Mitochondrial GFA2 Is Required for Synergid Cell Death in ...GFA2 Is Required for Cell Death 2217 FG1 to early FG5) and failed to cellularize. With category 1 mutants (14 of 39 mutants),

The Plant Cell, Vol. 14, 2215–2232, September 2002, www.plantcell.org © 2002 American Society of Plant Biologists

Mitochondrial GFA2 Is Required for Synergid Cell Deathin Arabidopsis

Cory A. Christensen,

1

Steven W. Gorsich, Ryan H. Brown,

2

Linda G. Jones, Jessica Brown,

2

Janet M. Shaw, and Gary N. Drews

3

Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840

Little is known about the molecular processes that govern female gametophyte (FG) development and function, andfew FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mu-tants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes governessentially every step of FG development. To identify genes involved in cell death during FG development, we analyzedthis mutant collection for lines with cell death defects. From this analysis, we identified one mutant,

gfa2

, with a defectin synergid cell death. Additionally, the

gfa2

mutant has a defect in fusion of the polar nuclei. We isolated the

GFA2

gene and show that it encodes a J-domain–containing protein. Of the J-domain–containing proteins in

Saccharomycescerevisiae

(budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix.GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast

mdj1

mutant, suggesting that GFA2is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

INTRODUCTION

The female gametophyte (FG) is a haploid structure thatplays an integral role in the angiosperm life cycle. It devel-ops within ovules and most often consists of an egg cell, acentral cell, two synergid cells, and three antipodal cells(Figure 1). The FG is important for many aspects of the an-giosperm reproductive process: in addition to giving rise tothe embryo and endosperm of the seed, the FG plays a rolein pollen tube guidance (Hülskamp et al., 1995; Ray et al.,1997; Shimizu and Okada, 2000; Higashiyama et al., 2001),fertilization (Russell, 1992, 1996), and the induction of seeddevelopment (Ohad et al., 1996; Chaudhury et al., 1997;Grossniklaus et al., 1998).

FG development requires many fundamental cellular pro-cesses, including mitosis, vacuole formation, cell wall for-mation, nuclear migration, nuclear fusion, and cell death(Figure 1). Thus, mutations that affect these processes arelikely to affect the FG, exhibit reduced transmission throughthe FG, and appear at reduced frequency in the sporophyte

generation. Consequently, genetic analysis of these cellularprocesses is likely to require gametophytic screens andanalysis of the gametophyte generation. An example of thisis cell death, which is important in the diploid sporophytegeneration for tissue development, senescence, and plantdefense (Martienssen, 1997; Buckner et al., 1998; Richberget al., 1998; Lam et al., 1999). However, because cell deathoccurs during the gametophytic phase (Figure 1), cell deathmutants may not be identified readily in sporophyte-orientedscreens (Martienssen, 1997).

Little is known about the molecular and genetic pro-cesses that govern FG development and the FG’s reproduc-tive functions. The number and identities of genes ex-pressed in the FG, and the proportion of these that areunique to the FG, are unknown. These genes presumablyregulate and mediate FG development and function. How-ever, for all but a few of these genes, specific functions inthe FG are unknown. Also, little is known about FG physiol-ogy. The physiological pathways that exist in the FG, andthe extent to which FG physiology is independent from thatof the surrounding sporophytic tissue, are unknown. Thus, itis not clear whether FG development and physiology aregoverned primarily by the haploid or the diploid genome.

As a step toward addressing these issues, several groupshave performed screens for gametophytic mutants in Arabi-dopsis and maize, and a number of FG mutations have beenidentified (Bonhomme et al., 1998; Christensen et al., 1998;Drews et al., 1998; Grossniklaus et al., 1998; Howden et al.,1998; Grini et al., 1999; Shimizu and Okada, 2000; Evans

1

Current address: Paradigm Genetics, Inc., 108 Alexander Drive,P.O. Box 14528, Research Triangle Park, NC 27709-4528.

2

Current address: Plant Biology Graduate Group, Section of Molec-ular and Cellular Biology, University of California, 1 Shields Avenue,Davis, CA 95616.

3

To whom correspondence should be addressed. E-mail [email protected]; fax 801-581-4668.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.002170.

2216 The Plant Cell

and Kermicle, 2001). However, relatively few of these muta-tions have been characterized at the phenotypic and molec-ular levels, and a specific function in the FG is known foronly few genes.

The present study had two main objectives. The first wasto determine the extent to which FG development and func-tion are controlled/mediated by FG-expressed genes and toidentify specific genes required by these developmental/re-productive steps. To address these issues, we have identi-fied and characterized a large collection of FG mutants. Thesecond objective was to initiate a molecular genetic analysisof cell death during FG development. To this end, we identi-fied and characterized a mutant,

gfa2

, that has a defect insynergid cell death during the reproductive process. Ouranalysis of the

GFA2

gene suggests a role for mitochondriain cell death in plants.

RESULTS

Analysis of 39 Female Gametophyte Mutants

To gain insight into the steps of FG development mediatedby FG-expressed genes, we identified and analyzed a largecollection of FG mutants. We identified 31 new FG mutants(

fem5

to

fem38

) from T-DNA– and transposon-mutagenizedlines (see Methods) and analyzed these along with the previ-ously identified

ctr1

mutant (Kieber and Ecker, 1994). Thesemutants are summarized in Table 1. Because the FG is hap-loid, complementation tests could not be performed to de-termine whether any of the FG mutations are allelic. How-ever, preliminary molecular analysis of these and seven

previously identified mutants (

gfa2

to

gfa5

and

fem2

to

fem4

; Table 1) has shown that for all mutants analyzed todate (22 of 39 mutants), the T-DNA/transposon insertionsites fall within different genomic regions (C.A. Christensen,R.H. Brown, and G.N. Drews, unpublished data), suggestingthat these mutations are not allelic.

To determine the penetrance of these mutations in the FG(i.e., the proportion of genotypically mutant FGs that fail totransmit the mutant allele), we crossed heterozygous mu-tants as females to wild-type males and scored the numberof heterozygous (kanamycin-resistant) and homozygouswild-type (kanamycin-sensitive) progeny (Table 2). To deter-mine whether these mutations also affect the male gameto-phyte, we crossed heterozygous mutants as males to wild-typefemales and scored the number of heterozygous (kanamy-cin-resistant) and homozygous wild-type (kanamycin-sensi-tive) progeny (Table 2). As shown in Table 2, of the new mu-tations,

fem8

,

fem9

,

fem17

, and

fem20

appear to affect theFG specifically.

