- Email: [email protected]
Die and let live – programmed cell death in plants
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Die and let live — programmed cell death in plants Eric Lam*, Dominique Pontier and Olga del Pozo 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: [email protected] Current Opinion in Plant Biology 1999, 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 recognition 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 suicidal 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 structural 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 regulators of animal PCD have been defined in the past 10 years, they revolved around only a small number of central 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 activities. 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 animal counterparts, plants must deal with suicidal cells and their contents in fundamentally different ways. A distinguishing 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 during TE development appears to catalyze the autolysis of the cell’s content in a systematic fashion after the synthesis 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 different 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
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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 common 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 disruption of the cytoskeleton early on in plant PCD has also been suggested by the finding that disruption of microfilaments 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 during 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 phosphorylation, calcium channel functions and reactive oxygen species (ROS) in HR cell death. Studies of disease-related cell death are less developed 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 particularly, the requirement of ethylene in disease-related cell death induction [14•]. Consistent with these observations, 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 contrast, 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 formation 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
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consistent with our recent observation that a senescencespecific 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 hydroxylase (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 transgenic 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 morphologically 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 signaling process to activate the HR pathway. Another likely signaling molecule for the activation of HR cell death is ROS (reactive oxygen species) [24,25•]. Two phases of ROS generation by plants are known to occur upon incompatible interactions with pathogens [24]. We have shown that ambient oxygen pressure is required for HR cell death while PR gene induction remains little affected by low oxygen pressure [26]. HR-like cell death and defense gene activation can also be induced ectopically by either transgenic expression of a gene encoding glucose oxidase [27] or suppression of endogenous catalase [28•]. The action of ROS may be augmented by nitric oxide (NO) to potentiate and propagate the cell death signals [25•]. It has been speculated for a number of years that ROS involved in activating the HR may be generated by an NADPH oxidase similar to the well-characterized ROS signaling system in neutrophils [24,25•]. Recent identification of six Arabidopsis genes homologous to the large subunit of the human NADPH oxidase should facilitate testing of this hypothesis [29,30]. In support of the involvement of NADPH oxidase as an activator of ROS for HR induction, recent work by Kawasaki et al. [31••] showed that the small GTP-binding protein Rac may mediate the HR response of rice in an ROS-dependent
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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 proliferation 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 production, 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.
inhibited by the serine protease inhibitor AEBSF (4-[2aminoethyl]-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 suggest 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 activation 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 interaction 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 membrane of the mitochondria [41]. They can also suppress or activate caspases through interaction with the Ced4/APAF1 regulator.
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 avirulent 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 proapoptotic Bax is found to activate HR-like cell death [38••]. These observations suggest the possibility that caspase-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 proteases 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
In plants, the fungal toxin victorin appears to induce cell death via inhibition of the mitochondrial enzyme glycine decarboxylase [14•]. More recently, the mitochondrial connection 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 mitochondria 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 directly 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 animals and genes with weak sequence homology to BI-1 have been identified in C. elegans and Arabidopsis sequence databases. Although its function is unclear at present, BI-1 may be an ancient regulator of cell death activation through the mitochondria. The mitochondrial connection in HR cell death has also been made at the level of the Alternative oxidase (AOX). AOX is not found in animals and its function as a heat generator in floral tissues such as the Voodoo Lily has been
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established. However, plants suppressed in AOX expression 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 dramatic amounts of ROS in their mitochondria upon treatment with AA. Moreover, they showed that AOX-deficient plants constitutively expressed defense gene markers whereas overexpressors of AOX repressed the basal expression levels of these genes. Thus, ROS generated in the mitochondria can lead to the induction of PR gene expression. 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 activate AOX expression [46••]. In addition, mutations or transgenes that lead to an impairment of heme biosynthesis result in spontaneous lesion formation [13,47,48]. The disruption of heme biosynthesis may lead to a loss of functional cytochromes and excess ROS generation from the mitochondria. Together with expected defects in ROS protectants 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 induction 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 complemented 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 tempting 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) infection. Future manipulation of AOX levels should lead to a better understanding of the role for this protein in determining 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.
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Ultimately, forward or reverse genetic definition of candidates 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 1.
