pathogen-induced programmed cell death in tobacco · programmed cell death during the...

1333Journal of Cell Science 110, 1333-1344 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS9537

Pathogen-induced programmed cell death in tobacco

Ron Mittler1,*, Lee Simon2 and Eric Lam1,†

1Center for Agricultural Molecular Biology, Foran Hall, Dudley Road, Rutgers The State University of New Jersey, Cook College,New Brunswick, NJ 08903-0231, USA2Waksman Institute, Rutgers The State University of New Jersey, Piscataway, New Jersey 08855-0759, USA

*Present address: Department of Plant Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel†Author for correspondence (e-mail: [email protected])

Sacrificing an infected cell or cells in order to preventsystemic spread of a pathogen appears to be a conservedstrategy in both plants and animals. We studied some of themorphological and biochemical events that accompanyprogrammed cell death during the hypersensitive responseof tobacco plants infected with tobacco mosaic virus.Certain aspects of this cell death process appeared to besimilar to those that take place during apoptosis in animalcells. These included condensation and vacuolization of thecytoplasm and cleavage of nuclear DNA to 50 kbfragments. In contrast, internucleosomal fragmentation,condensation of chromatin at the nuclear periphery andapoptotic bodies were not observed in tobacco plantsduring tobacco mosaic virus-induced hypersensitive

response. A unique aspect of programmed cell death duringthe hypersensitive response of tobacco to tobacco mosaicvirus involved an increase in the amount of monomericchloroplast DNA. Morphological changes to the chloroplastand cytosol of tobacco cells and increase in monomericchloroplast DNA occurred prior to gross changes in nuclearmorphology and significant chromatin cleavage. Ourfindings suggest that certain aspects of programmed celldeath may have been conserved during the evolution ofplants and animals.

Key words: Hypersensitive response, Pathogen, Programmed celldeath, TMV, Tobacco

SUMMARY

INTRODUCTION

Programmed cell death (PCD) is one of the key mechanismscontrolling cell proliferation, generation of developmentalpatterns, and defense of animals against viral pathogens andenvironmental insults (Ellis and Horvitz, 1986; Lakshmi et al.,1992; Raff, 1992; Schwartzman and Cidlowski, 1993). One ofthe most widely studied forms of PCD is apoptosis, a type ofPCD that displays a distinct set of physiological and morpho-logical features (Martin et al., 1994). Morphological hallmarksof apoptosis include the condensation of chromatin at thenuclear periphery and the condensation and vacuolization ofthe cytoplasm. These changes are followed by breakdown ofthe nucleus and fragmentation of the cell to form apoptoticbodies (Wyllie et al., 1984; Schwartzman and Cidlowski,1993). Among the many biochemical changes commonlyfound in cells undergoing apoptosis is the systematic frag-mentation and degradation of nuclear DNA (Bortner et al.,1995). Large fragments of 300 and/or 50 kb are first producedby endonucleolytic degradation of nuclear DNA (Oberhammeret al., 1993; Walker et al., 1993). These are further degradedby cleavage at linker DNA sites between nucleosomes resultingin DNA fragments that are multimers of about 180 bp (Wyllieet al., 1984). Degradation of nuclear DNA during apoptosis iscoordinated with activation of specific endonucleases that arethought to mediate chromatin cleavage (Gaido and Cidlowski,1991; Barry and Eastman, 1993; Peitsch et al., 1993; Arruti etal., 1994).

In plants, PCD is thought to be activated during the courseof several differentiation pathways and in response to attack bycertain pathogens (Dietrich et al., 1994; Greenberg et al., 1994;Hammond-Kosack et al., 1994; Levine et al., 1994, 1996;Pontier et al., 1994; Mittler et al., 1995; Jones and Dangl, 1996;Mittler and Lam, 1996; Ryerson and Heath, 1996; Wang et al.,1996). Activation of cell death, following recognition ofinvading pathogens, results in the formation of a zone of deadcells localized around the site of infection. Killing of cells atand around the site of infection, also called a hypersensitiveresponse (HR) lesion, is thought to participate in preventingsystemic proliferation of some pathogens. Several lines ofevidence suggest that death of plant cells during the HR resultsfrom the activation of a PCD pathway. This evidence stemsfrom studies that demonstrate the activation of HR cell deathby certain elicitors in the absence of a pathogen (He et al.,1993; Hammond-Kosack et al., 1994; Levine et al., 1994), byexpression of different foreign genes (summarized by Mittlerand Lam, 1996), and as a result of mutations in certain geneswhich are thought to be involved in the cell death pathway(Walbot et al., 1983; Woltor et al., 1993; Greenberg et al., 1994;Dietrich et al., 1994). In addition, cell death that occurs duringthe HR was shown to require active plant metabolism and todepend on the activity of the host transcription and translationmachinery (He et al., 1993, 1994). Therefore, cell death thatoccurs during the HR is not directly caused by the invadingpathogen but rather results from the activation of a plant-encoded pathway for PCD.

1334 R. Mittler, L. Simon and E. Lam

The interaction between tobacco plants (genotype NN) andtobacco mosaic virus (TMV) is one of the classical modelsystems for studying the HR (Whitham et al., 1994). Using anin situ assay for the detection of 3′ hydroxyl groups of degradednuclear DNA (TUNEL assay; Gavrieli et al., 1992), we foundthat nuclei of tobacco cells undergoing PCD, during the HR toTMV, contain degraded nuclear DNA (Mittler et al., 1995). Wealso identified several deoxyribonuclease activities that areinduced during TMV-activated and transgenically-triggeredPCD (Mittler and Lam, 1995). The activity of one of thesenucleases, NUC III, was found in isolated nuclei of cells under-going PCD and shown to be a Ca2+-dependent endonuclease(Mittler and Lam, 1995).

