1-Butanol triggers programmed cell death in Populus euphratica cell cultures

1-Butanol, which is a specific inhibitor of phospholipase D, usually inhibits phosphatidic acid (PA) production and blocks the PA-dependent signaling pathway under stress conditions. However, the effects of 1-butanol on plant cells under non-stress condition are still unclear. In this study, we report that 1-butanol induced a dose dependent cell death in poplar (Populus euphratica) cell cultures. In contrast, the control 2-butanol and ethanol had no effects on cell viability. 1-Butanol-treated cells displayed hallmark features of programmed cell death (PCD), such as shrinkage of the cytoplasm, DNA fragmentation, condensed or stretched chromatin and the activation of caspase-3-like protease. Exogenous application of PA markedly inhibited the 1-butanol-induced PCD. 1-Butanol also caused a burst of mitochondrial H₂O₂ ([H₂O₂]_mit) that was usually accompanied by a loss of mitochondrial membrane potential (?Ψm). Supplement of PA, antioxidant enzyme (catalase) and antioxidant (ascorbic acid) reversed this effect. Moreover, a significant increase of nitric oxide (NO) was observed in 1-butanol-treated poplar cells. This NO burst was suppressed by PA or inhibitors of NO synthesis. Further pharmacological experiments indicate that the burst of NO contributed to the 1-butanol-induced inhibition of antioxidant enzymes and subsequent H₂O₂-dependent PCD. In conclusion, we propose that 1-butanol is a potent inducer of PCD in plants and this process is regulated by the PA, NO and H₂O₂.     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

Interplay Among Nitric Oxide and Reactive Oxygen Species: A Complex Network Determining Cell Survival or Death

Programmed cell death (PCD) is an integrated cellular process occurring in plant growth, development, and defense responses to facilitate normal growth and development and better survival against various stresses as a whole. As universal toxic chemicals in plant and animal cells, reactive oxygen or nitrogen species (ROS or RNS), mainly superoxide anion (O₂^−•), hydrogen peroxide (H₂O₂) or nitric oxide (^•NO), have been studied extensively for their roles in PCD induction. Physiological and genetic studies have convincingly shown their essential roles. However, the details and mechanisms by which ROS and ^•NO interplay and induce PCD are not well understood. Our recent study on Cupressus lusitanica culture cell death revealed the elicitor-induced co-accumulation of ROS and ^•NO and interactions between ^•NO and H₂O₂ or O₂-^• in different ways to regulate cell death. ^•NO and H₂O₂ reciprocally enhanced the production of each other whereas ^•NO and O₂^−• showed reciprocal suppression on each other''s production. It was the interaction between ^•NO and O₂-^• but not between ^•NO and H₂O₂ that induced PCD, probably through peroxynitrite (ONOO⁻). In this addendum, some unsolved issues in the study were discussed based on recent studies on the complex network of ROS and ^•NO leading to PCD in animals and plants.Key Words: cell death, nitric oxide, reactive oxygen species, interaction, posttranslational modification     

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.     

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.     

The wound response in fresh-cut lettuce involves programmed cell death events

In this work, the involvement of programmed cell death (PCD) in the wound-induced postharvest browning disorder and senescence in butterhead lettuce (Lactuca sativa L.) fresh-cuts was studied. At the wounded (cut, bruised) sites, rapid browning, loss of chlorophyll and massive cell death, accompanied with accumulation of reactive oxygen species and increased electrolyte leakage occurred in a narrow strip of tissue adjacent the injury. The dead cell morphology (protoplast and nuclei shrinkage) together with the biochemical and physiological changes resembled necrotic PCD type. With a slight delay post-wounding, senescence associated with similar cell death features was initiated in distant non-wounded sites. In addition to necrotic PCD, both in wounded and senescing tissue, the appearance of empty cell corpses was observed, indicating that part of the cells might undergo vacuolar PCD (self-digestion of cellular content after vacuole collapse). The wounding-induced local cell death at the primary site of damage suggested that PCD may serve as a mechanism to seal-off the wound by building a physical barrier of dead cells. However, the cell death at sites remote from the wound suggests the distribution of long-distance senescence-inducing wound messengers. Trichomes in unwounded tissue often were the first to show H₂O₂ accumulation and dead cells; thereafter, the elevated H₂O₂ and cell death appeared in connecting cells and senescence progressed over larger areas. This suggests that trichomes may contribute to mediating the wound signalling leading to subsequent senescence. Our findings demonstrate that PCD is an integral part of the wound syndrome in fresh-cut lettuce.     