To determine the steps of FG development affected bythe mutations, we analyzed the terminal phenotypes usingconfocal laser scanning microscopy (CLSM) (Christensen etal., 1997, 1998) and divided the mutants into phenotypiccategories. To comprehensively define the spectrum of pos-sible phenotypes, we included seven previously analyzedmutants (

gfa2

to

gfa5

and

fem2

to

fem4

; Table 1) in our cat-egorization scheme. Together, the 39 FG mutants fall intofive phenotypic categories (Table 2). These phenotypic cate-gories along with wild-type development are summarized inFigure 1. Confocal images of the FG mutants are shown inFigure 2.

Category 1 and category 2 mutants had defects duringthe nuclear division phase of megagametogenesis (stages

Figure 1. Developmental Stages Affected in the FG Mutants.

Megagametogenesis has been described previously and divided into seven stages (Christensen et al., 1997). The haploid megaspore definesstage FG1. The megaspore undergoes three rounds of mitosis without cytokinesis, giving rise to an eight-nucleate syncytium (early-stage FG5).Immediately after the third mitosis, cell walls form (late-stage FG5). The central cell inherits two nuclei, called the polar nuclei, which fuse to formthe homodiploid secondary nucleus (stage FG6). Finally, the antipodal cells degenerate, producing a mature FG (stage FG7). Early during the fer-tilization process, one of the synergid cells is induced to undergo cell death (data not shown). Category designations show the developmentalstages affected in the FG mutants. In this figure, cytoplasm is shown in light gray, vacuoles are shown in black, nuclei are shown in dark gray,and nucleoli are represented as the white areas within the nuclei. ac, antipodal cell; cc, central cell; ec, egg cell; emb, embryo; end, endosperm;pn, polar nucleus; sc, synergid cell; sdc, seed coat; sn, secondary nucleus.

GFA2 Is Required for Cell Death 2217

FG1 to early FG5) and failed to cellularize. With category 1mutants (14 of 39 mutants), all or most (

�

60%) of the mu-tant FGs arrested at stage FG1. Arrested FGs either per-sisted (Figure 2H) or degenerated (Figure 2G) during ovuledevelopment. Mutant FGs that progressed beyond stageFG1 exhibited category 2 defects. With category 2 mutants(17 of 39 mutants), all or most (

�

60%) of the mutant FGsprogressed beyond stage FG1. Category 2 defects includedabnormal nuclear numbers and positions (Figures 2J to 2M)and developmental arrest at stages FG2 to early FG5 (Figure 2I).

Category 3 and category 4 mutants became cellularizedand had defects during the later phases of megagameto-genesis. Category 3 mutants (4 of 39 mutants) had defects

in cellular morphology, including abnormal nuclear positionswithin cells, misshapen cells, and unusual cell features. With

fem4

, the defects were quite pronounced and included ir-regular cell shapes and polarities of the egg and synergidcells (Christensen et al., 1998). With the other three category3 mutants, FG morphology was almost wild type:

fem7

(Fig-ure 2N) and

fem9

(data not shown) FGs had slightly reducedcytoplasm and slightly abnormal cell shape, and

fem8

FGshad many small vacuoles in the central cell cytoplasm (Fig-ure 2O). Category 4 mutants (2 of 39 mutants) had defects infusion of the polar nuclei. With both mutants, the polar nu-clei migrated properly, came to lie side by side, but failed tofuse.

gfa2

affected nuclear fusion only (Figure 2P), whereas

Table 1.

Mutants Discussed in This Article

Mutant Isolate No. Mutagen Ecotype Source

constitutive triple response (ctr1)

– X-ray L-

er

Kieber and Ecker, 1994

female gametophyte2 (fem2)

2462-5 T-DNA Ws Christensen et al., 1998


2465-35 T-DNA Ws Christensen et al., 1998


TJ160 T-DNA Col Christensen et al., 1998


TJ13 T-DNA Col This article












a308 Transposon L-

er

This article


a466 Transposon L-

er

This article


B271 Transposon L-

er

This article


A839 Transposon L-

er

This article


A845 Transposon L-

er

This article


E160 Transposon L-

er

This article




RB46 T-DNA Col This article



































gametophytic factor2 (gfa2)

102 T-DNA Ws Feldmann et al., 1997


13/21 T-DNA Ws Feldmann et al., 1997





Col, Columbia; L-

er

, Landsberg

erecta

; Ws, Wassilewskija.

2218 The Plant Cell

gfa3

affected both nuclear fusion (predominantly) and cellu-larization (Christensen et al., 1998).

Category 5 mutants (2 of 39 mutants) appeared normal atthe terminal developmental stage (stage FG7), suggestingthat megagametogenesis was not affected. This finding, to-gether with the reduced transmission of these mutationsthrough the FG (Table 2), suggests that category 5 muta-tions affect one of the FG’s reproductive functions (pollentube guidance, fertilization, etc.). Molecular analysis of the

T-DNA insertion site in

fem17

(C.A. Christensen, R.H.Brown, and G.N. Drews, unpublished data) suggests thatthe disrupted gene corresponds to the

FIS2

gene (Luo et al.,1999), which is required to repress endosperm developmentbefore fertilization (Chaudhury et al., 1997).

To investigate the role of

CTR1

, we analyzed the postpol-lination phenotypes of

ctr1

. In

ctr1/ctr1

siliques, 92% (45 of49) of ovules contained a pollen tube in the micropyle (97%in the wild-type control), indicating that pollen tube guidance

Table 2.

Penetrance, Female Gametophyte Specificity, and Phenotype

Mutant Penetrance in FG

a

Penetrance in MG

a

Terminal Phenotype

b

ctr1-2

58%

c

0%

c

Wild type (5)

fem2

44%

d

0%

d

Stage FG1

d

(1)

fem3

42%

d

44%

d

Stage FG1

d

(1)

fem4

100%

d

42%

d

Abnormal cellular morphology, stage FG5/6/7 (3)

fem5

87% (685) 72% (1799) Abnormal nuclear number, size, and position (2)

fem6

93% (455) 86% (811) Abnormal cellular morphology (3)

fem7

81% (831) 30% (614) Abnormal cellular morphology (3)

fem8

67% (441) 9%

e

(623) Abnormal cellular morphology (3)

fem9

42% (431) 12%

e

(857) Stage FG1 (1)

fem10

93% (1275) 92% (1347) Abnormal nuclear number and position (2)

fem11

54% (624) 100% (100) Stage FG1 to stage FG6 (2)

fem12

73% (1111) 100% (403) Stage FG1 (1)

fem13

84% (406) 100% (423) Abnormal nuclear number, cellularization; twinning (2)

