco cultivars (Nicotiana tabacum L. cv. Bel B and Bel W3) and their cell suspension cultures to quercinin, a novel elicitin produced by the oak pathogen Phytophthora quercina. N-terminal sequencing of the purified protein proved that it belongs to the basic β-elicitins with threonine on position 13. Both tobacco leaves and cells of the cultivar Bel W3 showed hypersensitive cell death after quercinin treatment. Leaves of Bel B also developed quercinin-induced necrosis but higher concentrations of quercinin were necessary as compared to Bel W3. Also Bel B cells showed cell death induction only at the highest quercinin concentration (20 nM). In cell suspension experiments we also measured the quercinin-induced oxidative burst, which occurred in both cultivars. H₂O₂ production in Bel B increased with increasing quercinin concentration and was inhibited only at the highest elicitin concentration (20 nM) whereas the oxidative burst in Bel W3 was completely abolished by 5 nM quercinin. Furthermore we demonstrated that neither H₂O₂ nor superoxide were responsible for cell death induction since neither the inhibitor diphenyleneiodonium (DPI) nor the enzymes catalase (CAT) and superoxide dismutase (SOD) influenced the hypersensitive reaction (HR) in Bel W3 cells. Due to the different response of Bel W3 and Bel B towards the P. quercina elicitin, our system represents an interesting tool to elucidate signaling pathways in tobacco leading to hypersensitive cell death.     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary.  Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H⁺ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H⁺ consumption for O₂ ^•− dismutation to H₂O₂. Also K⁺ influx was strongly depressed by MeJA, even transitorily reverting to K⁺ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H₂O₂ accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O₂ uptake by roots gave similar results. These and other results for additions of H₂O₂ or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H₂O₂ being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN⁻- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O₂ ^•− and H₂O₂, O₂ uptake, and peroxidase activity by roots. Received July 12, 2002; accepted October 2, 2002; published online May 21, 2003 RID="*"     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

Role of hydrogen peroxide and apoplastic peroxidase in tomato — Botrytis cinerea interaction

The aim of the research was to estimate the sensitivity of tomato tissue and spore from necrotrophic isolate of B. cinerea on H₂O₂. The influence of exogenic H₂O₂ and B. cinerea on plant tissue and on the activity of peroxidases (PO), catalase (CAT) and superoxide dismutase (SOD) in apoplastic tomato leaves fraction were investigated. It was proved that 40 mM H₂O₂ damaged the cells of a host, and inhibited in vitro germination of B.cinerea spores. Complete inhibition of germination was observed after the use 100 mM H₂O₂. In the presence of spores H₂O₂ was decomposed to H₂O and O₂. Trace activity of catalase was observed in a solution of spores used for inoculation. Necrosis which appeared on the leaves after 40 mM H₂O₂ treatment resembled hypersensitive response. On the leaves pretreated at this concentration the development of infection was observed. The H₂O₂ concentration harmful for the tissues, stimulated the PO activity measured with NADH — responsible for generation of ^·O ₂ ⁻ , as well as with syringaldazine (S) and ferulic acid (FA), substrates characteristics of forms lignifying and strengthening the cell wall. Clear increase in CAT activity, resulting from infection and early pretreatment with H₂O₂ was observed in apoplast. No effect on SOD activity was observed. A hypothesis may be put forward, that germinating spores produce enzymes which allow them to decompose H₂O₂ generated in apoplast, so there is little likelihood that B. cinerea can be directly inhibited by reactive oxygen forms (ROS) during initial stages of infection. Necrotic lesions resembling HR generated by exogenous H₂O₂ as well as induction of activity of apoplastic plant enzymes, particularly PO connected with strengthening and lignification of cell wall, were not sufficient factors to inhibit fungal expansion.     

Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis‐related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT‐triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease‐inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H₂O₂ and significant induction of some defense‐response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT‐triggered resistance responses, and enabled Xcv proliferation in infected leaves. H₂O₂ accumulation, cell‐death induction, and defense‐response gene expression were distinctly reduced in CaPR4c‐silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.     

No interaction between methyl jasmonate and ozone in Pima cotton: growth and allocation respond independently to both

Ozone (O₃) is damaging to plants, inducing signalling pathways involving antagonism between jasmonates and ethylene. These pathways mediate O₃ responses, particularly to acute exposure, and their manipulation protected several species against acute and chronic O₃. We use chronic daily exposure of up to 163 ppb O₃, and twice weekly application of up to 320 µg plant^?1 methyl jasmonate (MeJA) to test two hypothesizes: 1) a low rate of MeJA does not affect growth but increases O₃ sensitivity; 2) a high rate inhibits growth but reduces O₃ sensitivity. Both hypotheses were rejected. Growth declined with increases in both MeJA and O₃. MeJA at 40 µg plant^?1 caused no direct effect, and at 160 µg plant^?1 reduced growth similarly at all O₃. Neither rate altered O₃ sensitivity. These additive responses are not consistent with protection by MeJA in this system. They may reflect inter‐specific differences in signalling, since O₃ concentrations used here exceeded some reported acute exposures. Alternatively, parallel responses to O₃ and MeJA may suggest that O₃‐induced jasmonates play a developmental role in chronic response but no protective role in the absence of lesions characteristic of acute exposure. MeJA appears useful as a probe of these mechanisms.