The Plant Cell, Vol. 13, 1803–1818, August 2001, www.plantcell.org © 2001 American Society of Plant Biologists

The PET1-CMS Mitochondrial Mutation in Sunflower Is Associated with Premature Programmed Cell Death and

Cytochrome

c

Release

Janneke Balk

1

and Christopher J. Leaver

University of Oxford, Department of Plant Sciences, South Parks Road, Oxford OX1 3RB, United Kingdom

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinctform of programmed cell death (PCD), for example, through the release of cytochrome

c

, which activates a proteolyticenzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal de-velopment, including the penultimate stage of anther development, leading to dehiscence and pollen release. However,there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pol-len development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associatedwith mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and bio-chemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD ofthe tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleo-somal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochon-dria. In addition, immunocytochemical analysis revealed that cytochrome

c

was released partially from themitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The de-crease in cytochrome

c

content in mitochondria isolated from male sterile florets preceded a decrease in the integrityof the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, likemammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype isdiscussed with regard to cellular respiratory demand and PCD during normal anther development.

INTRODUCTION

Mammalian mitochondria have been shown to play a pivotalrole in the induction of apoptosis, a morphologically distinctform of programmed cell death (PCD) (reviewed in Cai et al.,1998; Green and Reed, 1998; Mignotte and Vayssiere,1998). In mammals, apoptosis is characterized by conden-sation of the nucleus and the cytoplasm, cleavage of nu-clear DNA into

�

180-bp oligonucleosomal units (DNAladdering), and packaging of the cell corpse in vesicles thatare ingested by phagocytes. PCD is a genetically basedpathway, in contrast to necrosis or “accidental” cell death,which is characterized by swelling of the cell, rupture of theplasma membrane, and spilling of the cell contents. It hasbeen shown in mammalian systems that mitochondria canplay a role in the induction of PCD by release of intermem-brane space components into the cytosol, including cyto-chrome

c

, a key component of the electron transport chain(Liu et al., 1996). The mechanism by which cytochrome

c

re-

lease occurs has yet to be established unequivocally, but itseems to be regulated by antiapoptotic and proapoptoticproteins of the Bcl-2 family (Green and Reed, 1998). Once inthe cytosol, cytochrome

c

activates a proteolytic cascademediated by caspases (cysteinylaspartate proteases), lead-ing to the oligonucleosomal cleavage of DNA (Enari et al.,1998) and the organized breakdown of the cell.

In plants, several tissues or whole organs undergo celldeath as part of their normal development, for example, inxylogenesis, anther dehiscence, and senescence, or in re-sponse to pathogens and environmental stresses (reviewedin Greenberg, 1996; Jones and Dangl, 1996; Beers, 1997;Pennell and Lamb, 1997; Richberg et al., 1998; Lam et al.,1999). In contrast to mammalian apoptosis, the genetic ba-sis underlying these developmental and (a)biotically inducedcell deaths in plants remains to be discovered. However,many of the cytological and biochemical markers of mam-malian apoptosis have been observed in plants, suggestingthat gene-specific or “programmed” cell death does exist.By analogy with mammalian apoptosis, plant mitochondriahave been suggested to play a pivotal role in the integrationof environmental and developmental signals that trigger celldeath (Jones, 2000, and references therein).

1

To whom correspondence should be addressed. E-mail janneke.balk@plant-sciences.ox.ac.uk; fax 44-1865-275144.

1804 The Plant Cell

To further investigate the potential link between mito-chondrial function and PCD in plants, we decided to reex-amine cytoplasmic male sterility (CMS) in sunflower, whichis characterized by the inability to produce viable pollen as-sociated with a mutation in the mitochondrial genome. Inhumans, several pathologies associated with mutations inthe mitochondrial genome have been linked to the role ofmitochondria in apoptosis (Wallace, 1999), and it is possiblethat detailed investigations of mitochondrial mutations inplants may reveal a similar link.

CMS has been described in

�

150 plant species (Kaul,1988), and, in combination with the associated nuclear re-storer of fertility genes, it has been used extensively in plantbreeding for the production of a range of F1 hybrid crops. InCMS plants, anther development may be severely affected,as is the case in homeotic-like mutants of tobacco and

Brassica

species (Kofer et al., 1991, 1992; Makaroff, 1995),but in most CMS mutants, anthers develop normally and mi-crosporogenesis is arrested during or soon after meiosis(Kaul, 1988). Generally, the tapetum undergoes cellular deg-radation during meiosis of the meiocytes, followed soon af-ter by the death of the immature microspores.

CMS is associated with the expression of novel openreading frames (ORFs) in the mitochondrial genome in mostspecies investigated to date (Schnable and Wise, 1998) or withthe deletion of mitochondrial genes in tobacco (Gutierres etal., 1997). The novel ORFs are thought to have originated byaberrant recombination events in the plant mitochondrialgenome. The DNA sequences of the novel ORFs often con-tain partial sequences of other mitochondrial genes andmay be cotranscribed with functional mitochondrial genes.PET1-CMS in sunflower was identified in an interspecific crossbetween

Helianthus petiolaris

and

H. annuus

(Leclercq,1969) and is associated with the expression of a novel mito-chondrial gene,

orf522

, which encodes a 15-kD polypeptide(Horn et al., 1991; Laver et al., 1991; Monéger et al., 1994).The

orf522

gene was created by a recombination event in-volving an inversion/insertion rearrangement 3

�

to the

atp1

gene (Köhler et al., 1991; Laver et al., 1991). The first 18amino acids of ORF522 are identical to ORFB, which maybe the plant homolog of yeast and mammalian ATP8, a sub-unit of the F

0

F

1

-ATPase (Gray et al., 1998).As part of the normal developmental sequence associ-

ated with pollen formation and release, several anther tis-sues undergo cell death in a precisely coordinated andtemporal progression (Goldberg et al., 1993; Wu and Cheung,2000). The tapetal cells lyse and release lipid componentsthat coat the pollen exine (Piffanelli and Murphy, 1998). Thestomium cells die and shear to allow release of the pollenfrom the anther locule. The endothecium and epidermal celllayers undergo cell death, and special cell wall structures inthe endothecium cells provide the mechanism whereby thelocules spring open at dehiscence. Recently, Papini et al.(1999) showed that the degradation of tapetal cells in twoangiosperms (

Lobivia rauschii

and

Tillandsia albida

) showscytological features characteristic of PCD when studied by

electron microscopy. These include shrinkage of the cell,condensation of chromatin, swelling of the endoplasmicreticulum, and the persistence of mitochondria. In addition,Wang et al. (1999) observed oligonucleosome-sized DNAcleavage in barley anthers at the end of the unicellular stageof pollen development.

Sunflower is a suitable system in which to study mito-chondrial biogenesis and function during flower develop-ment because the florets develop in a spatial and temporalsequence such that the older stages are found at the pe-riphery of the capitulum. The stages of pollen developmentas described by Horner (1977) have been related to budlength (Smart et al., 1994). Therefore, whorls of florets atsuccessive stages of development can be dissected easily,providing enough material for the isolation of mitochondriaand associated in situ microscopic analysis. Because themale and female parts of the flower are clearly separatedand pollen development takes place when the ovary is rela-tively undeveloped, florets enriched in anthers can be iso-lated easily.

