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Journal of Fruit and Ornamental Plant Research Vol. 13, 2005: 135-158

REVIEW

HYPERSENSITIVE CELL DEATH IN PLANTS – ITSMECHNISMS AND ROLE IN PLANT DEFENCE

AGAINST PATHOGENS

E l e n a T . I a k i m o v a 1 , L e c h M i c h a l c z u k 2

a n d E r n s t J . W o l t e r i n g 3

1Agrobioinstitute, 8 Dragan Tsankov Blvd., 1164, Sofia, Bulgaria,2Institute of Pomology and Floriculture

Pomologiczna 18, 96-100 Skierniewice, Poland3Wageningen University and Research Center, Agrotechnology & Food

Wageningen, The Netherlands

*Correspondence to: [email protected]

(Received November 11, 2005/Accepted February 2, 2006)

A B S T R A C T

This review is a recent update in the understanding of the hypersensitive response(HR) of plants with special consideration to the physiological and biochemicaldeterminants in different model systems. Hypersensitive response is reviewed asa form of programmed cell death (PCD) representing one of the mechanisms of plantdefence against diseases. Major signalling pathways and molecules that accompanythe HR, such as proteolytic cascades, oxidative events and ethylene that are supposedto play a key role in the plant’s cell death machinery are discussed. Special attentionis paid to the HR in fruit species. Studies on plant PCD are shown to provide a clue tobetter understanding disease resistance processes in plants and to establish theevolutionary aspects of PCD similarities through animal and plant kingdoms.

Key words: fruit plants, hypersensitive response, programmed cell death

Abbreviat ions: AOA, aminooxyacetic acid; APX, ascorbate peroxidase; AVG,aminoethoxyvinylglycine; CAT, catalase; CMV, cucumber mosaic virus; CPT,camptothecin; DHAR, dehydroascorbate reductase; DHMC, 2,5-dihydroxymethyl-cinnamate; DPI, diphenyl iodonium; GR, glutathione reductase; JA, jasmonic acid; LAR,local acquired resistance; MAPK, mitogen-activated protein kinase; MDHAR,monodehydroascorbate reductase; MeJA, methyljasmonate; NO, nitric oxide; PAL,phenylalanine ammonia-lyase; PMA, phorbol 12-myristate 13-acetate; PMSF,phenylmethylsulphonylfluoride; PPV, plum pox virus; ROS, reactive oxygen species; SA,

E.T. Iakimova et al.

J. Fruit Ornam. Plant Res. vol. 13, 2005: 135-158136

salicylic acid; SAR, systemic acquired resistance; SOD, superoxide dismutase; STS, silverthiosulphate; TCV, turnip crinkle virus; TDFs, transcript-derived fragments; TFP,trifluoperazine; TMV, tobacco mosaic virus; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling; TVTV, turnip vain clearing tobamo-virus;VPE, vacuolar processing enzyme; W-7, N-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide

INTRODUCTION

The term hypersensitive response(HR) historically comes from Stakman(1915) who used it to describe a rapidhost cell death in plants (oat, wheatand barley) infected with fungalpathogen Puccinia graminis. Modernstudies of this phenomenon wereinitiated in early 1960’ by Klement etal. (1964), who observed rapid,localised necrosis on tobacco leavesinfiltrated with plant pathogenicbacteria Pseudomonas syringae pv.syringae. Although since that timenumerous works have been done onpathogen-induced cell death in plants(for reviews see: Greenberg et al.,1994; He 1996; Dangl et al., 1996;Morel and Dangl, 1997; Gilchrist,1998; Heath, 2000; Shirasu andSchulze-Lefert, 2000; Goldbach et al.,2003; Greenberg and Yao, 2004;Khurana et al., 2005), it is far frombeing fully understood. Because it isassociated with disease resistance, HRremains the focus of interest for plantbiologists. Many studies are devoted tothis process in model plants such asArabidopsis thaliana, tobacco, rice,wheat, spruce, tomato, carrot, pepper,etc. The exploration of plant cell deathresponse to pathogens led to anassumption for a common players,cross talking in the struggle of theplant to survive. Thus, the devlopmentof an effective hypersensitive response

in any plant system relies, on aneffective and rapid signal transductionsystem.

This review aims to summarizesome of the recent findings on thephysiological, biochemical and mole-cular machineries of the HR. As theHR is a process of programmed celldeath (PCD) associated with plantreaction to pathogens, a description ofmain morphological and biochemicaldeterminants of PCD is also discussed.

1. Programmed cell death –definition and morphologicaldeterminants

Programmed cell death is func-tionally conserved and genedirectedwell-orchestrated process of cell self-destruction (Gilchrist, 1998). In animaland plant kingdoms, it is an activeprocess of cell suicide leading tocontrolled elimination of cells that areharmful, unwanted or misplaced inspecific structures and organs, andplays an essential role during develop-ment and morphogenesis. PCD isinvolved in defence mechanismsagainst infected or mutated cells and inresponse to environmental stresses,including abiotic and biotic stimuli.Deregulation of PCD is implicated invarious human diseases, includingbirth defects, ischemic vasculardiseases, neurodegenerative diseases(e.g. Alzheimer’s and Parkinson’s

Hypersensitive cell death in plants - its mechanisms and role….

J. Fruit Ornam. Plant Res. vol. 13, 2005: 135-158 137

diseases), autoimmune diseases, AIDS,cancer, Diabetes mellitus type I.

