Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses

Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD.     

Apoptosis: Programmed cell death in health and disease

Apoptosis is a normal physiological cell death process of eliminating unwanted cells from living organisms during embryonic and adult development. Apoptotic cells are characterised by fragmentation of nuclear DNA and formation of apoptotic bodies. Genetic analysis revealed the involvement of many death and survival genes in apoptosis which are regulated by extracellular factors. There are multiple inducers and inhibitors of apoptosis which interact with target cell specific surface receptors and transduce the signal by second messengers to programme cell death. The regulation of apoptosis is elusive, but defective regulation leads to aetiology of various ailments. Understanding the molecular mechanism of apoptosis including death genes, death signals, surface receptors and signal pathways will provide new insights in developing strategies to regulate the cell survival/death. The current knowledge on the molecular events of apoptotic cell death and their significance in health and disease is reviewed.     

A cellular suicide strategy of plants: vacuole-mediated cell death

Programmed cell death (PCD) occurs in animals and plants under various stresses and during development. Recently, vacuolar processing enzyme (VPE) was identified as an executioner of plant PCD. VPE is a cysteine protease that cleaves a peptide bond at the C-terminal side of asparagine and aspartic acid. VPE exhibited enzymatic properties similar to that of a caspase, which is a cysteine protease that mediates the PCD pathway in animals, although there is limited sequence identity between the two enzymes. VPE and caspase-1 share several structural properties: the catalytic dyads and three amino acids forming the substrate pockets (Asp pocket) are conserved between VPE and caspase-1. In contrast to such similarities, subcellular localizations of these proteases are completely different from each other. VPE is localized in the vacuoles, while caspases are localized in the cytosol. VPE functions as a key molecule of plant PCD through disrupting the vacuole in pathogenesis and development. Cell death triggered by vacuolar collapse is unique to plants and has not been seen in animals. Plants might have evolved a VPE-mediated vacuolar system as a cellular suicide strategy.     

Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.     

细胞程序性死亡与生态适应

细胞程序性死亡是多细胞有机生命周期中正常的组成部分,细胞程序性死亡过程的存在对生物体是一种保护机制。它是在生物进化过程中形成的,也是生物对环境的适应方式之一。     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

Interdigital cell death function and regulation: new insights on an old programmed cell death model

Interdigital cell death (ICD) is the oldest and best-studied model of programmed cell death (PCD) in vertebrates. The classical view of ICD function is the separation of digits by promotion of tissue regression. However, in addition, ICD can contribute to digit individualization by restricting interdigital tissue growth. Depending on the species, the relative contribution of either regression or growth-restricting functions of ICD to limb morphogenesis may differ. Under normal conditions, most cells appear to die by apoptosis during ICD. Accordingly, components of the apoptotic machinery are found in the interdigits, though their role in the initiation and execution of cell death is yet to be defined. Fgf8 has been identified as a survival factor for the distal mesenchymal cells of the limb such that ICD can initiate following specific downregulation of Fgf8 expression in the ectoderm overlying the interdigital tissue. On the other hand, Bmps may promote cell death directly by acting on the interdigital tissue, or indirectly by downregulating Fgf8 expression in the ectoderm. In addition, retinoic acid can activate ICD directly or through a Bmp-mediated mechanism. Interactions at different levels between these factors establish the spatiotemporal patterning of ICD activation. Defining the regulatory network behind ICD activation will greatly advance our understanding of the mechanisms controlling PCD in general.     

Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells

Abstract

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H₂O₂-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H₂O₂ induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H₂O₂-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

植物细胞程序化死亡的形态学和生理生化特征

植物细胞程序化死亡(PCD)是一种由基因控制的、主动的细胞死亡过程,它在植物正常生长发育过程中起着重要作用。发生程序化死亡的植物细胞在形态、生理生化方面表现出一些共性特点和个性特点,该文对这些特点进行了综述。     

Neural activity and survival in the developing nervous system

Recent evidence suggests that blockade of normal excitation in the immature nervous system may have profound effects on neuronal survival during the period of natural cell death. Cell loss following depression of electrical activity in the central nervous system (CNS) may explain the neuropsychiatric deficits in humans exposed to alcohol or other CNS depressants during development. Thus, understanding the role of electrical activity in the survival of young neurons is an important goal of modern basic and clinical neuroscience. Here we review the evidence from in vivo and in vitro model systems that electrical activity participates in promoting neuronal survival. We discuss the potential role of moderate elevations of intracellular calcium in promoting survival, and we address the possible ways in which activity and conventional trophic factors may interact.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues

Recent studies have expanded our view of the interactions between small molecule signals that regulate the hypersensitive response and other forms of cell suicide in plants. The mitochondrion has received increasing support as a mediator of at least some forms of programmed cell death in plants. In addition, new information provides a glimpse of how plant hormone signaling may be integrated with extensive autolysis, sensitivity to reactive oxygen intermediates and cell death.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.