Cysteine and serine proteases are prominent players in the control of developmental and pathogen-activated cell deaths in plants. Ethylene, salicylic acid, the small G-protein Rac, calcium and reactive oxygen species are recurring mediators of death signaling. Lastly, the mitochondrion has emerged in both plant and animal systems as a ‘central depot’ that interprets multiple signals and in some instances determines the fate of the cell.
Addresses
Biotech Center, Foran Hall, Cook College, 59 Dudley Road, Rutgers University, New Brunswick, NJ 08903, USA
*e-mail: lam@aesop.rutgers.edu
Current Opinion in Plant Biology1999, 2:502–507
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.
Abbreviations AA antimycin A
AEBSF 4-(2-aminoethyl)-benzenesulfonyl-flonyl-fluoride
AOX Alternative oxidase
BI-1 Bax inhibitor-1
HR hypersensitive response
NO nitric oxide
PCD programmed cell death
PR pathogenesis-related
ROS reactive oxygen species
SA salicylic acid
SHAM salicylhydroxamic acid
TE tracheary element
VDAC voltage dependent anion channel
Introduction
The study of programmed cell death (PCD) has seen a remarkable boom in the past 10 years since the early recog-nition in the 1970’s by Andrew Wyllie and his coworkers that a variety of animal cells die with similar morphological features which appeared to be the result of an active suici-dal process (for review see [1]). This renaissance of PCD research was brought about by the convergence of work on PCD with studies of animal genes involved in oncogenesis such as Bcl-2, and genetic research on cell death mutants by Bob Horvitz and his coworkers in the nematode Caenorhabditis elegans (reviewed in [1]). The realization that molecules with similar biochemical functions and structur-al domains are conserved in diverse organisms to regulate PCD convinced most skeptics that this process can indeed be studied at the molecular level with defined players. Remarkably, although many signaling pathways and regu-lators of animal PCD have been defined in the past 10 years, they revolved around only a small number of cen-tral players and their modes of action appeared to be variations on a few themes. The central enzymatic activity that has been most well-characterized is a growing family of cysteine proteases called caspases. They are synthesized
as zymogens that require specific cleavage and subsequent oligomerization in order to reveal their enzymatic activi-ties. Once activated via proteolysis, they appear to activate a cascade of proteases in a serial fashion that is reminescent of the classic blood clotting factor cascade.
PCD research in plants has also begun to blossom in the past five years [2,3]. There are three major reasons for our interest in this process in plants. First, it is a way of life for plants, just as it is for animals. Essential processes such as xylem differentiation and tapetal cell degeneration all involve PCD. Second, comparison of the mechanisms and molecules that are involved in the two kingdoms should enlighten us about the evolution of PCD and may help to determine the indispensible players in the chain of events that lead to cell death in eukaryotes. Third, from an applied perspective, the ability to regulate cell death in plants may have important applications in agriculture and post-harvest industries. For example, suppression of PCD induced by biotrophic pathogens could minimize disease symptoms and inhibition of cell death during senescence may prolong the shelf-life of crops and vegetables. In this review, we discuss work published in the past year that has begun to reveal some of the players that are involved in controling PCD in plant cells.
The demise of a cell — the phenomenon
What actually occurs when a plant cell commits suicide? In contrast to apoptosis in animals, the presence of a rigid wall in plant cells poses a distinct problem: because the dying cell cannot be packaged into small apoptotic bodies and then engulfed by its neighbors, as in the case for their ani-mal counterparts, plants must deal with suicidal cells and their contents in fundamentally different ways. A distin-guishing feature of plant cells is the prominant vacuole, which contains many catabolic enzymes such as proteases and nucleases. It is thus not surprising that this organelle appears to play an important role in tracheary element (TE) differentiation [4••] as well as degeneration of the aleurone in barley [5•]. The disruption of the vacuole dur-ing TE development appears to catalyze the autolysis of the cell’s content in a systematic fashion after the synthe-sis of the reticulated secondary cell wall [4••,6].