There are various mechanisms bywhich PCD is executed and thisdiversity reflects the definitions.According to Martin et al. (1994), PCDis a functional term used to describe“cell death as a normal part of the life ofmulticellular organisms” emphasisingthe role of developmental cell death.Priority to the genome is given byZhivotovsky et al. (1997), who describethe PCD as “genetically controlled celldeletion process”. Biochemistry hasbeen stressed by Laytragoon (1998),who defined PCD as a process wherebydevelopmental or environmental stimuliactivate “a specific set of events thatculminate in cell death”. Morphologicalsimilarities are found between animalcells undergoing apoptosis and suicidalplant cells, including condensation andshrinkage of the cytoplasm and nucleus,nuclear DNA fragmentation andformation of apoptotic-like bodies(Wang et al., 1996ab; de Jong et al.,2000). This implies an evolutionaryconservation of aspects of programmedcell death between animals and plants.

Three major types of programmedcell death, based mainly on morpho-logical changes, are recognised so far:apoptosis, autophagy and non-lysosomal cell death (reviewed in vanDoorn and Woltering, 2005). Apop-tosis, a term originally introduced byKerr et al. (1972), is characterised bya specific set of cellular morphologicalfeatures (Steller, 1995) such as plasmamembrane blebbing, disintegration ofcytoskeletal elements, condensation ofthe nucleus and the cytoplasm, inter-nucleosomal cleavage of DNA and

fragmentation of the cell intomembrane confined, DNA containingvesicles (apoptotic bodies). A dis-tinguishing feature of apoptosis is thatfragments of the dying cell areengulfed and digested by the lyso-somes of neighbouring (living) cells(e.g. phagocytes). This latter processhas, so far, not been observed in anyplant system, which indicates that trueapoptosis does not occur in plants.Autophagy (self-eating) is a majordegradation and recycling system ineukaryotic cells (Lum et al., 2005). It isfeatured by degradation of cellularcomponents by formation of autopha-golysosome (Yoshimori, 2004).Autophagy is associated with cellsurvival as well as cell death and itmay be a strategy to remobilise part ofthe cell contents prior to cell death.Autophagy was found to play a role inplant immune response, especially inpreventing of death and pathogenspread in cells adjacent to HR lesion(Greenberg, 2005). DevelopmentalPCD (which is associated with thenormal developmental processes) isinvolved in degeneration of specificcells and most of the examples ofdevelopmental PCD in plants conformto the definition of autophagic PCD(van Doorn and Woltering, 2005).Many morphological features thatwere earlier considered typical forapoptosis also occur in autophagic celldeath. The third morphological type ofPCD is called non-lysosomal (ornecrosis-like) PCD. It does not involvea role of the lysosome of the dying cellitself nor of other cells. It is associatedwith swelling of organelles andformation of “empty spaces” in the



cytoplasm, and has similarities tonecrosis (Baehrecke, 2005).Although not much detailed infor-mation is available on cell morpho-logy during the plant HR, mostly thisseems to conform to this non-lysosomal type of PCD (van Doornand Woltering, 2005). Necrosis isa cell death that is not accompaniedby chromatin condensation (Leistand Jäätelä, 2001) and is oftenclassified as aborted apoptosis.Although necrosis has been definedas an accidental and uncontrolled celldeath, there is growing evidence thatnecrotic and apoptotic forms of celldeath may share similarities (Guima-raes and Linden, 2004).

2. Plant PCD

Apart from the specificity ofmorphological characteristics, theprocess of PCD is accompanied bytypical biochemical events and simi-larities have been reported betweenanimal and plant programmed celldeath signalling.

In plants, as in animals, program-med cell death is essential machineryfor growth, development and playsa role in the response to stresses(Danon et al., 2000; Jones, 2001; Lam,2004). Cellular suicide is involved in,for example, xylogenesis (Fukuda,2000), aerenchyma formation (Drew etal., 2000), plant reproduction (Wu andCheung, 2000), leaf and petalsenescence (Quirino et al., 2000;Rubinstein, 2000), endosperm celldeath during germination (Fath et al.,2000; Young and Gallie, 2000).Furthermore, PCD plays an important

role in plant response to pathogens(Heath 2000; Shirasu and Schulze-Lefert, 2000; Greenberg and Yao,2004), and to diverse abiotic stressessuch as heat shock (Tian et al., 2000;McCabe and Leaver, 2000; Zhang etal., 2004), exposure to toxic chemicals(Sun et al., 1999), ozone (Overmyer etal., 2000; Rao et al., 2000; Pasqualiniet al., 2003, Overmyer et al., 2005),UV radiation (Danon and Gallois,1998) and hypoxia (Xu et al., 2004).Biochemical changes involving produ-ction of reactive oxygen species (Jabs,1999; Levine et al., 1994; Moeder etal., 2002), Ca release (Levine et al.,1996; Zuppini et al., 2003), proteolysis(Beers and Freeman, 1997; Xu andChye, 1999; de Jong et al., 2000), andethylene biosynthesis (Lund et al.,1998; Spencer et al., 2003; Wolteringet al., 2003) are found to activelyparticipate in the plant PCD signaltransduction.

