Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

TNFalpha and reactive oxygen species in necrotic cell death

Death receptors, including the TNF receptor-1 （TNF-RI）, have been shown to be able to initiate caspase-independent cell death. This form of ＂necrotic cell death＂ appears to be dependent on the generation of reactive oxygen species. Recent data have indicated that superoxide generation is dependent on the activation of NADPH oxidases, which form a complex with the adaptor molecules RIP1 and TRADD. The mechanism of superoxide generation further establishes RIP1 as the central molecule in ROS production and cell death initiated by TNFa and other death receptors. A role for the sustained JNK activation in necrotic cell death is also suggested. The sensitization of virus-infected cells to TNFα indicates that necrotic cell death may represent an alternative cell death pathway for clearance of infected cells.     

Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.     

The role of reactive oxygen species and autophagy in safingol-induced cell death

Safingol is a sphingolipid with promising anticancer potential, which is currently in phase I clinical trial. Yet, the underlying mechanisms of its action remain largely unknown. We reported here that safingol-induced primarily accidental necrotic cell death in MDA-MB-231 and HT-29 cells, as shown by the increase in the percentage of cells stained positive for 7-aminoactinomycin , collapse of mitochondria membrane potential and depletion of intracellular ATP. Importantly, safingol treatment produced time- and concentration-dependent reactive oxygen species (ROS) generation. Autophagy was triggered following safingol treatment, as reflected by the formation of autophagosomes, acidic vacuoles, increased light chain 3-II and Atg biomarkers expression. Interestingly, scavenging ROS with N-acetyl--cysteine could prevent the autophagic features and reverse safingol-induced necrosis. Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μ safingol treatment. In addition, Bcl-xL and Bax might be involved in the regulation of safingol-induced autophagy. Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression. Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.     

Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species

Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.     

Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue

The importance of reactive oxygen species for herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid)-induced cell death in roots was investigated. This was in order to understand its mode of action in grass species grown in the dark. Under these dark conditions, quinclorac suppressed the shoot and root growth of maize (Zea mays L. cv. Honey Bantam) in a concentration-dependent manner (50muM), although the inhibition level was less than that observed under growth conditions in the light. Analysis of cell viability using Evans blue or fluorescein diacetate-propidium iodide (FDA-PI) staining showed that the maize root cells significantly lost their viability after 14h root treatment with 10muM quinclorac, but not 10muM 2,4-dichlorophenoxyacetic acid (2,4-D). Determination of reactive oxygen species (ROS) in maize roots using a superoxide anion (O(2)(-))-specific indicator, dihydroethidium (DHE), indicated that 50muM quinclorac induced a high level of O(2)(-) production in maize roots after 14h root treatment than that of either the control (non-treated) or with 50muM 2,4-D. Moreover, either cell death or ethane evolution, an indicator of lipid peroxide formation, in maize root segments was significantly enhanced by 50muM quinclorac, but not by 50muM 2,4-D. On the other hand, the 50muM 2,4-D treatment induced much higher ethylene and cyanide production in the root segments than with the 50muM quinclorac. These results suggest that quinclorac-induced cell death in maize roots may be caused by ROS and lipid peroxidation, but not by ethylene and its biosynthetic pathway-related substances including cyanide, which have been thought to be the causative factor of quinclorac-induced phytotoxicity in susceptible grass weeds such as Echinochloa, Digitaria, and Setaria.     

A novel protein Jpk induces bacterial cell death through reactive oxygen species

Jpk, a trans-acting regulatory factor associating with the position-specific regulatory element of Hoxa-7, has been reported to induce cell death in both prokaryotic and eukaryotic cells upon overexpression. The N- and C-terminal deleted variants of Jpk were constructed and then the toxicity of each construct was analyzed by checking the viability of the cells and the concomitant morphological changes through electron microscopy following the expression. The N-terminus of Jpk harboring transmembrane domain seemed to be more toxic to bacterial cell than C-terminus and the morphology of bacterial cells expressing N-terminal Jpk was similar to that induced by full length Jpk. The toxicity caused by Jpk protein in bacterial cell was through the production of ROS, which was decreased by an antioxidant (DTT) in a concentration dependent manner. The finding described in this study provides valuable clues on the relationship between Jpk toxicity and ROS generation.     