fem14

79% (105) 100% (218) Abnormal nuclear number, position; collapse (2)

fem15

100% (99) 64% (64) Abnormal nuclear number and cellularization (2)

fem16

66% (206) 71% (298) No FG (1)

fem17

98% (66) 10%

e

(133) Wild type (5)

fem18

39% (305) 65% (694) Stage FG1 to stage FG2 (1)

fem19

100% (306) 100% (459) Abnormal nuclear number and position (2)

fem20

34% (154) 0% (413) Stage FG1, stage FG5 (2)fem21 100% (108) 100% (376) Stage FG3 to stage FG5 (2)fem22 100% (710) 99% (601) Stage FG4 to stage FG5 (2)fem23 98% (826) 43% (1024) Stage FG3 to stage FG5 (2)fem24 83% (504) 16% (726) Stage FG1, stage FG3 to stage FG5 (2)fem25 86% (342) 99% (847) Abnormal nuclear number, position; stage FG1 to FG2 (2)fem26 99% (260) 17% (796) Stage FG1 (1)fem29 17% (387) 71% (535) Stage FG1 (1)fem30 15% (183) 97% (774) Stage FG1 to stage FG5; abnormal nuclear number (2)fem31 66% (506) 88% (256) Stage FG1, stage FG3 (1)fem33 94% (174) 70% (214) Stage FG1, stage FG3 to stage FG4 (2)fem34 100% (300) 98% (360) Stage FG3 (2)fem35 35% (295) 31% (602) Stage FG1 (1)fem36 99% (315) 92% (246) Stage FG1, stage FG3 to stage FG4 (2)fem37 42% (328) 16% (668) Stage FG1 (1)fem38 51% (330) 53% (355) Stage FG1 (1)gfa2 99% (571) 86% (704) Unfused polar nucleid (4) gfa3 82% (281) 99% (365) Unfused polar nuclei, cellularization failured (4)gfa4 61% (1252) 100% (908) Stage FG1d (1)gfa5 79% (441) 84% (649) Stage FG1d (1)

a FG, female gametophyte; MG, male gametophyte. The number of progeny scored is in parentheses.b Phenotypic category is in parentheses.c Previously described by Kieber and Ecker (1994).d Previously described by Christensen et al. (1998).e Not significantly different from 0 at P � 0.05.


was not affected significantly. We next examined fertilizedctr1 embryo sacs at 15 to 48 h after pollination (HAP) withwild-type pollen. ctr1 seeds at 48 HAP (Figures 2S and 2T)resembled wild-type seeds at 15 HAP (Figures 2D and 2E):the embryo was single celled and relatively round and con-tained a central nucleus and many small vacuoles; the en-dosperm contained two to four nuclei; and one synergidwas degenerated. These data suggest that ctr1 FGs be-come fertilized but arrest very early in seed development.

Together, these data suggest that most (�95%) FG-expressed genes are required for megagametogenesis andthat these genes play a role in specific developmental steps(e.g., cell formation) and/or specific cellular processes (e.g.,nuclear fusion). Furthermore, these data suggest that a pro-portion (�5%) of FG-expressed genes play no role inmegagametogenesis and instead are required for one of theFG’s reproductive functions.

The gfa2 Mutation Affects Synergid Cell Death

In an effort to identify the genes involved in cell death duringFG development, we analyzed our mutant collection forlines that display cell death defects. Cell death occurs threetimes during FG development: first, immediately after meio-sis, three of the megaspores undergo cell death; second,late during megagametogenesis, the three antipodal cellsdegenerate (Figure 1); third, in response to pollination, oneof the synergid cells undergoes cell death (Christensen etal., 1997). As discussed above, the analysis of megagame-togenesis in each of our FG mutants did not reveal mutantswith defects in megaspore or antipodal cell death.

We also asked whether synergid cell death after pollina-tion is affected in our FG mutants. We focused on those mu-tants in which the egg apparatus (egg and synergid cells) ismorphologically normal: fem17/fis2 (discussed above), ctr1(Figures 2S and 2T), and gfa2 (Figure 2P). As discussedabove (Figures 2S and 2T) and by others (Chaudhury et al.,1997), the fertilization process appears to be normal in thectr1 and fem17/fis2 mutants, indicating that synergid celldeath is not affected in these mutants.

To determine whether the gfa2 mutation affects synergidcell death, we pollinated gfa2/GFA2 pistils with wild-typepollen and examined gfa2 embryo sacs (those with unfusedpolar nuclei) at 7 to 24 HAP. In the wild type (Wassilewskija[Ws-2] ecotype), one of the synergid cells was degeneratedby 7 HAP (Figure 2C) in essentially all (40 of 41) ovules. Incontrast to the wild type, with most (44 of 55) gfa2 FGs,both synergids were intact and showed no signs of degen-eration (Figures 2Q and 2R). All gfa2 embryo sacs that failedto undergo synergid cell death (i.e., those with two intactsynergids) showed no signs of fertilization (cf. Figures 2A to2E with Figures 2Q and 2R).

The synergid-cell-death defect of gfa2 could be a second-ary consequence of a failure to attract pollen tubes. To ad-dress this issue, we pollinated gfa2/GFA2 pistils with wild-

type pollen and scored the number of ovules with pollentubes in the micropyle. In gfa2/GFA2 siliques, �99% (148 of149) of ovules had a pollen tube in the micropyle (100% inthe wild-type control), indicating that the gfa2 mutation doesnot affect pollen tube guidance. Together, these data indi-cate that the GFA2 gene product is required for synergidcell death during the fertilization process.

GFA2 Encodes a Member of the DnaJ Protein Family

We used inverse PCR (Triglia et al., 1988) to identify theT-DNA insertion site in gfa2 mutants. The insertion site cor-responded to locus At5 g48030 from BAC clone MDN11 onchromosome 5. Introducing a wild-type copy of this geneinto the gfa2 mutant restored transmission of the gfa2 allelethrough the FG and rescued the nuclear fusion defect (datanot shown), indicating that locus At5 g48030 corresponds tothe GFA2 gene. To determine GFA2 gene structure, we iso-lated a GFA2 cDNA clone and compared its sequence withthat of the genomic sequence. As shown in Figure 3, theGFA2 gene consists of 18 exons, has an open reading frameof 1368 bp, and is predicted to encode a protein of 456amino acids. The T-DNA insert in the gfa2 mutant fell withinthe second exon, likely creating a null allele (Figure 3).

