A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.     

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.     

An endo-1,4-β-glucanase expressed at high levels in rapidly expanding tissues

Plant developmental processes involving modifications to cell wall structure, such as cell expansion, organ abscission and fruit ripening, are accompanied by increased enzyme activity and mRNA abundance of endo-1,4--glucanases (EGases). An EGase cDNA clone, Ce14, isolated from tomato (Lycopersicon esculentum) has been shown to be identical to a tomato pistil-predominant EGase cDNA, TPP18. In addition to its previously reported expression during certain stages of early pistil development, Ce14 mRNA was also detected at high levels in the growing zones of etiolated hypocotyls (about 2.5-fold less than in pistils) and in young expanding leaves (about 3.5-fold less than in pistils). The abundance of Ce14 mRNA declined precipitously in older tissues as cells became fully expanded, and was barely detectable in mature vegetative tissues. Ce14 mRNA abundance was also low in abscission zones, and did not increase as abscission progressed. In fruit, Ce14 mRNA was present at low levels during fruit expansion, but was essentially absent during subsequent fruit development and ripening. Treatment of etiolated hypocotyls with ethylene or high concentrations of auxin sufficient to induce rapid lateral cell expansion and hypocotyl swelling also brought about an approximate doubling of Ce14 mRNA abundance, suggesting that Ce14 mRNA accumulation may be promoted directly or indirectly by ethylene. Thus, accumulation of Ce14 mRNA was found to be correlated with rapid cell expansion in pistils, hypocotyls and leaves.     

Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid

Oxalic acid is thought to be a key factor of the early pathogenicity stage in a wide range of necrotrophic fungi. Studies were conducted to determine whether oxalate could induce programmed cell death (PCD) in Arabidopsis thaliana suspension cells and to detail the transduction of the signalling pathway induced by oxalate. Arabidopsis thaliana cells were treated with millimolar concentrations of oxalate. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements. Involvement of the anion channel and ethylene in the signal transduction leading to PCD was determined by using specific inhibitors. Oxalic acid induced a PCD displaying cell shrinkage and fragmentation of DNA into internucleosomal fragments with a requirement for active gene expression and de novo protein synthesis, characteristic hallmarks of PCD. Other responses generally associated with plant cell death, such as anion effluxes leading to plasma membrane depolarization, mitochondrial depolarization, and ethylene synthesis, were also observed following addition of oxalate. The results show that oxalic acid activates an early anionic efflux which is a necessary prerequisite for the synthesis of ethylene and for the PCD in A. thaliana cells.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H₂O₂) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO₄. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H₂O₂ were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.     

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death

The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis. Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax. AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms. Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively. We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain. A Pseudomonas syringae pv. tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans. Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD.     

Cadmium toxicity in cultured tomato cells--role of ethylene, proteases and oxidative stress in cell death signaling

Our aim was to investigate the ability of cadmium to induce programmed cell death in tomato suspension cells and to determine the involvement of proteolysis, oxidative stress and ethylene. Tomato suspension cells were exposed to treatments with CdSO(4) and cell death was calculated after fluorescein diacetate staining of the living cells. Ethylene was measured in a flow-through system using a laser-driven photo acoustic detector; hydrogen peroxide was determined by chemiluminescence in a ferricyanide-catalysed oxidation of luminol. We have demonstrated that cadmium induces cell death in tomato suspension cells involving caspase-like proteases, indicating that programmed cell death took place. Using range of inhibitors, we found that cysteine and serine peptidases, oxidative stress, calcium and ethylene are players in the cadmium-induced cell death signaling. Cadmium-induced cell death in tomato suspension cells exhibits morphological and biochemical similarities to plant hypersensitive response and to cadmium effects in animal systems.     

Signal transduction events in aluminum-induced cell death in tomato suspension cells

In this study, some of the signal transduction events involved in AlCl(3)-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 microM AlCl(3) showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation. Cell death was effectively inhibited by protease and human caspase inhibitors indicating a cell death execution mechanism with similarities to animal apoptosis. Cell death was suppressed by application of antoxidants and by inhibitors of phospholipase C (PLC), phospholipase D (PLD) and ethylene signalling pathways. The results suggest that low concentrations of heavy metal ions stimulate both PLC and PLD signalling pathways leading to the production of reactive oxygen species (ROS) and subsequent cell death executed by caspase-like proteases.     

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.     

Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins

Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction.     

小麦淀粉胚乳发育期间的程序性细胞死亡

小麦淀粉胚乳在发育过程中经历程序性细胞死亡(PCD).小麦淀粉胚乳的DNA在发育的特定阶段呈现梯状电泳条带,用乙烯处理使DNA片段化发生的时间提前,而且ABA处理虽然不能推迟DNA片段化的发生时间,但能减弱DNA片段化的程度.小麦淀粉胚乳细胞在PCD过程中出现某些动植物细胞凋亡的共同的结构变化特征,但也有一些独特的结构变化.如染色质凝聚后仅少数染色质块发生趋边化;细胞核在PCD过程中最先开始衰退,细胞核解体时胞质中有丰富的细胞器,细胞核解体后细胞并未死亡,在胞质中仍在合成和积累淀粉和储藏蛋白,直到细胞被淀粉充满,细胞才死亡;不形成凋亡小体,死亡的淀粉胚乳细胞成为营养物质的储藏库.因此小麦淀粉胚乳细胞的PCD是一种特殊形式的PCD.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H₂O₂) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H₂O₂ accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H₂O₂ production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H₂O₂ accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.