Amplification of the gamma-irradiation-induced cell death pathway by reactive oxygen species in human U937 cells

Given the critical involvement of reactive oxygen species (ROS) in cell death, their hierarchical status in the cell pathway has been analyzed by many investigators. However, it has been shown that ROS can act either upstream or downstream of various death mediators depending on experimental settings. To investigate whether the contrasting relationships may exist in a single model system, human U937 cells were irradiated with lethal doses of gamma-rays. This resulted in a promotion of mitochondrial ROS production, which was found to be induced via sequential actions of c-Jun N-terminal kinase (JNK), Bax, and caspase-3. Interestingly, the induced ROS, in turn, re-activated JNK, Bax, and caspase-3 in the same model system. Consistently, the blockade of Bax action by RNA interference or Bcl-2 overexpression abolished the activation of JNK induced after, but not before, the production of ROS. Bcl-2 overexpression also blocked the translocation of Bax from the cytosol to the mitochondria only after the induction of ROS. Functional analyses revealed that the initial ROS-independent activations of JNK, Bax, and caspase-3 are not sufficient for cell death, and thus, should be re-activated by ROS in order to kill the cells. These findings suggest that ROS do not simply mediate the lethal action of gamma-irradiation, but actually amplify it by forming a feedback loop between a downstream effector caspase and the upstream initiation signals leading to the activation of JNK. This role for ROS appears to allow Bcl-2 to block the signaling events, which are initially induced upstream.     

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.     

Autophagy,programmed cell death and reactive oxygen species in sexual reproduction in plants

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.     

Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34⁺lin^?) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G₀ and G₂ phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.     

JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species

c-Jun N-terminal kinase (JNK), or stress-activated protein kinase, is an important member of the mitogen-activated protein kinase superfamily, the members of which are readily activated by many environmental stimuli. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important groups of free radicals that are capable of eliciting direct damaging effects or acting as critical intermediate signaling molecules, leading to oxidative and nitrosative stress and a series of biological consequences. Recently there has been an increasing amount of research interest focusing on the regulatory role of JNK activation in ROS-and RNS-induced cellular responses. In this review we will first summarize and discuss some recent findings regarding the signaling mechanisms of ROS-or RNS-mediated JNK activation. Second, we will talk about the role of JNK in ROS-or RNS-mediated cell death (both apoptosis and necrosis). Finally, we will analyze the emerging evidence for the involvement of ROS and RNS as mediators in tumor necrosis factor alpha-induced apoptosis. Taken together, the accumulating knowledge about the ROS/RNS-induced JNK signaling pathway has greatly advanced our understanding of the complex processes deciding the cellular responses to environmental stress.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.     

