Constitutive caspase-like machinery executes programmed cell death in plant cells

The morphological features of programmed cell death (PCD) and the molecular machinery involved in the death program in animal cells have been intensively studied. In plants, cell death has been widely observed in predictable patterns throughout differentiation processes and in defense responses. Several lines of evidence argue that plant PCD shares some characteristic features with animal PCD. However, the molecular components of the plant PCD machinery remain obscure. We have shown that plant cells undergo PCD by constitutively expressed molecular machinery upon induction with the fungal elicitor EIX or by staurosporine in the presence of cycloheximide. The permeable peptide caspase inhibitors, zVAD-fmk and zBocD-fmk, blocked PCD induced by EIX or staurosporine. Using labeled VAD-fmk, active caspase-like proteases were detected within intact cells and in cell extracts of the PCD-induced cells. These findings suggest that caspase-like proteases are responsible for the execution of PCD in plant cells.     

Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa

Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.     

Vacuolar proteases livening up programmed cell death

The molecular identity of the key executioners involved in controlling plant programmed cell death (PCD) has been elusive. In a recent paper published in Science, Hatsugai and coworkers reported that a well-characterized protease called VPE from the plant cell vacuole can cleave caspase-specific substrates and is required for cell death activation by tobacco mosaic virus. This work provides clear evidence for the importance of the vacuole in plant PCD and a novel regulatory function for this organelle as well as for VPE proteases.     

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

Caspase-like proteases regulate aluminum-induced programmed cell death in peanut

Recent evidence has proved that caspase protease activities are detected in both mammals and plants during programmed cell death (PCD). The characteristics and functions of caspase-like proteases play important roles in understanding the mechanisms of PCD in plants. In this work, we report firstly the involvement of caspase-like protease activities and effects in aluminum (Al) stress in two contrasting peanut genotypes. Caspase-like activities in the root tip cells of ‘Zhonghua 2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant) were detected using synthetic caspase substrates during Al-triggered PCD. Caspase-1-, -2-, -3-, -4-, -5-, -6-, -8- and -9-like proteases were found in peanut root tip cells. VDQQDase (caspase-2-like) and WEHD (caspase-5-like) were the first detected in the plants, and almost all of the caspase-like proteases were activated during Al-induced PCD, especially caspase-3-like and caspase-1-like, which was higher in ‘Zhonghua 2’ than in ‘99-1507’. The highest activity levels of caspase-3- and caspase-1-like proteases occurred 8 and 4 h after 100 µM Al treatment, respectively. Compared with 100 µM AlCl₃ treatment alone, specific caspase-3 protease inhibitor Ac-DEVD-CHO inhibited the increase of caspase-3-like protease activity, Al content, Hsr203j expression, cell death and DNA fragmentation, and the decrease in root growth induced by 100 µM AlCl₃ treatment, but it was more obvious in ‘Zhonghua 2’ than in ‘99-1507’. In conclusion, there were different caspase-like proteases in root tips of peanut, and caspase-3-like protease was a crucial executioner in Al-induced PCD. Its effects in the ‘Zhonghua 2’ genotype were higher than in ‘99-1507’. An improved model of the mechanism of Al-induced PCD and Al tolerance differences in different genotypes is proposed.     

Redox regulation in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.     

The lace plant: a novel model system to study plant proteases during developmental programmed cell death in vivo

Programmed cell death (PCD) plays a major role in plant development and defense throughout the plant kingdom. Within animal systems, it is well accepted that caspases play a major role in the PCD process, although no true caspases have yet to be identified in plants. Despite this, vast amounts of evidence suggest the existence of caspase-like proteases in plants. The lace plant (Aponogeton madagascariensis) forms perforations in a predictable pattern between longitudinal and transverse veins over its entire leaf surface via PCD. Due to the thin nature of the leaf, allowing for long-term live cell imaging, a perfected method for sterile culture, as well as the feasibility of pharmacological experiments, the lace plant provides an excellent model to study developmental PCD. In this review, we report the suitability of the lace plant as a novel organism to study proteases in vivo during developmentally regulated cell death.     

Caspase-like proteases and their role in programmed cell death in plants

The investigations performed over recent few years have proved the existence of caspase-like proteases in plants. Three groups of caspase-like proteases: metacaspases, legumain family proteases (VPEs) and saspases have been identified and characterized in plants so far. A considerable amount of evidence supports the role of these enzymes in programmed cell death (PCD) occurring during plant development, their organ senescence as well as hypersensitive response (HR) after pathogen attack. Current knowledge of these enzyme molecular and biochemical structures is summarized in the paper. The homology of caspase-like proteases to animal caspases has been also indicated. Some future perspectives of research concerning the signal pathway during PCD, the regulation of activity and mode of action of these proteases are presented in the article.     

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS- induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.     

Phytaspases: Aspartate-specific proteases involved in plant cell death

Structures and properties of the recently found aspartate-specific cell death-related plant proteases called phytaspases are reviewed and compared to those of caspases, animal apoptotic proteases. Caspases (cysteine-dependent proteases) and phytaspases (serine-dependent proteases) dramatically differ in structure, although manifest a similar substrate specificity and a play a similar role in the programmed cell death. Distinctions in the structural organization of animal and plant death proteases were shown to define differences in the regulation strategies of functioning of these proteolytic enzymes in the two kingdoms.     