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H₂O₂)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.     

Cell death induced by sodium nitroprusside and hydrogen peroxide in tobacco BY-2 cell suspension

The interplay between nitric oxide (NO) and reactive oxygen species can lead to an induction of cell death in plants. The aim of our work was to find out if cyanide released from sodium nitroprusside (SNP; a donor of NO) could be involved in the cell death induction, which is triggered by SNP and H₂O₂. Cell suspension of Nicotiana tabacum L. (line BY-2) was treated with 0.5 mM SNP, 0.5 mM potassium ferricyanide (PFC; analogue of sodium nitroprusside which can not release NO) and/or by 0.5 mM glucose with 0.5 U cm⁻³ glucose oxidase (GGO; a donor system of H₂O₂). The cell death was induced only by combination of SNP and GGO. Thus cyanide released was not involved in the induction of cell death. However, SNP showed toxic effect because of decrease in activities of intracellular oxidoreductases and esterases. The cell death caused by SNP and GGO occurred within 12 h. During cell death either length or width of the cell increased. Central vacuole was formed in 20 to 40 % of cells. Most of the dead cells showed a condensed cytoplasm. Two hallmarks of programmed cell death (PCD), chromatin condensation and blebbing of nuclear periphery, were observed. However, oligonucleosomal fragmentation of DNA, another hallmark of PCD, was not detected.     

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.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Programmed cell death in seeds of angiosperms

During the diversi fication of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death(PCD) has played a fundamental role. However,examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus,suspensor and endosperm in those representative examples of seeds studied to date.     

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.     

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H₂O₂). In this study, the functions of NO and H₂O₂ after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H₂O₂ induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H₂O₂ generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H₂O₂ in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H₂O₂ to activate CuZnSOD and APX, which further decreased H₂O₂ level and reduced the cell death caused by wounding.     

Effects of exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> on the content of endogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript>, activities of catalase and hydrolases,and cell ultrastructure in tobacco leaves

It was shown that tobacco leaf treatment with 100 mM H₂O₂ increased their content of endogenous H₂O₂ and activities of catalase and hydrolases (acid phosphatase, proteases, and RNase) and also caused various changes in the cell structure. In this case, programmed cell death (PCD) occurred in some cells, which was observed as chromatin condensation, cytoplasm collapse, etc. In the meantime, many cells displayed organelle activation rather than PCD. It is suggested that cells that undergo H₂O₂-dependent PCD release signaling molecules inducing protective mechanisms against oxidative stress in neighboring cells not exhibiting PCD.     

Antioxidant system in programmed cell death of sycamore (<Emphasis Type="Italic">Acer pseudoplatanus</Emphasis> L.) cultured cells

Reactive oxygen species (ROS) have pleiotropic effects in plants. ROS can lead to cellular damage and death or play key roles in control and regulation of biological processes, such as programmed cell death (PCD). This dual role of ROS, as toxic or signalling molecules, is possible because plant antioxidant system (AS) is able to achieve a tight control over ROS cellular levels, balancing properly their production and scavenging. AS response in plant PCD has been clearly described only in the hypersensitive response in incompatible plant–pathogen interactions and in the senescence process and has not been completely unravelled. In sycamore (Acer pseudoplatanus L.) cultured cells PCD can be induced by Fusicoccin (Fc), Tunicamycin (Tu), and Brefeldin A (Ba). These chemicals induce comparable PCD time course and extent, while H₂O₂ production is detectable only in Fc- and, to a lesser extent, in Ba-treated cells. In this paper the AS has been investigated during PCD of sycamore cells, measuring the effects of the three inducers on the cellular levels of non-enzymatic and enzymatic antioxidants. Results show that the AS behaviour is different in the PCD induced by the three chemicals. In Fc-treated cells AS is mainly devoted to decrease the concentration of toxic intracellular H₂O₂ levels. On the contrary, in cells treated with Tu and Ba, the cell redox state is shifted to a more reduced state and the enzymatic AS is partially down-regulated, allowing ROS to act as signalling molecules.     