Our initial characterization of the molecular mechanismsinvolved in PCD that occurs during the HR of tobacco plantsto TMV suggests that some similarities exist between thisprocess and PCD in animal cells. We therefore examined themorphological changes that accompany TMV-induced HR celldeath in tobacco using transmission electron microscopy(TEM) and the mode of nuclear and chloroplast DNA degra-dation using field inversion gel electrophoresis (FIGE).

MATERIALS AND METHODS

Plant material and pathogen infectionWild-type tobacco plants (Nicotiana tabacum cv Xanthi nc NN)were grown under continuous illumination provided by cool-whitefluorescent lamps (200 µmol m−2 second−1). Fully expanded youngleaves of 5- to 6-week-old plants were infected with TMV strain U1and kept at 30°C for 4 days under continuous light (200 µmol m−2

second−1). PCD was induced by shifting of TMV-infected andcontrol uninfected plants from 30°C to 25°C and cell death wasassayed by measuring ion leakage from leaf discs as previouslydescribed (Mittler et al., 1995, Mittler and Lam, 1995). PCD wasalso induced by infecting leaves of tobacco plants grown at 25°Cwith Pseudomonas syringae pv. phaseolicola (NPS3121) asdescribed by Lindgren et al. (1986). As a control for PCD inducedby bacteria, leaves were infiltrated with a Hrp− derivative ofNPS3121 (NPS4000; Lindgren et al., 1986). Necrotic cell death wasinduced by incubating leaves of wild-type plants at −20°C for 1 hourand thawing these leaves at 25°C for 0.5 or 1 hours. Plant materialwas collected at various time points and flash-frozen in liquidnitrogen. Intact chloroplasts were isolated according to the methodof Orozco et al. (1986).

Light microscopyFor structural studies, 2 to 3 mm thick stem sections obtained fromthe internode regions located directly above the infected leaves, orleaf pieces (approximately 2 to 3 mm wide and 6 to 8 mm long)taken from systemically infected leaves located directly above theinfected leaves, were fixed in 2.5% glutaraldehyde, 0.1 M sodiumphosphate buffer, pH 7.0, or 10% formaldehyde, 5% acetic acid,40% ethanol for 3 hours, dehydrated through a graded ethanol series(25, 50, 75, and 100% for 20 minutes at each step), and incubatedovernight in 100% ethanol (alternatively, tissue was stained with0.1% eosin in 95% ethanol overnight and washed twice, 10 minuteseach, in 100% ethanol). The dehydrated tissue was then takenthrough a graded xylene series (25, 50, 75, and 100% in ethanol for1 hour at each step). Finally the tissue was embedded in paraffin(Paraplast+, Fisher Scientific) by a paraffin graded series (25, 50,75, and 100%, in xylene, 3 hours each step, at 59°C). Tissues wereinfiltrated in 100% paraffin overnight at 59°C and sectioned on aReichert-Jung 2040 retractable rotary microtome at a thickness of 5

µm. Sections were mounted on slides, deparaffinized, stained withhematoxylin (Fisher Scientific) according to the manufacturer’sinstructions, and observed by light microscopy with a Nikon EF-Doptiphot epifluorescence microscope.

Transmission electron microscopyStems obtained at different time points following activation of celldeath were sampled for TEM. Cross-sections of stem tissue wereobtained from the internode regions located directly above theinfected leaves. Cross-sections (about 1 mm width) were fixed for 3hours in either 2.5% glutaraldehyde, 1% sucrose, 0.1 M sodiumphosphate buffer, pH 7.0, or 2.5% glutaraldehyde, 0.1 M sodiumphosphate buffer, pH 7.0, at 22°C, with gentle agitation. Afterwashing in 0.1 M sodium phosphate buffer, the tissue was post-fixedfor 2 hours with 1% osmium tetroxide, 0.1 M sodium phosphatebuffer, pH 7.0, at 22°C, with gentle agitation. The samples were thenwashed with distilled water, dehydrated in a graded ethanol seriesand embedded in Spurr (1969) epoxy resin. Ultrathin longitudinalsections obtained with a diamond knife in a Reichert OMU3microtome were stained with uranyl acetate and lead citrate.Sections were observed and photographed at 80 kV in a JEOL100CX TEM.

Field inversion gel electrophoresisTissue samples (0.2 g) obtained at different time points following thetemperature shift were ground to a fine powder in liquid nitrogen. Thepowder was dissolved in 1 ml 1× ET buffer (10 mM Tris-HCl, pH 8.0,50 mM EDTA) or 1 ml 500 mM EDTA, 10 mM Tris-HCl, pH 8.0,preheated to 50°C and mixed with 1 ml 1.4% (W/V) low melting pointagarose (BRL) in 1× ET preheated to 50°C. Agarose plugs werepoured in a Bio-Rad standard mold. Plugs were incubated for 3 hoursin 10 mM Tris-HCl, pH 8.0, 0.5 M EDTA, 1% sarcosyl and 1 mg/mlproteinase K (Promega), at 52-54°C with gentle agitation. Plugs werethen washed with 1× ET and stored at 4°C in 1× ET (Guidet andLangridge, 1992). FIGE was performed with a Bio-Rad CHEFMapper apparatus using 1% agarose (BRL) gels and 0.5× TBE buffer(45 mM Tris-borate, 1 mM EDTA, pH 8.0). Electrophoretic separa-tion was performed with a 6 V/cm voltage gradient at an angle of120°, and a linear ramping factor with alternating pulse time of 5.3 to20.5 seconds over the course of 18 hours. Following electrophoreticseparation the gels were stained with ethidium bromide and pho-tographed. FIGE gels were loaded based on equal amounts of tissue(0.025g fresh weight/lane).

