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Cysteine and serine proteases are prominent players in thecontrol of developmental and pathogen-activated cell deaths inplants. Ethylene, salicylic acid, the small G-protein Rac,calcium and reactive oxygen species are recurring mediators ofdeath signaling. Lastly, the mitochondrion has emerged in bothplant and animal systems as a ‘central depot’ that interpretsmultiple signals and in some instances determines the fate ofthe cell.

AddressesBiotech Center, Foran Hall, Cook College, 59 Dudley Road, RutgersUniversity, New Brunswick, NJ 08903, USA*e-mail: lam@aesop.rutgers.edu

Current Opinion in Plant Biology 1999, 2:502–507

1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.

AbbreviationsAA antimycin AAEBSF 4-(2-aminoethyl)-benzenesulfonyl-flonyl-fluorideAOX Alternative oxidaseBI-1 Bax inhibitor-1HR hypersensitive responseNO nitric oxidePCD programmed cell deathPR pathogenesis-relatedROS reactive oxygen speciesSA salicylic acidSHAM salicylhydroxamic acidTE tracheary elementVDAC voltage dependent anion channel

IntroductionThe study of programmed cell death (PCD) has seen aremarkable boom in the past 10 years since the early recog-nition in the 1970’s by Andrew Wyllie and his coworkersthat a variety of animal cells die with similar morphologicalfeatures which appeared to be the result of an active suici-dal process (for review see [1]). This renaissance of PCDresearch was brought about by the convergence of work onPCD with studies of animal genes involved in oncogenesissuch as Bcl-2, and genetic research on cell death mutantsby Bob Horvitz and his coworkers in the nematodeCaenorhabditis elegans (reviewed in [1]). The realization thatmolecules with similar biochemical functions and structur-al domains are conserved in diverse organisms to regulatePCD convinced most skeptics that this process can indeedbe studied at the molecular level with defined players.Remarkably, although many signaling pathways and regu-lators of animal PCD have been defined in the past10 years, they revolved around only a small number of cen-tral players and their modes of action appeared to bevariations on a few themes. The central enzymatic activitythat has been most well-characterized is a growing familyof cysteine proteases called caspases. They are synthesized

as zymogens that require specific cleavage and subsequentoligomerization in order to reveal their enzymatic activi-ties. Once activated via proteolysis, they appear to activatea cascade of proteases in a serial fashion that is reminescentof the classic blood clotting factor cascade.

PCD research in plants has also begun to blossom in thepast five years [2,3]. There are three major reasons for ourinterest in this process in plants. First, it is a way of life forplants, just as it is for animals. Essential processes such asxylem differentiation and tapetal cell degeneration allinvolve PCD. Second, comparison of the mechanisms andmolecules that are involved in the two kingdoms shouldenlighten us about the evolution of PCD and may help todetermine the indispensible players in the chain of eventsthat lead to cell death in eukaryotes. Third, from anapplied perspective, the ability to regulate cell death inplants may have important applications in agriculture andpost-harvest industries. For example, suppression of PCDinduced by biotrophic pathogens could minimize diseasesymptoms and inhibition of cell death during senescencemay prolong the shelf-life of crops and vegetables. In thisreview, we discuss work published in the past year that hasbegun to reveal some of the players that are involved incontroling PCD in plant cells.

The demise of a cell — the phenomenonWhat actually occurs when a plant cell commits suicide? Incontrast to apoptosis in animals, the presence of a rigid wallin plant cells poses a distinct problem: because the dyingcell cannot be packaged into small apoptotic bodies andthen engulfed by its neighbors, as in the case for their ani-mal counterparts, plants must deal with suicidal cells andtheir contents in fundamentally different ways. A distin-guishing feature of plant cells is the prominant vacuole,which contains many catabolic enzymes such as proteasesand nucleases. It is thus not surprising that this organelleappears to play an important role in tracheary element(TE) differentiation [4••] as well as degeneration of thealeurone in barley [5•]. The disruption of the vacuole dur-ing TE development appears to catalyze the autolysis ofthe 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 cellsdestined for PCD is likely to be a common strategy forplants. However, the sequence of events and the resultingcellular morphological changes may differ depending onthe particular inductive death signal [2,3,6–9]. This may bea consequence of the role that cell death may have in dif-ferent situations. In the case of developmental cell deathsuch as senescence and TE differentiation, the recycling ofthe dead cell’s content may be an important ‘purpose’. Inthe case of cell death related to pathogen containment, cell