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Groover A, Jones AM: Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 1999, 119:375-384. In vitro differentiation of tracheary element (TE) was studied. The heterotrimeric G-protein activator mastoparan and a calcium ionophore were found to accelerate hormone-dependent programmed cell death. Vacuole collapse and cessation of cytoplasmic streaming preceded nuclear DNA fragmentation, suggestive of an autolytic process of programmed cell death. Interestingly, these authors found that addition of 1% (w/v) trypsin activated vacuole collapse and nuclear DNA fragmentation in a calciumdependent manner whereas exogenous soybean trypsin inhibitor has the opposite effect of inhibiting TE differentiation. A 40 kDa secreted protease that can be inhibited with soybean trypsin inhibitor was found to correlate with TE differentiation. 5. •
Bethke PC, Lonsdale JE, Fath A, Jones RL: Hormonally regulated programmed cell death in barley aleurone cells. Plant Cell 1999, 11:1033-1045. This paper describes the careful microscopic analysis of gibberellic acid induced aleurone cell death with isolated protoplasts. Cell death is preceded by an abrupt loss of membrane integrity and vacuolization of cytoplasmic vesicles. Signaling by cGMP is suggested by the effects of LY83583, a guanylyl cyclase inhibitor, on DNA degradation and programmed cell death of aleurone cells. The authors concluded that gibberellic acid activated aleurone cell death is likely to occur via autolysis rather than apoptosis. 6.
Fukuda H: Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol Plant Mol Biol 1996, 47:299-325.
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Gilchrist DG: Programmed cell death in plant disease: the purpose and promise of cellular suicide. Annu Rev Phytopathol 1998, 36:393-414.
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Mittler R, Simon L, Lam E: Pathogen-induced programmed cell death in tobacco. J Cell Sci 1997, 110:1333-1344.
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Young TE, Gallie DR: Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol Biol 1999, 39:915-926.
10. Pontier D, Gan S, Amasino RM, Roby D, Lam E: Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Mol Biol 1999, 39:1243-1255. 11. O’Brian IEW, Baguley BC, Murray BG, Morris BAM, Ferguson IB: • Early stages of the apoptotic pathway in plant cells are reversible. Plant J 1998, 13:803-814. Using flow cytometry, these authors reported large changes in chromatin condensation during cell death induction of tobacco protoplasts by chemical treatments. Annexin V binding, indicative of phosphatidylserine exposure, as well as chromatin condensation appeared to be reversible at the early stage of the cell death process in this assay system. 12. Skalamera D, Heath MC: Changes in the cytoskeleton accompanying infection-induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi. Plant J 1998, 16:191-200.
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13. Buckner B, Janick-Buckner D, Gray J, Johal GS: Cell-death mechanisms in maize. Trends Plant Sci 1998, 3:218-223. 14. Navarre DA, Wolpert TJ: Victorin induction of an • apoptotic/senescence-like response in oats. Plant J 1999, 11:237-249. Cell death induction by the host-selective fungal toxin victorin in susceptible oat leaves resulted in chromosomal laddering, similar to those previously reported for Alternaria alternate toxin and tomato. Victorin interacts with subunits of the photorespiratory enzyme glycine decarboxylase (GDC) and effectively inhibits this mitochondrial enzyme in vivo. Parallels between victorin induced cell death and senescence are drawn. An interesting phenotype is the cleavage of the 14 amino-terminal amino acid residues from the large subunit of ribulose 1,5-bisphosphate carboxylase (LSU) and its inhibition by a variety of treatments, including cysteine protease inhibitors such as E-64 and calpeptin. This work suggests the plant mitochondria may be the site where an apoptotic signal can be activated by victorin inhibition of photorespiration through the GDC. Ethylene also appears to be required for victorin induced cell death and LSU cleavage. 15. Lund ST, Stall RE, Klee HJ: Ethylene regulates the susceptible response to pathogen infection in tomato. Plant Cell 1998, 10:371-382. 16. Lawton KA, Potter SL, Uknes S, Ryals J: Acquired resistance signal transduction in Arabidopsis is ethylene independent. Plant Cell 1994, 6:581-588. 17.
He CJ, Morgan PW, Drew MC: Transduction of an ethylene signal is required for cell death and lysis in the root cortex of maize during aerenchyma formation induced by hypoxia. Plant Physiol 1996, 112:463-472.