Plant mitochondrial pathway leading to programmed cell death

Programmed cell death (PCD) is a finely tuned process of multicellular organisms. In higher plants, PCD regulates many developmental processes and the response of host plants to incompatible pathogens (hypersensitive response). Four types of PCD have been described in plants, mainly associated to vacuole rupture, that is followed by the appearance of the typical PCD hallmarks (i.e. nuclear DNA fragmentation and cell shrinkage). However, in some cases vacuole collapse is preceded by an early alteration of other subcellular organelles, such as mitochondria. In particular, the central role played by mitochondria in PCD has been largely recognised in animal cells. This review deals with the involvement of mitochondria in the manifestation of plant PCD, in comparison to that described in animal PCD. The main hallmark, connecting animal and plant PCD via mitochondria, is represented by the release of cytochrome c and possibly other chemicals such as nucleases, which may be accomplished by different mechanisms, involving both swelling and non-swelling of the organelles.     

PGPR regulate caspase-like activity,programmed cell death,and antioxidant enzyme activity in paddy under salinity

The response of two root associated bacteria Pseudomonas pseudoalcaligenes and Bacillus pumilus were studied in the (salt-sensitive) rice GJ17 cultivar to salinity under controlled environmental growth conditions for protection of plant from adverse effect of salinity. Salinity affects the growth of salt-sensitive cultivar, but inoculation of plant growth promoting rhizobacteria (PGPR) reduces the harmful effect of salinity. The present study states that PGPR helps to reduce lipid peroxidation and superoxide dismutase activity in salt-sensitive GJ17 cultivar under salinity and play an important role in the growth regulation for positive adaptation of plants to salt stress. This study shows that inoculation of paddy (Oryza sativa) with such bacteria could provide salt-tolerant ability by reducing the toxicity of reactive oxygen species by reducing plant cell membrane index, cell caspase-like protease activity, and programmed cell death and hence resulted in increase cell viability. As these isolates remain associated with the roots, the effects of tolerance against salinity are observed here. Results also indicate that isolated PGPR strain help in alleviating up to 1.5 % salinity stress as well as improve tolerance.     

Evidence for programmed cell death and activation of specific caspase-like enzymes in the tomato fruit heat stress response

The tomato (Lycopersicon esculentum) fruit is the best available model to study the stress response of fleshy fruit. Programmed cell death (PCD) plays an important role in stress responses in mammals and plants. In this study, we provide evidence that PCD is triggered in the tomato fruit heat stress response by detection of the sequential diagnostic PCD events, including release of cytochrome c, activation of caspase-like proteases and the presence of TUNEL-positive nuclei. Investigating the time course of these events for 12 h after heat treatment indicated that cytochrome c release and caspase-like protease activation occurred rapidly and were consistent with the onset of DNA fragmentation. In addition, LEHDase and DEVDase enzymes were specifically activated in tomato fruit pericarp during the heat treatment and recovery time. There was no significant activation of YVADase or IETDase proteases. Preincubation of pericarp discs with the broad-spectrum, cell-permeable caspase inhibitor Z-VAD-FMK, suppressed heat-induced cell death measured by trypan blue, accompanied by a decrease in LEHDase and DEVDase activities. Gui-Qin Qu and Xiang Liu contributed equally to this work.     

Organisation and regulation of the cytoskeleton in plant programmed cell death

Programmed cell death (PCD) involves precise integration of cellular responses to extracellular and intracellular signals during both stress and development. In recent years much progress in our understanding of the components involved in PCD in plants has been made. Signalling to PCD results in major reorganisation of cellular components. The plant cytoskeleton is known to play a major role in cellular organisation, and reorganization and alterations in its dynamics is a well known consequence of signalling. There are considerable data that the plant cytoskeleton is reorganised in response to PCD, with remodelling of both microtubules and microfilaments taking place. In the majority of cases, the microtubule network depolymerises, whereas remodelling of microfilaments can follow two scenarios, either being depolymerised and then forming stable foci, or forming distinct bundles and then depolymerising. Evidence is accumulating that demonstrate that these cytoskeletal alterations are not just a consequence of signals mediating PCD, but that they also may have an active role in the initiation and regulation of PCD. Here we review key data from higher plant model systems on the roles of the actin filaments and microtubules during PCD and discuss proteins potentially implicated in regulating these alterations.     

Plant programmed cell death and the point of no return

The point of no return during programmed cell death (PCD) is defined as the step beyond which the cell is irreversibly committed to die. Some plant cells can be saved before this point by inducing the formation of functional chloroplasts. A visibly senescent tissue will then become green again and live for months or years. The mechanism of this reversal is only partially known. The point of no return in fungi and animals is often associated with lack of mitochondrial function. In plant cells that do not regreen, there is no evidence for PCD reversal that results in a long life. It is unclear why chloroplast-containing cells, in contrast to those with only mitochondria, have long lives after PCD reversal.