Transfer of DNA from FIGE gels and hybridizationDNA in FIGE gels was depurinated with UV light or 0.25 N HCl.Gels were then washed with distilled water and incubated in 0.4 NNaOH, 1.5 M NaCl for 20 minutes. DNA was transferred to a nylonmembrane (Zeta-Probe GT, Bio-Rad) by the capillary transfer methodusing 0.4 N NaOH, 1.5 M NaCl for 48 hours. The membrane wasbriefly washed in 2× SSC (300 mM NaCl, 30 mM Na-citrate, pH 7.0)and baked in a vacuum oven at 80°C for 45 minutes. Hybridizationwas carried out at 55°C using Image (USB Biochemicals) hybridiz-ation solution. A mixture of six probes corresponding to six differenttobacco genes (PR-1a, cytosolic APX, rbcS, TGA-1a, PR-2 and PR-3) was used to detect cleavage of nuclear DNA. 18 S rDNA probe wasobtained as previously described (Mittler et al., 1995). A chloroplastintergenic fragment from the maize rbcL-atpB divergent transcriptionunit (Lam et al., 1988) was used as a chloroplast specific probe anda tobacco mitochondrial DNA probe that contains sequences encodingthe ATPase subunit 9 and rps13 genes was used as a mitochondriaspecific probe (Bland et al., 1986). DNA probes were labeled with arandom-primed labeling kit (Promega) using [32P]dATP. Total DNAwas also isolated and analyzed with conventional 1.5% agarose gelsas previously described (Mittler and Lam, 1995). DNA transferredfrom these gels was analyzed with the different probes as describedabove.

1335Pathogen-induced PCD in tobacco

RESULTS

Ultrastructural changes in tobacco cells undergoingPCDThe activation of cell death and defense mechanisms in tobaccoplants infected with TMV is inhibited at 30°C (Whitham et al.,1994). Therefore, tobacco plants infected with TMV at 30°Ccannot restrict the spread of the virus by an HR lesion andbecome systemically infected. Upon shifting of systemicallyinfected plants from 30°C to 25°C the inhibition of TMV-induced PCD is removed and massive cell death in systemi-cally infected tissues occurs. In leaves and stems of systemi-cally infected tobacco plants, cell death is initiated andpropagated through parenchyma cell layers (Goodman andNovacky, 1994; Mittler et al., 1995). For ultrastructural studieswe used stem tissue. As shown in Fig. 1A,B,C, this tissuecontained many cells at different stages of collapse whichprovides a gradient of cells at various stages of cell death.Thus, in contrast to leaf tissue in which most cells undergo celldeath simultaneously (Fig. 1D,E,F) the use of stem tissueenabled us to study subtle changes in the morphology of fixedcells from the same cell layer and within the same section (alsosee Mittler et al., 1995). In addition, parenchyma stem cellslocated at the first and second layers directly adjacent to theepidermis of young stems serve a role which is analogous tothat of leaf mesophyll cells. Thus, ultrastructural changes inthese cells are likely to resemble morphological changes thatoccur in leaf mesophyll cells undergoing the HR, which werethe subject of earlier studies (summarized by Goodman andNovacky, 1994). Using an in situ TUNEL assay we found that,similar to nuclei of animal cells undergoing PCD, nuclei oftobacco stem cells undergoing TMV-induced PCD contain a

Fig. 1. Cross sectionsthrough stems (A,B,C) orleaves (D,E,F) fixed 0(A,D), 24 (B,E) and 48(C,F) hours after inductionof PCD (a shift from 30°Cto 25°C of TMV-infectedplants). Cells at differentstages of cell death can beseen at the border oflesions, indicated by arrowsin B,C. E, epidermis; P,parenchyma. Bars: 40 µm(in C for A,B,C and in Ffor D,E,F).

high level of 3′-OH groups of degraded nuclear DNA (Mittleret al., 1995). Therefore, we examined whether nuclei of stemcells undergoing PCD exhibit any of the characteristic mor-phological changes observed in nuclei of animal cells duringapoptosis. TMV-infected plants shifted from 30°C to 25°Cwere compared with healthy uninfected plants shifted from30°C to 25°C and with TMV-infected plants grown at 30°C.None of the morphological alterations found during TMV-induced PCD were observed in these control tissues. As acontrol for cell death not caused by activation of a PCDpathway, we examined tobacco stems subjected to a freeze-thaw treatment. We refer to this type of death as ‘necrosis’since it results from a massive, non-programmed and rapidinjury that clearly does not require the activation of any cellularprotein. Unlike toxin- or chemically-induced cell death thatmay result in the activation of a PCD mechanism (Chang et al.,1989; Schwartzman and Cidlowski, 1993), this type of rapiddeath is not likely to induce a PCD pathway.

Longitudinal sections of systemically infected stem tissuefixed 48 hours after temperature shift were examined. Asshown in Fig. 2A,B,C, nuclei of tobacco cells undergoingPCD appeared not to contain condensed chromatin localizedto the nuclear periphery. Nor did these nuclei appear tofragment or to form vesicles with nuclear debris. Thechromatin in tobacco nuclei undergoing TMV-induced PCDappeared to form discrete, darkly-stained patches distributedthroughout the nuclei (Fig. 2B). These patches probablyrepresent condensation, although they may simply be theresult of redistribution or reorganization of chromatin. Fig.2C shows a nucleus at a late stage of cell death. The cellshown in Fig. 2C appears smaller than cells at earlier stagesof HR PCD. Both by light microscope (Fig. 1; Mittler et al.,


1995) and by electron microscope observations cells at latestages of PCD appeared to collapse into a relatively smallflattened shape. At this stage almost no lightly stainedchromatin can be seen inside the nuclear envelope. Thenucleoli seemed to remain intact throughout the death process

and appeared very condensed at late stages of cell death (Fig.2C). The nuclear envelope also appeared to remain intact andcan be seen even in cells at late stages of HR cell death (arrowin Fig. 2C). In contrast to these changes, nuclei of cellssubjected to a freeze-thaw treatment appeared very disor-