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.Key words: caspase-like activation, FRET, programmed cell death, reactive oxygen species, ultraviolet-CUltraviolet-C has been shown to be a very convenient trigger to induce PCD in plants and protoplasts.^1,2 Others have shown that UV induction of plant PCD requires light and that caspase-like proteolytic activation is induced in this process.¹ Our recent works have shown that ROS mainly localizing in mitochondria and chloroplasts are produced in a light dependent manner during the early stages of UV stress, and that ROS production and mitochondrial dysfunction play important roles during UV-induced Arabidopsis PCD (Fig. 1).² We also found that if the Arabidopsis plants, which were kept at light for 1 h after UV irradiation then were moved to the dark and kept for 60 h, showed no evident plant death phenomena (unpublished data), though burst of ROS has appeared after UV exposure and subsequent 1 h light irradiation.² In contrast, seedlings developed an obvious bleaching when kept in light for 60 h after UV treatment. These findings prompt us to carry out further investigations to dig out the role of ROS in the execution of this type of cell death, and to ask whether the produced ROS in the early stages is involved in the activation of caspase-like protease.Open in a separate windowFigure 1Hypothetical model of the signal transduction pathways in the plant programmed cell death induced by UV-C overexposure. After UV and light treatment a quick burst of ROS appear in the region of mitochondria and chloroplasts, then the mitochondria undergo functional dysfunction, which ultimately leads to cell death. Caspase-like activation and nucleus damage are also involved in the control of this type cell death. Solid line means the issues have been detected. Dotted line and question marks indicate events that have not been detected in this process. For detailed explanation, see the text.It has been reported that ROS is required for the release of cytochrome c (cyt c) and subsequent activation of caspase-like proteases during heat-shock induced plant PCD, and the addition of caspase inhibitors (zVAD-fmk or AC-DEVD-CHO) can prevent the degradation of cyt c and protect the plant cells from cell death.³ Thus these findings suggest that ROS can trigger the release of cyt c, but do not cause cell death, which requires caspase-like activation.³ Conversely, caspase inhibitors have also shown to effectively block the oxidative burst and the plant cell death induced by camptothecin incubation.⁴ These studies suggest the complex relationship between ROS production and caspase activation during execution of plant PCD event. The ROS production and the mitochondrial dysfunction during UV-induced plant PCD have been illustrated in our research. We have suggested the occurrence of MTP disruption during UV stress; however, whether cyt c is released from mitochondria has not been assessed (Fig. 1). The important roles of cyt c release and subsequent caspase activation have been suggested in various types of programmed cell death including mammal and plant cells.^3,5,6 It will be a very challenging work to detect whether cyt c is released from mitochondria and is involved in the caspase-like proteolytic activation, and to further elucidate the relationship between ROS production and caspase-like activation in UV-induced plant PCD (Fig. 1).The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD.⁷ Using synthetic fluorogenic caspase-3 substrate, DEVD cleavage activity was detected during UV or heat shock-induced apoptosis of plant cells, and caspase inhibitors were able to suppress these types of cell death.^1,3 Caspase-like activities have also been detected in plant hypersensitive response (HR) triggered by tobacco mosaic virus (TMV), or plant PCD induced by chemicals like camptothecin.^8,9 All these experiments suggest the existence of functional caspase proteolytic activity in plant cells undergoing PCD. However, most of these results are from in vitro analysis using synthetic fluorogenic substrates or synthetic peptide inhibitor to caspases, this demand us to further dig out the plant caspase encoding gene and to real-time detect the caspase-like activity in vivo.Another of our ongoing work is aiming to detect the caspase-3-like proteolytic activation in living plant cells during UV-induced plant PCD, which is achieved by using the fluorescence resonance energy transfer (FRET) technique. FRET is the phenomenon whereby a fluorescent molecule—the donor—transfers energy by a nonradiative (through space) mechanism to a neighboring chromophore - the acceptor.¹⁰ FRET as a powerful technique to monitor compartmentation and subcellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells has gained increasing importance for biotechnological applications during the last few years.¹¹ During the past few years FRET technique has been successfully used to monitor interactions and distances between molecules in living plant cells.^12–14 Presently, we have constructed a recombinant caspase substrate to monitor caspase-3-like protease activation in single living plant protoplast in real time. This recombinant is composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (ECFP-DEVD-EYFP) as the papers demonstrated. 15 Arabidopsis mesophyll protoplasts have been successfully transiently transfected with our recombinant plasmid for expression of ECFP-DEVD-EYFP fusion proteins under control of the CaMV 35S promoter according to a modified procedure (as described previously, ref. ¹⁶). Preliminary experimental results have proved the feasibility of this method to real-time detect the caspase-like activation in living plant cells during UV-induced plant PCD.Using this FRET probe, we may real-time detect the caspase-like activation during UV-induced plant PCD, and elucidate the relationship between ROS production and caspase-like activation as well as verify our hypothesis that whether ROS is necessary for the activation of caspase-like proteases during this process. So the role of ROS in the execution of this type cell death can be further investigated. These subsequent researches will certainly increase our knowledge about the signal transduction pathways in UV-induced Arabidopsis PCD.     

Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species

The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased susceptibility to AAL-toxin due to the knockout of a gene involved in sphingolipid metabolism. Genetic screening of mutagenized loh2 seeds resulted in the isolation of AAL-toxin-resistant mutant atr1.Atr1 displays a wild type phenotype when grown on soil but it develops less biomass than loh2 on media supplemented with 2% and 3% sucrose. Atr1 was also more tolerant to the reactive oxygen species-generating herbicides aminotriazole (AT) and paraquat. Microarray analyses of atr1 and loh2 under AT-treatment conditions that trigger cell death in loh2 and no visible damage in atr1 revealed genes specifically regulated in atr1 or loh2. In addition, most of the genes strongly downregulated in both mutants were related to cell wall extension and cell growth, consistent with the apparent and similar AT-induced cessation of growth in both mutants. This indicates that two different pathways, a first controlling growth inhibition and a second triggering cell death, are associated with AT-induced oxidative stress.