To study the role of mitochondria in plant PCD, we havecharacterized anther development in fertile and PET1-CMSsunflower with respect to morphological and biochemicalmarkers of PCD. We have shown that in plants carrying thePET1-CMS mitochondrial mutation, first the tapetal cellsand then the young microspores lost their shape and con-densed concomitant with oligonucleosomal cleavage of nu-clear DNA. Initially, mitochondria persisted and, in tapetalcells, released cytochrome

c

into the cytosol. The release ofcytochrome

c

preceded cellular condensation, DNA frag-mentation, and a decrease in the integrity of the outer mito-chondrial membrane and the respiratory control ratio. Thesedata provide strong evidence that PET1-CMS cytoplasmcauses premature induction of PCD in the tapetum, leadingto death of the microspores, which results in the male sterilephenotype, and suggest that plant mitochondria are in-volved in the induction of PCD.

RESULTS

Cytological Features of Anther Development in the Fertile and Sterile Sunflower Lines

Several reports have compared anther development inPET1-CMS sunflower and the isonuclear fertile line at theresolution of light and electron microscopy (Horner, 1977;Laveau et al., 1989; Smart et al., 1994). However, these re-ports focus on the initial cytological abnormalities duringanther development in the sterile line to provide an under-standing of the underlying causes of the CMS phenotype.We performed a light microscopic study of the later stagesof cellular degradation in PET1-CMS anthers compared withfertile anthers to determine whether the dying tissues exhibitfeatures of necrosis or PCD.

Programmed Cell Death during CMS Anther Development 1805

Figure 1. Anther Development in Florets of the Fertile and Sterile Sunflower Lines.

Four stages of anther development in the fertile line and the corresponding stages of development in the sterile line were compared using histo-logical staining. The images are of cross-sections through single locules.(A) Fertile line, premeiosis.(B) Sterile line, premeiosis.(C) Fertile line, pachytene.(D) Sterile line, pachytene.(E) Fertile line, tetrad stage.(F) Sterile line, tetrad stage.(G) Fertile line, stage 6 according to Horner (1977): young microspores.(H) Sterile line, stage 6 as in (G).en, endothecium cell(s); ep, epidermal cell(s); m, meiocytes; ml, middle layer; ms, microspore(s); t, tapetal cell(s). Bars � 20 �m.

1806 The Plant Cell

In histologically stained anther sections observed by lightmicroscopy, the first sign of abnormal development inPET1-CMS sunflower became evident at pachytene, whenthe tapetal cells began to enlarge (Figures 1C and 1D; seealso Horner, 1977). This was followed by condensation ofthe tapetal cytoplasm at the tetrad stage (Figure 1F), loss ofregular cell shape, and separation of the chromatin into de-lineated masses (cf. Figures 5K and 5L).

At the tetrad stage, the microspores in the sterile line ap-peared normal, although they were compressed in the cen-ter of the locule (Figure 1F). The middle layer in the sterileline was enlarged, whereas this cell layer had disappearedfrom the fertile anthers at this stage (Figure 1E). When theflorets of the PET1-CMS line had reached a length that cor-responds to stage 6 (young microspores) during fertile de-velopment (Horner, 1977; Smart et al., 1994), the tapetalcells were further condensed and the microspores were de-graded into a lump of condensed material, crushed by theenlarged middle layer (Figure 1H).

In the fertile line, the tapetal cytoplasm stained darker atthe tetrad stage (cf. Figures 1C and 1E), the cell wallsstarted to disappear (Horner, 1977), and the cells becameplasmodial. When the microspores had developed a spikyexine (Figure 1G), the tapetal cytoplasm stained less in-tensely and the cells became irregular in shape, with large,diffuse nuclei. The cells and nuclei of the endothecium andepidermal cells condensed from the tetrad stage onward.

Premature Death of Anther Tissues in the Sterile Line Is Associated with Oligonucleosomal Cleavage of DNA

A distinct feature of PCD is the cleavage of nuclear DNA intooligonucleosome-sized fragments (

�

180 bp). To determineat which stage of anther development and in which tissuesthe fragmentation of nuclear DNA occurs, fertile and sterileanthers were compared using the TUNEL assay (TdT-medi-ated dUTP nick-end labeling). This is an in situ techniquethat measures the fragmentation of DNA by incorporatingfluorescein 12–dUTP at the free 3

�

-OH ends (Figure 2, brightgreen to yellow). The stages of development and cytologywere identified by differential interference contrast micros-copy (data not shown) to avoid the use of fluorescent DNAdyes that have partially overlapping emission spectra withfluorescein.

At premeiosis, there was no difference in TUNEL stainingbetween fertile and sterile anthers (Figures 2A and 2B).However, in the sterile anthers at pachytene (Figure 2D), thenuclei of all anther tissues except for the meiocytes stainedmore intensely than in their fertile equivalents, suggestingthat DNA fragmentation had been initiated. When the sterileflorets reached the tetrad stage (Figure 2G), the nuclei of allanther tissues, including the microspores (the meiotic prod-ucts of the meiocytes), showed clear DNA fragmentation.

At the tetrad stage in fertile anthers, the cells forming thestomium contained condensed nuclei and fragmented DNA

(Figure 2F). Nuclear DNA fragmentation in fertile antherswas not investigated beyond the tetrad stage because fertileanther development was used as a reference to anther de-velopment in the sterile line. However, Wang et al. (1999)have reported that toward the end of the unicellular stage ofpollen development in barley, the nuclei of tapetal cells andthe tissues of the anther wall were TUNEL positive and oli-gonucleosomal DNA cleavage was observed.

The in situ TUNEL assay does not discriminate betweenrandom fragmentation and oligonucleosome-sized cleavageof DNA. To discriminate between the former, which is asso-ciated with necrosis, and the latter, which is characteristic ofPCD, total DNA was extracted from fertile and sterile floretsat the stages of anther development approximating thoseinvestigated using the TUNEL assay and analyzed by aga-rose gel electrophoresis with ethidium bromide staining. Theresults shown in Figure 3 indicate PCD-specific laddering ofDNA in sterile florets at the tetrad stage, correlating with theprogressive indication of DNA fragmentation in sterile an-thers observed using the TUNEL assay. The lowest frag-ment corresponded approximately to the size of DNAcontained within nucleosomes (z180 bp). No induction ofDNA cleavage was observed in the fertile florets at the tet-rad stage, despite the appearance of TUNEL-positive nucleiin the stomia, which apparently were too few in number todetect by agarose gel electrophoresis and ethidium bromidestaining.