The classic form of apoptosisinvolves key cell death executioners,which are cysteinyl-aspartic-proteasesnamed caspases (Grütter, 2000).A sequence of caspase-activated prote-olytic cascade, involving initiator cas-pases and down-stream activation ofexecutioner caspases, leads to apo-ptotic phenotype (Hengartner, 2000).Although true structural homologuesof animal caspases have not yet beenidentified in plants, there are accu-mulating evidences that cysteineproteases, showing functional simila-rity to caspases, participate in theprogrammed cell death in plants.Caspases can be selectively inhibitedby small peptides mimicking thesubstrate recognition site and such



peptides can also be used to determinecaspase activity. Using such anapproach, caspase-like activity hasbeen detected following the infectionof tobacco with bacteria and a virus(Del Pozo and Lam, 1998; 2003). Byadministration of specific inhibitors,a role of caspase-like proteases inapoptotic-like cell death of tomatosuspension cells in response to treat-ment with camptothecin (CPT, analkaloid from Camptoteca acuminata)and the fungal toxin Fumonisin B1 (deJong et al., 2000; Woltering et al.,2002) is established.

Two groups of proteases aresuggested to function in caspase-likemanner in plants: vacuolar processingenzymes (VPEs) and metacaspases(Woltering, 2004; Sanmartin et al.,2005). In addition, two subtilin-likeSer proteases (SAS-1 and SAS-2),named saspases, were purified andcharacterized to exhibit caspase-likehydrolytic activity (Warren andWolpert 2004). VPEs play a role incell death and are important factorsaffecting plant susceptibility topathogens. A study with Arabidopsisthaliana showed that VPE-gamma hascaspase-like activity and that mutantswith reduced activity showed lessercell death rate and increasedsusceptibility to pathogens (Pseudo-monas syringae, Botrytis cinerea,turnip mosaic virus) (Rojo et al.,2004). The involvement of VPE is alsoreported in TMV-induced hyper-sensitive cell death in tobacco(Hatsugai et al., 2004) and mycotoxin-induced cell death (Kuroyanagi et al.,2005).

3. HR definition, morphologicalfeatures and elicitation

Plants have elaborated sophis-ticated and efficient system to count-eract the spread of pathogen invasion.The hypersensitive response is a formof cell death associated with plantresistance to pathogen infection andoccurs at incompatible and sometimesin compatible plant-pathogen inter-actions (Morel and Dangl, 1997). HRis characterized by a rapid, localizeddeath of tissues at the site of infection,limiting further pathogen multipli-cation and spread (Gilchrist, 1998;Heath, 2000; Shirasu and Schulze-Lefert, 2000). During this response, theplant sacrifices some of its cells tocircle the invading pathogen witha layer or a ring of dead plant cells thusinhibiting the growth of the pathogenby killing of infected and non-infectedcells and by producing a physicalbarrier.

During the HR, dying plant cellsstrengthen their cell walls bydepositing different phenolic com-pounds, synthesizing diverse toxiccompounds called phytoalexins, andaccumulating proteins with antimicro-bial activity called pathogenesis-related proteins (Dangl et al., 1996).The understanding of the pathogen-plant interactions that result in the HRmay lead to the discovery of effectivemethods for disease control (Dangl etal., 1996).

Hypersensitive cell death is gene-tically controlled and occurs when theplant genotype recognizes a specificisolate of a pathogen. The reactioninvolves activation of host defence-related genes (Suh et al., 2003) and



various defence responses (Greenberget al., 1994; Lamb and Dixon, 1997).

The morphological and ultra-structural changes that accompanyPCD during plant-microorganisminteractions have been studied in onlya few model pathogens and plants.Cessation of cytoplasmic streamingfollowed by protoplast dismantling andby cleavage of nuclear DNA has beenobserved as an early event during HRin the epidermis of cowpea (Vignaunguiculata) that had been infected bythe rust fungus Uromyces vigna and incell death induced by the barleypowdery mildew fungus. PCD inplant-pathogen interactions does showsome features of apoptosis but no clearevidence for the formation of apo-ptotic-like bodies has been found andno phagocytosis has been observed inthe primary lesion. It seems that HRcell death mostly belongs to the non-lysosomal category of cell death(Woltering and van Doorn, 2005). Inthe light of the recent understanding ofthe role of autophagy in plant immuneresponses, Lui et al. (2005) haveobserved formation of autopha-gosomes in cells adjacent to theprimary infection and in distantsystemic tissues. The blocking ofautophagy resulted in increased localvirus propagation, which demonstratesthat autophagy plays a prominent rolein plant immune response.

Evidences suggesting that HRresults from PCD process are: theactivation of cell death in the absenceof pathogens by mutations in certaingenes thought to be involved in the celldeath pathway, the activation of celldeath upon recognition of elicitors

produced by the pathogen, and theactivation of the HR by expression oftransgenes in plants (Heath, 2000). Thefact that cell death resembling the HRcan be activated in the absence ofa pathogen strongly suggests that thistype of cell death is not directly causedby the invading pathogen but ratherresults from the activation of a host-encoded pathway for PCD. Perhaps themost persuasive evidence that the HRis a PCD process is the existence ofmutants that spontaneously activate theHR in the absence of a pathogen(Dangl et al., 1996). These mutants areoften referred to as “disease lesionmimics” and the mutations that causethe appearance of HR lesions in theabsence of a pathogen are thought tooccur in plant genes that control PCD,thus presenting a powerful tool for thestudy of HR in plants. Disease lesionmimic mutants have been isolated fromtomato, maize, barley, rice, andArabidopsis.