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death may be used to rapidly ‘isolate’ the infected cellsand efficient transport of the dead cell’s content may besecondary [10]. In most cases of plant PCD, vacuolizationof the cytoplasm and disruption of the tonoplast are com-mon events that appear to precede mass disruption of themitochondria and nucleus. On the other hand, recent flowcytometry studies with tobacco protoplasts suggest thatnuclear DNA condensation could be activated before thefirst irreversible step of PCD [11•]. Evidence for disrup-tion of the cytoskeleton early on in plant PCD has alsobeen suggested by the finding that disruption of microfila-ments by cytochalasin E could suppress cell death inducedby fungal pathogens in cowpea [12] and that cytoplasmicstreaming is arrested during TE differentiation [4••].

What’s new in PCD signaling in plants?The signaling processes that activate PCD in plantspromise to be as diverse as those that have been found inanimals. One of the most well-characterized systems todate is the differentiation of Zinnia TEs in vitro. Earlierwork with the Zinnia TE system has established thatwounding and hormonal balance (auxin and cytokinintreatments) are critical signaling components for initiatingthe transformation of mesophyll cells to TE [6]. Morerecently, the involvement of calcium, heterotrimericG-proteins and extracellular proteases has beensuggested [4••].

In contrast to other plant PCD systems, a large body ofliterature has accumulated on the study of cell death dur-ing plant pathogen interactions [2,3,7,13]. Two majortypes of cell death can result when plants are inoculatedwith a pathogen: the rapid hypersensitive response (HR),which is typically associated with resistance, and a slowerform of death that is the result of disease. Previous workhas implicated the involvement of protein phosphoryla-tion, calcium channel functions and reactive oxygenspecies (ROS) in HR cell death. Studies of disease-relat-ed cell death are less developed as it is not obvious thatthey resulted from a suicide process. However, recentwork with the fungal toxin victorin has implicated anactive participation of the dying cells and more particu-larly, the requirement of ethylene in disease-related celldeath induction [14•]. Consistent with these observa-tions, ethylene insensitivity in tomato was found tosuppress cell death due to infection with biotrophicpathogens [15]. These observations raised the possibilitythat ethylene action may be involved in the slower celldeath that is manifested during plant diseases. In con-trast, resistance response does not appear to requireethylene action [16]. Interestingly, ethylene may also beinvolved in developmental cell death such as endospermdegeneration in wheat and during maize aerenchyma for-mation induced by hypoxia [9,17]. Taking into account itsclassic role in tissue senescence, it appears that ethylenemay be an important signal for slower forms of cell deathduring which efficient recycling of the dead cell’s contentmight be important for the plant. This interpretation is

consistent with our recent observation that a senescence-specific marker gene, SAG12, is activated in cellsneighboring HR lesions, where chlorotic symptoms ofcell death are often observed [10].

Salicylic acid (SA) has emerged in the past several years asa positive feedback regulator of cell death during the HR[2,3]. This was first suggested in the study of disease lesionmimics in Arabidopsis [18], where spontaneous cell deathin the mutants lsd6 and lsd7 was repressed by transgenicexpression of a gene encoding a bacterial salicylate hydrox-ylase (NahG), which converts SA to catechol. Two newdisease lesion mimic mutants, ssi1 and acd6, appeared toshow the same dependence on SA [19•,20••]. Interestingly,ozone induced cell death in Arabidopsis has also beenshown to be potentiated by SA [21]. A cell wall associatedprotein kinase has recently been suggested to be involvedin suppressing cell death signaling by SA [22•].