Fig. 2. Longitudinal sections through stem cells fixed 0 and 48 hoursafter induction of PCD (a shift from 30°C to 25°C of TMV-infectedplants) or 20 minutes after a freeze-thaw cycle showingmorphological changes in nuclei of cells undergoing PCD or necroticdeath. (A,B,C) Changes in nuclear morphology observed inparenchyma stem cells at different stages of TMV-induced PCD.(A) Early changes in nuclear morphology showing no condensationof nuclear material. (B) Apparent condensation of nuclear materialinto discrete patches distributed throughout the nucleus. (C) Nucleusand surrounding cell at a late stage of HR cell death (arrow points tonuclear envelope). The cell appears to have collapsed and is smallerthan cells at earlier stages of PCD. Degraded chloroplasts can beseen on both sides of the nucleus. (D) Morphological changes in aparenchyma stem cell fixed 20 minutes after a freeze-thaw cycle.Only cell debris are visible, the arrow points to the remains of thenucleolus. (E) Nucleus of a TMV-infected cell kept at 30°C (0hours). (F) Nucleus of an uninfected cell subjected to a temperatureshift (48 hours). Bars, 5 µm.

ganized (Fig. 2D). Vesicles with a variety of sizes and theapparent remains of chloroplasts were evident. Chromatin, aswell as a defined nuclear and/or cell envelope, could not beeasily identified. The nucleoli in these cells (arrow in Fig. 2D)did not appear as condensed as the nucleoli found in cellsduring late stages of PCD (Fig. 2C). The nucleus shown inFig. 2B is from a cell located directly adjacent to thecollapsed layer (shown in Fig. 2C). The nucleus shown in Fig.2A is from a cell located adjacent to the cell layer that thenucleus shown in Fig. 2B is taken from. Thus, Fig. 2A,B,Crepresent nuclei from three different layers: a collapsed layer(Fig. 2C), a layer adjacent to the collapsed layer (Fig. 2B),and a layer which is one layer away from a collapsed layer(Fig. 2A). Fig. 2E shows the nucleus of a TMV-infected cellat 30°C. At this temperature the activation of PCD isinhibited.

The cytoplasm of tobacco cells undergoing TMV-inducedPCD is shown in Fig. 3. Compared with the cytoplasm ofhealthy, uninoculated cells shifted from 30°C to 25°C (Fig. 3A)and cytoplasm of TMV-infected plants grown at 30°C (data notshown), the cytoplasm of tobacco cells undergoing PCDappeared condensed and contained vesicular structures (Fig.3B). Some of these vesicles apparently originated from theplant vacuole membrane. In the majority of cells studied,degradation of the vacuole membrane occurred prior to the dis-ruption of the plasma membrane (data not shown). No apparentapoptotic bodies were detected in plant tissue undergoing PCDin our system (data not shown). Changes to the plasmamembrane are shown in Fig. 3C. These included invaginationof the membrane and possibly formation of vesicles. Vesiclesthat appeared to form from the plasma membrane containeddense material attached to the interior surface of the membrane(Fig. 3C). This dense material may indicate accumulation oftannins or other phenolic compounds on the exterior part of theplasma membrane. Alternatively, these vesicles may betargeted to the plasma membrane and may be involved insecretion of proteins, antimicrobial compounds or othermaterials.

Ultrastructural changes in chloroplasts are shown in Figs2A,B, 3D,E. Compared with chloroplasts of healthy uninfectedcells shifted from 30°C to 25°C (Fig. 3A), and in agreementwith previous reports (Weintraub and Ragetli, 1964; Goodmanand Novacky, 1994), chloroplasts of plant cells undergoing

TMV-induced PCD contained large starch granules (Fig.2A,B). A number of these chloroplasts contained a mass ofdense material that was located directly adjacent to thethylakoid stacks, and was surrounded by a membrane (Fig.3D,E). These masses of dense material probably representalterations and/or reorganization of the thylakoid membranes,although we have not ruled out the possibility that they resultfrom the aggregation of other material. In agreement withprevious reports (Graca and Martin, 1975; Goodman andNovacky, 1994), and with a relatively small number of excep-tions, the gross morphology of mitochondria appeared morestable than chloroplasts during the HR cell death process.

In all cells studied, morphological changes in the cytoplasm(i.e. vacuolization and condensation) and chloroplast (i.e. accu-mulation of starch granules) occurred prior to ultrastructuralchanges in the nuclei (compare Fig. 2E to A, in which thenuclei appeared almost normal while the cytoplasm and chloro-plasts appear to undergo morphological changes associatedwith the HR).

Based on light microscope observations of systemicallyinfected stem tissue at 24 hours post cell death activation,approximately 12% of parenchyma stem cells appeared toundergo TMV-induced PCD. Of these approximately 30%exhibited morphological changes such as vacuolization andcondensation of cytoplasm. The other 70% of dying or deadcells appeared collapsed and contained cellular debris with nodefined cell membrane. These cells likely represent late stagesof cell death and may have previously contained vesicles andcondensed cytoplasm. At 48 hours post cell death activationapproximately 20% of parenchyma stem cells appeared toundergo cell death. Of these approximately 16% exhibitedmorphological changes whereas the rest appeared collapsedwith no or little content. An example of such tissues is shownin Fig. 1B,C.