The self-ingestion approach to the dismantling of cells destined for PCD is likely to be a common strategy for plants. However, the sequence of events and the resulting cellular morphological changes may differ depending on the particular inductive death signal [2,3,6–9]. This may be a consequence of the role that cell death may have in dif-ferent situations. In the case of developmental cell death such as senescence and TE differentiation, the recycling of the dead cell’s content may be an important ‘purpose’. In the case of cell death related to pathogen containment, cell
Die and let live — programmed cell death in plants
death may be used to rapidly ‘isolate’ the infected cells and efficient transport of the dead cell’s content may be secondary [10]. In most cases of plant PCD, vacuolization of the cytoplasm and disruption of the tonoplast are com-mon events that appear to precede mass disruption of the mitochondria and nucleus. On the other hand, recent flow cytometry studies with tobacco protoplasts suggest that nuclear DNA condensation could be activated before the first irreversible step of PCD [11•]. Evidence for disrup-tion of the cytoskeleton early on in plant PCD has also been suggested by the finding that disruption of microfila-ments by cytochalasin E could suppress cell death induced by fungal pathogens in cowpea [12] and that cytoplasmic streaming is arrested during TE differentiation [4••].
What’s new in PCD signaling in plants?
The signaling processes that activate PCD in plants promise to be as diverse as those that have been found in animals. One of the most well-characterized systems to date is the differentiation of Zinnia TEs in vitro. Earlier work with the Zinnia TE system has established that wounding and hormonal balance (auxin and cytokinin treatments) are critical signaling components for initiating the transformation of mesophyll cells to TE [6]. More recently, the involvement of calcium, heterotrimeric G-proteins and extracellular proteases has been suggested [4••].
In contrast to other plant PCD systems, a large body of literature has accumulated on the study of cell death dur-ing plant pathogen interactions [2,3,7,13]. Two major types of cell death can result when plants are inoculated with a pathogen: the rapid hypersensitive response (HR), which is typically associated with resistance, and a slower form of death that is the result of disease. Previous work has implicated the involvement of protein phosphoryla-tion, calcium channel functions and reactive oxygen species (ROS) in HR cell death. Studies of disease-relat-ed cell death are less developdisease-relat-ed as it is not obvious that they resulted from a suicide process. However, recent work with the fungal toxin victorin has implicated an active participation of the dying cells and more particu-larly, the requirement of ethylene in disease-related cell death induction [14•]. Consistent with these observa-tions, ethylene insensitivity in tomato was found to suppress cell death due to infection with biotrophic pathogens [15]. These observations raised the possibility that ethylene action may be involved in the slower cell death that is manifested during plant diseases. In con-trast, resistance response does not appear to require ethylene action [16]. Interestingly, ethylene may also be involved in developmental cell death such as endosperm degeneration in wheat and during maize aerenchyma for-mation induced by hypoxia [9,17]. Taking into account its classic role in tissue senescence, it appears that ethylene may be an important signal for slower forms of cell death during which efficient recycling of the dead cell’s content might be important for the plant. This interpretation is
consistent with our recent observation that a senescence-specific marker gene, SAG12, is activated in cells neighboring HR lesions, where chlorotic symptoms of cell death are often observed [10].
Salicylic acid (SA) has emerged in the past several years as a positive feedback regulator of cell death during the HR [2,3]. This was first suggested in the study of disease lesion mimics in Arabidopsis [18], where spontaneous cell death in the mutants lsd6 and lsd7 was repressed by transgenic expression of a gene encoding a bacterial salicylate hydrox-ylase (NahG), which converts SA to catechol. Two new disease lesion mimic mutants, ssi1 and acd6, appeared to show the same dependence on SA [19•,20••]. Interestingly, ozone induced cell death in Arabidopsis has also been shown to be potentiated by SA [21]. A cell wall associated protein kinase has recently been suggested to be involved in suppressing cell death signaling by SA [22•].
The specific requirement of calcium signaling in HR cell death had been suggested through studies using calcium channel blockers [2,3,7]. The recent work of Heo et al. [23••] suggested that this dependence on calcium may involve specific isoforms of calmodulin. HR-like cell death, pathogenesis-related (PR) gene induction and broad spectrum disease resistance are activated by trans-genic expression of two soybean calmodulin-encoding genes. These phenotypes are not SA dependent and the level of SA is not altered by the transgenes. Thus, distinct pathways can give rise to phenotypes that are morphologi-cally very similar. As these soybean genes are induced during the defense response, they may participate in the long term signaling for resistance rather than the initial sig-naling process to activate the HR pathway.
manner. Rac is known to regulate mammalian NADPH oxidase as well.