PCD can be induced in plants bytoxins produced by a number ofpathogens – harpins from Pseudo-monas syringae, Erwinia amylovora,Xanthomonas campestris, the fungaltoxin victorin, xylanase fromTrichoderma viridae (He, 1996; Grantand Mansfield, 1999; Lam et. al.,1999), Alternaria alternata AALtoxin, the fungal toxin Fumonisin B1(FUM) from Fusarium moniliforme(Wang et al., 1996a), fungal toxincryptogein from Phytophthoracryptogea (Hirasawa et al., 2005).Also, plant viruses such as tobaccomosaic virus (TMV) are reported toelicit PCD (del Pozo and Lam, 2003).Purified elicitors, such as Fumonisin



B1 or harpins can trigger HR, inducingphysiological changes associated withdisease resistance (Numberger et al.,1994).

For colonizing and parasitizingtheir hosts, pathogens have evolvedmultiple mechanisms, including viru-lence factors that compromise theintegrity and function of the plant cell.One of them are harpins, which are cellmembrane associated proteins, invol-ved in type III secretion system ofpathogens and are encoded by genes ofthe hrp clusters (Samuel et al., 2005).Bacterial genes encoding the compo-nents of type III secretion system areinduced when bacteria penetrate theplant and type III proteins are believedto promote the disease by altering thenormal physiology of the plant tobenefit the pathogen (Abramovitch andMartin, 2004). It has been shown thatspraying plants with harpin coordina-tely induces systemic resistance topathogens and micro-HR and that bothsignal components EDS1 and NDR1,which are required for the function ofsome R genes are necessary for thedevelopment of resistance (Peng et al.,2003).

4. Biochemical features of the HR

Results of molecular and bio-chemical studies support the hypo-thesis that caspase-like enzymes areinvolved in the HR. These include thesuppression of HR by synthetic peptidecaspase inhibitors and the observedincrease of caspase-like proteaseactivity in plant cells undergoing HR-PCD (Lam et al., 2001). Additionalplayers that may be similar to some ofthose controlling PCD in animals are

small GTP-binding proteins of the Rasclass and cystein-sensitive proteases.Study of Hatsugai et al. (2004) hasshown that VPE and the cellularvacuole control the cellular suicide thatis essential for HR in response toTMV.

Major players involved in theactivation of the HR are reactiveoxygen species (ROS), nitric oxide(NO), calcium and proton pumps,mitogen-activated protein kinases(MAPKs), and salicylic acid (SA). It isbelieved that the initial recognition ofthe pathogen by a plant receptor (partof the gene-for-gene response) acti-vates a signal transduction pathwaythat involves the translocation of Ca2+

and protons across the plasmamembrane into the cytosol, proteinphosphorylation/dephosphorylation,activation of enzymes that generateROS such as NADPH-oxidase andperoxidases, and accumulation of NOand SA. From challenged, HR-developing cells, some particulardiffusible molecules known as stressphytohormones (SA, jasmonic acid(JA) and ethylene), may play animportant signalling function inestablishing resistance both locally(local acquired resistance, LAR) andsystemically (systemic acquired resis-tance, SAR).

When attacked by incompatiblepathogens, plants respond by acti-vating a variety of defence responses,including ROS-generating enzymecomplex (Bolwell, 1999). The increaseof cellular concentration of ROS isa key event in plant and animal PCDand occurs as a result of many stresses(Laloi et al., 2004; Neill et al., 2002),



but an oxidative burst is an essentialprerequisite for induction of planthypersensitive cell death (Levine et al.,1994; Lamb and Dixon, 1997; Jabs,1999). Under normal conditions, ROSare cleared from the cell by the actionof superoxide dismutase (SOD),catalase, or glutathione peroxidase.Under stress conditions, ROS enhancethe lipid catabolism resulting inperoxidation of polyunsaturated fattyacids in the cell membranes that in turnleads to structural decomposition,change in permeability, and inducesdamage by alterations of essentialproteins, and DNA. CPT-induced PCDin tomato cell culture is accompaniedby a release of ROS into the culturemedium and it has been demonstratedthat accumulation of H2O2 and celldeath can be effectively suppressed byaddition of NADPH oxidase inhibitordiphenyl iodonium (DPI) and caspaseinhibitors (de Jong et al., 2002;Woltering et al., 2003). In addition, thefinding that catalase inhibits CPT-induced cell death suggests that H2O2

is responsible for the induction of PCD(de Jong et al., 2002).

Upon pathogen recognition, one ofthe earlier cell reactions is opening ofspecific ion channels, and the for-mation of superoxide and H2O2

(Kasparovsky et al., 2003). It has beenshown that H2O2 participates in thecross-linking of cell wall structuralproteins and functions as a local triggerof PCD in pathogen challenged cells.In addition, H2O2 acts as a diffusiblesignal (Hung et al., 2005) and inducesgenes encoding cellular protectants inadjacent cells when soybean cells havebeen elicited by glucan elicitor isolated

from mycelial walls of the fungalpathogen Phytophthora megasperma f.sp. glycinea (Levine et al., 1994).