18. Weymann K, Hunt M, Uknes S, Neuenschwander U, Lawton K, Steiner HY, Ryals J: Suppression and restoration of lesion formation in Arabidopsis lsd mutants. Plant Cell 1995, 7:2013-2022. 19. Shah J, Kachroo P, Klessig DF: The Arabidopsis ssi1 mutation • restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent. Plant Cell 1999, 11:191-206. The authors describe a semidominant mutant of Arabidopsis that constitutively expresses PR genes in an NPR1 independent manner. Spontaneous HR-like cell death lesions are observed and the phenotypes are SA dependent as revealed through NahG expression. This work suggests that SA is required, although not sufficient, to induce HR cell death. 20. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT: •• A gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses and cell growth. Plant Cell 1999, 11:191-206. This report documents the characterization of a new lesion mimic mutant in Arabidopsis, acd6. It is caused by a dominant mutation that activates cell death and resistance to bacterial pathogen; however, the plant is unable to respond with an HR upon challenge with an avirulent bacteria and phytoalexin synthesis is not activated. Interestingly, the phenotype of acd6 is SA dependent, as it can be reversed by NahG expression, but is partially NPR1 independent. A novel phenotype of the acd6 mutant in comparison to other previously described lesion mimics is the observation of abnormal cell growth and enlargement that is SA dependent. A model is presented whereby SA potentiates both cell death and cell growth and/or proliferation and the acd6 mutation effectively decreases the threshold of response for SA signaling to these processes. 21. Rao M, Davis KR: Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid. Plant J 1999, 17:603-614. 22. He ZH, He D, Kohorn BD: Requirement for the induced expression • of a cell wall associated receptor kinase for survival during the pathogen response. Plant J 1998, 14:55-63. Suppression of a cell wall associated receptor kinase by antisense technology and a dominant-negative mutant resulted in heightened sensitivity to 2,2-dichloroisonicotinic acid (INA) whereas overexpression of this kinase resulted in higher tolerance for SA treatment. 23. Heo WD, Lee SH, Kim MC, Kim JC, Chung WS, Chun HJ, Lee KJ, •• Park CY, Park HC, Choi JY, Cho MJ: Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses. Proc Natl Acad Sci USA 1999, 96:766-771. The expression of two isoforms encoded by SCaM4 and SCaM5 of the calmodulin gene family from soybean was found to respond to pathogen and fungal elicitors. Overexpression of these genes in transgenic tobacco resulted in the appearance of hypersensitive-response-like phenotypes including spontaneous cell death in older mature leaves, pathogen-related
gene expression and broad spectrum resistance to various types of pathogens. Surprisingly, salicylic acid (SA) levels are not affected by these transgenes suggesting that they act via SA-independent pathways to induce lesions and resistance. This work provides evidence that intracellular calcium levels can play important roles in the control of cell death activation in plants via specific calmodulin isoforms that are transcriptionally regulated. Moreover, it clearly demonstrates the existence of SA-independent cell death and resistance pathways. 24. Lamb C, Dixon RA: The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:251-275. 25. Van Camp W, Van Montagu M, Inze D: H2O2 and NO: redox signals • in disease resistance. Trends in Plant Sci 1998, 3:330-334. A succinct review on the role of ROS and various plant signaling components such as nitric oxide, ethylene and salicylic acid on the activation of cell death and disease resistance. The authors provide a good summary of the temporal behavior of various markers of cell death and defense, as well as a working model integrating recent observations of the various signaling components. 26. Mittler R, Shulaev V, Seskar M, Lam E: Inhibition of programmed cell death in tobacco plants during a pathogen-induced hypersensitive response at low oxygen pressure. Plant Cell 1996, 8:1991-2001. 27.
Kazan K, Murray FR, Goulter KC, Llewellyn DJ, Manners JM: Induction of cell death in transgenic plants expressing a fungal glucose oxidase. Mol Plant–Microbe Int 1998, 11:555-562.
28. Chamnongpol S, Willekens H, Moeder W, Langebartels C, • Sandermann H, Van Montagu M, Inze D, Van Camp W: Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc Natl Acad Sci USA 1998, 95:5818-5823. Expression of catalase in tobacco plants was suppressed by transgenic expression of antisense transcripts. High light treatment of these plants results in activation of hypersensitive-response-like cell death as well as defense gene markers and disease resistance, presumably due to the production of H2O2. These responses were shown to be systemic and the induction of cell death can be uncoupled from disease resistance and defense markers, with sublethal levels of ROS (reactive oxygen species) inducing pathogenesis-related gene expression and resistance without concomitant cell death. 29. Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JJ: Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J 1998, 14:365-370. 30. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs. Plant Cell 1998, 10:255-266. 31. Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H, •• Shimamoto K: The small GTP-binding protein Rac is a regulator of cell death in plants. Proc Natl Acad Sci USA 1999, 96:10922-10926. The role of the small GTP-binding protein OsRac1 was examined by expression of its constitutively active or dominant-negative mutations in transgenic rice cell cultures and plants. Expression of the transgene encoding the constitutively active mutant of OsRac1 resulted in ROS (reactive oxygen species) production and programmed cell death which correlated with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) staining. Involvement of the NADPH oxidase is suggested by inhibition of these phenotypes by diphenylene iodonium. Conversely, transgenic expression of a dominant-negative variant of OsRac1 in a rice lesion mimic mutant resulted in suppression of ROS generation and cell death induction by a rice blast fungus and calyculin A, a protein phosphatase I inhibitor. This work provides the first clear evidence for the involvement of Rac in the regulation of plant NADPH oxidase in ROS generation and cell death induction. It complements nicely the identification of multiple plant genes encoding the large subunit of the plant NADPH oxidase and suggests that analogous to the neutrophil NADPH oxidase, the plant enzyme is also regulated by Rac and phosphorylation. 32. Reichheld JP, Vernoux T, Lardon F, Van Montagu M, Inze D: Specific checkpoints regulate plant cell cycle progression in response to oxidative stress. Plant J 1999, 17:647-656. 33. Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF, • Lynn D, Dow MJ, Roberts K, Martin C: Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants. Plant Cell 1998, 10:1801-1816. An extensive study characterizing the phenotypic defects resulting from downregulation of phenolic acid metabolism in transgenic tobacco through the transgenic expression of the gene encoding the transcription factor AmMYB308. Concomitant with the decrease of phenolic compounds,