How does ROS lead to the activation of cell death and how is it managed in the cell? Reichheld et al. [32] suggest that alteration of the cell cycle may be one aspect of death induction by ROS. Interestingly, the role of SA in cell pro-liferation as well as cell death was postulated to explain some of the abnormal leaf morphologies observed in the acd6 mutant [20••]. Thus, SA and ROS may cooperate to arrest the cell cycle and tip the balance toward cell death. As plants are likely to be exposed to a variety of agents or environmental conditions that can activate ROS produc-tion, a buffering system that can deal with normal fluctuations in ROS level should be in place to prevent chance activation of PCD. The work of Tamagnone et al. [33•] showed that transgenic expression of a transcription regulator leads to plant cells that are hyper-responsive to pathogen challenge, abnormal cell expansion and PCD in the leaf palisade cells, and increase in lipid peroxidation. Addition of phenolic precursors can rescue cell cultures derived from these transgenic plants from cell death and abnormal morphologies, thus showing nicely that these phenotypes are reversible. This work provided evidence that phenolic products could play an important role in ROS homeostasis during normal plant development.
The nuts and bolts of the death engine
The identities of the key executioners of plant cells remain elusive, whereas in animal systems a large number of caspases and their regulators have been defined in the past five years [34•,35••]. Caspase-like proteolytic activity has been observed to be transiently activated in plants synchronized to undergo the HR [36••]. Peptide inhibitors of caspases can abolish HR cell death induced by aviru-lent bacteria without affecting the induction of defense genes significantly. Furthermore, transgenic expression of the gene encoding a broad range caspase inhibitor from Baculovirus, p35, also leads to the delay of HR cell death in tobacco (O del Pozo, E Lam, unpublished data). More recently, Mitsuhara et al. [37•] reported that in tobacco expression of transgenes encoding the pro-survival cell death regulators Bcl-xL and Ced-9 can delay HR cell death as well as death induced by UVB and paraquat, whereas expression of the transgene encoding the pro-apoptotic Bax is found to activate HR-like cell death [38••]. These observations suggest the possibility that cas-pase-like proteases and a Ced-4/APAF1 like regulatory switch may operate in plants to control cell death [35••]. However, alternative explanations for these results are possible (see below).
In addition to caspases, other cysteine and serine proteas-es may also be involved in HR cell death. Solomon et al. [39•] found that expression of the cysteine protease inhibitor cystatin can suppress ROS and bacteria-induced cell death in soybean cell cultures, while activation of cell death by the xylanase from Trichoderma viride can be
inhibited by the serine protease inhibitor AEBSF (4-[2-aminoethyl]-benzenesulfonyl-flonyl-fluoride), but not by caspase inhibitors [40]. Activation of cell death by victorin in oat also appears to be mediated by cysteine proteases sensitive to E-64 and calpeptin [14•]. These studies sug-gest that proteases with different specificities may be used to regulate cell death induced by different agents in different species. Involvement of intracellular cysteine proteases [6] as well as an extracellular serine protease [4••] have also been proposed to regulate cell death acti-vation during TE differentiation.
In recent years, the role of mitochondria in controlling cell death activation has been recognized in the animal field. Cytochrome c leakage from this organelle appears to mediate many signaling pathways for PCD. Once in the cytosol, cytochrome c can activate caspases through inter-action with Ced-4/APAF1 in conjunction with dATP [35••]. In addition, disruption of electron flow through the electron transport chain of the mitochondria is likely to lead to accumulation of reducing equivalents that can result in ROS. Bcl-2 related proteins can either activate or repress the leakage of cytochrome c through the voltage dependent anion channel (VDAC) located in the mem-brane of the mitochondria [41]. They can also suppress or activate caspases through interaction with the Ced-4/APAF1 regulator.
In plants, the fungal toxin victorin appears to induce cell death via inhibition of the mitochondrial enzyme glycine decarboxylase [14•]. More recently, the mitochondrial con-nection to PCD in plants was implicated by the work of Lacomme and Santa Cruz [38••]. Bax was found to activate HR-like cell death and its localization to plant mitochon-dria was required. Bax expression was also known to induce cell death in yeast with similar dependence on its targeting to the mitochondria [42]. Thus, it appears that this organelle may be a conserved site where death signals can be generated in eukaryotes. This may act via leakage of cytochrome c, which has not been demonstrated in plant PCD or in yeast. It may also be mediated by other cell death factors that bypass the caspase switch and act direct-ly at the nuclear level [43••]. This type of cell death activation may be responsible for caspase-independent PCD pathways that can be modulated by Bcl-2 [44]. Interestingly, using the phenotype of Bax expressing yeast, a novel gene called Bax inhibitor (BI)-1 was cloned from human cells [42]. BI-1 appears to be well conserved in ani-mals and genes with weak sequence homology to BI-1 have been identified in C. elegansand Arabidopsissequence databases. Although its function is unclear at present, BI-1 may be an ancient regulator of cell death activation through the mitochondria.