The specific requirement of calcium signaling in HR celldeath had been suggested through studies using calciumchannel blockers [2,3,7]. The recent work of Heo et al.[23••] suggested that this dependence on calcium mayinvolve specific isoforms of calmodulin. HR-like celldeath, pathogenesis-related (PR) gene induction andbroad spectrum disease resistance are activated by trans-genic expression of two soybean calmodulin-encodinggenes. These phenotypes are not SA dependent and thelevel of SA is not altered by the transgenes. Thus, distinctpathways can give rise to phenotypes that are morphologi-cally very similar. As these soybean genes are inducedduring the defense response, they may participate in thelong term signaling for resistance rather than the initial sig-naling process to activate the HR pathway.

Another likely signaling molecule for the activation of HRcell death is ROS (reactive oxygen species) [24,25•]. Twophases of ROS generation by plants are known to occurupon incompatible interactions with pathogens [24]. Wehave shown that ambient oxygen pressure is required forHR cell death while PR gene induction remains littleaffected by low oxygen pressure [26]. HR-like cell deathand defense gene activation can also be induced ectopical-ly by either transgenic expression of a gene encodingglucose oxidase [27] or suppression of endogenous catalase[28•]. The action of ROS may be augmented by nitricoxide (NO) to potentiate and propagate the cell death sig-nals [25•]. It has been speculated for a number of years thatROS involved in activating the HR may be generated byan NADPH oxidase similar to the well-characterized ROSsignaling system in neutrophils [24,25•]. Recent identifica-tion of six Arabidopsis genes homologous to the largesubunit of the human NADPH oxidase should facilitatetesting of this hypothesis [29,30]. In support of theinvolvement of NADPH oxidase as an activator of ROS forHR induction, recent work by Kawasaki et al. [31••]showed that the small GTP-binding protein Rac maymediate the HR response of rice in an ROS-dependent

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manner. Rac is known to regulate mammalian NADPHoxidase as well.

How does ROS lead to the activation of cell death and howis it managed in the cell? Reichheld et al. [32] suggest thatalteration of the cell cycle may be one aspect of deathinduction by ROS. Interestingly, the role of SA in cell pro-liferation as well as cell death was postulated to explainsome of the abnormal leaf morphologies observed in theacd6 mutant [20••]. Thus, SA and ROS may cooperate toarrest the cell cycle and tip the balance toward cell death.As plants are likely to be exposed to a variety of agents orenvironmental conditions that can activate ROS produc-tion, a buffering system that can deal with normalfluctuations in ROS level should be in place to preventchance activation of PCD. The work of Tamagnone et al.[33•] showed that transgenic expression of a transcriptionregulator leads to plant cells that are hyper-responsive topathogen challenge, abnormal cell expansion and PCD inthe leaf palisade cells, and increase in lipid peroxidation.Addition of phenolic precursors can rescue cell culturesderived from these transgenic plants from cell death andabnormal morphologies, thus showing nicely that thesephenotypes are reversible. This work provided evidencethat phenolic products could play an important role in ROShomeostasis during normal plant development.

The nuts and bolts of the death engineThe identities of the key executioners of plant cellsremain elusive, whereas in animal systems a large numberof caspases and their regulators have been defined in thepast five years [34•,35••]. Caspase-like proteolytic activityhas been observed to be transiently activated in plantssynchronized to undergo the HR [36••]. Peptide inhibitorsof caspases can abolish HR cell death induced by aviru-lent bacteria without affecting the induction of defensegenes significantly. Furthermore, transgenic expression ofthe gene encoding a broad range caspase inhibitor fromBaculovirus, p35, also leads to the delay of HR cell deathin tobacco (O del Pozo, E Lam, unpublished data). Morerecently, Mitsuhara et al. [37•] reported that in tobaccoexpression of transgenes encoding the pro-survival celldeath regulators Bcl-xL and Ced-9 can delay HR celldeath 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 regulatoryswitch may operate in plants to control cell death [35••].However, alternative explanations for these results arepossible (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 proteaseinhibitor cystatin can suppress ROS and bacteria-inducedcell death in soybean cell cultures, while activation of celldeath by the xylanase from Trichoderma viride can be