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abnormal development of the leaf palisade layer is correlated with premature programmed cell death as detected by TUNEL staining. This ‘early senescence’ phenotype could be rescued in cell cultures by supplying exogenous phenolic precursors. Interestingly, the hypersensitive response is activated much faster in these transgenic plants and this response correlated with a heightened level of lipid peroxidation. These and earlier results suggest that phenolic compounds act as important buffers against the effects of ROS (reactive oxygen species) in plants. 34. Aravind L, Dixit VM, Koonin EV: The domains of death: evolution of • the apoptosis machinery. Trends Biochem Sci 1999, 24:47-53. A thorough and provocative review of the various types of cell death regulators that have been defined in animal systems. Interesting comparisons between molecules that are or may be involved in cell death regulation from plants, animals and prokaryotes are presented and the evolutionary implications summarized. 35. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by •• caspase family proteinases. J Biol Chem 1999, 274:20049-20052. A well-written and comprehensive summary of the state-of-the-art knowledge on the structure, function and regulation of caspases, the growing family of cysteine proteases that specializes in controlling cell death activation. 36. del Pozo O, Lam E: Caspases and programmed cell death in the •• hypersensitive response of plants to pathogens. Curr Biol 1998, 8:1129-1132. This work showed that synthetic peptide inhibitors of animal caspases can suppress hypersensitive response (HR) cell death induced by avirulent bacteria when co-infiltrated into tobacco leaves. The induction of two HR cell death gene markers was inhibited whereas pathogenesis-related gene induction was not affected by these inhibitors, thus showing that plant–pathogen signaling remains intact and that defense gene activation can be uncoupled from cell death. Transient induction of caspase-like proteolytic activity was detected in extracts from leaf tissues during tobacco mosaic virus (TMV)-induced HR that was synchronized by temperature shift. This work constitutes the first report of caspase-like protease activities in plants and provided evidence for their participation in HR cell death activation. 37. •
Mitsuhara I, Malik KA, Miura M, Ohashi Y: Animal cell-death suppressors Bcl-xL and Ced-9 inhibit cell death in tobacco plants. Curr Biol 1999, 9:775-778. This work showed that expression in transgenic tobacco of the genes encoding the pro-survival cell death regulators Bcl-x L and Ced-9 from animal systems can delay cell death induced by ultraviolet light, paraquat and pathogen challenge. Although the mechanism responsible for these observations is unclear at present, the results suggest that these regulators may suppress one or more evolutionarily conserved cell death switches in plants. 38. Lacomme C, Santa Cruz S: Bax-induced cell death in tobacco is •• similar to the hypersensitive response. Proc Natl Acad Sci USA 1999, 96:7956-7961. The gene encoding the pro-apoptotic regulator Bax from mammalian systems 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 bacterial pathogen was shown to depend on protein synthesis and multiple proteases 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 J 1999, 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. Nature 1999, 399:483-487.
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42. Shaham S, Shuman MA, Herskowitz I: Death-defying yeast identify novel apoptosis genes. Cell 1998, 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. Nature 1999, 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 Chem 1998, 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 USA 1999, 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 antisense 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 hypersensitive to treatment with antimycin A (AA) — a specific inhibitor of Complex III of the oxidative electron transport chain — and rapid cell death is activated concomitant with production of dramatic levels of ROS in the mitochondria. Overexpression of AOX suppresses induction of ROS by AA treatment as well as suppressing basal levels of PR-1 expression. This work provides the first evidence that interference with electron transport in plant mitochondria 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 Cell 1998, 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-dichloroisonicotinic acid (INA) in tobacco. Cyanide treatment can reverse the effects of NahG expression by inhibiting tobacco mosaic virus (TMV) cellto-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-dependent manner, but does not affect resistance to bacterial or fungal pathogens. This work provided evidence that SA-mediated cell death activation 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 Cell 1998, 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 J 1999, 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 syringae pv tomato. Plant Physiol 1999, 120:529-538. AOX (alternative oxidase) expression was found to be induced rapidly in the infected leaves by challenge with avirulent, but more slowly with virulent, bacterial pathogens in Arabidopsis. In addition, the rapid but not the slower rise in AOX is dependent on salicylic acid but independent of NPR1. Furthermore, ethylene sensing through ETR1 is absolutely required for AOX induction. This work suggests that the local induction of AOX involves ethylene production during the hypersensitive response HR and that AOX could serve as a protectant for ROS (reactive oxygen species) damage to the cells at or near the infection site.
