PERSPECTIVE DOI: 10.1038/s41477-017-0020-x Reassessing apoptosis in plants Martin Dickman1,2*, Brett Williams3*, Yurong Li1,2, Paul Figueiredo1,4,5 and Thomas Wolpert6,7 Cell death can be driven by a genetically programmed signalling pathway known as programmed cell death (PCD). In plants, PCD occurs during development as well as in response to environmental and biotic stimuli. Our understanding of PCD regulation in plants has advanced significantly over the past two decades; however, the molecular machinery responsible for driving the system remains elusive. Thus, whether conserved PCD regulatory mechanisms include plant apoptosis remains enigmatic. Animal apoptotic regulators, including Bcl-2 family members, have not been identified in plants but expression of such regulators can trigger or suppress plant PCD. Moreover, plants exhibit nearly all of the biochemical and morphological features of apoptosis. One difference between plant and animal PCD is the absence of phagocytosis in plants. Evidence is emerging that the vacuole may be key to removal of unwanted plant cells, and may carry out functions that are analogous to animal phagocytosis. Here, we provide context for the argument that apoptotic-like cell death occurs in plants. ulticellular organisms possess intrinsic and extrinsic neurodegenerative diseases, such as Alzheimer’s and Parkinson’s programs for cell suicide. Programmed cell death (PCD) (too much PCD)16,17. Mis genetically controlled either by so-called death receptors Apoptosis is the most intensively studied form of PCD and in or intrinsic signals that direct the cell to eliminate itself during animals occurs by two major pathways, extrinsic and intrinsic, development and in response to pathogens or environmental distinguished by the origin of the death signal. The extrinsic path- insults. It is thought that PCD arose during evolution in response to way begins with cell surface receptors that bind to death ligands viruses to prevent virion replication, thereby sparing surrounding to trigger an intracellular signalling pathway that results in death. cells1. Hallmarks of PCD have been observed in bacteria In contrast, the intrinsic pathway is controlled by signals generally and yeast, presumably selected for by viruses or other evolutionary emanating from mitochondria. Cells undergoing apoptosis display pressures2,3. The presence of prokaryote-derived chloroplasts and characteristic morphological and biochemical features that include mitochondria in plant cells, while yet to be shown as essential to chromatin condensation, DNA cleavage (ladders), TUNEL (termi- plant PCD, are major sources of reactive oxygen species (ROS) and nal deoxynucleotidyl transferase dUTP nick-end labelling)-positive have been implicated in plant PCD regulation4–7, with the latter nuclei, formation of apoptotic bodies, externalization of phos- organelle playing a crucial role in animal PCD. phatidylserine, and phagocytosis by macrophages (Table 1)18–20. Genes controlling PCD are conserved across wide evolutionary From these initial hallmark responses, it appeared that apoptosis distances from Caenorhabditis elegans to humans1,8. However, triggered the orderly dismantling and eventual death of the cell whether functional conservation extends to plants, and if so to in a linear fashion (Fig. 1). It has since become apparent there are what degree, is unclear and has been continuously debated in the a number of ways that cells can be programmed to die, including literature. PCD has physiological roles in many plant processes non-apoptotic forms of PCD such as ferroptosis, pyroptosis and and several reports suggest similarities to animal PCD9–15. Here, we necroptosis21. Thus, PCD can be viewed as a continuum, a fluid tar- discuss similarities and differences between plant and animal PCD get likely to increase in number and complexity. including evidence for and against the existence of a plant apoptotic response. Because this debate is still unresolved, we will use the Plant PCD is similar but different term ‘apoptotic-like’ when discussing potentially related forms of Like animals, plants use a suite of regulatory pathways to program PCD in plants. the demise of cells13,22–25. Plant PCD has been observed during development (for example, xylem formation, seed maturation, Defining cell death leaf senescence and several plant reproductive processes) and in There are many ways cells can die and not all cell death regulators plant–pathogen interactions (for example, Sclerotinia sclerotiorum, function equally. PCD is a continuum of cell death processes Cochliobolus victoriae, tobacco mosaic