inhibited by the serine protease inhibitor AEBSF (4-[2-aminoethyl]-benzenesulfonyl-flonyl-fluoride), but not bycaspase inhibitors [40]. Activation of cell death by victorinin oat also appears to be mediated by cysteine proteasessensitive to E-64 and calpeptin [14•]. These studies sug-gest that proteases with different specificities may beused to regulate cell death induced by different agents indifferent species. Involvement of intracellular cysteineproteases [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 celldeath activation has been recognized in the animal field.Cytochrome c leakage from this organelle appears tomediate many signaling pathways for PCD. Once in thecytosol, cytochrome c can activate caspases through inter-action with Ced-4/APAF1 in conjunction with dATP[35••]. In addition, disruption of electron flow through theelectron transport chain of the mitochondria is likely tolead to accumulation of reducing equivalents that canresult in ROS. Bcl-2 related proteins can either activate orrepress the leakage of cytochrome c through the voltagedependent anion channel (VDAC) located in the mem-brane of the mitochondria [41]. They can also suppress oractivate caspases through interaction with the Ced-4/APAF1 regulator.

In plants, the fungal toxin victorin appears to induce celldeath via inhibition of the mitochondrial enzyme glycinedecarboxylase [14•]. More recently, the mitochondrial con-nection to PCD in plants was implicated by the work ofLacomme and Santa Cruz [38••]. Bax was found to activateHR-like cell death and its localization to plant mitochon-dria was required. Bax expression was also known toinduce cell death in yeast with similar dependence on itstargeting to the mitochondria [42]. Thus, it appears thatthis organelle may be a conserved site where death signalscan be generated in eukaryotes. This may act via leakageof cytochrome c, which has not been demonstrated in plantPCD or in yeast. It may also be mediated by other celldeath factors that bypass the caspase switch and act direct-ly at the nuclear level [43••]. This type of cell deathactivation may be responsible for caspase-independentPCD 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 fromhuman cells [42]. BI-1 appears to be well conserved in ani-mals and genes with weak sequence homology to BI-1have been identified in C. elegans and Arabidopsis sequencedatabases. Although its function is unclear at present, BI-1may be an ancient regulator of cell death activationthrough the mitochondria.

The mitochondrial connection in HR cell death has alsobeen made at the level of the Alternative oxidase (AOX).AOX is not found in animals and its function as a heat gen-erator in floral tissues such as the Voodoo Lily has been

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established. However, plants suppressed in AOX expres-sion remain largely normal until stressed by inhibitors ofthe mitochondrial electron transport chain such asantimycin 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 upontreatment with AA. Moreover, they showed that AOX-defi-cient plants constitutively expressed defense gene markerswhereas overexpressors of AOX repressed the basal expres-sion levels of these genes. Thus, ROS generated in themitochondria can lead to the induction of PR gene expres-sion. Furthermore, these observations suggest thatinterruption of normal electron transport functions leads torapid generation of ROS and the role of AOX may be to actas a safety valve to ‘siphon’ off the reducing intermediatesfrom the clogged electron transport chains. Consistent withthis idea is the observation that treatments with inhibitorsof oxidative electron transport such as AA and cyanide acti-vate AOX expression [46••]. In addition, mutations ortransgenes that lead to an impairment of heme biosynthe-sis result in spontaneous lesion formation [13,47,48]. Thedisruption of heme biosynthesis may lead to a loss of func-tional cytochromes and excess ROS generation from themitochondria. Together with expected defects in ROS pro-tectants that require heme as their prosthetic group such ascatalases and peroxidases, it is plausible that in these plantsROS can easily reach a threshold sufficient to activate celldeath spontaneously. Induction of AOX has been observedunder various stress conditions and a recent study inArabidopsis showed that rapid localized AOX induction byavirulent bacterial pathogens requires SA [49••]. On theother hand, ethylene is absolutely required for AOX induc-tion by pathogens. This work suggests that localizedinduction of AOX may act to protect the cells at or near theinfection sites against ROS damage. This work comple-mented the studies of Chivasa and Carr [46••] who showedthat cyanide treatment, which activates AOX synthesis, canreverse the spreading cell death phenotype observed inNahG expressing tobacco plants. The effects of cyanidecan be reversed by salicylhydroxamic acid (SHAM), aninhibitor of AOX. Obviously, the results with chemicalinhibitors have to be interpreted cautiously since SHAMcan also inhibit other enzymes such as peroxidases that mayfunction to protect against ROS. Nevertheless, it is tempt-ing to speculate that AOX may be involved in cell deathregulation in the development of spreading lesions such asthose observed during tobacco mosaic virus (TMV) infec-tion. Future manipulation of AOX levels should lead to abetter understanding of the role for this protein in deter-mining lesion size and disease resistance.