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Die and let live — programmed cell death in plants Eric Lam*, Dominique Pontier and Olga del Pozo 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: [email protected] Current Opinion in Plant Biology 1999, 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 recognition 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 suicidal 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 structural 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 regulators of animal PCD have been defined in the past 10 years, they revolved around only a small number of central 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 activities. 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 animal counterparts, plants must deal with suicidal cells and their contents in fundamentally different ways. A distinguishing 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 during TE development appears to catalyze the autolysis of the cell’s content in a systematic fashion after the synthesis 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 different 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
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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 common 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 disruption of the cytoskeleton early on in plant PCD has also been suggested by the finding that disruption of microfilaments 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 during 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 phosphorylation, calcium channel functions and reactive oxygen species (ROS) in HR cell death. Studies of disease-related cell death are less developed 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 particularly, the requirement of ethylene in disease-related cell death induction [14•]. Consistent with these observations, 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 contrast, 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 formation 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
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consistent with our recent observation that a senescencespecific 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 hydroxylase (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 transgenic 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 morphologically 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 signaling process to activate the HR pathway. Another likely signaling molecule for the activation of HR cell death is ROS (reactive oxygen species) [24,25•]. Two phases of ROS generation by plants are known to occur upon incompatible interactions with pathogens [24]. We have shown that ambient oxygen pressure is required for HR cell death while PR gene induction remains little affected by low oxygen pressure [26]. HR-like cell death and defense gene activation can also be induced ectopically by either transgenic expression of a gene encoding glucose oxidase [27] or suppression of endogenous catalase [28•]. The action of ROS may be augmented by nitric oxide (NO) to potentiate and propagate the cell death signals [25•]. It has been speculated for a number of years that ROS involved in activating the HR may be generated by an NADPH oxidase similar to the well-characterized ROS signaling system in neutrophils [24,25•]. Recent identification of six Arabidopsis genes homologous to the large subunit of the human NADPH oxidase should facilitate testing of this hypothesis [29,30]. In support of the involvement of NADPH oxidase as an activator of ROS for HR induction, recent work by Kawasaki et al. [31••] showed that the small GTP-binding protein Rac may mediate the HR response of rice in an ROS-dependent
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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 proliferation 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 production, 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.
inhibited by the serine protease inhibitor AEBSF (4-[2aminoethyl]-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 suggest 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 activation 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 interaction 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 membrane of the mitochondria [41]. They can also suppress or activate caspases through interaction with the Ced4/APAF1 regulator.
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 avirulent 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 proapoptotic Bax is found to activate HR-like cell death [38••]. These observations suggest the possibility that caspase-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 proteases 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
In plants, the fungal toxin victorin appears to induce cell death via inhibition of the mitochondrial enzyme glycine decarboxylase [14•]. More recently, the mitochondrial connection 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 mitochondria 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 directly 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 animals and genes with weak sequence homology to BI-1 have been identified in C. elegans and Arabidopsis sequence databases. Although its function is unclear at present, BI-1 may be an ancient regulator of cell death activation through the mitochondria. The mitochondrial connection in HR cell death has also been made at the level of the Alternative oxidase (AOX). AOX is not found in animals and its function as a heat generator in floral tissues such as the Voodoo Lily has been
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established. However, plants suppressed in AOX expression 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 dramatic amounts of ROS in their mitochondria upon treatment with AA. Moreover, they showed that AOX-deficient plants constitutively expressed defense gene markers whereas overexpressors of AOX repressed the basal expression levels of these genes. Thus, ROS generated in the mitochondria can lead to the induction of PR gene expression. 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 activate AOX expression [46••]. In addition, mutations or transgenes that lead to an impairment of heme biosynthesis result in spontaneous lesion formation [13,47,48]. The disruption of heme biosynthesis may lead to a loss of functional cytochromes and excess ROS generation from the mitochondria. Together with expected defects in ROS protectants 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 induction 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 complemented 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 tempting 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) infection. Future manipulation of AOX levels should lead to a better understanding of the role for this protein in determining 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.
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Ultimately, forward or reverse genetic definition of candidates 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 1.
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Richberg MH, Aviv DH, Dangl JL: Dead cells do tell tales. Curr Opin Plant Biol 1998, 1:480-485.