established. However, plants suppressed in AOX expres-sion remain largely normal until stressed by inhibitors of the mitochondrial electron transport chain such as antimycin A (AA). The recent paper by Maxwell et al. [45••] showed that AOX suppressed plants produce rapid and dra-matic amounts of ROS in their mitochondria upon treatment with AA. Moreover, they showed that AOX-defi-cient plants constitutively expressed defense gene markers whereas overexpressors of AOX repressed the basal expres-sion levels of these genes. Thus, ROS generated in the mitochondria can lead to the induction of PR gene expres-sion. Furthermore, these observations suggest that interruption of normal electron transport functions leads to rapid generation of ROS and the role of AOX may be to act as a safety valve to ‘siphon’ off the reducing intermediates from the clogged electron transport chains. Consistent with this idea is the observation that treatments with inhibitors of oxidative electron transport such as AA and cyanide acti-vate AOX expression [46••]. In addition, mutations or transgenes that lead to an impairment of heme biosynthe-sis result in spontaneous lesion formation [13,47,48]. The disruption of heme biosynthesis may lead to a loss of func-tional cytochromes and excess ROS generation from the mitochondria. Together with expected defects in ROS pro-tectants that require heme as their prosthetic group such as catalases and peroxidases, it is plausible that in these plants ROS can easily reach a threshold sufficient to activate cell death spontaneously. Induction of AOX has been observed under various stress conditions and a recent study in Arabidopsis showed that rapid localized AOX induction by avirulent bacterial pathogens requires SA [49••]. On the other hand, ethylene is absolutely required for AOX induc-tion by pathogens. This work suggests that localized induction of AOX may act to protect the cells at or near the infection sites against ROS damage. This work comple-mented the studies of Chivasa and Carr [46••] who showed that cyanide treatment, which activates AOX synthesis, can reverse the spreading cell death phenotype observed in NahG expressing tobacco plants. The effects of cyanide can be reversed by salicylhydroxamic acid (SHAM), an inhibitor of AOX. Obviously, the results with chemical inhibitors have to be interpreted cautiously since SHAM can also inhibit other enzymes such as peroxidases that may function to protect against ROS. Nevertheless, it is tempt-ing to speculate that AOX may be involved in cell death regulation in the development of spreading lesions such as those observed during tobacco mosaic virus (TMV) infec-tion. Future manipulation of AOX levels should lead to a better understanding of the role for this protein in deter-mining lesion size and disease resistance.
Conclusions
The mitochondrion has emerged recently as a conserved site where cell death signals in the form of ROS and protein factors can be generated. Our understanding of its role in plant PCD will be aided by comparative analyses with results from animal and yeast fields, in addition to the wealth of genomic information that is rapidly accumulating.
Ultimately, forward or reverse genetic definition of candi-dates for cell death regulation will be required to sort out the complex interplay between the varieties of regulators that are likely to control this important process.
Acknowledgements
Research on cell death in our laboratory is funded in part by the New Jersey Commission on Science and Technology, and a competitive research grant from the United States Department of Agriculture. Sharing of preprints and unpublished information by Jean Greenberg, Ko Shimamoto and Yuko Ohashi is gratefully acknowledged.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
••of outstanding interest
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The gene encoding the pro-apoptotic regulator Bax from mammalian sys-tems is expressed in tobacco by a tobacco mosaic virus (TMV) viral vector and found to induce hypersensitive-response-like phenotypes such as cell death and pathogenesis-related gene expression. Mutational analyses show that the likely target site of Bax in plants is the mitochondria and that domains required for Bax dimerization are required for optimal cell death induction. This work provides evidence that plant mitochondria can be the site of origin for signals leading to hypersensitive-response-like phenomena. 39. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A: The
• involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants.Plant Cell
1999, 11:431-444.
Cell death induction in soybean cells by oxidative stress or avirulent bacter-ial pathogen was shown to depend on protein synthesis and multiple pro-teases are induced. Ectopic expression of the soybean cysteine protease inhibitor cystatin resulted in suppression of programmed cell death. This work showed that a cysteine protease or proteases that can interact with cystatin may be good candidates for activities that are required to execute the death signal upon oxidative stress and during the HR in this system. 40. Yano A, Suzuki K, Shinshi H: A signaling pathway, independent of
the oxidative burst, that leads to hypersensitive cell death in cultured tobacco cells includes a serine protease.Plant J1999, 18:105-109.