First Signs of Abnormal Development in the Tapetum of Sterile Anthers Are Associated with the Release of Cytochrome

c

from the Mitochondria

Several reports have demonstrated the translocation of cy-tochrome

c

from the mitochondria into the cytosol upon in-duction of PCD (Balk et al., 1999; Stein and Hansen, 1999;Sun et al., 1999). Therefore, the localization of cytochrome

c

during anther development was investigated in the fertileand sterile sunflower lines using a monoclonal antibodyagainst rat cytochrome

c

(7H8.2C12). The epitope recog-nized by the antibody has been mapped (Jemmerson et al.,1991), and the amino acid sequence of the epitope is identi-cal in rat and sunflower cytochrome

c

. Cross-reactivity wasconfirmed by protein gel blot analysis of cytochrome

c

puri-fied from sunflower seedlings (data not shown). The identityof purified cytochrome

c

was confirmed by its spectrum(Margoliash and Walasek, 1967) and internal sequence data(see Methods). The 7H8.2C12 monoclonal antibody wastested for use in immunocytochemistry by protein gel blotanalysis of subcellular fractions of sunflower tissues. Theantibody labeled an

�

12-kD protein in sunflower mitochon-dria (Figure 4). The antibody cross-reacted weakly with a50-kD protein in the cytosolic fraction when used for proteingel blot analysis, but in situ, a mitochondria-like, punctatelabeling pattern was observed. No cross-reaction with chlo-roplast proteins was detected.

Programmed Cell Death during CMS Anther Development 1807

Localization of porin, an integral outer mitochondrial mem-brane protein, was used as a control for mitochondrial den-sity and distribution. A monoclonal antibody against maizeporin (PM035) recognized three proteins of 28, 30, and 31 kDin sunflower mitochondria (Figure 4). These proteins are likelyto be isoforms, as have been reported to exist in wheat (Elkeleset al., 1995). Thin sections of anthers from successive stagesof development were labeled with the antibodies as indicated

in Figure 5 and visualized with a fluorescein isothiocyanate(FITC)–conjugated secondary antibody (Figure 5, yellow-green). Nucleic acids were stained with propidium iodide (red)to confirm the stage of anther development.

In both fertile and sterile anthers at premeiosis (Figures 5Ato 5C), cytochrome

c

and porin accumulated in the meio-cytes, corresponding to the high mitochondrial numbers re-ported previously (Smart et al., 1994). At pachytene, the

Figure 2. DNA Fragmentation in Florets of the Fertile and Sterile Sunflower Lines.

Three stages of anther development in the fertile line and the corresponding stages of development in the sterile line were compared with re-spect to nuclear DNA fragmentation using the TUNEL assay (bright green to yellow is positive stain).(A) Fertile line, premeiosis.(B) Sterile line, premeiosis.(C) Fertile line, pachytene.(D) Sterile line, pachytene.(E) As a negative control, the terminal deoxynucleotidyl transferase enzyme was omitted from the labeling mixture. Sterile line, pachytene.(F) Fertile line, tetrad stage.(G) Sterile line, tetrad stage.m, meiocyte(s); ms, microspores; st, stomium cells; t, tapetal cell(s). Bars � 20 �m.

1808 The Plant Cell

localization pattern of porin in sterile anthers (Figure 5F) wassimilar to the localization pattern of cytochrome

c

in fertileanthers (Figure 5D), most likely reflecting a high mitochon-drial density in the meiocytes. However, when sections ofsterile anthers at pachytene were labeled for cytochrome

c

(Figure 5E), in addition to the punctate labeling in the meio-cytes the tapetal cell layer was found to be brightly stained.At higher magnification (Figure 5J), the labeling pattern ofcytochrome

c

in the tapetal cells of sterile anthers at pachy-tene appeared both punctate and diffuse, in contrast to theentirely diffuse pattern observed in PCD-induced HeLa cells(Bossy-Wetzel et al., 1998). This finding suggests that onlypart of the cytochrome

c

is released into the cytosol.The total intensity of the fluorescence associated with cy-

tochrome

c

in the tapetum of sterile anthers at pachyteneappeared much greater than that for the comparable sec-tions of fertile anthers (Figure 5D), which is likely to be attrib-utable to the increased accessibility of the antigen ratherthan to a sudden increase in the amount of cytochrome

c

.Similarly, Varkey et al. (1999) described “display” of the oth-erwise hidden cytochrome

c

epitope in

Drosophila melano-gaster

nurse cells undergoing PCD. In

D. melanogaster

,however, cytochrome

c

is thought to remain bound to theouter mitochondrial membrane upon release from the inter-membrane space, whereas our data suggest that at leastpart of the cytochrome

c

is translocated into the cytosol.At the tetrad stage, the cytological degradation of tapetal

cells in the sterile line became apparent (Figures 5H and 5L).

In the tapetal cells of both fertile and sterile anthers, thefluorescence associated with cytochrome

c

was increasedand appeared granular/diffuse (Figures 5G and 5H). In con-trast, the localization pattern of porin was still punctate,suggesting that mitochondria were not yet degraded com-pletely (Figure 5I; data shown for sterile anthers only). In fer-tile anthers, cytochrome

c

was localized mostly around thenucleus of the young microspores (Figures 5G and 5K), cor-responding to the localization of mitochondria (Dickinsonand Li, 1988; Smart et al., 1994). A similar localization pat-tern could be seen for porin in sterile anthers (Figure 5I),whereas in sterile anthers the fluorescence associated withcytochrome

c

was much reduced in the microspores (Fig-ures 5H and 5L).

To semiquantify the amount of cytochrome

c

associatedwith mitochondria during fertile and sterile anther develop-ment, mitochondria were prepared from the upper, mostlyanther-containing part of florets at premeiosis, meiosis, andtetrad stage. Small samples of the mitochondrial fractionwere fixed and processed for electron microscopy to con-firm that the population of mitochondria isolated displayedthe ultrastructural characteristics described by Laveau et al.(1989) and Smart (1994) (data not shown). Mitochondrialproteins were separated by SDS-PAGE, and the relativeamount of cytochrome

c

was determined by protein gel blotanalysis and densitometry. Figure 6 shows the relativeamount of cytochrome

c

per milligram of mitochondrial pro-

Figure 3. Oligonucleosomal Cleavage of Total DNA in Florets of theFertile and Sterile Sunflower Lines.

Agarose gel electrophoresis of total DNA stained with ethidium bro-mide. The developmental stages correspond to those stages usedfor the TUNEL assay (Figure 2). Pre, premeiosis; Mei, meiosis; Tet,tetrad stage.

Figure 4. Characterization of the Monoclonal Antibodies Used for inSitu Labeling.

Protein gel blot analysis using monoclonal antibodies against rat cy-tochrome c (7H8.2C12) and maize porin (PM035) on 30 �g of mito-chondrial (Mit), chloroplast (Chl), and cytosolic (Cts) proteins fromflorets (mitochondria and cytosol) or leaves (chloroplast) of the fertilesunflower line.