Calcium is an important element inelicitor-mediated cell suicide signal-ling. Manipulations of proton andcalcium homeostasis are shown toactivate the HR. These include bio-chemical studies and the expression ofa proton channel that is localized to theplasma membrane and is reported toactivate the HR (Dominique et al.,2002). La3+ exerts inhibitory effect onCa channels and can completely blockthe cytoplasm shrinkage of culturedcarrot cells triggered by ethanol, heatshock and H2O2 (McCabe et al., 1997).In tomato suspension cells La3+

treatment prevents Fumonisin B1- andCPT-stimulated cell death, thusindicating that Ca2+ is implicated inCPT-triggered suicidal cascade (deJong et al., 2002; Iakimova et al.,2004). Crosstalk between pathways ofproteolysis, ROS, calcium andethylene is suggested at apoptotic-likecell death studied in two modelsystems: in chemical-treated suspen-sion tomato cells and in Pseudomonassyringae pv. tabaci challenged tobaccoleaves (de Jong et al., 2002; Iakimovaet al., 2004) and in tobacco leavessimultaneously infiltrated with Pseu-domonas syringae pv tabaci andcaspase inhibitors (Richael et al.,2001).

The combination of enhancedproduction of ROS and NO is shownto activate the HR in the absence ofa pathogen, and SA is demonstrated tofacilitate the formation of ROS as wellas to inhibit two of the key ROSremoval enzymes in plants: catalase



and ascorbate peroxidase (Conrath etal., 1995; Delledonne et al., 2001;Durner et al., 1997; Durner andKlessig, 1995). These processes arethought to be orchestrated by a cascadeof MAPKs (del Pozo et al., 2004;Pedley and Martin, 2004; Menke et al.,2005). The examination of a non-hosthypersensitive response and a diseaseinduced by Pseudomonas syringae pv.phaseolicola and P. syringae pv.tabaci in tobacco has revealed anincrease of NO 40 min following thechallenge with the pathogen, which issome 5 h before the initiation of visibletissue collapse (Mur et al., 2005).

Many plant species are resistant tothe majority of microbial invaders,a phenomenon that is termed non-hostresistance. At non-host resistance, theimportance of signalling pathwaysinvolving ROS, NO, alteration ofcytoplasmic Ca2+ levels and post-trans-lationally regulated MAPK activity isalso demonstrated, in addition to theirrole at incompatible plant-pathogeninteractions. It has been shown thatactivation of inducible plant defenceresponses is probably brought about bythe recognition of pathogen-associatedmolecular pat-terns (PAMP) that arecharacteristic of a whole class ofmicrobial organisms (Nürnberger andLipka, 2005). By characterisation ofexpressed sequence tags (ESTs), 14HR-specific ESTs involved in theexecution of the HR at β-megasperminelicitation of tobacco cells areestablished. Half of the ESTs exhibithomology with genes encodinga receptor-like kinase protein, proteinsinvolved in the regulation of plasmamembrane structure, proteins of the

ubiquitin/16S proteasome proteolyticsystem, RNA binding proteins, anda protein hypothesized to be a trueregulator of the HR (Ghannam et al.,2005).

cDNA–AFLP is a technique that isextensively used for construction ofgenetic linkage maps and is anefficient method for identification ofstress-induced genes. It allowslocalization of genes that confer resis-tance to viruses, nematodes, fungi orbacteria (Savelkoul et al., 1999). BycDNA–AFLP, transcripts, whoseexpression is rapidly altered duringinfection with fungal pathogenCladosporium fulvum expressing theAvr9 gene at the defence response oftobacco cell culture, are displayed.cDNA clones have been obtained forACRE (Avr9/Cf-9 rapidly elicited)genes. The amino acid sequence ofsome ACRE proteins shows homologyto known proteins such as calciumbinding protein, 13-lipoxygenase, anda RING-H2 zinc finger protein(Durrant et al., 2000).

Study on the involvement of NO inplant resistance to pathogens hasrevealed that key features, such asdefence gene expression and theactivation of a HR in synergy withROS are dependent on NO production.By using cDNA-AFLP, the inves-tigation of the changes in theexpression profiles of Arabidopsisthaliana following infiltration with theNO donor sodium nitroprusside, hasdetected altered expression patterns for120 of the approximately 2,500cDNAs examined. Sequence analysisshows homologies with genes involvedin signal transduction, disease resis-



tance and stress response, photo-synthesis, cellular transport, and basicmetabolism. Comparison of theexpression profiles with data obtainedby microarray has revealed that manyof the identified genes, modulated byNO, have been previously reported tobe modulated during plant diseases(Polverari et al., 2003).

Cassava genes differentiallyexpressed during the hypersensitivereaction of leaves in response to anincompatible Pseudomonas syringaepathovar have also been isolated by theapplication of cDNA-AFLP. Seventy-eight transcript-derived fragments(TDFs) showing differential expres-sion (75% up-regulated, 25% down-regulated) have been identified. Manyencode putative homologues of knowndefence-related genes involved insignalling (e.g. calcium transport andbinding, ACC oxidases and a WRKYtranscription factor), cell wall streng-thening (e.g. cinnamoyl coenzyme Areductase and peroxidase), program-med cell death (e.g. proteases, 26Sproteosome), antimicrobial activity(e.g. proteases and -1,3-glucanases)and the production of antimicrobialcompounds (e.g. DAHP (3-deoxy-arabinoheprulosonate-7-phosphate)synthase and cytochrome P450s)(Kemp et al., 2005). The regulation ofdefence gene expression is involved ininduced disease resistance. Theseinclude the plant-specific WRKYfamily of transcription factors definedby a DNA binding domain thatcontains the highly conserved aminoacid sequence WRKYGQK. WRKYfactors have been implicated in plantdefence, plant senescence, and

response to various environmentalstresses. WRKY70 appears to playa central role in controlling which typeof defence response (jasmonate- orsalicylate-mediated) is activated inresponse to pathogen attack. HighWRKY70 levels activate expression ofsystemic acquired resistance-relatedgenes while repressing jasmonic acid-responsive gene expression and viceversa (Li et al., 2004).

