Autophagy regulates programmed cell death during the plant innate immune response

The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD.     

Accelerated cell death in Podospora autophagy mutants

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The DeltaPaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent protein-PaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in DeltaPaATG1 and DeltaPaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in DeltaPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.     

Programmed cell death in parasitic protozoans that lack mitochondria

In multicellular organisms and in all protozoans harbouring mitochondria, the pathways leading to programmed cell death (PCD) are localized in the mitochondria. Intriguingly, unicellular parasites devoid of mitochondria such as Trichomonas vaginalis and Giardia intestinalis undergo a form of cell death resembling apoptosis, the most frequent form of PCD. This reinforces the idea that PCD must have evolved before the evolution of multicellularity. Moreover, this leads to the hypothesis of an early emergence of death pathways in eukaryotes preceding mitochondrial endosymbiosis and brings into question the central role of mitochondria in PCD.     

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     

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore ( Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c , FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.     

Apoptotic-like regulation of programmed cell death in plants

In plants, apoptotic-like programmed cell death (PCD) can be distinguished from other forms of plant cell death by protoplast condensation that results in a morphologically distinct cell corpse. In addition, there is a central regulatory role for the mitochondria and the degradation of the cell and its contents by PCD associated proteases. These distinguishing features are shared with animal apoptosis as it is probable that plant and animal cell death programmes arose in a shared unicellular ancestor. However, animal and plant cell death pathways are not completely conserved. The cell death programmes may have been further modified after the divergence of plant and animal lineages leading to converged, or indeed unique, features of their respective cell death programmes. In this review we will examine the features of apoptotic-like PCD in plants and examine the probable conserved components such as mitochondrial regulation through the release of apoptogenic proteins from the mitochondrial intermembrane space, the possible conserved or converged features such as “caspase-like” molecules which drive cellular destruction and the emerging unique features of plant PCD such as chloroplast involvement in cell death regulation.     

Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner

Plant genomes harbor autophagy-related (ATG) genes that encode major components of the eukaryotic autophagic machinery. Autophagy in plants has been functionally linked to senescence, oxidative stress adaptation and the nutrient starvation response. In addition, plant autophagy has been assigned negative ('anti-death') and positive ('pro-death') regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. The role of autophagy in plant disease and basal immunity to microbial infection has, however, not been studied in detail. We have employed a series of autophagy-deficient genotypes of the genetic model plant Arabidopsis thaliana in various infection systems. Genotypes lacking ATG5, ATG10 or ATG18a develop spreading necrosis and enhanced disease susceptibility upon infection with toxin-producing pathogens preferring a necrotrophic lifestyle. These findings suggest that autophagy positively controls the containment of host tissue integrity upon infections by host-destructive microbes. In contrast, autophagy-deficient genotypes exhibit markedly increased immunity to infections by biotrophic pathogens through altered homeostasis of the plant hormone salicylic acid, thus suggesting an additional negative regulatory role of autophagy in plant basal immunity. In sum, our findings suggest that the role of plant autophagy in immunity cannot be generalized, and depends critically on the lifestyle and infection strategy of invading microbes.     

Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A₁ or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.     

Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A 1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.     

Autophagy and apoptosis in Chinese hamster ovary cell culture

Upon nutrient deprivation during Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. However, only apoptosis has drawn attention in the field of CHO cell culture. Numerous studies on engineering genes or supplementing essential nutrients or chemical additives to culture media to overcome cell death induced by various stimuli have been limited to apoptosis. Recently, autophagic morphologies were demonstrated by the processing of LC3 into the 16 kDa LC3-II form, and the accumulation of multiple autophagosomes in CHO cell culture. Therefore, it seems worthwhile to revisit the issue of cell death in CHO cell culture with the concept of autophagy in mind, in order to achieve a maximum production of foreign proteins by protecting cells from both types of PCD.     

Autophagy is required for gamete differentiation in the moss Physcomitrella patens

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.     