ConclusionsThe mitochondrion has emerged recently as a conservedsite where cell death signals in the form of ROS and proteinfactors can be generated. Our understanding of its role inplant PCD will be aided by comparative analyses withresults from animal and yeast fields, in addition to thewealth 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 outthe complex interplay between the varieties of regulatorsthat are likely to control this important process.

AcknowledgementsResearch on cell death in our laboratory is funded in part by the New JerseyCommission on Science and Technology, and a competitive research grantfrom the United States Department of Agriculture. Sharing of preprints andunpublished information by Jean Greenberg, Ko Shimamoto and YukoOhashi is gratefully acknowledged.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Raff M: Cell suicide for beginners. Nature 1998, 396:119-122.

2. Pennell RI, Lamb C: Programmed cell death in plants. Plant Cell1997, 9:1157-1168.

3. 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 het-erotrimeric G-protein activator mastoparan and a calcium ionophore werefound to accelerate hormone-dependent programmed cell death. Vacuolecollapse and cessation of cytoplasmic streaming preceded nuclear DNAfragmentation, suggestive of an autolytic process of programmed celldeath. Interestingly, these authors found that addition of 1% (w/v) trypsinactivated vacuole collapse and nuclear DNA fragmentation in a calcium-dependent manner whereas exogenous soybean trypsin inhibitor has theopposite effect of inhibiting TE differentiation. A 40 kDa secreted proteasethat can be inhibited with soybean trypsin inhibitor was found to correlatewith 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 acidinduced aleurone cell death with isolated protoplasts. Cell death is preced-ed by an abrupt loss of membrane integrity and vacuolization of cytoplasmicvesicles. Signaling by cGMP is suggested by the effects of LY83583, aguanylyl cyclase inhibitor, on DNA degradation and programmed cell deathof aleurone cells. The authors concluded that gibberellic acid activated aleu-rone 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.

7. Gilchrist DG: Programmed cell death in plant disease: thepurpose and promise of cellular suicide. Annu Rev Phytopathol1998, 36:393-414.

8. Mittler R, Simon L, Lam E: Pathogen-induced programmed celldeath in tobacco. J Cell Sci 1997, 110:1333-1344.

9. Young TE, Gallie DR: Analysis of programmed cell death in wheatendosperm reveals differences in endosperm developmentbetween cereals. Plant Mol Biol 1999, 39:915-926.

10. Pontier D, Gan S, Amasino RM, Roby D, Lam E: Markers forhypersensitive response and senescence show distinct patternsof 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 chromatincondensation during cell death induction of tobacco protoplasts by chemi-cal treatments. Annexin V binding, indicative of phosphatidylserine exposure,as well as chromatin condensation appeared to be reversible at the earlystage of the cell death process in this assay system.