4. ••
Groover A, Jones AM: Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 1999, 119:375-384. In vitro differentiation of tracheary element (TE) was studied. The heterotrimeric G-protein activator mastoparan and a calcium ionophore were found to accelerate hormone-dependent programmed cell death. Vacuole collapse and cessation of cytoplasmic streaming preceded nuclear DNA fragmentation, suggestive of an autolytic process of programmed cell death. Interestingly, these authors found that addition of 1% (w/v) trypsin activated vacuole collapse and nuclear DNA fragmentation in a calciumdependent manner whereas exogenous soybean trypsin inhibitor has the opposite effect of inhibiting TE differentiation. A 40 kDa secreted protease that can be inhibited with soybean trypsin inhibitor was found to correlate with TE differentiation. 5. •
Bethke PC, Lonsdale JE, Fath A, Jones RL: Hormonally regulated programmed cell death in barley aleurone cells. Plant Cell 1999, 11:1033-1045. This paper describes the careful microscopic analysis of gibberellic acid induced aleurone cell death with isolated protoplasts. Cell death is preceded by an abrupt loss of membrane integrity and vacuolization of cytoplasmic vesicles. Signaling by cGMP is suggested by the effects of LY83583, a guanylyl cyclase inhibitor, on DNA degradation and programmed cell death of aleurone cells. The authors concluded that gibberellic acid activated aleurone cell death is likely to occur via autolysis rather than apoptosis. 6.
Fukuda H: Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol Plant Mol Biol 1996, 47:299-325.
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Gilchrist DG: Programmed cell death in plant disease: the purpose and promise of cellular suicide. Annu Rev Phytopathol 1998, 36:393-414.
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Mittler R, Simon L, Lam E: Pathogen-induced programmed cell death in tobacco. J Cell Sci 1997, 110:1333-1344.
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Young TE, Gallie DR: Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol Biol 1999, 39:915-926.
10. Pontier D, Gan S, Amasino RM, Roby D, Lam E: Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Mol Biol 1999, 39:1243-1255. 11. O’Brian IEW, Baguley BC, Murray BG, Morris BAM, Ferguson IB: • Early stages of the apoptotic pathway in plant cells are reversible. Plant J 1998, 13:803-814. Using flow cytometry, these authors reported large changes in chromatin condensation during cell death induction of tobacco protoplasts by chemical treatments. Annexin V binding, indicative of phosphatidylserine exposure, as well as chromatin condensation appeared to be reversible at the early stage of the cell death process in this assay system. 12. Skalamera D, Heath MC: Changes in the cytoskeleton accompanying infection-induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi. Plant J 1998, 16:191-200.
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13. Buckner B, Janick-Buckner D, Gray J, Johal GS: Cell-death mechanisms in maize. Trends Plant Sci 1998, 3:218-223. 14. Navarre DA, Wolpert TJ: Victorin induction of an • apoptotic/senescence-like response in oats. Plant J 1999, 11:237-249. Cell death induction by the host-selective fungal toxin victorin in susceptible oat leaves resulted in chromosomal laddering, similar to those previously reported for Alternaria alternate toxin and tomato. Victorin interacts with subunits of the photorespiratory enzyme glycine decarboxylase (GDC) and effectively inhibits this mitochondrial enzyme in vivo. Parallels between victorin induced cell death and senescence are drawn. An interesting phenotype is the cleavage of the 14 amino-terminal amino acid residues from the large subunit of ribulose 1,5-bisphosphate carboxylase (LSU) and its inhibition by a variety of treatments, including cysteine protease inhibitors such as E-64 and calpeptin. This work suggests the plant mitochondria may be the site where an apoptotic signal can be activated by victorin inhibition of photorespiration through the GDC. Ethylene also appears to be required for victorin induced cell death and LSU cleavage. 15. Lund ST, Stall RE, Klee HJ: Ethylene regulates the susceptible response to pathogen infection in tomato. Plant Cell 1998, 10:371-382. 16. Lawton KA, Potter SL, Uknes S, Ryals J: Acquired resistance signal transduction in Arabidopsis is ethylene independent. Plant Cell 1994, 6:581-588. 17.
He CJ, Morgan PW, Drew MC: Transduction of an ethylene signal is required for cell death and lysis in the root cortex of maize during aerenchyma formation induced by hypoxia. Plant Physiol 1996, 112:463-472.