BLAST searches against the SWISS-PROT database us-ing the predicted GFA2 protein sequence revealed homol-ogy with bacterial DnaJ (HSP40) proteins. As shown in Fig-ure 4, within the region of homology (residues 90 to 456),GFA2 is 37% identical and 53% similar to Escherichia coliDnaJ. DnaJ proteins are defined by the presence of an�70–amino acid J-domain, which contains the highly con-served HPD tripeptide (Kelley, 1998). GFA2 has this domainand, like many other DnaJ family members, also contains aGly/Phe-rich domain, a Cys-rich zinc-finger domain, and a lesswell-conserved C-terminal domain (Figure 4). The J-domainand the Gly/Phe-rich domain are thought to mediate the in-teractions between DnaJ and its cochaperone DnaK, andthe zinc-finger and C-terminal domains are thought to medi-ate the interactions between DnaJ and its substrate proteins(Hartl, 1996; Bukau and Horwich, 1998; Kelley, 1998; Fink,1999).

The GFA2 Gene Is Expressed Throughout the Plant

We performed two assays to determine where the GFA2gene is expressed within the plant. First, we used reversetranscriptase–PCR to assay the presence of GFA2 RNA invarious plant organs. Total RNA purified from roots, rosetteleaves, stems, open flowers, and apical tips (including un-opened flowers) was used as a template to make single-stranded cDNA by reverse transcription, and primers spe-cific for GFA2 and actin were used to amplify cDNAs fromeach of the RNA populations. As shown in Figure 5, PCRbands representing the spliced transcripts of both genes

2220 The Plant Cell

Figure 2. Phenotypic Analysis of the FG Mutants.

(A) and (B) Wild-type FG at the terminal developmental stage (stage FG7). The egg is pear shaped, has a nucleus at its chalazal end, and has asingle large vacuole occupying the remainder of the cell. Both synergid cells are present, and the secondary nucleus is undivided.(C) Wild-type (ecotype Ws) seed at 7 HAP containing a single-celled zygote, an undivided endosperm nucleus (primary endosperm nucleus), onedegenerated synergid cell, and one persistent synergid cell (data not shown).(D) and (E) Wild-type (ecotype Ws) seed at 15 HAP. The zygote is relatively round, has several small vacuoles, and has a centrally located nu-cleus. One of the synergids is degenerate, and the primary endosperm nucleus has divided.(F) Wild-type (ecotype Ws) seed at 48 HAP containing a four-celled proembryo (two cells are not projected in this image) and multiple peripheralendosperm nuclei.(G) fem16 FG that has collapsed.(H) fem12 FG arrested at the one-nucleate stage (stage FG1).(I) fem12 FG arrested at the four-nucleate stage.(J) fem12 FG containing three micropylar nuclei and one chalazal nucleus.(K) fem13 FG containing two nuclei in the position of the egg nucleus.


were amplified from each RNA population, indicating thatGFA2 RNA is present in each organ.

Second, we transformed plants with a GFA2::�-gluc-uronidase (GUS) reporter construct and analyzed GUS ex-pression in seedlings and whole flowering plants. As shownin Figures 6A and 6B, strong GUS activity was detected inall organs. GUS activity also was detected in ovules duringFG development (Figure 6B). Together, these data indicatethat the GFA2 gene is expressed throughout the plant.

GFA2 Is Similar to Yeast Mdj1p and Localizesto Mitochondria

We compared GFA2 to the J-domain proteins from Saccha-romyces cerevisiae and found that Mdj1p is the yeast pro-tein most similar to GFA2 (data not shown). Mdj1p is a mito-chondria-targeted DnaJ family member that functions as achaperone in the mitochondrial matrix (Rowley et al., 1994;Westermann et al., 1996; Duchniewicz et al., 1999). This se-quence similarity suggests that GFA2 might be a mitochon-dria-targeted protein. Furthermore, the GFA2 N terminushas the characteristics of a mitochondrial targeting se-quence (Figure 4) (Gavel et al., 1988; Roise, 1997).

To determine whether GFA2 is targeted to mitochondria,we transformed plants with a GFA2–green fluorescent pro-tein (GFP) translational fusion construct driven by the 35Spromoter of Cauliflower mosaic virus (35S::GFA2-GFP). As acontrol, we also transformed plants with a GFP that lacks atargeting sequence (35S::GFP) (von Arnim et al., 1998). Withboth constructs, transgenic plants appeared normal. Con-trol plants containing the 35S::GFP construct exhibited GFPfluorescence in the cytoplasm and nucleus (Figure 6C), inagreement with previous studies (Grebenok et al., 1997; von

Arnim et al., 1998). By contrast, in plants containing the35S::GFA2-GFP construct, GFP fluorescence was detectedin multiple small intracellular compartments (Figure 6D). Thispattern was seen in all tissues examined and colocalizedwith a mitochondrion-specific dye, Mitotracker (Figures 6Eto 6G). Together, these data indicate that the GFA2 proteinlocalizes to mitochondria.

GFA2 Partially Complements a Yeast mdj1 Deletion

The sequence similarity and mitochondrial localization sug-gest that GFA2 is the Arabidopsis ortholog of yeast MDJ1.To test this possibility further, we determined whether GFA2protein can substitute for Mdj1p in yeast by expressingGFA2 in an mdj1 yeast strain. The yeast strains used in thisstudy are listed in Table 3. To generate an MDJ1 null allele,we replaced the entire MDJ1 coding region with the HIS3gene. We refer to this allele as mdj1::HIS3. As expectedfrom previous studies (Rowley et al., 1994; Duchniewicz etal., 1999), mdj1::HIS3 cells at 25�C failed to grow on thenonfermentable carbon source, glycerol, but grew slowly onthe fermentable carbon source, dextrose. This petite pheno-type is consistent with a defect in mitochondrial function. Inaddition, at 37�C, mdj1::HIS3 cells failed to grow on bothcarbon sources, indicating that Mdj1p function also is re-quired for survival at high temperature (Duchniewicz et al.,1999).

We introduced an expression vector containing the GFA2cDNA (pRS416-MET25-GFA2) into heterozygous yeast cells(MDJ1/mdj1::HIS3). As a control, we transformed heterozy-gous cells (MDJ1/mdj1::HIS3) with the pRS416-MET25 vec-tor alone. Transformed heterozygous diploids then weresporulated, and tetrads were dissected onto glycerol- or

Figure 2. (continued).