12. Skalamera D, Heath MC: Changes in the cytoskeletonaccompanying infection-induced nuclear movements and thehypersensitive 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-deathmechanisms 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 susceptibleoat leaves resulted in chromosomal laddering, similar to those previouslyreported for Alternaria alternate toxin and tomato. Victorin interacts withsubunits of the photorespiratory enzyme glycine decarboxylase (GDC) andeffectively inhibits this mitochondrial enzyme in vivo. Parallels between vic-torin induced cell death and senescence are drawn. An interesting pheno-type is the cleavage of the 14 amino-terminal amino acid residues from thelarge subunit of ribulose 1,5-bisphosphate carboxylase (LSU) and its inhibi-tion by a variety of treatments, including cysteine protease inhibitors such asE-64 and calpeptin. This work suggests the plant mitochondria may be thesite where an apoptotic signal can be activated by victorin inhibition of pho-torespiration through the GDC. Ethylene also appears to be required for vic-torin induced cell death and LSU cleavage.

15. Lund ST, Stall RE, Klee HJ: Ethylene regulates the susceptibleresponse to pathogen infection in tomato. Plant Cell 1998,10:371-382.

16. Lawton KA, Potter SL, Uknes S, Ryals J: Acquired resistance signaltransduction in Arabidopsis is ethylene independent. Plant Cell1994, 6:581-588.

17. He CJ, Morgan PW, Drew MC: Transduction of an ethylene signalis required for cell death and lysis in the root cortex of maizeduring aerenchyma formation induced by hypoxia. Plant Physiol1996, 112:463-472.

18. Weymann K, Hunt M, Uknes S, Neuenschwander U, Lawton K,Steiner HY, Ryals J: Suppression and restoration of lesionformation 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 constitu-tively expresses PR genes in an NPR1 independent manner. SpontaneousHR-like cell death lesions are observed and the phenotypes are SA depen-dent as revealed through NahG expression. This work suggests that SA isrequired, 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 incontrolling cell death, defenses and cell growth. Plant Cell 1999,11:191-206.

This report documents the characterization of a new lesion mimic mutant inArabidopsis, acd6. It is caused by a dominant mutation that activates celldeath and resistance to bacterial pathogen; however, the plant is unable torespond with an HR upon challenge with an avirulent bacteria and phy-toalexin synthesis is not activated. Interestingly, the phenotype of acd6 is SAdependent, as it can be reversed by NahG expression, but is partially NPR1independent. A novel phenotype of the acd6 mutant in comparison to otherpreviously described lesion mimics is the observation of abnormal cellgrowth and enlargement that is SA dependent. A model is presented where-by SA potentiates both cell death and cell growth and/or proliferation andthe acd6 mutation effectively decreases the threshold of response for SAsignaling to these processes.

21. Rao M, Davis KR: Ozone-induced cell death occurs via two distinctmechanisms in Arabidopsis: the role of salicylic acid. Plant J1999, 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 tech-nology and a dominant-negative mutant resulted in heightened sensitivityto 2,2-dichloroisonicotinic acid (INA) whereas overexpression of thiskinase 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 ofplant disease resistance responses. Proc Natl Acad Sci USA1999, 96:766-771.

The expression of two isoforms encoded by SCaM4 and SCaM5 of thecalmodulin gene family from soybean was found to respond to pathogenand fungal elicitors. Overexpression of these genes in transgenic tobaccoresulted in the appearance of hypersensitive-response-like phenotypesincluding spontaneous cell death in older mature leaves, pathogen-related

gene expression and broad spectrum resistance to various types ofpathogens. Surprisingly, salicylic acid (SA) levels are not affected by thesetransgenes suggesting that they act via SA-independent pathways toinduce lesions and resistance. This work provides evidence that intracellu-lar calcium levels can play important roles in the control of cell death acti-vation in plants via specific calmodulin isoforms that are transcriptionallyregulated. Moreover, it clearly demonstrates the existence of SA-indepen-dent 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 compo-nents such as nitric oxide, ethylene and salicylic acid on the activation ofcell death and disease resistance. The authors provide a good summaryof the temporal behavior of various markers of cell death and defense, aswell as a working model integrating recent observations of the varioussignaling components.