Cleavage of chromatin and changes to chloroplastDNA in tobacco cells undergoing TMV-induced HRcell deathWe previously reported that nuclei of stem cells undergoingPCD contain degraded DNA (Mittler et al., 1995). However,DNA isolated from stems undergoing HR cell death andanalyzed by conventional agarose gel electrophoresis did notappear to be fragmented in a manner that indicated internu-cleosomal fragmentation. Thus, no DNA ladder of fragmentsthat are multimers of 180 bp was detected (Mittler and Lam,1995). We therefore tested whether the DNA in nuclei of stemcells undergoing HR cell death is fragmented to largefragments of about 300 and 50 kb that are associated withseveral cases of PCD in animal systems (Oberhammer et al.,1993; Walker et al., 1993; Bortner et al., 1995). Total DNA wasisolated from infected and uninfected stems following a tem-perature shift and subjected to analysis by FIGE. As shown inFig. 4A, DNA degradation was detected by FIGE in DNAobtained from systemically infected stem tissue at 24 and 48hours post activation of cell death. In contrast, no systematicdegradation was observed when DNA in agarose plugs wassubjected to conventional agarose gel electrophoresis (Fig.4B). These findings are in agreement with our previous obser-vation that at 24 and 48 hours nuclei of stem cells contain frag-mented DNA and that no internucleosomal fragmentation isdetected following activation of TMV-induced PCD (Mittler et


al., 1995; Mittler and Lam, 1995). However, it was not clearwhether the fragmentation observed by FIGE is specific forPCD, and although fragments of about 50 kb appear to beformed, total DNA isolated from stems undergoing HR celldeath contained DNA fragments with a rather large range of

sizes. This apparent heterogeneity in DNA fragments isolatedfrom stem cells and analyzed by FIGE (Fig. 4A) may be theresult of the presence in each sample of parenchyma cells atvarious stages of HR cell death. Moreover, stems containxylem and phloem cells that may undergo an independent,


Fig. 3. Longitudinal sections through stem cells fixed 48 hours after atemperature shift of infected and uninfected stem cells showingultrastructural alterations to the cytoplasm. (A) Cytoplasm of controlplants shifted from 30°C to 25°C. Chloroplast and mitochondria canbe seen adjacent to the cell wall. (B) Condensation and vacuolizationof cytoplasm during PCD. Vesicles that appear to form from thevacuole membrane and mitochondria can be seen within thecondensed cytosol. (C) Invagination and apparent formation ofcytoplasmic vesicles from the plasma membrane of cells undergoingPCD. In contrast to vesicles that appeared to form from the vacuolemembrane, shown in B, vesicles associated with the plasmamembrane contained darkly-stained material attached to their interiorsurface. (D,E) Accumulation of dense material and/or structuralalterations to the thylakoid membranes of chloroplasts during PCD(arrows). PM, plasma membrane; V, vacuole; W, cell wall. Bar, 5 µm.

developmentally controlled PCD. Therefore, although they arebetter suited for morphological studies, stems appear to be apoor specimen for biochemical analysis.

In order to obtain a relatively homogeneous population ofcells undergoing TMV-induced PCD, systemically infectedleaves, shifted from 30°C to 25°C and sampled without theirvein tissues, was used for biochemical studies. This tissue wasmostly composed of mesophyll and epidermal cells. Based onlight microscope observation, and in contrast to stem cells, themajority of systemically infected leaf mesophyll cells undergoHR cell death in a relatively synchronized manner (Fig.1D,E,F). Similar to nuclei of stem cells undergoing PCD(Mittler et al., 1995), nuclei of leaf mesophyll cells undergo-ing TMV-induced PCD contain degraded DNA as revealed bythe TUNEL assay (D. D. Dunigan, personal communication).Leaf tissue undergoing TMV-induced PCD was compared withtissue obtained from control plants shifted from 30°C to 25°Cand from TMV-infected plants grown at 30°C. No degradationof cellular DNA was observed in these control tissues. Cell

Fig. 4. Degradation of total DNA isolated from stems undergoingTMV-induced PCD. (A) FIGE of total DNA from TMV-infected(TMV) and control (WT) tobacco stems following a temperatureshift from 30°C to 25°C. (B) Conventional agarose gelelectrophoresis of DNA from the same plugs as in A. Lambda ladder(Promega) and 1 kb ladder (BRL) were used to estimate size of DNAfragments. Temperature shift and DNA isolation and analysis wereperformed as described in Materials and Methods. Size of molecularmarkers is given at left in kb. h, hour; TMV, tobacco mosaic virusinfected; WT, wild type.

death was quantitated by measuring ion leakage from leaf discsobtained at different time points following the temperatureshift (Greenberg et al., 1994; Mittler and Lam, 1995). Asshown in Fig. 5A, ion leakage was detected at 24 and 48 hoursfollowing the temperature shift. As a control for necrotic celldeath, leaves of uninoculated healthy plants shifted from 30°Cto 25°C for 48 hours were subjected to a freeze-thaw cycle andsampled at 0.5 and 1 hour after thawing (Fig. 5). As shown inFig. 5B, cleavage of total DNA obtained from leaves occurredlate during HR cell death (48 hours). Degradation of DNA 48hours following the temperature shift resulted in the formationof DNA fragments of about 50 kb. No 300 kb fragments wereobserved during this PCD process. Similar observations weremade with samples prepared by dissolving the ground tissue insolutions containing 50 or 500 mM EDTA. Thus, we believethat these DNA fragments are not artifacts produced bynuclease activities during sample preparation. In contrast to thelate appearance of 50 kb fragments during PCD, fragments ofabout 150 kb were detected as early as 6 hours following thetemperature shift. Necrotic cell death was accompanied byrapid appearance of 50 kb fragments. However, the 150 kbfragments were not observed in these samples. DNA isolatedas previously described (Mittler and Lam, 1995) was alsoanalyzed with conventional agarose gels. As shown in Fig. 5B,no internucleosomal fragmentation was observed during PCDor necrotic cell death.

Because total DNA isolated from leaves contains nuclear,chloroplast and mitochondrial DNA, we transferred the DNAshown in Fig. 5B (left) to a nylon membrane and used nuclear,chloroplast and mitochondria specific probes to analyze thedegradation pattern of DNA in these different cellular com-partments. As shown in Fig. 5C almost all of the nuclear DNApresent in total DNA isolated from leaves 48 hours followingthe temperature shift was cleaved to fragments of about 50 kb.A probe for the 18 S rDNA genes was also used. This probewas applied in an attempt to detect changes in DNA primarilyassociated with the nucleoli. However, no difference wasobserved between the pattern of DNA degradation using the 18S rDNA probe or the nuclear cDNA probes. Although 50 kbfragments were observed in control and early time points inFig. 5B, these fragments did not hybridize significantly withthe nuclear specific probes. However, strong hybridization wasobserved between these fragments and the mitochondriaspecific probe, suggesting that these fragments observed in theearlier time points consisted primarily of mitochondrial DNA.Very specific hybridization was observed between the chloro-plast specific probe, the 150 kb fragment and a fragment ofabout 300 kb (Fig. 5C). Because the monomeric size of thetobacco chloroplast genome is 155.8 kb (Shinozaki et al.,1986), these 150 and 300 kb fragments were most likelycomposed of monomers and dimers of chloroplast DNA.