(L) fem5 FG containing two large nuclei.(M) fem5 FG containing four micropylar nuclei and two chalazal nuclei.(N) fem7 FG with reduced cytoplasm associated with the nuclei and fuzzy nuclear margins. The second synergid was present but is not pro-jected in this image.(O) fem8 FG containing a large number of small vacuoles in the central cell cytoplasm and a disordered egg apparatus.(P) gfa2 FG with unfused polar nuclei.(Q) and (R) gfa2 FG at 24 HAP. Both synergids are intact, the egg cell appears to be unfertilized (it is pear shaped, has a nucleus at its chalazalend, and has a single large vacuole), and the polar nuclei appear to be unfertilized (they are unfused and remain near the chalazal end of the eggcell). gfa2 embryo sacs at 7 to 15 HAP have similar morphology.(S) and (T) ctr1-2 seed at 48 HAP showing early signs of embryogenesis. This seed has three endosperm nuclei, a single-celled zygote, a degen-erated synergid, and a persistent synergid.(A), (C), (D), (F) to (Q), and (S) are CLSM images. (B), (E), (R), and (T) are depictions. All CLSM images are projections of several optical sectionstaken at the terminal developmental stage or at the HAP indicated. In the CLSM images, cytoplasm is shown in gray, vacuoles are shown inblack, and nucleoli are shown in white. In the depictions, cytoplasm is shown in light gray, vacuoles are shown in black, nuclei are shown in darkgray, and nucleoli are shown in white. In all images, the micropylar pole is at the bottom and the chalazal pole is at the top. cn, chalazal nuclei;dsc, degenerate synergid cell; ec, egg cell; ecn, egg cell nucleus; en, endosperm nucleus; fg, female gametophyte; mn, micropylar nuclei; n, nu-cleus; pe, proembryo; pen, primary endosperm nucleus; pn, polar nucleus; sc, synergid cell; scn, synergid cell nucleus; sn, secondary nucleus;su, suspensor; WT, wild type; z, zygote; zn, zygote nucleus.

2222 The Plant Cell

dextrose-containing plates and grown at 25, 30, and 37�C.As shown in Figure 7, mdj1::HIS3 cells containing the GFA2expression vector grew slightly faster than mdj1::HIS3 cellscontaining the empty vector at 25�C (cf. rows 3 and 4) and30�C (cf. rows 7 and 8).

In addition, the presence of GFA2 rescued the lethality ofmdj1::HIS3 at 37�C (cf. rows 11 and 12). However, the pres-ence of the GFA2 expression vector did not restore the abil-ity of mdj1::HIS3 cells to grow on glycerol (data not shown).These data (summarized in Table 4) indicate that Arabidop-sis GFA2 can partially substitute for Mdj1p in yeast. Thesedata, along with the sequence similarity and mitochondriallocalization, suggest very strongly that GFA2 is the orthologof yeast MDJ1 and that GFA2 protein functions as a chaper-one in the mitochondrial matrix.

The gfa2 Mutation Does Not Affect Viability

The data presented above suggest that the gfa2 mutationcauses defects in mitochondrial function. Thus, the gfa2 de-fects could be a secondary consequence of decreased met-abolic activity. To assess this possibility, we asked whethergfa2 FGs express genes associated with central cell differ-entiation. We analyzed the expression of a FIE reporter geneconstruct (FIE::GFP) whose expression is initiated in thecentral cell at approximately the time of polar nuclei fusion(Yadegari et al., 2000). We examined ovules from plants thatwere heterozygous for the gfa2 mutation and homozygousfor FIE::GFP.

As expected, approximately half (39 of 70) of the ovulescontained a FG with unfused polar nuclei. All of the ovulesexamined (n � 98) expressed FIE::GFP in the central cell(data not shown). We also examined ovules from plants het-erozygous for the gfa2 mutation and containing a FIE-GFPfusion protein construct (FIE::FIE-GFP). GFP expressedfrom this construct is localized in the central cell nucleus(Kinoshita et al., 2001). As shown in Figures 6H and 6I, theFIE::FIE-GFP construct is localized to the nucleus and is ex-pressed at the same level in mutant and wild-type FGs.

gfa2 FGs exhibited several other indicators of cell viabilityand metabolic activity. First, the failure of polar nuclei fusionwas the only defect observed during megagametogenesis(Figure 2P). In the wild type, many other energy-requiringsteps (e.g., cell wall formation) occurred at approximatelythe same time as nuclear fusion. Yet, none of these stepsappeared to be affected in the gfa2 mutant (Figure 2P). Fur-thermore, the nuclei, vacuoles, and cell membranes in gfa2FGs were intact and showed no signs of degradation (Fig-ures 2P and data not shown). Second, as discussed above,gfa2 FGs attracted pollen tubes. The presence of a func-tional FG is essential for pollen tube attraction to an ovule(Hülskamp et al., 1995; Ray et al., 1997; Shimizu and Okada,2000; Higashiyama et al., 2001). Third, within emasculatedflowers, gfa2 FGs persisted as long as wild-type FGs (data

Figure 3. GFA2 Gene Sequence.

Exons are indicated with uppercase letters. A 33-bp deletion associ-ated with the site of the T-DNA insert is indicated by strikethrough.The position of the T-DNA left border is indicated above thestrikethrough. Plant DNA on the other side of the T-DNA insert is ad-jacent to the internal T-DNA sequence. The predicted GFA2 proteincontains two in-frame ATG codons (marked in boldface and under-lined) in its N terminus. The first Met codon is expected to initiatetranslation because it satisfies the consensus sequence criteria for atranslation initiation codon (Kozak, 1991). Some of the exon-intronboundaries derived from the cDNA sequence differ from the annota-tion of the At5 g48030 locus shown in the MIPS Arabidopsis thalianadatabase (MATDB; http://mips.gsf.de/proj/thal/db/index.html). Mis-called exons and splice sites relative to the annotation for At5g48030 are underlined. The underlined regions are indicated as ex-ons or introns according the structure of the GFA2 cDNA.


not shown). Together, these data suggest that gfa2 FGs areviable and metabolically active.

DISCUSSION

FG-Expressed Genes are Required throughoutFG Development

One of the objectives of this study was to identify and char-acterize a large collection of mutants so that we could inferthe developmental and reproductive steps regulated andmediated by FG-expressed genes. Our analysis has shownthat a large number of FG mutants can be recovered (dis-cussed below) and that these mutants have defectsthroughout development. Many of these mutations appear

to affect specific processes and can be ordered within theFG developmental pathway (Figure 1). These data indicatethat megagametogenesis is controlled/mediated extensivelyby FG-expressed genes and that FG-expressed genes con-trol/mediate essentially every step of FG development. Theone developmental step for which no mutants were recov-ered is degeneration of the antipodal cells, possibly be-cause mutations that affect this step occur infrequently ordo not affect FG viability.