41. Shimizu S, Narita M, Tsujimoto Y:Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.Nature1999, 399:483-487.
42. Shaham S, Shuman MA, Herskowitz I:Death-defying yeast identify novel apoptosis genes.Cell1998, 92:425-427.
43. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
•• Mangion J, Jacotot E, Costantini P, Loeffler M et al.: Molecular characterization of mitochondrial apoptosis-inducing factor.
Nature1999, 397:441-445.
A flavoprotein that has homology to bacterial oxidoreductases was purified from mouse liver mitochondria. It is a mitochondrial protein that is released by the transition pore which can be regulated by Bcl-2. Purified apoptosis inducing factor (AIF) can induce apoptotic phenotypes in isolated nuclei as well as activate cytochrome c release from mitochondria in whole cells with subsequent caspase activation. AIF thus may function directly in inducing nuclear events as well as serving to amplify cell death signals. It may be involved in caspase-independent cell death signaling as AIF activity in microinjected cells is insensitive to the broad range inhibitor Z–VAD–fmk. (Z—Val—Ala—Asp—fluoromethylketone).
44. Okuno S, Shimizu S, Ito T, Nomura M, Hamada E, Tsujimoto Y, Matsuda H: Bcl–2 prevents caspase-independent cell death.
J Biol Chem1998, 273:34272-34277.
45. Maxwell DP, Wang Y, McIntosh L: The alternative oxidase lowers
•• mitochondrial reactive oxygen production in plant cells.
Proc Natl Acad Sci USA1999, 96:8271-8276.
Using a transgenic strategy, the level of the alternative oxidase (AOX) enzyme was increased or decreased in tobacco plants using sense or anti-sense transcript expression, respectively. In the absence of stress, plants deficient in AOX show detectable levels of ROS (reactive oxygen species) in their mitochondria and expression of PR-1. These plants are also hyper-sensitive to treatment with antimycin A (AA) — a specific inhibitor of Complex III of the oxidative electron transport chain — and rapid cell death is activat-ed concomitant with production of dramatic levels of ROS in the mitochon-dria. Overexpression of AOX suppresses induction of ROS by AA treatment as well as suppressing basal levels of PR-1expression. This work provides the first evidence that interference with electron transport in plant mitochon-dria can lead to significant ROS generation and this process is regulated by AOX. Furthermore, ROS generated from plant mitochondria can activate cell death and defense gene markers coordinately, analogous to that observed during the hypersensitive response.
46. Chivasa S, Carr JP: Cyanide restores N gene-mediated resistance
•• to tobacco mosaic virus in transgenic tobacco expressing salicylic acid hydroxylase.Plant Cell1998, 10:1489-1498.
Antimycin A and cyanide treatments were found to induce expression of
AOX to the same level as salicylic acid (SA) and its analog 2,2-dichloro-isonicotinic acid (INA) in tobacco. Cyanide treatment can reverse the effects of NahG expression by inhibiting tobacco mosaic virus (TMV) cell-to-cell movement and lesion proliferation, as well as virus replication. Interestingly, the effect of cyanide can be reversed by treatment with SHAM (salicylhydroxamic acid), an inhibitor of AOX. Treatment with SHAM alone can block SA-dependent resistance to TMV, induce PR-1 in an SA-depen-dent manner, but does not affect resistance to bacterial or fungal pathogens. This work provided evidence that SA-mediated cell death acti-vation that is required for optimal virus restriction probably involves AOX. However, the effects of SHAM and cyanide on resistance may be more complex to interpret at present.
47. Hu G, Yalpani N, Briggs SP, Johal GS: A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.Plant Cell1998, 10:1095-1105.
48. Molina A, Volrath S, Guyer D, Maleck K, Ryals J, Ward E: Inhibition of protoporphyrinogen oxidase expression in Arabidopsis causes a lesion-mimic phenotype that induces systemic acquired resistance.Plant J1999, 17:667-678.
49. Simons BH, Millenaar FF, Mulder L, Van Loon LC, Lambers H:
•• Enhanced expression and activation of the alternative oxidase during infection of Arabidopsis with Pseudomonas syringaepv tomato.Plant Physiol1999, 120:529-538.
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