18. Weymann K, Hunt M, Uknes S, Neuenschwander U, Lawton K, Steiner HY, Ryals J: Suppression and restoration of lesion formation in Arabidopsis lsd mutants. Plant Cell 1995, 7:2013-2022. 19. Shah J, Kachroo P, Klessig DF: The Arabidopsis ssi1 mutation • restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent. Plant Cell 1999, 11:191-206. The authors describe a semidominant mutant of Arabidopsis that constitutively expresses PR genes in an NPR1 independent manner. Spontaneous HR-like cell death lesions are observed and the phenotypes are SA dependent as revealed through NahG expression. This work suggests that SA is required, although not sufficient, to induce HR cell death. 20. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT: •• A gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses and cell growth. Plant Cell 1999, 11:191-206. This report documents the characterization of a new lesion mimic mutant in Arabidopsis, acd6. It is caused by a dominant mutation that activates cell death and resistance to bacterial pathogen; however, the plant is unable to respond with an HR upon challenge with an avirulent bacteria and phytoalexin synthesis is not activated. Interestingly, the phenotype of acd6 is SA dependent, as it can be reversed by NahG expression, but is partially NPR1 independent. A novel phenotype of the acd6 mutant in comparison to other previously described lesion mimics is the observation of abnormal cell growth and enlargement that is SA dependent. A model is presented whereby SA potentiates both cell death and cell growth and/or proliferation and the acd6 mutation effectively decreases the threshold of response for SA signaling to these processes. 21. Rao M, Davis KR: Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid. Plant J 1999, 17:603-614. 22. He ZH, He D, Kohorn BD: Requirement for the induced expression • of a cell wall associated receptor kinase for survival during the pathogen response. Plant J 1998, 14:55-63. Suppression of a cell wall associated receptor kinase by antisense technology and a dominant-negative mutant resulted in heightened sensitivity to 2,2-dichloroisonicotinic acid (INA) whereas overexpression of this kinase resulted in higher tolerance for SA treatment. 23. Heo WD, Lee SH, Kim MC, Kim JC, Chung WS, Chun HJ, Lee KJ, •• Park CY, Park HC, Choi JY, Cho MJ: Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses. Proc Natl Acad Sci USA 1999, 96:766-771. The expression of two isoforms encoded by SCaM4 and SCaM5 of the calmodulin gene family from soybean was found to respond to pathogen and fungal elicitors. Overexpression of these genes in transgenic tobacco resulted in the appearance of hypersensitive-response-like phenotypes including spontaneous cell death in older mature leaves, pathogen-related
gene expression and broad spectrum resistance to various types of pathogens. Surprisingly, salicylic acid (SA) levels are not affected by these transgenes suggesting that they act via SA-independent pathways to induce lesions and resistance. This work provides evidence that intracellular calcium levels can play important roles in the control of cell death activation in plants via specific calmodulin isoforms that are transcriptionally regulated. Moreover, it clearly demonstrates the existence of SA-independent cell death and resistance pathways. 24. Lamb C, Dixon RA: The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:251-275. 25. Van Camp W, Van Montagu M, Inze D: H2O2 and NO: redox signals • in disease resistance. Trends in Plant Sci 1998, 3:330-334. A succinct review on the role of ROS and various plant signaling components such as nitric oxide, ethylene and salicylic acid on the activation of cell death and disease resistance. The authors provide a good summary of the temporal behavior of various markers of cell death and defense, as well as a working model integrating recent observations of the various signaling components. 26. Mittler R, Shulaev V, Seskar M, Lam E: Inhibition of programmed cell death in tobacco plants during a pathogen-induced hypersensitive response at low oxygen pressure. Plant Cell 1996, 8:1991-2001. 27.
Kazan K, Murray FR, Goulter KC, Llewellyn DJ, Manners JM: Induction of cell death in transgenic plants expressing a fungal glucose oxidase. Mol Plant–Microbe Int 1998, 11:555-562.
28. Chamnongpol S, Willekens H, Moeder W, Langebartels C, • Sandermann H, Van Montagu M, Inze D, Van Camp W: Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc Natl Acad Sci USA 1998, 95:5818-5823. Expression of catalase in tobacco plants was suppressed by transgenic expression of antisense transcripts. High light treatment of these plants results in activation of hypersensitive-response-like cell death as well as defense gene markers and disease resistance, presumably due to the production of H2O2. These responses were shown to be systemic and the induction of cell death can be uncoupled from disease resistance and defense markers, with sublethal levels of ROS (reactive oxygen species) inducing pathogenesis-related gene expression and resistance without concomitant cell death. 29. Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JJ: Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J 1998, 14:365-370. 30. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs. Plant Cell 1998, 10:255-266. 31. Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H, •• Shimamoto K: The small GTP-binding protein Rac is a regulator of cell death in plants. Proc Natl Acad Sci USA 1999, 96:10922-10926. The role of the small GTP-binding protein OsRac1 was examined by expression of its constitutively active or dominant-negative mutations in transgenic rice cell cultures and plants. Expression of the transgene encoding the constitutively active mutant of OsRac1 resulted in ROS (reactive oxygen species) production and programmed cell death which correlated with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) staining. Involvement of the NADPH oxidase is suggested by inhibition of these phenotypes by diphenylene iodonium. Conversely, transgenic expression of a dominant-negative variant of OsRac1 in a rice lesion mimic mutant resulted in suppression of ROS generation and cell death induction by a rice blast fungus and calyculin A, a protein phosphatase I inhibitor. This work provides the first clear evidence for the involvement of Rac in the regulation of plant NADPH oxidase in ROS generation and cell death induction. It complements nicely the identification of multiple plant genes encoding the large subunit of the plant NADPH oxidase and suggests that analogous to the neutrophil NADPH oxidase, the plant enzyme is also regulated by Rac and phosphorylation. 32. Reichheld JP, Vernoux T, Lardon F, Van Montagu M, Inze D: Specific checkpoints regulate plant cell cycle progression in response to oxidative stress. Plant J 1999, 17:647-656. 33. Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF, • Lynn D, Dow MJ, Roberts K, Martin C: Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants. Plant Cell 1998, 10:1801-1816. An extensive study characterizing the phenotypic defects resulting from downregulation of phenolic acid metabolism in transgenic tobacco through the transgenic expression of the gene encoding the transcription factor AmMYB308. Concomitant with the decrease of phenolic compounds,