In addition, we identified a mutant class that includes ctr1and fem17, in which megagametogenesis is not affecteddetectably. This, together with the fact that these mutationsexhibit reduced transmission through the FG, suggests thatthe affected genes are required for one of the FG’s repro-ductive functions. Consistent with this notion, the T-DNA in-sertion in fem17 (C.A. Christensen, R.H. Brown, and G.N.Drews, unpublished data) disrupts the previously identifiedFIS2 gene (Luo et al., 1999), which plays a role in the induc-tion of endosperm development (Chaudhury et al., 1997).

The CTR1 gene encodes a Raf Ser/Thr protein kinase thatis involved in ethylene signal transduction (Kieber et al.,1993). Seed development becomes arrested soon after thefertilization of ctr1 FGs with wild-type pollen (Figure 2S).These data suggest either that maternal expression (i.e., FGexpression) of the CTR1 gene is required for embryo and/orendosperm development after fertilization or that the CTR1gene is imprinted paternally and required after fertilizationfor embryo and/or endosperm development (Chaudhury andBerger, 2001).

In the three independent screens we performed, mutantfrequency was 0.5 to 0.7% among T-DNA/transposon lines(see Methods). However, this number is an underestimatebecause lines containing multiple independently segregat-ing inserts were not considered in segregation distortionscreens. In our T-DNA screens, approximately one-third ofthe lines contained multiple inserts; thus, the corrected fre-quency of FG mutants was �1.0%. Mutant frequencies sim-ilar to this have been reported by other groups (Moore et al.,1997; Bonhomme et al., 1998; Howden et al., 1998). In asaturation screen, 180,000 T-DNA insertions must be screened

Figure 4. Alignment of the Predicted Protein Sequences for Arabi-dopsis GFA2, Yeast Mdj1p, and E. coli DnaJ.

The J domain (residues 90 to 156) and the zinc-finger domain (resi-dues 238 to 294) are double underlined. The Gly/Phe-rich domain(residues 157 to 237) lies between these two domains. Sequencesimilarity is indicated by shading. Relative to DnaJ, the predictedGFA2 sequence contains an 89-residue extension at the N terminusthat has several characteristics of a mitochondrial targeting se-quence: an overrepresentation of Arg, Ala, and Ser residues (25 of89 residues); a paucity of Asp and Glu residues (3 of 89 residues);and an ability to form an amphiphilic �-helix (residues 7 to 16 mayform an amphiphilic �-helix region) (Gavel et al., 1988; Roise, 1997).

Figure 5. Reverse Transcriptase–PCR Analysis of GFA2 Expression.

2224 The Plant Cell

to achieve a 95% probability of mutating any gene of me-dian size (Krysan et al., 1999), and approximately three al-leles per gene are isolated on average. Using these threenumbers, we estimate that �600 (0.01 � 180,000 � 0.33)independent FG loci could be identified via mutation.

The gfa2 Mutation Affects Cell Death andNuclear Fusion

A second objective of this study was to initiate a geneticanalysis of cell death during FG development. Cell deathplays a prominent role in FG development: soon after meio-sis, three of the meiotic products (megaspores) undergo celldeath; late during megagametogenesis, the three antidop-dal cells degenerate; and soon after pollination, one of thesynergid cells undergoes cell death. Although we did notidentify mutants with defects in megaspore or antipodal celldeath, one mutant, gfa2, exhibited a defect in synergid celldeath. In the wild type, most FGs contain a degeneratingsynergid cell at 7 HAP (Figure 2C), whereas in most gfa2FGs, a degenerating synergid cell is not observed even at24 HAP (Figure 2Q). Megaspore and antipodal cell deathboth occur normally in gfa2 mutants, suggesting that syner-gid cell death may occur via a different mechanism thanmegaspore and antipodal cell death.

The synergid cell death process has been described in anumber of species. An exact time course has not been re-ported; however, the process invariably involves a dramaticdecrease in cell volume, collapse of the vacuoles, and com-plete disintegration of the plasma membrane and most or-ganelles (Jensen and Fisher, 1968; Cass and Jensen, 1970;van Went, 1970; Mogensen, 1972; van Went and Cresti,1988; Huang et al., 1993). Synergid cell death has not beendescribed in Arabidopsis. Thus, the extent to which syner-gid cell death in Arabidopsis resembles synergid cell deathin other species remains unknown. It also is not clear which

Figure 6. GFA2::GUS Expression in the Wild Type, GFA2-GFP Pro-tein Localization in the Wild Type, and FIE::FIE-sGFP Expression inthe gfa2 Mutant.

(A) and (B) Histochemical assay for the expression of theGFA2::GUS transgene in the whole plant (A) and the flower (B).Longer histochemical staining showed expression in the petals.(C) Trichome from a transgenic plant expressing 35S::GFP.(D) Trichome from a transgenic plant expressing 35S::GFA2-GFP.(E) to (G) Mitotracker-stained root hair from a transgenic plant ex-pressing 35S::GFA2-GFP.(E) Mitotracker fluorescence.(F) GFP fluorescence.(G) Merger of red and green channel fluorescence.(H) and (I) Expression of the FIE::FIE-GFP construct in the wild type(H) and the gfa2 mutant (I).pn, polar nucleus; sn, secondary nucleus.

Table 3. Yeast Strains Used in This Study

Strain Name Genotype

JSY4037 MATa/� ura3-52/ura3-52 his3200/his3200leu21/leu21 trp163/trp163 lys2202/lys2202mdj1::HIS3/MDJ1 with pRS416-MET25-GFA2

JSY4042 MATa/� ura3-52/ura3-52 his3200/his3200leu21/leu21 trp163/trp163 lys2202/lys2202mdj1::HIS3/MDJ1 with pRS416-MET25

JSY4046 MATa ura3-52 his3200 leu21 trp163 lys2202mdj1::HIS3 with pRS416-MET25-GFA2

JSY4048 MATa ura3-52 his3200 leu21 trp163 lys2202MDJ1 with pRS416-MET25-GFA2

JSY4058 MATa ura3-52 his3200 leu21 trp163 lys2202MDJ1 with pRS416-MET25

JSY4059 MATa ura3-52 his3200 leu21 trp163 lys2202mdj1::HIS3 with pRS416-MET25


step of synergid cell death is affected in gfa2 mutants. Toresolve these issues, we are analyzing synergid cell death inwild-type and gfa2 FGs using transmission electron micros-copy.