Programmed Cell Death during CMS Anther Development 1809

tein averaged over four experiments. The same protein gelblots were stripped and relabeled with a monoclonal anti-body against maize E1

�

, which is a subunit of pyruvate de-hydrogenase, an enzyme complex in the mitochondrialmatrix. In contrast to the monoclonal antibody against porin(Figure 4), the E1

�

antibody recognized a single polypeptideand therefore is more suitable for quantification. When therelative amount of cytochrome

c

was normalized for E1

�

levels, similar results were obtained, as shown in Figure 6,indicating that any changes found are not caused by differ-ing impurities of the mitochondrial preparations (data notshown).

During fertile anther development, the levels of cyto-chrome

c

associated with mitochondria were similar at pre-meiosis and meiosis but decreased at the tetrad stage(Figure 6). In sterile florets, the mitochondrial levels of cyto-chrome

c

at premeiosis were similar to the levels in fertileflorets, whereas at meiosis, the relative amount of cyto-chrome

c

was significantly lower (0.001

�

P

�

0.01) than infertile florets. At the tetrad stage, the mitochondrial cyto-chrome

c

levels in sterile florets decreased further to a levelthat was similar to that found in fertile florets.

To confirm that cytochrome

c

was translocated from themitochondria into the cytosol, cytosolic extracts were ana-lyzed by protein gel blot analysis. However, because it wasestimated based on specific enzyme activity assays that 1part of mitochondria (mg of protein) is proportional to morethan 10 parts of cytosolic protein (mg) extract, the bulk ofprotein was removed by ammonium sulfate precipitation toenable the detection of cytochrome

c

by protein gel blotanalysis. Low amounts of cytochrome

c

then could be de-tected in the 60 to 80% ammonium sulfate precipitate of thecytosolic protein fractions at the later stages of anther de-velopment in sterile florets but not at any stage of anther de-velopment in fertile florets (data not shown).

Decrease in Mitochondrial Cytochrome

c

in Sterile Anthers Precedes a Decline in Outer Membrane Integrity and Respiratory Control Ratio

Mitochondria from successive stages of anther develop-ment also were used to investigate whether the significantdecrease in mitochondrial cytochrome

c

in sterile anthers atmeiosis was the result of a reduction in the integrity of theouter mitochondrial membrane and whether the reducedlevels of cytochrome

c

had an effect on respiration. Therespiratory assays were conducted on mitochondrial prepa-rations from two independent experiments that were rep-resentative of the four experiments used to quantify thecytochrome

c

content (Figure 6). The outer mitochondrialmembrane integrity assay is based on the accessibility ofcomplex IV (cytochrome

c

oxidase) to exogenous cyto-chrome

c

that is reduced by ascorbate. The outer mitochon-drial membrane integrity was similar in mitochondria fromfertile and sterile anthers at premeiosis and meiosis but was

reduced significantly (0.01

�

P

�

0.05) in mitochondria fromsterile anthers at the tetrad stage (Figure 7A).

To investigate whether the decrease in mitochondrial cy-tochrome

c

is associated with downregulation of theamount and/or activity of cytochrome

c

oxidase and notwith a sudden, PCD-associated release, cytochrome

c

oxi-dase activity was measured in isolated mitochondria. Nosignificant decrease in cytochrome

c

oxidase activity wasobserved during anther development in either the fertile orthe sterile line, suggesting that the decrease in cytochrome

c

is not caused by downregulation of the cytochrome

c

oxi-dase pathway (Figure 7B). In contrast, Bino et al. (1986) ob-served a lower activity of cytochrome

c

oxidase in CMSpetunia just after the onset of cellular degradation and inCMS-S maize before degeneration of the microspores com-pared with their respective isonuclear fertile lines. However,the enzyme activity measured reflects the maximum activityin the presence of excess cytochrome

c

substrate, whichmay not reflect the situation in vivo. Unfortunately, it was notpossible to estimate the absolute amounts of cytochrome

c

oxidase by protein gel blot analysis because there are noantibodies available that cross-react with sunflower cyto-chrome

c

oxidase subunits.The release of cytochrome

c

from the mitochondria islikely to affect the activity of the respiratory chain and, con-sequently, ATP synthesis. To obtain a measure of this effect,the oxygen consumption rate of isolated mitochondria wasdetermined with and without ADP. The ratio is known as therespiratory control ratio (RCR) and reflects the degree ofcoupling of ATP synthesis to electron transport. Figure 7Cshows that the RCR was similar in mitochondria isolatedfrom fertile and sterile anthers at premeiosis and meiosis butthat there was a significant reduction in RCR at the tetradstage compared with meiosis in the sterile sunflower line.The RCR also was reduced in mitochondria from fertile an-thers at the tetrad stage, but not significantly.

Mitochondrial Biogenesis in Anthers

In PET1-CMS sunflower, the CMS-specific mitochondrialprotein ORF522 is expressed in all tissues, but the deleteri-ous phenotype associated with its expression is limited tothe anthers. Because this is true for many types of CMS in-vestigated to date, two hypotheses are commonly proposedthat could explain the tissue specificity of the CMS pheno-type: (1) there exists an anther-specific compound that in-teracts with the CMS-associated polypeptide, and (2) therespiratory demands are highest in the developing tapetumand/or meiocytes. We have shown a dramatic increase inmitochondrial gene expression during anther development insunflower (Smart et al., 1994), which was consistent with theobservation by Lee and Warmke (1979), who showed, usingelectron microscopy and quantitative analysis, that duringmeiosis in maize the mitochondrial number per cell increases20- and 40-fold in meiocytes and tapetal cells, respectively.

1810 The Plant Cell

Figure 5. Immunolocalization of Cytochrome c in Florets of the Fertile and Sterile Sunflower Lines.

Three stages of anther development in the fertile line were compared with the corresponding stages in the sterile line with respect to the in situlocalization of cytochrome c. Thin sections from PEG-embedded plant material were labeled with monoclonal antibodies against rat cytochromec or, as depicted for the sterile line only, with monoclonal antibodies against porin, an integral outer mitochondrial membrane protein. The sec-tions were secondarily labeled with a FITC-conjugated antibody (yellow/green), and nucleic acids were stained with propidium iodide (red) to fa-cilitate identification of the developmental stage.(A), (B), and (C) Premeiosis.(D), (E), and (F) Pachytene.(G), (H), and (I) Tetrad stage.(A), (D), and (G) show localization of cytochrome c during anther development in the fertile line. (B), (E), and (H) show localization of cytochromec during anther development in the sterile line. (C), (F), and (I) show localization of porin during anther development in the sterile line.(J) Subcellular localization of cytochrome c in the sterile line at pachytene shown at higher magnification.(K) Cytochrome c localization and propidium iodide staining in the fertile line at the tetrad stage shown at higher magnification. The arrowheadpoints to the chromatin of a tapetal cell.(L) Cytochrome c localization and propidium iodide staining in the sterile line at the tetrad stage shown at higher magnification. The arrowheadpoints to the chromatin of a dying tapetal cell.cyt c, cytochrome c; m, meiocyte(s); t, tapetal cell(s); ms, microspore(s). Bars � 20 �m.