The chloroplast genome is thought to exist as a mixture ofmultimeric forms (Deng et al., 1989). When DNA extractedfrom isolated chloroplasts is subjected to FIGE, a variety ofmultimeric forms is observed with the predominant speciesbeing the 150 kb monomer. However, Deng et al. (1989)indicated that the process of chloroplast isolation may causethe random linearization of chloroplast DNA that may occur atDNA membrane attachment sites. Therefore, it is possible thatthe chloroplast genome exists as a large concatemer and not asa mixture of multimeric forms. Because monomeric or dimeric

1340 R. Mittler, L. Simon and E. LamC

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Fig. 5. Cleavage of nuclear DNA and changes to the chloroplast genome during PCD. (A) Ion leakage form leaf tissue during TMV-inducedPCD and following freeze/thaw-induced necrosis. Leaf discs were sampled at different time points following a temperature shift of systemicallyinfected tissue from 30°C to 25°C (HR cell death), and following a freeze-thaw treatment (Necrosis). Conductivity is expressed in micromhos(µmhos). (B) FIGE (left) and conventional gel electrophoresis (right) of DNA from tobacco leaves undergoing HR and necrotic cell death.DNA was obtained at different time points following a temperature shift of systemically infected leaves from 30°C to 25°C (HR cell death), andfollowing a freeze-thaw treatment (Necrosis) as described in Materials and Methods. Lambda ladder (λL, Promega) and 1 kb ladder (M, BRL)were used as molecular size markers. (C) Cleavage of nuclear, mitochondrial and chloroplast DNA during HR cell death and followingnecrosis. DNA transferred from the FIGE gel, shown in B, was hybridized with nuclear or organelle specific probes. DNA transfer andconditions for hybridization are described in Materials and Methods. Size of molecular markers is given at left in kb. C, control; h, hour; HR,hypersensitive response.

chloroplast DNA was not detected in total DNA isolated fromthe 0 hour or control samples (Fig. 5B,C), we determinedwhether DNA extracted from isolated chloroplasts obtainedfrom the 0 hour time point contained these forms of chloro-plast DNA. As shown in Fig. 6, DNA extracted from thesechloroplasts contained monomeric and dimeric forms of thechloroplast genome. Thus, the process of chloroplast isolationcan apparently generate monomeric and dimeric forms of thechloroplast genome. These forms were not found in total DNAisolated from tissues at the same time point (Figs 5B,C, 6).These findings suggest that the amount of monomeric chloro-plast DNA, detected by FIGE, increased during TMV-inducedPCD. This increase was detected as early as 6 hours followingactivation of PCD, and was not observed following necroticcell death (Fig. 5).

The plant mitochondrial genome is thought to exist as amixture of a master chromosome and a variety of subgenomicfragments. However, 80% of mitochondrial DNA released fromisolated plant mitochondria does not migrate during FIGE(Fauron et al., 1995). As shown in Fig. 5C, the extent of hybrid-ization of the mitochondria specific probe to the 50 kb fragmentsis increased at early stages of cell death (6 to 24 hours). Thisobservation suggests that more mitochondrial DNA is releasedfrom the agarose plugs of these time points compared to controlor 0 hours. Alternatively, the number of subgenomic fragmentsmay increase during early stages of PCD.

Cleavage of nuclear DNA during bacteria-inducedPCDWe extended our study of DNA degradation during PCD that


Fig. 6. FIGE and DNA blot analysis of DNA isolated fromchloroplasts. (A) FIGE of total DNA and DNA extracted fromisolated chloroplasts at 0 and 24 hours following a temperature shiftof TMV-infected plants from 30°C to 25°C. (B) DNA blot analysisof DNA from the FIGE gel shown in A with a chloroplast specificprobe. DNA transfer and conditions for hybridization are describedin Materials and Methods. Size of molecular markers is given at leftin kb. Chloroplasts were isolated according to the method of Orozcoet al. (1986). Isolated chloroplasts with equal chlorophyll contentwere mixed with low melting point agarose in 2× ET, molded intoplugs, treated and subjected to FIGE as described in Materials andMethods.

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(µm

hos)

Fig. 7. Fragmentationof nuclear DNAduring bacteria-induced PCD. (A) Ionleakage form leaftissue followingbacteria-induced HRcell death. Leaveswere infiltrated withwild type (NPS3121)or Hrp− strain(NPS4000) ofPseudomonassyringae pv.phaseolicola andsampled 18 hours postinfiltration. Conductivity is expressed in micromhos (µmhos).(B) Degradation of DNA during bacteria-induced HR. Total DNAwas isolated from leaves, treated as described for A, and subjected toFIGE. Following electrophoretic separation DNA was transferred toa nylon membrane and probed with nuclear and chloroplast specificprobes as described in Materials and Methods. (C) FIGE of DNAisolated from leaf tissues at different time points following infectionwith an HR-inducing bacteria (NPS3121). A Lambda DNA ladder(Promega) was used to estimate size of DNA fragments. Size ofmolecular markers is given at left in kb. h, hour.

accompanies the HR to the interaction between tobacco plantsand bacterial pathogens that induce the HR. Ultrastructuralstudies of bacteria-induced HR death revealed that this type ofcell death share some common features with TMV-inducedPCD. These include vacuolization of the cytoplasm andcollapse of cells with no apparent formation of apoptoticbodies (Roebuck et al., 1978; Goodman and Novacky, 1994;Bestwick et al., 1995). Pseudomonas syringae pv. phaseolicolais a bean pathogen that induces an HR response in tobacco(Lindgren et al., 1986). As shown in Fig. 7A infiltration ofleaves with a phaseolicola strain that induces the HR(NPS3121) triggers PCD. In contrast, infiltration of leaves witha P.s. phaseolicola strain that is Hrp− (NPS4000) does notinduce the HR (Fig. 7A). Cell death triggered by the Hrpcluster is accompanied by the formation of 50 kb fragments(Fig. 7B,C). However, in contrast to cell death that is inducedby TMV no significant increase in the amount of monomericchloroplast DNA, detected by FIGE, was observed during thiscell death process.