26. Mittler R, Shulaev V, Seskar M, Lam E: Inhibition of programmedcell death in tobacco plants during a pathogen-inducedhypersensitive response at low oxygen pressure. Plant Cell 1996,8:1991-2001.

27. Kazan K, Murray FR, Goulter KC, Llewellyn DJ, Manners JM: Inductionof cell death in transgenic plants expressing a fungal glucoseoxidase. 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 intransgenic tobacco. Proc Natl Acad Sci USA 1998, 95:5818-5823.

Expression of catalase in tobacco plants was suppressed by transgenicexpression of antisense transcripts. High light treatment of these plantsresults in activation of hypersensitive-response-like cell death as well asdefense gene markers and disease resistance, presumably due to the pro-duction of H2O2. These responses were shown to be systemic and theinduction of cell death can be uncoupled from disease resistance anddefense markers, with sublethal levels of ROS (reactive oxygen species)inducing pathogenesis-related gene expression and resistance without con-comitant cell death.

29. Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE,Jones JJ: Six Arabidopsis thaliana homologues of the humanrespiratory burst oxidase (gp91phox). Plant J 1998, 14:365-370.

30. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: Aplant homolog of the neutrophil NADPH oxidase gp91phox subunitgene encodes a plasma membrane protein with Ca2+ bindingmotifs. 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 expres-sion of its constitutively active or dominant-negative mutations in transgenicrice cell cultures and plants. Expression of the transgene encoding the con-stitutively active mutant of OsRac1 resulted in ROS (reactive oxygenspecies) production and programmed cell death which correlated withTUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) staining. Involvement of the NADPH oxidase is suggested by inhi-bition of these phenotypes by diphenylene iodonium. Conversely, transgenicexpression of a dominant-negative variant of OsRac1 in a rice lesion mimicmutant resulted in suppression of ROS generation and cell death inductionby a rice blast fungus and calyculin A, a protein phosphatase I inhibitor. Thiswork provides the first clear evidence for the involvement of Rac in the reg-ulation of plant NADPH oxidase in ROS generation and cell death induction.It complements nicely the identification of multiple plant genes encoding thelarge subunit of the plant NADPH oxidase and suggests that analogous tothe neutrophil NADPH oxidase, the plant enzyme is also regulated by Racand phosphorylation.

32. Reichheld JP, Vernoux T, Lardon F, Van Montagu M, Inze D: Specificcheckpoints regulate plant cell cycle progression in response tooxidative 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 cellmorphology in leaves of transgenic tobacco plants. Plant Cell1998, 10:1801-1816.

An extensive study characterizing the phenotypic defects resulting fromdownregulation of phenolic acid metabolism in transgenic tobacco throughthe transgenic expression of the gene encoding the transcription factorAmMYB308. Concomitant with the decrease of phenolic compounds,

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Die and let live — programmed cell death in plants Lam, Pontier and del Pozo 507