Programmed Cell Death during CMS Anther Development 1811

However, while mitochondrial numbers increase rapidlyduring pollen development, their volumes decrease associ-ated with a loss of internal membrane structures (Lee andWarmke, 1979; Dickinson and Li, 1988). Therefore, mito-chondrial number may not be a good measure of mitochon-drial biogenesis on a per cell basis. Thus, we performed aquantitative analysis of mitochondrial protein using a fluo-rescent label and optical sectioning by confocal microscopyin meiocytes and tapetal cells at premeiosis and at pachy-tene. The data were compared with data from root peridermcells, a vegetative tissue with high respiratory activity. Thicktissue sections (30 to 70 �m) were labeled using an anti-body against mitochondrial CPN60 (Robertson et al., 1995),a mitochondrial chaperonin involved in protein import andassembly and hence mitochondrial biogenesis (Prasad etal., 1990). CPN60 labeling was visualized with a FITC-conju-gated secondary antibody. The integrated CPN60-specificfluorescence (signal) was expressed per cell (Figure 8A) andper unit volume (Figure 8B).

The CPN60 signal increased approximately sevenfold inmeiocytes and 20-fold in tapetal cells from premeiosis to themeiotic metaphase and was highest in tapetal cells atmetaphase. These increases were comparable to the ap-proximately eightfold and �12-fold increases, respectively,found by Lee and Warmke (1979) in maize during the samedevelopmental period. The CPN60 signal per cell in rootperiderm cells was similar to that found in premeiotic meio-cytes. Because of the dramatic difference in cell sizes, theCPN60 signal per unit volume in root periderm cells actuallywas comparable to that in meiocytes at pachytene. Becauseof an increase in cell size, the increase in CPN60 signal perunit volume during meiosis was far less dramatic than theincrease per cell (approximately twofold and 1.5-fold, re-spectively) for meiocytes and tapetal cells, respectively. Incomparison, Lee and Warmke (1979) found a 4.5-fold andthreefold increase, respectively, in mitochondrial numberper unit volume in maize.

DISCUSSION

PET1-CMS Mutation Is Associated with Premature PCD in Anther Tissues

To study the role of mitochondria in PCD in plants, we havecharacterized anther development in PET1-CMS sunflowerand compared that with anther development in the fertileline. Plants carrying the PET1-CMS cytoplasm are malesterile because of the death of the tapetum and meiocytesnear the end of meiosis (Leclercq, 1969; Horner, 1977).

Anther tissues undergo cell death as part of their normaldevelopmental program leading to dehiscence. Recently,Papini et al. (1999) showed that the degradation of tapetalcells in two angiosperms (L. rauschii and T. albida) shows

cytological features characteristic of PCD when studied byelectron microscopy. In addition, Wang et al. (1999) ob-served oligonucleosomal DNA cleavage in barley anthers atthe end of the unicellular stage of pollen development. Insunflower, we observed the onset of PCD leading to antherdehiscence at the tetrad stage, when the nuclei of the sto-mium condensed and the nuclear DNA was fragmented.Also, the endothecium and epidermal cells started to con-dense. In contrast to fertile development, in PET1-CMS sun-flower, first the tapetum and then the microspores lost theirregular cell shape and condensed starting at the pachytenestage. The cells of the middle layer and later the endothe-cium and epidermis enlarged.

The TUNEL assay showed that the nuclear DNA of all an-ther tissues was fragmented at the tetrad stage and, asshown by agarose gel electrophoresis and ethidium bro-mide staining, that this fragmentation was in fact oligonu-cleosomal cleavage. The chromatin of the tapetal cells atthe tetrad stage separated into delineated masses. Despitethe cell collapse and fragmentation of DNA, the mitochon-dria were not degraded completely, as shown by in situ lo-calization of porin. The latter finding confirms the electronmicroscopic observation by Horner (1977) that “most or-ganelles seem to be intact, even though the mitochondriaare somewhat pleomorphic with some evidently degenerat-ing.” Similarly, Laveau et al. (1989) observed that “the mito-chondria and plastids seem normal at the first signs ofabnormal development, but soon after the mitochondriashow a loss of cristae and a lighter matrix both in tapetalcells and meiocytes.”

Cellular condensation, oligonucleosomal cleavage, thespecific appearance of the chromatin, and the initial persistence

Figure 6. Relative Cytochrome c Content in Isolated Mitochondriafrom Florets of the Fertile and Sterile Sunflower Lines.

The relative amount of cytochrome c was determined by protein blotanalysis and densitometry, working within a linear range of 20 to 40�g of mitochondrial proteins as determined experimentally. The dataare the average values of four independent experiments (SE), foreach of which mitochondrial preparations were derived from four toeight sunflower heads. Statistical analysis was performed using Stu-dent’s t test on sample means (** 0.001 � P � 0.01).

1812 The Plant Cell

of the mitochondria all are features of mammalian apopto-sis, a morphologically distinct form of PCD. In contrast towhat is seen in apoptosis, the nuclei of the dying cells didnot condense and the cells were not broken down into apop-totic bodies that could be phagocytosed by neighboringcells. The latter effect, however, is observed rarely in plants,although apoptotic body-like structures have been ob-served during the PCD induced by mycotoxins (Wang et al.,1996).

The PET1-CMS phenotype is associated with a mutation inthe mitochondrial genome leading to the expression of anovel polypeptide, ORF522. Although there is no direct evi-dence that ORF522 causes the death of the male reproduc-tive tissues, overexpression of ORF522 has been shown tobe lethal to Escherichia coli (Nakai et al., 1995). Therefore, ourdemonstration that the premature death of tapetal cells in thePET1-CMS line exhibited many features of apoptosis stronglysuggests that mitochondria are involved in plant PCD.

Cytochrome c Release

A link between plant mitochondria and PCD is supported byour observation that in tapetal cells, cytochrome c was par-tially released from the mitochondria into the cytosol duringboth fertile anther development and anther development inthe sterile line. In fertile anthers, the release of cytochrome cwas observed at the tetrad stage, whereas in the sterile line,cytochrome c release took place at pachytene coincidentwith the first cytological signs of abnormal development, inagreement with our hypothesis that the PET1-CMS cyto-plasm induces premature PCD. However, cytochrome c re-lease into the cytosol was not observed in all tissues withcharacteristics of apoptosis (e.g., the stomium during fertiledevelopment and the microspores in the sterile line). In thelatter tissues, the mitochondrial level of cytochrome c wasreduced without a corresponding immunolabeling of the cy-tosol, which could account for the difficulty of detecting cy-tochrome c in the cytosolic protein fraction of florets. Thissuggests that in these cells, cytochrome c was degradedrapidly either inside the mitochondria or upon translocationinto the cytosol. Complete degradation of the mitochondriacould be ruled out because immunolabeling with anti-porinantibodies showed a mitochondria-like, punctate pattern inthe microspores.