DISCUSSION

Nuclei of tobacco stem cells undergoing pathogen-inducedPCD were found to contain degraded DNA by an in situTUNEL assay (Mittler et al., 1995). Previous ultrastructuralstudies with leaf mesophyll cells indicated that nuclei of cellsundergoing TMV-induced PCD maintain their structuralintegrity even at late stages of cell death and that they assumea granular appearance (Hayashi and Matsui, 1965; Graca andMartin, 1975; Goodman and Novacky, 1994). However, thesestudies did not report the sequence of changes in nuclear mor-

phology during PCD. Our TEM observations show a changein the morphology of nuclear material during PCD (Fig. 2).This change appears to be a condensation of nuclear materialinto discrete patches distributed throughout the nucleus that isfollowed by condensation of the nucleoli and by an apparentchange in the staining of chromatin that may indicatebreakdown of nuclear material. Nuclei exhibiting these mor-phological changes were observed in stem cells located in andaround lesions. Stem cells localized in a similar manner werepreviously reported to exhibit positive TUNEL staining(Mittler et al., 1995), indicating that these changes may beassociated with the observed degradation of nuclear DNA.Morphological changes in plant nuclei undergoing PCD duringthe maturation of xylem vessels and during the differentiationof phloem cells resembled some of the changes observed inthis study (Esau, 1972; Lai and Srivastava, 1976). In general,the morphology of the nuclear material in plant cells appearsto change prior to the disruption of the nuclear envelope. Thisprocess does not involve condensation of nuclear materialspecifically in the nuclear periphery or fragmentation of nuclei.


In contrast to these differences from apoptosis in animal cells,the nucleoli in both plants and animals appeared to remainintact during most of the cell death process (Fig. 2C; Falcieriet al., 1994). Recently, Wang et al. (1996) reported that nucleiof tomato protoplasts undergoing toxin-induced cell death arefragmented in a manner that is similar to apoptosis in animalcells. Although this observation was not confirmed by TEM, itraises the possibility that nuclei of plant cells undergoingcertain types of PCD can display apoptosis-like fragmentationof nuclear materials.

We further investigated the morphological changes in thecytosol of stem cells during the HR of tobacco plants infectedwith TMV. We found that some of these changes appear similarto certain characteristic changes found in the cytosol of animalcells during apoptosis. These included condensation and vac-uolization of the cytoplasm and relatively high stability ofmitochondria. Our findings are in agreement with those ofGraca and Martin (1975) who reported that the cytoplasm ofleaf mesophyll cells becomes condensed and that mitochondriamaintained their structural integrity during late stages of celldeath. However, previous studies did not report the vacuoliza-tion of cytoplasm during TMV-induced cell death in tobacco.Vacuolization of cytoplasm was reported in leaf mesophyllcells undergoing bacteria-induced cell death (Roebuck et al.,1978). Changes in the cytoplasm of tobacco cells occurredprior to ultrastructural changes in the nuclei. These findingssuggest that, similar to some cases of PCD in animals(Jacobson et al., 1994; Earnshaw, 1995), the cytoplasm ofplants may play an important role during PCD. In contrast tothese similarities, tobacco cells undergoing TMV-induced PCDdid not form apoptotic bodies. This may not be surprising sinceplant cells contain walls that are likely to prevent engulfmentof apoptotic bodies by neighboring cells. Thus, the content ofthe dying plant cell is likely ‘recycled’ through some mecha-nisms that may be distinct from those involved in animal PCD.

One of the most widely studied biochemical events thatoccurs during PCD in animal cells is the fragmentation ofnuclear DNA. Two different mechanisms of chromatincleavage are activated during PCD: cleavage to large 300and/or 50 kb fragments (Oberhammer et al., 1993; Walker etal., 1993) and internucleosomal fragmentation (Wyllie et al.,1984). However, in certain instances cleavage of nuclear DNAto large DNA fragments may occur in the absence of internu-cleosomal fragmentation (Oberhammer et al., 1993; Beere etal., 1995; Bortner et al., 1995). Nuclei of tobacco cells under-going TMV-induced PCD contain degraded nuclear DNA(Mittler et al., 1995). However, no internucleosomal fragmen-tation was detected in DNA isolated from tobacco tissue under-going PCD (Mittler and Lam, 1995). In the present work, wetested whether the mode of chromatin cleavage in tobaccoresembles cases of PCD in animals that only display cleavageof chromatin to large DNA fragments. Using FIGE, we foundthat chromatin of tobacco cells undergoing PCD in response toTMV and HR-inducing bacteria is cleaved to fragments ofabout 50 kb (Figs 5, 7). Cleavage of chromatin occurred lateduring the HR (Figs 1D,E,F, 5). This observation is consistentwith our previous findings that degradation of nuclear DNAdetected with the in situ TUNEL assay occurred late duringcell death (Mittler et al., 1995). Our findings suggest that themode of DNA degradation during TMV-induced PCD intobacco appears to involve cleavage of chromatin to 50 kb