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abnormal development of the leaf palisade layer is correlated with premature programmed cell death as detected by TUNEL staining. This ‘early senescence’ phenotype could be rescued in cell cultures by supplying exogenous phenolic precursors. Interestingly, the hypersensitive response is activated much faster in these transgenic plants and this response correlated with a heightened level of lipid peroxidation. These and earlier results suggest that phenolic compounds act as important buffers against the effects of ROS (reactive oxygen species) in plants. 34. Aravind L, Dixit VM, Koonin EV: The domains of death: evolution of • the apoptosis machinery. Trends Biochem Sci 1999, 24:47-53. A thorough and provocative review of the various types of cell death regulators that have been defined in animal systems. Interesting comparisons between molecules that are or may be involved in cell death regulation from plants, animals and prokaryotes are presented and the evolutionary implications summarized. 35. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by •• caspase family proteinases. J Biol Chem 1999, 274:20049-20052. A well-written and comprehensive summary of the state-of-the-art knowledge on the structure, function and regulation of caspases, the growing family of cysteine proteases that specializes in controlling cell death activation. 36. del Pozo O, Lam E: Caspases and programmed cell death in the •• hypersensitive response of plants to pathogens. Curr Biol 1998, 8:1129-1132. This work showed that synthetic peptide inhibitors of animal caspases can suppress hypersensitive response (HR) cell death induced by avirulent bacteria when co-infiltrated into tobacco leaves. The induction of two HR cell death gene markers was inhibited whereas pathogenesis-related gene induction was not affected by these inhibitors, thus showing that plant–pathogen signaling remains intact and that defense gene activation can be uncoupled from cell death. Transient induction of caspase-like proteolytic activity was detected in extracts from leaf tissues during tobacco mosaic virus (TMV)-induced HR that was synchronized by temperature shift. This work constitutes the first report of caspase-like protease activities in plants and provided evidence for their participation in HR cell death activation. 37. •
Mitsuhara I, Malik KA, Miura M, Ohashi Y: Animal cell-death suppressors Bcl-xL and Ced-9 inhibit cell death in tobacco plants. Curr Biol 1999, 9:775-778. This work showed that expression in transgenic tobacco of the genes encoding the pro-survival cell death regulators Bcl-x L and Ced-9 from animal systems can delay cell death induced by ultraviolet light, paraquat and pathogen challenge. Although the mechanism responsible for these observations is unclear at present, the results suggest that these regulators may suppress one or more evolutionarily conserved cell death switches in plants. 38. Lacomme C, Santa Cruz S: Bax-induced cell death in tobacco is •• similar to the hypersensitive response. Proc Natl Acad Sci USA 1999, 96:7956-7961. The gene encoding the pro-apoptotic regulator Bax from mammalian systems 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 bacterial pathogen was shown to depend on protein synthesis and multiple proteases 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 J 1999, 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. Nature 1999, 399:483-487.
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42. Shaham S, Shuman MA, Herskowitz I: Death-defying yeast identify novel apoptosis genes. Cell 1998, 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. Nature 1999, 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 Chem 1998, 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 USA 1999, 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 antisense 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 hypersensitive to treatment with antimycin A (AA) — a specific inhibitor of Complex III of the oxidative electron transport chain — and rapid cell death is activated concomitant with production of dramatic levels of ROS in the mitochondria. Overexpression of AOX suppresses induction of ROS by AA treatment as well as suppressing basal levels of PR-1 expression. This work provides the first evidence that interference with electron transport in plant mitochondria 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 Cell 1998, 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-dichloroisonicotinic acid (INA) in tobacco. Cyanide treatment can reverse the effects of NahG expression by inhibiting tobacco mosaic virus (TMV) cellto-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-dependent manner, but does not affect resistance to bacterial or fungal pathogens. This work provided evidence that SA-mediated cell death activation 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 Cell 1998, 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 J 1999, 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 syringae pv tomato. Plant Physiol 1999, 120:529-538. AOX (alternative oxidase) expression was found to be induced rapidly in the infected leaves by challenge with avirulent, but more slowly with virulent, bacterial pathogens in Arabidopsis. In addition, the rapid but not the slower rise in AOX is dependent on salicylic acid but independent of NPR1. Furthermore, ethylene sensing through ETR1 is absolutely required for AOX induction. This work suggests that the local induction of AOX involves ethylene production during the hypersensitive response HR and that AOX could serve as a protectant for ROS (reactive oxygen species) damage to the cells at or near the infection site.