In mitochondria isolated from successive stages of sterileanther development in sunflower, cytochrome c release pre-ceded a decrease in outer mitochondrial membrane integrityand respiratory control ratio, which is in agreement with ourprevious results obtained from mitochondria isolated fromcucumber cotyledons undergoing heat-induced PCD (Balket al., 1999). These data raise the question of how cyto-chrome c is released from the mitochondria. The mecha-nism of cytochrome c release during mammalian apoptosisis a topic of ongoing debate; however, two mechanisticprinciples have been proposed (Green and Reed, 1998).

Figure 7. Respiratory Properties of Isolated Mitochondria from Flo-rets of the Fertile and Sterile Sunflower Lines.

Standard oxygen electrode assays were used to determine outermembrane integrity (A), cytochrome c oxidase activity (B), and re-spiratory control ratio (C). The data are averages of two independentexperiments (SE) of those used for Figure 6. Statistical analysiswas performed using Student’s t test on sample means (* 0.01 � P �0.05).

Programmed Cell Death during CMS Anther Development 1813

One is that the mitochondrial matrix swells as a result of ei-ther hyperpolarization or permeability transition of the innermitochondrial membrane and that, because the inner mem-brane is folded, the increased volume of the matrix rupturesthe outer mitochondrial membrane. The other possibility isthat a transient pore is formed in the outer mitochondrialmembrane, either by proapoptotic proteins on their own orby interaction with porin.

Our assay for outer mitochondrial membrane integrity ismore likely to measure the rupture of the outer membrane,because the transient pores might close upon isolation ofthe mitochondria and resuspension in isoosmotic buffer. Forexample, Kluck et al. (1999) reported that two proapoptoticproteins, Bid and Bax, cause complete release of cyto-chrome c but only a limited permeabilization of the outermembrane in isolated Xenopus mitochondria using an assaysimilar to that used in our studies. Therefore, our datasuggest that during anther development in PET1-CMS sun-flower, cytochrome c is released from the tapetal mito-chondria via transient pores rather than by swelling of thematrix and rupture of the outer mitochondrial membrane.This is in agreement with the normal appearance of mito-

chondria as observed by electron microscopy at the onsetof tapetum degradation (Horner, 1977; Laveau et al., 1989).The mitochondria isolated from sterile florets at the tetradstage, however, did show a reduction in outer mitochondrialmembrane integrity as well as a reduced respiratory controlratio. These data suggest that at the tetrad stage, both theouter and inner mitochondrial membranes in part of the mi-tochondrial population have lost their integrity, which is notsurprising given the state of cellular degradation visible inhistologically stained sections.

Tissue-Specific Phenotype of CMS

How does the PET1-CMS cytoplasm cause the prematuredeath of anther tissues? The male sterile phenotype of thePET1-CMS mutants is associated with the expression ofORF522, a 15-kD hydrophobic protein that is bound to mi-tochondrial membranes (Horn et al., 1996). Any physiologi-cal differences between the PET1-CMS line and theisonuclear fertile line remain to be demonstrated (Hustedt,1995), but based on its mitochondrial localization, ORF522is assumed to affect respiration. Although ORF522 is ex-pressed in all tissues, it may cause a relatively minor degreeof mitochondrial dysfunction that results in premature celldeath in anther tissues only. One explanation for this tissueand developmental specificity is that respiratory demandsare highest in the developing tapetum and/or meiocytes,which is supported by the observation that mitochondrialnumbers per cell increased dramatically during meiosis inmaize (Lee and Warmke, 1979) and Cosmos bipinnatus(Dickinson and Li, 1988) and by the anther-specific accumu-lation of transcripts of respiratory genes (Smart et al., 1994;Zabaleta et al., 1998). By quantifying the fluorescent signalassociated with a biogenesis-associated mitochondrial pro-tein (CPN60), we have confirmed this dramatic increase dur-ing anther development in sunflower. However, if the CPN60signal per unit volume is considered, the values for root tis-sues are equal to or higher than those found for anther tis-sues. Therefore, high respiratory demands might not be theonly reason for the anther-specific phenotype of CMS.

The alternative hypothesis of an anther-specific com-pound proposed by Flavell (1974), could play a role in theform of the “ready-to-be-activated” PCD machinery in-volved in anther dehiscence. Although the genetic basis ofPCD remains to be determined, anther-specific proteaseactivities have been characterized in lily (DeGuzman andRiggs, 2000), and a cysteine endopeptidase is expressed inthe circular cell cluster, connective and stomium, in to-bacco (Koltunow et al., 1990). In addition, tapetum-specificserine proteases have been cloned from lily (Taylor et al.,1997) and pine (Walden et al., 1999), and a tapetum-specificgene (A9) with similarity to cysteine protease inhibitors hasbeen identified in several plant species (Paul et al., 1992;Walden et al., 1999). Therefore, the combination of high re-spiratory demands and the tissue-specific expression of

Figure 8. Quantification of Mitochondrial CPN60 in Several Sun-flower Tissues.

Thick sections of several sunflower tissues were labeled with an anti-body against CPN60, and the fluorescent signal of the secondary anti-body was quantified using confocal microscopy and optical sectioning.The relative CPN60 signal is expressed per cell (A) and per unit volume(B). The data are average values of at least three cells (SE).

1814 The Plant Cell

PCD-associated proteins—activated prematurely by mito-chondrial components in the cytosol—could explain the tis-sue-specific phenotype of CMS. Also, the timing of celldeath in the tapetum is critical for the development of func-tional pollen.

The ORF522 protein shares 18 amino acids at its N ter-minus with ORFB. Recently, ORFB, which is encoded inall plant mitochondrial genomes studied to date, wasfound to share a low degree of sequence and structuralhomology with ATP8 (Gray et al., 1998), a subunit of theFoF1-ATP synthases in Reclinomonas, fungi, and mam-mals. Because the N terminus of ORFB is important forassembly (Hadikusumo et al., 1988), the presence ofORF522 might affect the assembly of FoF1-ATPase. Inter-estingly, FoF1-ATPase is thought to play a role in apoptosis(reviewed in Matsuyama and Reed, 2000) in mammals andpossibly yeast, although the precise mechanism remains tobe determined. Interesting in this respect is a report byHernould et al. (1998) describing the death of the tapetumfollowed by the meiocytes in tobacco transformed with anunedited copy of mitochondrion-targeted ATP9.

The many CMS-associated mitochondrial mutations in arange of plant species have proved a very useful tool for thestudy of mitochondrial function and the role of nucleargenes therein. We have shown that CMS in sunflowercauses premature PCD associated with the release of cyto-chrome c during anther development. CMS, therefore, couldbe used to identify mitochondrial components that are ableto induce cell death and cytosolic components that are in-volved in the breakdown of the cell (e.g., proteases and en-donucleases).

METHODS

Materials

Sunflower (Helianthus annuus) seed were provided by Rhône Pou-lenc Agrochimie (France). Fertile (RPA842B) and sterile (RPA842A)lines are isonuclear containing the sunflower nuclear genotype. Thefertile (maintainer) line carries the sunflower cytoplasm and the sterileline carries the H. petiolaris cytoplasm. Plants were grown in non-heated greenhouses during the summer. Male florets were harvestedafter �40 days. A male floret corresponds to the upper part of thesunflower floret, which contains the anthers. Flowering occurred af-ter �50 days.