The gfa2 mutation also affects the fusion of the polar nu-clei during megagametogenesis (Figure 2P). Fusion of thepolar nuclei begins with contact of the endoplasmic reticu-lum (ER) membranes that are continuous with the outer nu-clear membranes of the two nuclei. Fusion of the ER mem-branes results in outer nuclear membranes that arecontinuous. Finally, the inner nuclear membranes come intocontact and merge (Jensen, 1964; Schulz and Jensen,1973; Sumner and Van Caeseele, 1990; Huang and Russell,1992a). In gfa2 mutants, the outer nuclear membranes ofthe polar nuclei come into contact but do not fuse (Figure2P and data not shown), suggesting that the GFA2 geneproduct is required for the membrane fusion step.

GFA2 Is a Mitochondrial DnaJ Protein

GFA2 encodes a member of the DnaJ protein family, whichfunction as chaperones alone or in association with DnaK(HSP70) partners. Chaperone activity prevents the aggrega-tion or misfolding of nascent polypeptides, assists in the as-

sembly/disassembly of protein complexes, prevents non-productive interactions with other cellular components, andprevents protein aggregation during stress (Hartl, 1996;Bukau and Horwich, 1998; Kelley, 1998; Fink, 1999). Of theyeast DnaJ family members, GFA2 is most similar to mito-chondrially localized Mdj1p. GFA2 protein localized to mito-chondria (Figures 6E to 6G), and the GFA2 gene partiallycomplemented a yeast mdj1 deletion (Figure 7, Table 4). To-gether, these data suggest very strongly that GFA2 is theArabidopsis ortholog of MDJ1.

Disruption of the MDJ1 gene leads to mitochondrial ge-nome loss and reduced oxidative phosphorylation and ATPproduction by mitochondria (Rowley et al., 1994; Prip-Buuset al., 1996; Westermann et al., 1996; Duchniewicz et al.,1999). The gfa2 mutation most likely causes defects in mito-chondrial function similar to those caused by the mdj1 mu-tation (Rowley et al., 1994; Prip-Buus et al., 1996; Westermannet al., 1996; Duchniewicz et al., 1999). Thus, the gfa2 defectscould be a secondary consequence of reduced metabolicactivity.

Several lines of evidence argue against this possibility.First, the nuclear fusion defect is the only defect detectedduring megagametogenesis (Figures 2P to 2R) (Christensenet al., 1998). Nuclear division, nuclear migration, cell wallformation, and cell differentiation all occur normally. If gfa2

Figure 7. GFA2 Complementation of the Yeast mdj1 Mutant Growth Defect.

Cultures of the four possible combinations of the MDJ1 or mdj1::HIS3 allele and the expression vectors pRS416-MET25-GFA2 or pRS416-MET25 were diluted serially (1:10) and grown at 25, 30, and 37�C on dextrose.

2226 The Plant Cell

FGs were energy deficient, pleiotropic effects on these otherenergy-requiring processes would be expected. Second,gfa2 FGs do not appear degraded and persist within unfer-tilized ovules as long as wild-type FGs. Third, gfa2 FGs aremetabolically active: they attract pollen tubes and expressgenes at the same level as wild-type cells (Figures 6H and6I). Decreased metabolic activity would be expected if gfa2FGs were energy deficient. Together, these observationssuggest that the cell death and nuclear fusion defects arenot attributable simply to a lack of respiratory activity andreduced ATP levels.

These observations also suggest that the surroundingsporophytic tissue provides metabolites to the FG. As dis-cussed above, oxidative phosphorylation and ATP produc-tion most likely are reduced in gfa2 FGs. If this is the case,then the apparently normal metabolic activity of gfa2 FGssuggests that this energy deficiency is rescued by the provi-sion of metabolites by the surrounding sporophytic tissue.

Role of GFA2 in Cell Death

In animals, mitochondria play a central role in cell death(Green and Reed, 1998; Wang, 2001). This is establishedmost clearly in mammals, in which the mitochondrial inter-membrane space contains several cell death activators(e.g., cytochrome c) and effectors (e.g., nucleases). In re-sponse to a variety of cell death stimuli, the cell death acti-vators/effectors are released from mitochondria (Hengartner,2000; Wang, 2001). The cell death activators promotecell death by activating caspases that are largely responsi-ble for the morphological changes associated with celldeath (Hengartner, 2000). Much less is known about celldeath in plants, and a role for mitochondria is not firmly es-tablished (Lam et al., 1999; Jones, 2000).

In this study, we show that the GFA2 gene is required forsynergid cell death and that the absence of GFA2 proteinmost likely compromises mitochondrial function. These datasuggest very strongly that mitochondria play a role in celldeath in plants. Several other studies support this conclusion.

First, in several experimental systems, the release of cyto-chrome c from mitochondria has been shown to precedecell death (Balk et al., 1999; Stein and Hansen, 1999; Sun etal., 1999; Balk and Leaver, 2001).

Second, Bax, a cell death–promoting member of the Bcl-2

protein family, triggers cell death when expressed in plantcells. Deletion of the C-terminal hydrophobic tail (transmem-brane domain) eliminates this activity, and fusion of thetransmembrane domain to GFP targets GFP to mitochon-dria, suggesting that mitochondrial targeting of Bax is nec-essary for its death-promoting activity in plants (Lacommeand Santa Cruz, 1999).

Third, victorin, a fungal toxin that binds mitochondrial Glydecarboxylase, induces cell death in treated tissue (Navarreand Wolpert, 1999).

Finally, mutants or experimental manipulations that leadto increased levels of reactive oxygen species, which areproduced primarily in the mitochondrion, can induce celldeath or the expression of genes associated with cell death(Hu et al., 1998; Maxwell et al., 1999; Molina et al., 1999).

Given that GFA2 functions as a chaperone within the mi-tochondrial matrix, it is unlikely to play a direct role in celldeath. More likely, the gfa2 mutation affects cell death indi-rectly by compromising the folding of proteins that are in-volved directly in the cell death process. For example,GFA2-deficient mitochondria may fail to undergo the mor-phological and biochemical changes required to release cy-tochrome c in response to cell death stimuli. To investigatethese issues further, we are analyzing mitochondrial mor-phology and cytochrome c localization during synergid celldeath in wild-type and gfa2 FGs.

Role of GFA2 in Nuclear Fusion

Essentially nothing is known about the molecular aspects ofnuclear fusion in plants. However, genetic and biochemicalstudies in yeast have identified a number of proteins requiredfor nuclear fusion during karyogamy, which occurs via a pro-cess similar to nuclear fusion in plants (Rose, 1996). All of theidentified proteins localize to the ER, and most of them arecomponents of the translocon (Rose, 1996; Brizzio et al.,1999). Although a direct requirement for these translocon com-ponents in membrane fusion has been demonstrated, a spe-cific role of the translocon in nuclear fusion remains unclear.