Respiratory burst oxidase homologue‐dependent H2O2 and chloroplast H2O2 are essential for the maintenance of acquired thermotolerance during recovery after acclimation

Thermotolerance is improved by heat stress (HS) acclimation, and the thermotolerance level is “remembered” by plants. However, the underlying signalling mechanisms remain largely unknown. Here, we showed NADPH oxidase‐mediated H₂O₂ (NADPH‐H₂O₂), and chloroplast‐H₂O₂ promoted the sustained expression of HS‐responsive genes and programmed cell death (PCD) genes, respectively, during recovery after HS acclimation. When spraying the NADPH oxidase inhibitor, diphenylene iodonium, after HS acclimation, the NADPH‐H₂O₂ level significantly decreased, resulting in a decrease in the expression of HS‐responsive genes and the loss of maintenance of acquired thermotolerance (MAT). In contrast, compared with HS acclimation, NADPH‐H₂O₂ declined but chloroplast‐H₂O₂ further enhanced during recovery after HS over‐acclimation, resulting in the reduced expression of HS‐responsive genes and substantial production of PCD. Notably, the further inhibition of NADPH‐H₂O₂ after HS over‐acclimation also inhibited chloroplast‐H₂O₂, alleviating the severe PCD and surpassing the MAT of HS over‐acclimation treatment. Due to the change in subcellular H₂O₂ after HS acclimation, the tomato seedlings maintained a constant H₂O₂ level during recovery, resulting in stable and lower total H₂O₂ levels during a tester HS challenge conducted after recovery. We conclude that tomato seedlings increase their MAT by enhancing NADPH‐H₂O₂ content and controlling chloroplast‐H₂O₂ production during recovery, which enhances the expression of HS‐responsive genes and balances PCD levels, respectively.     

Hydrogen peroxide is involved in nitric oxide‐induced cell death in maize leaves

Recent evidence indicates that nitric oxide (NO) plays an important role in plant hypersensitive cell death. Here, we report that NO treatment led to rapid cell death and induced hydrogen peroxide (H₂O₂) accumulation in maize leaves. We also show that NO induced the expression of Zmrboh genes. Pharmacological study suggests that NO‐induced cell death is in part mediated via H₂O₂. In addition, semi‐quantitative RT‐PCR revealed that NO induced expression of the systemic acquired resistance (SAR) genes, ZmPR1 and ZmPR5.     

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.     

Defence gene expression in soybean is linked to the status of the cell death program

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H₂O₂ or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.     

MEK/ERK inhibitor U0126 enhanced salt stress-induced programmed cell death in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> suspension-cultured cells

Programmed cell death (PCD) is an active cellular suicide that occurs both in animals and plants throughout development and in response to abiotic or biotic stress. In contrast to plant hypersensitive response-like cell death, little is known about the molecular machinery that regulates the halophyte plant PCD under high salinity stress. Since mitogen-activated protein kinases (MAPKs) are involved in plant response/tolerance to salt stress, and plant MAPK genes belong to the extracellular signal-regulated kinase (ERK) subfamily, we have investigated the role of ERK-like enzymes in high salinity stress-induced cell death in Thellungiella halophila. The data showed that ERK-like enzymes were early (10 min) and transiently activated under 300 mM NaCl stress. Pretreatment with 10 μM U0126, a special MEK/ERK inhibitor, resulted in a small but statistically significant increase of the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive nuclei in contrast to salt alone. The effects of U0126 on H₂O₂ production and cytochrome c (cyt c) release were also investigated. We found that the pretreatment with U0126 accelerated H₂O₂ production as well as cyt c release, and eventually enhanced cell death. The results suggest that ERK-like enzymes in Thellungiella halophila may act as a positive regulator of salt tolerance, as illustrated by pretreatment with U0126 which enhanced cell death under high salinity stress.