The monoclonal antibody against rat cytochrome c (clone7H8.2C12) was purchased from Pharmingen (San Diego, CA). Themonoclonal antibodies against porin (PM035) and the E1� subunit ofthe pyruvate dehydrogenase complex (PM030), both from maize,were purchased from GT Monoclonal Antibodies (Lincoln, NE).

Fixation, Embedding, and Sectioning

Plant material was fixed in 50% (v/v) ethanol, 4% (v/v) formaldehyde,and 5% (v/w) glacial acetic acid for 30 min at room temperature and

washed twice with 70% (v/v) ethanol for 15 min. The tissue wasstored in 70% (v/v) ethanol at 4C until further use. Fixed plant mate-rial was dehydrated through an ethanol series of 75, 85, 95, and100% (v/v) and embedded in polyethylene glycol (PEG), 1500 grade(Fisher Scientific), at 56C. The PEG blocks were sectioned into 7-�msections, spread on Superfrost Plus slides (Merck), and baked on ahot plate at 40C overnight. The slides were placed in MilliQ water for30 sec to remove the PEG and air dried. The sections were then re-hydrated through an ethanol series of 100, 90, 70, and 50% (v/v) andMilliQ water for 2 min each before further procedures (see below) orhistological staining with Safranin O and Fast Green according toLangdale (1994). All in situ data were obtained from serial sections offlorets of the same sunflower line and developmental stage in two in-dependent experiments (including plant material).

TdT-Mediated dUTP Nick-End Labeling

Seven-micrometer sections were washed in PBS (160 mM NaCl, 2.7mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4) for 5 min and incu-bated in 20 �g/mL proteinase K in 100 mM Tris-HCl, pH 8.0, and 50mM Na2EDTA (100 �L per slide in a humid chamber). The sectionswere washed in PBS for 5 min and fixed in 4% (w/v) paraformalde-hyde in PBS for 10 min. The sections were washed again in PBS for5 min, and the 3�-OH ends of DNA were labeled with fluorescein 12–dUTP using the apoptosis detection system fluorescein (Promega),according to the supplier’s instructions. The fluorescent signal wasviewed using an Axiophot microscope (Zeiss, Jena, Germany) andphotographed using the camera supplied (Zeiss) and Provia Fuji-chrome 400 film (Fuji, Tokyo, Japan).

DNA Isolation and Agarose Gel Electrophoresis

Male florets (0.5 g) were ground in liquid nitrogen. The frozen pow-der was added to 15 mL of extraction buffer (100 mM Tris-HCl, pH8.0, 50 mM EDTA, 500 mM NaCl, and 10 mM �-mercaptoethanol)followed by the addition of 1 mL of 20% (w/v) SDS. After incubationat 65C for 10 min, 5 mL of potassium acetate was added and thesample was incubated on ice for 20 min. The proteins were pelletedby centrifugation at 17,400g at 4C for 20 min. The supernatantwas filtered through Miracloth (Calbiochem) into a tube containing10 mL of cold isopropanol and incubated on ice for 30 min. The nu-cleic acids were pelleted by centrifugation at 17,400g for 15 min.The pellet was air dried and resuspended in 0.7 mL of 50 mM Tris-HCl, pH 8.0, and 10 mM EDTA. The resuspension was centrifugedat maximum speed in a microcentrifuge to remove nondissolveddebris. Then, one-tenth volume of 3 M sodium acetate and two-thirds volume of cold isopropanol were added to the supernatant,and the samples were incubated on ice for 15 min. The nucleic ac-ids were pelleted at maximum speed in a microfuge for 10 min at4C, dried, and resuspended in 10 mM Tris-HCl, pH 8.0, and 1 mMEDTA. RNA was digested in the presence of 0.2 mg/mL RNase Afor 30 min at 37C. DNA (10 �g/lane) was separated on 1.5% aga-rose gels in 1 � Tris-acetate-EDTA containing 0.1 �g/mL ethidiumbromide.

Immunocytochemistry

Seven-micrometer sections were washed in PBS for 2 min and cov-ered in primary antibody diluted 1:100 in PBS and 0.5% (w/v) BSA

Programmed Cell Death during CMS Anther Development 1815

and incubated at 4C in a humid chamber overnight. The slides werewashed three times, first in PBS and 0.5% (w/v) BSA, then in PBSand 0.01% (v/v) Tween 20, and finally in PBS. The sections were in-cubated with fluorescein isothiocyanate (FITC)–conjugated rabbitanti-mouse IgG in PBS (Sigma; dilution according to the supplier’sinstructions) at room temperature in a humid chamber for 1 hr. Theslides were washed as described above. The nucleic acids werestained with 10 �g/mL propidium iodide for 5 min. The sections weremounted in citifluor/PBS and sealed with nail polish. The fluorescentsignal was viewed using an MRC 1024 confocal system (Bio-Rad)and a BX50WI fixed-stage upright microscope (Olympus, Tokyo, Ja-pan) with a �60 1.2–numerical aperture oil immersion lens.

Isolation of Mitochondria

Male florets were ground using a mortar and pestle on ice in coldgrinding buffer containing 0.4 M mannitol, 25 mM 3-(N-morpholino)-propanesulfonic acid–KOH, pH 7.8, 1 mM EGTA, 0.1% (w/v) BSA,and 40 mM �-mercaptoethanol. All further procedures were per-formed at 4C. Cell debris was pelleted by a quick centrifugation stepduring which the rotor was stopped as soon as it reached 6000g. Thesupernatant was removed and recentrifuged at 12,000g for 15 min topellet the mitochondria. The supernatant (cytosol) was stored at 80C for further analysis. The crude mitochondrial pellet was resus-pended in wash buffer containing 0.4 M mannitol, 5 mM 3-(N-mor-pholino)-propanesulfonic acid–KOH, pH 7.5, 1 mM EGTA, and 0.1%(w/v) BSA and loaded onto step gradients of 13.5 and 27% (v/v) Per-coll (Amersham Pharmacia Biotech) in resuspension buffer contain-ing 0.4 M mannitol, 10 mM Tricine-KOH, pH 7.2, and 1 mM EGTA.The gradients were spun at 30,000g for 45 min in a swinging-bucketrotor. The buff-colored fraction at the 13.5 to 27% interface was col-lected and washed in resuspension buffer by centrifugation at12,000g for 15 min. The pellet was resuspended in resuspensionbuffer, and the protein concentration was determined using the Coo-massie Plus protein assay reagent (Pierce Chemical Co.) accordingto the supplier’s instructions.

Purification of Cytochrome c

Cytochrome c was purified from mitochondria isolated from etiolatedsunflower seedlings according to the method of Diano and Martinez(1971). Amino acid sequences were generated from two tryptic pep-tides, AVIWEENTLYD and MVFPGLK, that match the published se-quence (Ramshaw et al., 1970).