Sphingolipid signalling is anotherimportant player in the HR. FumonisinB1 and AAL toxin are shown todisrupt sphingolipid metabolism byinhibiting ceramide synthase, thusinducing subsequent PCD (Spassievaet al., 2002). The HR is also associatedwith altered mitochondrial functions.Inhibition of ATP synthesis, blockageof cytochrome C release, conversion ofelectron transport from cytochromoxidase pathway to alternative oxidasepathway, uncoupling of oxidativephosphorylation, depolarization ofmitochondrial membranes and openingof mitochondrial pores are amongdevastating changes leading to celldeath at pathogen attack (Lam et al.,2001). Such changes have been foundin HR induced by Erwinia amylovora,by T-toxin of Colchliobolus hetero-strophus and by victorin from C.victoriae (see Xie and Chen, 2000).

5. Role of ethylene in the HR

Although the processes of plantPCD share similarity to animal PCD,the control of cell death in plantsinvolves plant-specific regulators. Inaddition to common suicidal cascades,it has been demonstrated in a numberof experimental plant systems that the



plant hormone ethylene plays animportant role in programmed celldeath and senescence.

The role of ethylene in pathogen-induced cell death is evaluated inethylene insensitive (never-ripe) NR-tomatoes. Following infection of thesemutants, greatly reduced cell death isobserved, indicating ethylene invo-lvement in programmed cell death(Lund et al., 1998). Ethylene signallingis found to play a role in the cell deathinduced by the mycotoxin FumonisinB1 in Arabidopsis and tomato (Asai etal., 2000; Moore et al., 1999; Mur etal., 2003).

Formation of HR-like lesions andenhanced production of ROS are foundto occur in response to abiotic celldeath inducer such as ozone (O3),which interplays with ethylene(Overmyer et al., 2000; 2005; Neill etal., 2003). Evidence exist that O3

triggers HR in Bel W3 tobacco plants,which is accompanied by H2O2

accumulation (Pasqualini et al., 2003).Ethylene synthesis and perception havebeen found necessary for active H2O2

production and cell death resulting invisible injury in tomato (Lycopersiconesculentum) leaves exposed to ozoneand a selective response is supposed inthe regulation of the spread of celldeath (Moeder et al., 2002).

ROS and ethylene are alsorecently reported to participate insusceptibility to cauliflower mosaicvirus (CaMV), turnip crinkle virus(TCV), cucumber mosaic virus(CMV), turnip vein clearing tobamo-virus (TVTV) (see Love et al., 2005).

Cell death in maize endospermcould be hastened by treatment with

ethylene and blocked by ethyleneinhibitors (Young and Gallie, 2000). Inoat mesophyll cells, the administrationof inhibitors of ethylene biosynthesisand action – aminooxyacetic acid(AOA) and silver thiosulphate (STS)effectively inhibited victorin-inducedPCD, involving RUBISCO cleavage,DNA laddering and changes inmitochondrial permeability (Curtis andWolpert, 2004). Microarray study ofAAL toxin-treated tobacco reveals thatgenes responsive to reactive oxygenspecies, ethylene and a number ofproteases are among the earliest to beup-regulated, suggesting that anoxidative burst, production of ethyleneand proteolysis play a role in theactivation of the cell death (Gechev etal., 2004). In Taxus chinensis cellsuspension ethylene enhances celldeath induced by a fungal elicitor fromAspergillus niger (Qin and Lan, 2004).Overproduced ethylene correlatesclosely with expression of lethalsymptoms and apoptotic-like changesin hybrid tobacco seedlings and thelethality can be suppressed by ethylenesynthesis inhibitors AOA and amino-ethoxyvinylglycine (AVG) (Yamadaand Marubashi, 2003).

Additional evidence supportingthe participation of ethylene in celldeath signalling cascade comes fromthe study on Arabidopsis thalianadouble mutants. Crosses of the lesionmimic mutant accelerated cell death 5(acd 5) and ethylene insensitive 2 (ein2), in which ethylene signalling isblocked, show decreased cell death(Greenberg et al., 2000). A study withtomato cell suspension showed thatethylene is an essential factor in CPT-



induced PCD (de Jong et al., 2002).Simultaneous application of CPT andethylene greatly enhances CPT-induced cell death, apparently asa result of the increased production ofH2O2. Two partly overlapping celldeath pathways are proposed. Thesecomprise one pathway involvingcaspase-like proteases that requireslow levels of ethylene and onecaspase-independent pathway opera-tive at high ethylene levels. The latterpathway presumably acts throughMAPK-like proteins that are notessential in PCD at basal ethyleneconcentrations (de Jong et al., 2002).

6. HR in fruit species

The physiological and biochemicalevents that participate in the HR offruit species exposed to pathogenattack are still poorly investigated,although many studies focus onmolecular resistance mechanisms andbiotechnology of resistant cultivars.