fragments that is followed by a non-specific degradation whichresults in a random pattern of DNA fragments. These do notdisplay the characteristic DNA ladder when separated by con-ventional agarose gels. It should be noted that not all cases ofPCD in animals display internucleosomal DNA fragmentation(Oberhammer et al., 1993; Schwartz et al., 1993; Beere et al.,1995; Bortner et al., 1995). DNA ladders have been interpretedas an indication that DNA is being digested at linker sitesbetween nucleosomes. However, if the proteins comprising thenucleosomes are degraded by specific or nonspecific proteasesduring or before the induction or activation of nucleases, nucle-osomal DNA ladders may not form and the pattern of DNAdegradation will be random. Regardless of the formation ofDNA fragments that are multimers of 180 bp, the appearanceof 50 kb fragments in tissues undergoing PCD is consistentwith our previous observations with the TUNEL stainingtechnique (Mittler et al., 1995). Recently, Levine et al. (1996)also reported that bacteria-induced PCD of cultured soybeancells is not accompanied by internucleosomal cleavage.However, Ryerson and Heath (1996) and Wang et al. (1996)reported that fungus-induced cell death in cowpea and toxin-induced death of tomato protoplasts is accompanied by inter-nucleosomal degradation of nuclear DNA. PCD in differentplant systems may therefore occur via different mechanismsthat exhibit distinct biochemical and morphological character-istics. Levine et al. (1996) also reported that bacteria-inducedPCD of cultured soybean cells is accompanied by theformation of 50 kb fragments. These results are consistent withour observations with intact tobacco leaves (Fig. 7).

The mode of cellular DNA degradation during pathogen-induced PCD was further studied with different nuclear, mito-chondrial and chloroplast specific probes (Fig. 5). An increasein the level of monomeric chloroplast DNA, detected by FIGE,was found to accompany TMV-induced PCD. The reason forthis monomerization is not clear, however, it is possible thatmodifications to the chloroplast membranes may causebreakage of the chloroplast genome at membrane attachmentsites (Deng et al., 1989). Alternatively, the presence of largestarch granules in cells undergoing TMV-induced PCD (Fig. 2;Weintraub and Ragetli, 1964; Goodman and Novacky, 1994)may cause breakage of chloroplast DNA during the prepara-tion of samples for FIGE. The increase in monomeric chloro-plast DNA occurred early during the HR to TMV and was notobserved during necrosis. Therefore, this process may serve asan early marker for TMV-induced PCD. However, in contrastto the increase in monomeric chloroplast DNA observedfollowing TMV-induced PCD in tobacco, PCD that is inducedby bacteria was not accompanied by similar changes in thelevel of monomeric chloroplast DNA. The chloroplasts of cellsundergoing necrotic death induced by a freeze/thaw treatmentor bacteria-induced PCD do not contain large starch granules(Fig. 2D; Goodman and Novacky, 1994), suggesting a correla-tion between the accumulation of large starch granules andbreakage or monomerization of chloroplast DNA in samplesprepared for FIGE. Further studies are required in order toestablish whether monomerization of the chloroplast genomeis unique to TMV-induced PCD in tobacco or whether it mayoccur during other cell death processes in plants. We recentlyfound that transgenically-triggered PCD (Mittler et al., 1995)is also accompanied by the appearance of DNA fragments ofabout 150 and 50 kb (data not shown). Thus, the presence of


the pathogen may not be required for these observed changesin the integrity of cellular DNA.

Cell death, induced by freezing and thawing, was alsoaccompanied by formation of 50 kb DNA fragments (Fig. 5).However, the relative hybridization of these fragments with thenuclear, mitochondrial and 18 S rDNA specific probes suggeststhat the composition of these fragments may be different fromthe composition of the 50 kb fragments observed during PCD.The fragments observed following necrosis seem to consistmostly of mitochondrial and nucleolar DNA. Formation of 50kb fragments was also reported during necrotic cell death ofanimal cells (Bortner et al., 1995). Therefore, it is possible thatformation of DNA fragments of about 50 kb may not bespecific to PCD per se. Further studies are required in order toresolve this question in both plants and animals. A possibleexplanation for the formation of 50 kb fragments duringnecrosis may be that the treatment used to induce necrotic celldeath in this study, i.e. freezing and thawing, caused the releaseof hydrolytic enzymes from the vacuole. These may be respon-sible for the degradation of nuclear material observedfollowing this treatment. An additional possibility is thatrupture of the vacuole membrane caused a rapid acidificationof the cytosol and nuclei which resulted in activation ofnucleases localized to these compartments. The mechanismthat may explain the observed fragmentation of DNA followingthe freeze/thaw treatment may also be responsible for some ofthe DNA degradation observed during the HR since theintegrity of the vacuole membrane was found to be compro-mised before that of the cell membrane (data not shown).Therefore, the degradation of nuclear DNA observed followingmechanical injury may in part be mediated by a mechanismthat is also responsible for DNA fragmentation during the HR.

Many of the morphological characteristics that accompanyTMV-induced PCD in tobacco were similar to those that occurduring PCD in Dictyostelium discoideum (Cornillon et al.,1994). These included condensation and vacuolization of thecytoplasm and focal chromatin condensation. However, incontrast to the degradation of nuclear DNA to 50 kb fragmentsin plants, no nuclear DNA fragmentation was observed in Dic-tyostelium. The similarities in morphological changes thataccompany pathogen-induced PCD in plants, PCD in Dic-tyostelium and apoptosis in animals suggest that some aspectsof PCD may have been conserved during the evolution ofeukaryotic organisms.

We thank Drs Daniel Klessig, Charles S. Levings, Peter B.Lindgren and Barbara A. Zilinskas for gifts of plasmids and bacterialstrains. We gratefully acknowledge Drs Eileen White and Peter Dayfor critical comments and Dr David D. Dunigan for communicationof unpublished results. This work was supported in part by the NewJersey Commission of Science and Technology, the US Departmentof Agriculture and by Waksman Institute Busch Funds.

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(Received 9 August 1996 - Accepted 21 February 1997)

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