Isolation of Chloroplasts

All procedures were performed at 4C. Young sunflower leaves werehomogenized with a Polytron (Kinematica AG, Lucerne, Switzerland)in a minimal amount of SRM (0.165 M sorbitol and 25 mM Hepes-KOH, pH 8.0). The homogenate was filtered through two layers of Mir-acloth (Calbiochem) and centrifuged at 3200g for 2 min. The pelletwas resuspended in SRM and loaded onto 35% (v/v) Percoll (Amer-sham Pharmacia Biotech) pads in SRM. After centrifugation at 1400gfor 7.5 min, the pellet was resuspended in SRM and washed by cen-trifugation at 3200g for 2 min. The pellet was resuspended in a smallvolume of SRM. The purity of the chloroplast sample was assessedunder the light microscope, and the protein concentration was mea-

sured using the Coomassie Plus protein assay reagent (Pierce Chem-ical Co.). The chloroplasts were frozen at 80C until further use.

Protein Gel Blot Analysis

Protein samples stored at 80C were separated by SDS-PAGE[15% (w/v) monomer and 2.6% (w/w) cross-linking agent] and trans-ferred to nitrocellulose (0.2 �m; Schleicher and Schuell) by semidryelectroblotting. The membrane was labeled with the 7H8.2C12 mon-oclonal antibody against rat cytochrome c as described by Bossy-Wetzel et al. (1998). For labeling with the porin and E1� monoclonalantibodies, the membrane was blocked against nonspecific bindingby incubation in PBS and 0.1% (v/v) Tween 20 (PBS-T) plus 1% (w/v) BSA for 1 hr. The membrane was then incubated in the primary an-tibody diluted 1:500 in block buffer at 4C overnight. The membranewas washed three times in PBS-T, incubated with horseradish per-oxidase–conjugated sheep anti-mouse Ig (Amersham Life Science),and diluted 1:10,000 in PBS-T for 1 hr. After four washes, labelingwas detected by chemiluminescence (DuPont–New England NuclearLife Science Products) and exposure to Kodak BioMax ML film.

The signal corresponding to cytochrome c was quantified usingdensitometry. The linear range of mitochondrial protein per lane (20to 40 �g) and film exposure time (1 to 5 min) were determined exper-imentally. The films were scanned and analyzed using a Fluor-SMulti-Imager and Multi-Analyst software (both from Bio-Rad).

Oxygen Electrode Assays

All respiratory studies were performed by polarographic measure-ment of oxygen in the aqueous phase using a Clark-type oxygenelectrode (Hansatech, King’s Lynn, Norfolk, UK) linked to a chart re-corder (Rikadenki, Tokyo, Japan). Freshly isolated mitochondria (50to 100 �g) were assayed in 1 mL of 0.3 M mannitol, 10 mM Tes-KOH,pH 7.5, 3 mM MgSO4, 10 mM NaCl, 5 mM KH2PO4, and 0.1% (w/v)BSA. Outer mitochondrial membrane integrity was measured as theratio of oxygen consumption in the presence of 50 �g/mL cyto-chrome c and 5 mM ascorbate with or without the addition of 0.05%(w/v) Triton X-100. Maximal cytochrome c oxidase activity was con-sidered to be the rate of oxygen consumption in the presence of Tri-ton X-100 in the outer membrane integrity assay. In a separate assay,10 mM succinate and 0.2 mM ATP were added to the mitochondria,followed by the addition of 0.1 mM ADP to measure the rate of oxy-gen consumption in state 3. The oxygen consumption in state 4 wasmeasured when the ADP was depleted as judged by a reduction inoxygen consumption rate. The respiratory control ratio was calcu-lated as the ratio of state 3 to state 4 oxygen consumption rate.

Quantitative Analysis of CPN60

Fixed tissue was sectioned into 30- to 70-�m sections using a vi-bratome (series 1000; General Scientific, Redhill, Surrey, UK) in Mil-liQ water using Wilkinson razor blades. The sections were transferredto eight-well slides coated with 2% (v/v) 3-aminopropyltriethoxysi-lane in acetone and subsequently with 2.5% (v/v) glutaraldehyde inPBS. The sections were air dried and then digested with 2% (w/v)cellulase and 1% (w/v) driselase in 25 mM Tris-HCl, pH 7.5, 140 mMNaCl, and 3 mM KCl (TBS) for 1 to 1.5 hr. The slides were washed inTBS for 5 min and incubated with CPN60 antibody (Robertson et al.,1995) diluted 1:1000 in TBS plus 1% (w/v) BSA and 0.01% (w/v)

1816 The Plant Cell

Tween 20 overnight at 4C. The slides were washed in TBS for 5 minand incubated with goat anti-rabbit FITC-conjugated antibody for 1hr at room temperature in the dark. The slides were washed againand incubated with propidium iodide (Molecular Probes, Eugene,OR) at a concentration of 10 �g/mL for 20 min to stain nucleic acids,facilitating the determination of the developmental stage.

After washing for 5 min, the slides were mounted in citifluor in glyc-erol/PBS. The labeling was viewed using an MRC 600 confocal sys-tem (Bio-Rad) and a Diaphot inverted microscope (Nikon, Tokyo,Japan) with a �60 1.4–numerical aperture oil immersion lens (laser,488 nm; filters, DE1 and R1). All images used for quantitative analysiswere made under identical settings (ND0, PMT1 3, PMT2 4, Kalman3, Zoom 1.6, normal scanning speed). Z-series of 1-�m steps weremade for each tissue. The FITC signal was integrated using the soft-ware provided (COMOS; Bio-Rad). Bleaching of the signal was notdetected during the time used for scanning. No significant levels oflabeling above background were detected in the control, in which theprimary antibody was omitted. To avoid differences in signal attribut-able to attenuation, cells from just under the cut surface were se-lected for analysis.

The value for the total CPN60 signal per cell was obtained with thefollowing equation: �(Px � Sx) (�P � BG), where Px is the numberof pixels per optical section x, Sx is the average signal per opticalsection x, and BG is the average background signal (measured in thenucleus; n � 3). The CPN60 signal per unit volume was calculated asthe total CPN60 signal per cell divided by �Px.

ACKNOWLEDGMENTS

We thank Paul McCabe for encouraging this project, Lee Sweetlovefor critical reading of the manuscript, Tony C. Willis for protein se-quencing, and Cledwyn Merriman for processing of the samples forelectron microscopy. This work was supported by the Gatsby Chari-table Foundation, the Biotechnology and Biological Science Re-search Council, and Lincoln College, Oxford (J.B.).

Received March 21, 2001; accepted May 22, 2001.

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DOI 10.1105/TPC.010116 2001;13;1803-1818Plant Cell

Janneke Balk and Christopher J. Leaver ReleasecCell Death and Cytochrome

The PET1-CMS Mitochondrial Mutation in Sunflower Is Associated with Premature Programmed

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