virus (TMV) and Botrytis ranging from highly orchestrated apoptosis at one end of the cinerea), as well as during environmental stresses (for example, spectrum to necrosis at the other. While an oversimplification, these drought, heat, cold and hypersalinity)6,26–33. class distinctions are important because the form of cell death plays The detection of animal apoptotic-like hallmarks in plants led a key role in dictating whether or not the outcome is beneficial to Fukuda et al. to classify plant PCD into three distinct categories, the organism. Inappropriate PCD regulation, as might be expected, (1) apoptotic-like cell death, (2) senescence-associated death, and can be disastrous and misregulation of PCD pathways can have (3) vacuole-mediated cell death34. Similarly, Reape and McCabe dis- dire consequences, such as cancers (too little PCD), or devastating cussed apoptotic-like cell death in reference to tapetal development 1 Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, Texas 77843, USA. 2 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA. 3 Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane 4001 QLD, Australia. 4 Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843, USA. 5 Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Texas A&M University, Bryan, Texas 77807, USA. 6 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA. 7 Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon 97331, USA. *e-mail: [email protected]; [email protected] NATURE PLANTS | www.nature.com/natureplants © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. PERSPECTIVE NATURE PLANTS with several differences of opinion. For a spirited discussion see Table 1 | Examples of apoptotic features associated with animal Madeo and Salvesen, who outline their criteria and views for and or plant cell death against the inclusion of metacaspases as caspases46,47. Apoptotic feature Animals Plants Refs. A feature associated with animal apoptosis is the generation of Cell shrinkage Yes Yes 11 apoptotic bodies. Apoptotic bodies are globular vesicles containing fragmented DNA that are detectable upon treatment with the DNA Chromatin condensation Yes Yes 96 stain propidium iodide in TUNEL-positive nuclei50. Once formed, Phosphatidylserine externalization Yes Yes 97 apoptotic bodies can be engulfed by phagocytes for ‘clean removal’ DNA laddering Yes Yes 4,30,52,53,98 (without inflammation)51. The presence of a rigid cell wall sur- TUNEL-positive nuclei Yes Yes 4,53,96 rounding plant cells presents a key difference from animal cells. As a (DNA cleavage) formidable barrier, the presence of the plant cell wall argues against Caspases or protease cell death Yes Yes 46,99,100 the necessity for breakdown of the cell into apoptotic-like bodies. signalling cascades Further, plants do not have phagocytes and cell walls block engulf- ment and removal of apoptotic cells by adjacent cells24. A reason- Mitochondria permeabilization and Yes Yes 6 able conclusion therefore, is that apoptotic bodies may be specific to depolarization animal PCD24. However, apoptotic-like bodies have been observed Cytochrome c release Yes Yes 64–66 in plant cells challenged with both abiotic and biotic elicitors52,53. Cytochrome-c-dependent Yes No 62 Figure 2 shows the presence of apoptotic body-like structures in activation of cell death tomato cells treated with a mycotoxin, Fumonisin B1 (a well-known 53 Apoptotic bodies Yes Yes 11,52 inducer of apoptosis) (Fig. 2a). Similar observations have been made in tomato cells subjected to heat stress52. Additionally, mem- Vacuole leakage and fusion with No Yes 75,79,101 plasmalemma brane blebbing, an established marker of animal apoptosis and the precursor to apoptotic body formation, was observed in carrot cell Note that the subset of features that are manifested during cell death can change depending on 54 the cell type and specific stimuli or circumstances that are supporting or promoting the cell death suspensions . Experiments demonstrated that at 55 °C the cells dis- process. Table reproduced from ref. 18. play a mechanistically different apoptotic-like cell death compared to cells incubated at higher temperatures, which displayed features indicative of necrotic cell death. Cells incubated at 55 °C, but not 85 °C, display apoptotic-like body formation. These observations in Lobivia rauschii10. Conversely and based largely on morphologi- illustrate that the type and strength of the signal can determine cal criteria, van Doorn et al. posed two classes of PCD, autolytic and which form of cell death ensues. However, it should be cautioned non-autolytic cell death23–25. These