In order to analyze mechanismsleading to compatible or incompatibleinteractions, early plant molecularevents have been investigated in twogenotypes of Malus with contrastingsusceptibility to fire blight, afterconfrontation with either E. amylovoraor the incompatible tobacco pathogenPseudomonas syringae pv. tabaci(Venisse et al., 2002). Defencemechanisms, such as generation of anoxidative burst and accumulation ofpathogenesis-related proteins, havebeen elicited in both resistant andsusceptible genotypes by the twopathogens at similar rates. Thiselicitation is linked to the functional

hypersensitive reaction and patho-genicity (hrp) gene cluster of E.amylovora. A delayed induction ofseveral genes of various branchpathways of the phenylpropanoidmetabolism is recorded in tissues ofthe susceptible genotype challengedwith the virulent wild-type strain of E.amylovora. In other plant-bacteriainteractions, including interactionswith the hrp secretion mutants thesegenes have been quickly induced. Theauthors suggest the existence of hrp-independent elicitors of defence in thefire blight pathogen as well as hrp-dependant mechanisms of suppressionof these non-specific inductions(Venisse et al., 2002).

In susceptible pear seedlings E.amylovora is also shown to generatean oxidative stress similar to the oneinduced by the incompatible P.syringae pv. tabaci (Venisse et al.,2001). Typical HR lesions andoxidative burst are observed in leavesof non-host tobacco plants infiltratedwith Asian pear pathogen Erwiniapyrifoliae as well. This indicates thatROS generation is necessary for thepathogen to kill the plant cell.

Grapevine (Vitis vinifera L., cv.Limberger) leaves and cell suspensionhave been induced to undergo a formof cell death that mimics the HR bytreatment with methyl jasmonate(MeJA) (Repka, 2002). Characteristicfeatures of apoptosis in animal andplant cells, such as typical changes innuclear morphology, fragmentation ofthe nucleus and protoplast collapsehave been observed to accompany thechemical-induced cell death. Local andectopic treatment of grapevine leaves



with phenylmethylsulphonylfluoride(PMSF), leupeptin, and especially witha specific inhibitor of cysteineproteases, E-64, have been found toinhibit MeJA-induced cysteineprotease activity and to block PCDtriggered by MeJA. These resultsindicate that proteolysis plays a crucialrole in MeJA-induced apoptotic-likecell death in grapevine leaves.

HR induction is reported in lemonseedlings infected with Alternariaalternata and a participation ofphenylpropanoid pathway, whichresults in de novo biosynthesis of thephytoalexin scoparone, is found to bea part of the HR. For elucidating someof the signalling elements thatparticipate in the development of HRin these plants, several compounds thatare known as activators or inhibitors ofsignal transduction elements in plantsor in animal cells have beenadministrated. Treatment of lemonseedlings with either cholera toxin orphorbol 12-myristate 13-acetate(PMA), in the absence of A. alternatainduce phenylalanine ammonia-lyase(PAL) and the synthesis of phytoalexinscoparone, suggesting the participationof G-protein and of serine/threoninekinase, respectively, in signal trans-duction. The use of trifluoperazine(TFP), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide),staurosporine, lavendustin A or 2,5-dihydroxymethyl-cinnamate (DHMC)have prevented PAL induction as wellas scoparone biosynthesis in responseto the fungal inoculation, thus sug-gesting the participation of calmodulin,serine/threonine and tyrosine proteinkinases in signal transduction in Citrus

limon in response to A. alternata(Ortega et al., 2002).

According to Lee and Hwang(2005), the inoculation of primarypepper leaves with an avirulent strainof Xanthomonas campestris pv.vesicatoria induces systemic acquiredresistance (SAR) in the non-inoculated, secondary leaves and thisSAR response is accompanied by thesystemic expression of the defence-related genes, a systemic micro-oxidative burst generating H2O2, andthe systemic induction of both ion-leakage and callose deposition in thenon-inoculated, secondary leaves.Some defence-related genes includingthose encoding PR-1, chitinase,osmotin, peroxidase, PR10, thionin,and SAR8.2 have been markedlyinduced in the systemic leaves. Theconspicuous systemic accumulation ofH2O2 and the strong increase inperoxidase activity in the pepper leavesis suggested to play a role in the celldeath process in the systemic micro-HRs, leading to the induction of theSAR.

An association of the antioxidantenzyme machinery with the resistanceof apricot (Prunus armeniaca L.) tosharka disease caused by plum poxvirus (PPV) is reported. The activity ofcatalase (CAT), dehydroascorbatereductase (DHAR), total superoxidedismutase (SOD), ascorbate peroxidase(APX), glutathione reductase (GR) andmonodehydroascorbate reductase(MDHAR) have been affected. Thedifferent behaviour of SODs (H2O2-generating enzymes) and APX (anH2O2-removal enzyme) in resistant andsusceptible cultivars suggests an



important role for H2O2 in the responseto PPV of the resistant cultivarGoldrich, in which no change in APXactivity is observed. The ability of theinoculated resistant cultivar to induceSOD1 and SOD2 as well as theimportant increase of DHAR suggestsa relationship between these activitiesand resistance to PPV (Hernández etal., 2001). Plum K4 hybrid is reportedhypersensitive against PPV isolate CG(Kegler et al., 2001) but there arealmost no published results about themechanism of the HR at PPVinfection. By inoculation with twoPPV strains (D- and M- strain),phenotypic features associated with thehypersensitivity of European plums(Prunus x domestica L.) against PPVhave recently been described.Biochemical and histochemical inves-tigations were used to explain themacroscopic visible characteristics ofhypersensitivity. In the graft union ofcombination of the hypersensitivecultivar Jojo with budwood infectedwith PPV, cell collapse has beendetected at the barrier between the twopartners. An accumulation of phenoliccompounds has been shown to precedethe cell death (Neumüller et al., 2005).