abnormal development of the leaf palisade layer is correlated with prematureprogrammed cell death as detected by TUNEL staining. This ‘early senes-cence’ phenotype could be rescued in cell cultures by supplying exogenousphenolic precursors. Interestingly, the hypersensitive response is activatedmuch faster in these transgenic plants and this response correlated with aheightened level of lipid peroxidation. These and earlier results suggest thatphenolic 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 regu-lators that have been defined in animal systems. Interesting comparisonsbetween molecules that are or may be involved in cell death regulationfrom plants, animals and prokaryotes are presented and the evolutionaryimplications 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 knowl-edge on the structure, function and regulation of caspases, the growing fam-ily 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 caspasescan suppress hypersensitive response (HR) cell death induced by aviru-lent bacteria when co-infiltrated into tobacco leaves. The induction of twoHR cell death gene markers was inhibited whereas pathogenesis-relatedgene induction was not affected by these inhibitors, thus showing thatplant–pathogen signaling remains intact and that defense gene activationcan be uncoupled from cell death. Transient induction of caspase-likeproteolytic activity was detected in extracts from leaf tissues duringtobacco mosaic virus (TMV)-induced HR that was synchronized by tem-perature shift. This work constitutes the first report of caspase-like pro-tease activities in plants and provided evidence for their participation inHR 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 genesencoding the pro-survival cell death regulators Bcl-xL and Ced-9 from ani-mal systems can delay cell death induced by ultraviolet light, paraquatand pathogen challenge. Although the mechanism responsible for theseobservations is unclear at present, the results suggest that these regula-tors may suppress one or more evolutionarily conserved cell deathswitches 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 sys-tems is expressed in tobacco by a tobacco mosaic virus (TMV) viral vectorand found to induce hypersensitive-response-like phenotypes such as celldeath and pathogenesis-related gene expression. Mutational analyses showthat the likely target site of Bax in plants is the mitochondria and thatdomains required for Bax dimerization are required for optimal cell deathinduction. This work provides evidence that plant mitochondria can be thesite 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 Cell1999, 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 proteaseinhibitor cystatin resulted in suppression of programmed cell death. Thiswork showed that a cysteine protease or proteases that can interact withcystatin may be good candidates for activities that are required to executethe death signal upon oxidative stress and during the HR in this system.

40. Yano A, Suzuki K, Shinshi H: A signaling pathway, independent ofthe oxidative burst, that leads to hypersensitive cell death incultured tobacco cells includes a serine protease. Plant J 1999,18:105-109.

41. Shimizu S, Narita M, Tsujimoto Y: Bcl-2 family proteins regulate therelease of apoptogenic cytochrome c by the mitochondrialchannel VDAC. Nature 1999, 399:483-487.

42. Shaham S, Shuman MA, Herskowitz I: Death-defying yeast identifynovel 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 purifiedfrom mouse liver mitochondria. It is a mitochondrial protein that is releasedby the transition pore which can be regulated by Bcl-2. Purified apoptosisinducing factor (AIF) can induce apoptotic phenotypes in isolated nuclei aswell as activate cytochrome c release from mitochondria in whole cells withsubsequent caspase activation. AIF thus may function directly in inducingnuclear events as well as serving to amplify cell death signals. It may beinvolved in caspase-independent cell death signaling as AIF activity inmicroinjected 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 anti-sense transcript expression, respectively. In the absence of stress, plantsdeficient 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 ComplexIII 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 treatmentas well as suppressing basal levels of PR-1 expression. This work providesthe first evidence that interference with electron transport in plant mitochon-dria can lead to significant ROS generation and this process is regulated byAOX. Furthermore, ROS generated from plant mitochondria can activate celldeath and defense gene markers coordinately, analogous to that observedduring 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 ofAOX to the same level as salicylic acid (SA) and its analog 2,2-dichloro-isonicotinic acid (INA) in tobacco. Cyanide treatment can reverse theeffects 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 alonecan block SA-dependent resistance to TMV, induce PR-1 in an SA-depen-dent manner, but does not affect resistance to bacterial or fungalpathogens. 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 morecomplex to interpret at present.

47. Hu G, Yalpani N, Briggs SP, Johal GS: A porphyrin pathwayimpairment is responsible for the phenotype of a dominantdisease 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 ofprotoporphyrinogen oxidase expression in Arabidopsis causes alesion-mimic phenotype that induces systemic acquiredresistance. 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 pvtomato. Plant Physiol 1999, 120:529-538.

AOX (alternative oxidase) expression was found to be induced rapidly in theinfected leaves by challenge with avirulent, but more slowly with virulent,bacterial pathogens in Arabidopsis. In addition, the rapid but not the slowerrise in AOX is dependent on salicylic acid but independent of NPR1.Furthermore, ethylene sensing through ETR1 is absolutely required for AOXinduction. This work suggests that the local induction of AOX involves ethyl-ene production during the hypersensitive response HR and that AOX couldserve as a protectant for ROS (reactive oxygen species) damage to the cellsat or near the infection site.

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