EDS1-mediated activation of autophagy regulates <Emphasis Type="Italic">Pst</Emphasis> DC3000 (<Emphasis Type="Italic">AvrRps4</Emphasis>)-induced programmed cell death in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Autophagy is a conserved intracellular process through which cytoplasmic components are degraded and recycled under stress conditions. In the innate immunity of higher plants, autophagy has either pro-survival or pro-death functions in pathogen-induced programmed cell death (PCD). In aged leaves, autophagy negatively regulates PCD by eliminating redundant salicylic acid. However, in young leaves, the specific pro-death mechanisms of autophagy and signaling pathways related to the autophagic process have not been elucidated. Here, we demonstrate that enhanced disease susceptibility 1 (EDS1) mediated the activation of autophagy and played a key role in the pro-death mechanism of autophagy during avirulent Pst DC3000 (AvrRps4) infection. The path through which autophagosomes enter the vacuole was blocked. Additionally, formation of the ATG12–ATG5 complex and the level of enzymatic activity associated with ATG8 cleavage decreased in eds1 mutants. The expression of EDS1 in atg5 mutants was also much lower than that in wild-type plants during pathogen-triggered PCD. These findings implied that EDS1 may regulate autophagy by affecting the activities of the two ubiquitin-like protein-conjugating pathways. Moreover, autophagy may regulate immunity-related PCD by affecting the expression of EDS1 in young plants. Our results provide important insights into the mechanisms of EDS1 in autophagy during infection with avirulent Pst DC3000 (AvrRps4) in Arabidopsis.     

Inhibition of autophagy reduces the rate of fluoride-induced LS8 apoptosis via regulating ATG5 and ATG7

Excessive fluoride affects ameloblast differentiation and tooth development. The fate of fluorinated ameloblasts is determined by multiple signaling pathways in response to a range of stimuli. Both autophagy and apoptosis are involved in the regulation of dental fluorosis as well as in protein synthesis and enamel mineralization. Emerging evidence suggests that autophagy and apoptosis are interconnected and that their interaction greatly influences cell death. However, the effect of autophagy on apoptosis in fluoride-treated ameloblasts is unclear. Here, we employed an in vitro cellular model of fluorosis in mouse ameloblast-like LS8 cells and induced autophagy using sodium fluoride (NaF). Our findings suggest that NaF treatment induces autophagy in LS8 cells, and ATG5 and ATG7 are important molecules involved in this process. We also showed that NaF treatment reduced cell viability in Atg5/7 siRNA and autophagy inhibitor-treated LS8 cells. More importantly, NaF-induced apoptosis can be reversed by inhibiting early stage of autophagy. In conclusion, our study shows that autophagy is closely related to dental fluorosis, and inhibition of autophagy, especially ATG5/7, reduces fluoride-induced cell death and apoptosis.     

Bcl-2 family members localize to tobacco chloroplasts and inhibit programmed cell death induced by chloroplast-targeted herbicides

In mammalian cells, apoptosis is often mediated via organelles. While apoptotic-like cell death occurs in plants, the mechanistic details are unresolved. Transgenic tobacco plants have been generated that harbour selected animal anti-apoptotic genes. Subcellular fractionation followed by western blot analysis indicated that chloroplasts serve as a location for these animal anti-apoptotic proteins in addition to the established mitochondrial location. To explore the functional significance of this observation, tobacco plants were treated with three chloroplast-directed herbicides. Wild-type plants died and exhibited features associated with apoptosis. Transgenic plants survived and did not show any apoptotic-like characteristics. Moreover, the herbicide-induced apoptotic-like cell death was light requiring. It was concluded that chloroplasts may be involved in mediating certain types of plant programmed cell death.     

Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans

Programmed cell death (PCD) is an essential and highly orchestrated process that plays a major role in morphogenesis and tissue homeostasis during development. In humans, defects in regulation or execution of cell death lead to diabetes, neurodegenerative disorders, and cancer. Two major types of PCD have been distinguished: the caspase-mediated process of apoptosis and the caspase-independent process involving autophagy. Although apoptosis and autophagy are often activated together in response to stress, the molecular mechanisms underlying their interplay remain unclear. Here we show that BEC-1, the C. elegans ortholog of the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin 1, is essential for development. We demonstrate that BEC-1 is necessary for the function of the class III PI3 kinase LET-512/Vps34, an essential protein required for autophagy, membrane trafficking, and endocytosis. Furthermore, BEC-1 forms a complex with the antiapoptotic protein CED-9/Bcl-2, and its depletion triggers CED-3/Caspase-dependent PCD. Based on our results, we propose that bec-1 represents a link between autophagy and apoptosis, thus supporting the view that the two processes act in concerted manner in the cell death machinery.     

Compensatory mechanisms and the type of injury determine the fate of cells with impaired macroautophagy

The relationship between the degradative process of autophagy and cellular death pathways remains unclear. Macroautophagy may potentially function to prevent or promote cell death, and both effects have been reported in studies of cells with a block in macroautophagy. To better delineate the function of macroautophagy in cell death, we contrasted the responses to death stimuli in wild-type and atg5(-/-) murine embryonic fibroblasts. We have reported that a knockout of the critical macroautophagy gene ATG5 sensitizes cells to death receptor ligand-induced death from Fas and tumor necrosis factor-alpha. Death occurs by caspase-dependent apoptosis resulting from activation of the mitochondrial death pathway. In contrast, atg5(-/-) cells are more resistant to death induced by oxidative stress from menadione or UV light. This resistance was associated with an upregulation of chaperone-mediated autophagy. Inhibition of this form of autophagy sensitizes cells to death from menadione, suggesting that the compensatory upregulation of chaperone-mediated autophagy, and not the loss of macroautophagy, prevents death from menadione. These findings demonstrate that the effects of a loss of macroautophagy on the cellular death response differ depending on the mechanism of cellular injury and the compensatory changes in other forms of autophagy.     

植物细胞程序性死亡的分类和膜通透性调控蛋白研究进展

程序性细胞死亡是由基因调控的贯穿于真核细胞生理和发育过程的细胞自杀行为。动物细胞的程序性死亡分成3类凋亡、自噬和坏死;线粒体和溶酶体分别在前两个过程中起关键作用。关于植物细胞程序性死亡的分类还存在很多争议,焦点是植物是否有细胞凋亡这种形式,核心问题是植物细胞的线粒体外膜上没有Bcl-2家族的膜通透性调控蛋白。近年,程序性细胞死亡也在细菌中发现,LrgAB家族的膜通透性调控蛋白起着重要作用。最近的研究表明,植物叶绿体外被膜上也有LrgAB家族的同源蛋白,它们在控制叶绿体发育和程序性细胞死亡方面起重要作用。因此,叶绿体在植物细胞死亡调控中的作用应该更加受到关注。     

Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development

Accumulating evidence supports the idea that necrotrophic plant pathogens interact with their hosts by controlling cell death. Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with a broad host range (>400 species). Previously, we established that oxalic acid (OA) is an important pathogenicity determinant of this fungus. In this report, we describe a mechanism by which oxalate contributes to the pathogenic success of this fungus; namely, that OA induces a programmed cell death (PCD) response in plant tissue that is required for disease development. This response exhibits features associated with mammalian apoptosis, including DNA laddering and TUNEL reactive cells. Fungal mutants deficient in OA production are nonpathogenic, and apoptotic-like characteristics are not observed following plant inoculation. The induction of PCD by OA is independent of the pH-reducing abilities of this organic acid, which is required for sclerotial development. Moreover, oxalate also induces increased reactive oxygen species (ROS) levels in the plant, which correlate to PCD. When ROS induction is inhibited, apoptotic-like cell death induced by OA does not occur. Taken together, we show that Sclerotinia spp.-secreted OA is an elicitor of PCD in plants and is responsible for induction of apoptotic-like features in the plant during disease development. This PCD is essential for fungal pathogenicity and involves ROS. Thus, OA appears to function by triggering in the plant pathways responsible for PCD. Further, OA secretion by Sclerotinia spp. is not directly toxic but, more subtly, may function as a signaling molecule.     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.