Cytological differences have beenfound in postharvest (detached) and inplanta (attached) fruits of pepperplants (Capsicum annuum) cv.Jejujaerae (susceptible) and Capsicumbaccatum cv. PBC80 (resistant),inoculated with the anthracnosepathogen Colletotrichum gloeo-sporioides. Cytological features ofPCD are observed in the resistantpepper fruit with postharvest

inoculation and, these have beencharacterized by positive responses toterminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling(TUNEL) and DNA laddering(hallmarks of apoptosis). The PCD-positive responses occurred around theinoculation sites early in in plantawound inoculation of the resistantpepper. Nuclear modifications andstructural changes of hypersensitivityare also observed in the resistant fruits,including separation of the plasmamembrane from the cell wall,dilation of the endoplasmic reti-culum, accumulation of electron-dense inclusions in vacuoles, andcytoplasmic vacuolization accom-panying fragmentation of thecytoplasm (Kim et al., 2004).

Using a differential hybridizationtechnique, pathogen-induced peppercyclophilin (CACYP1) cDNA clonewas isolated from a pepper cDNAlibrary from HR lesions of leavesinfected with Xanthomonas campestrispv. vesicatoria. The deduced aminoacid sequence of this CACYP1 showsa high level of homology with those ofcyclophilin proteins from tomatoes,potatoes, beans and Arabidopsis. Inaddition, the accumulation ofCACYP1 mRNA has been stronglyinduced in pepper plants byColletotrichum gloeosporioides infe-ction. At the incompatible interactions,a drastic induction of the CACYP1mRNA has been demonstrated tooccur in the bacteria inoculated leavescompared to that of the compatibleinteraction (Kong et al., 2001).



7. Manipulation of the HR is an“open door” towards to the controlof diseases in plants.

The interest for improving theresistance of crops to pathogens hasremarkably stimulated the researchon the identification of signalsproduced in plant-pathogen inter-actions and on the biochemical andmolecular steps required for theactivation of defence mechanisms.At pathogen invasion, the process ofPCD is linked to initiation ofresistance (Yu et al., 1998). Theidentification and characterization ofgenes and metabolic pathwaysinvolved in the HR provides a clue tobetter understanding the process ofPCD in plants and helps to establishthe similarities between animal andplant kingdoms. Selective inhibitionor induction of HR can be suc-cessfully employed for therapeuticmodulation of plant diseases causedby necrotrophic or biotrophic patho-gens. Transgenic expression ofanimal anti-apoptotic genes in plantsprovides broad-spectrum diseaseresistance against compatibleobligate pathogens (Dickman et al.,2001). Similarly, plant- and animal-derived pro-apoptotic genes can beengineered to be expressed in plantsfor activating HR-linked resistantdisease response against biotrophicpathogens (Khurana et al., 2005). Inaddition, the use of selectivesynthetic pathogen-inducible promo-ters has a high potential forengineering enhanced and durabledisease resistance thus avoiding socalled “runaway” cell death (Gurrand Rushton, 2005). HR is involved

in incompatible plant-pathogen inter-actions but besides its role in theinfected cells, the HR may coor-dinate the defence response inneighbouring cells and local acquiredresistance. Since the cell deathsassociated with disease symptomsand HR presumably share commonmechanisms, the investigation onunderlying biochemical pathwaysand molecular mechanisms in wildtype and transgenic plants may shedmore light into resistance mecha-nisms in plants.

Acknowledgements. The workon this paper was partially supportedby a grant from the Ministry ofEducation and Science, NationalScience Fund, Bulgaria, Project: VU-B-9/05 (for literature search).

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NADWRAŻLIWA ŚMIERĆKOMÓREK ROŚLINNYCH –JEJ MECHANIZMY I ROLA W REAKCJI OBRONNEJ

ROŚLIN PRZED PATOGENAMI

E l e n a T . I a k i m o v a , L e c h M i c h a l c z u ki E r n s t J . W o l t e r i n g

S T R E S Z C Z E N I E

W pracy tej przedstawiono najnowsze osiągnięcia w badaniach nad reakcjąnadwrażliwości (hypersensitive response – HR) u roślin, ze szczególnym uwzględ-nieniem fizjologicznych i biochemicznych uwarunkowańw różnych systemachmodelowych. Reakcja nadwrażliwości jest rozpatrywana jako jedna z formprogramowanej śmierci komórki (programmed cell death – PCD), będącąjednymz mechanizmów obrony roślin przed chorobami. Omawiane są główne drogitransdukcji sygnału oraz związki chemiczne związane z reakcjąnadwrażliwości, takiejak kaskada proteolityczna, reakcje oxydacyjne i etylen, które przypuszczalnie mająpodstawowe znaczenie w mechanizmie śmierci komórki. Specjalnąuwagępoświę-cono reakcji nadwrażliwości w roślinach sadowniczych. Badania nad programowanąśmierciąkomórki pozwalają lepiej zrozumiećmechanizm odporności roślin nachoroby.

Słowa kluczowe: rośliny sadownicze, reakcja nadwrażliwości, programowana śmierćkomórki

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