The mitochondrial localization of GFA2 raises the ques-tion of what role this protein plays in nuclear fusion. As dis-cussed above, the nuclear fusion defect does not appear tobe attributable simply to a lack of respiratory activity. Onepossibility is that functional mitochondria are required for

Table 4. Complementation of Yeast mdj1::HIS3 Growth Defects by Arabidopsis GFA2

25°C 30°C 37°C

Construct Glycerol Dextrose Glycerol Dextrose Glycerol Dextrose

MDJ1 pRS416-MET25

MDJ1 pRS416-MET25-GFA2

mdj1::HIS3 pRS416-MET25 � � � �

mdj1::HIS3 pRS416-MET25-GFA2 � � �


nuclear fusion in plants for reasons other than energy pro-duction. For example, nuclear membrane fusion may requirediffusible factors derived from one of the mitochondrion’sother metabolic activities, including hemes, cytochromes,carbon skeletons, and thymidylates (Mackenzie and McIntosh,1999; Kushnir et al., 2001).

Alternatively, mitochondria may provide factors via physicalcontact with the outer nuclear membrane (Lichtscheidl et al.,1990). For example, lipid composition, which is important formembrane trafficking within the secretory pathway (Burger,2000; Huijbregts et al., 2000; Huttner and Schmidt, 2000), maybe an important factor in membrane fusion, and mitochondrial-nuclear contact may be required to shuttle lipids between thetwo compartments (Staehelin, 1997). The analysis of additionalnuclear fusion mutants as well as mitochondrial mutants in Ar-abidopsis and yeast should help to clarify the possible role ofthe mitochondrion in nuclear fusion in these two organisms.

Synergid Cell Death in Arabidopsis

The timing and induction of synergid cell death appears to bevariable among species. In some species, synergid cell deathrequires pollination and does not occur if pollination is pre-vented (Christensen et al., 1997; Huang and Russell, 1992b;Jensen et al., 1983), which suggests that synergid degenerationis not a feature of the megagametogenesis developmental pro-gram in these species. By contrast, in other species, synergidcell death occurs before pollination and clearly is not depen-dent on pollination, which suggests that synergid degenerationis an integral aspect of the megagametogenesis developmen-tal program in these species (Huang and Russell, 1992b). Thus,different species may use different mechanisms to induce syn-ergid cell death.

In Arabidopsis, synergid cell death does not occur in theabsence of pollination (Christensen et al., 1997), and a de-generating synergid cell is apparent soon after pollination(within 7 HAP; Figure 2C). These observations suggest verystrongly that synergid cell death is induced by pollinationor the presence of pollen tubes within the female tissue(Christensen et al., 1997). At present, it is not knownwhether synergid degeneration in Arabidopsis occurs beforeor upon pollen tube arrival at the FG. Thus, it is unclearwhether synergid cell death is triggered by a long-range sig-nal or upon FG–pollen tube contact.

In some species, the synergid degenerates only after pol-len tube arrival, and degeneration is thought to occur via amechanical process: the release of pollen tube contents intothe synergid cell may cause a massive increase in volumeand pressure, resulting in a bursting of the synergid mem-brane (van Went and Willemse, 1984; Willemse and vanWent, 1984; Russell, 1992; Higashiyama et al., 2000). Thegfa2 mutation should not affect a mechanical process suchas this, suggesting that synergid degeneration in Arabidop-sis entails a physiological response by the synergid cell.

The gfa2 phenotype suggests that synergid degeneration

is not required for pollen tube attraction. The synergid cellsare the source of a pollen tube attractant (Higashiyama etal., 2001), and synergid cell death could, in principle, be im-portant for attractant release (van Went and Willemse, 1984;Willemse and van Went, 1984; Russell, 1992; Cheung,1996). In gfa2 FGs, synergid cell death failed to occur andovules containing gfa2 FGs attracted pollen tubes, suggest-ing that the attractant was released by intact synergid cells.These data are consistent with those showing that at leastone intact synergid is required for pollen tube attraction(Higashiyama et al., 2001).

Summary

We have shown that screens for FG mutants can identifygenes important for many developmental and cellular pro-cesses, including cellularization, nuclear fusion, cell death,and the induction of seed development. One example of thisis GFA2, which is required for cell death of the synergid cell.We have shown that GFA2 protein localized to mitochondriaand that the gfa2 mutation most likely affected mitochon-drial function. These data suggest very strongly that mito-chondria are involved in cell death in plants. Thus, the roleof mitochondria as important regulators of cell death ap-pears to be conserved evolutionarily in plants, mammals(Green and Reed, 1998), and Caenorhabditis elegans (Chenet al., 2000; Parrish et al., 2001).

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana growth conditions were as described previ-ously, except that plants were grown in Scott’s Terra-lite soil mix(Maysville, OH) (Christensen et al., 1997). We obtained seeds fromtransgenic plants for two FIE reporter construct lines. Line 24 con-tains 4.9 kb of FIE promoter fused to sGFP (FIE::sGFP) (Yadegari etal., 2000), and line 11 contains 1.6 kb of FIE promoter fused to theFIE cDNA and sGFP (FIE::FIE-sGFP) (Kinoshita et al., 2001).

Plant Transformation

T-DNA constructs were introduced into Agrobacterium tumefaciensstrain ASE by freeze-thaw transformation (Chen et al., 1994). Arabi-dopsis plants were transformed by the floral dip method (Clough andBent, 1998). Transformed progeny were selected by sowing surface-sterilized T1 seeds on 50 �g/mL kanamycin in Murashige and Skoog(1962) (MS) basal medium with Suc and agar (M-9274; Sigma).

Mutant Screen

Mutagenized individuals were transferred to soil, grown, andscreened for reduced seed set or the presence of abnormal seeds

2228 The Plant Cell

(white or brown seeds). Selfed seeds were collected from plants ex-hibiting reduced seed set, and the ratio of kanamycin-resistant tokanamycin-sensitive progeny was determined. With lines exhibiting aresistant-to-sensitive ratio of 1.5:1, �20 individuals were trans-ferred to soil and screened to determine if reduced seed set coseg-regated with kanamycin resistance. Two individuals were used in re-ciprocal crosses with the wild type, and lines exhibiting significantlyreduced transmission of kanamycin through the female gametophyte(FG) were selected for further analysis. Backcrossed individuals weretransplanted to soil for repeated reciprocal crosses (2 individuals),repeated cosegregation analysis (50 individuals), and analysis of theFG terminal phenotype (2 individuals). The theoretical aspects of thisscreen have been described previously (Christensen et al., 1998;Drews et al., 1998).

We performed three independent screens. In the first screen, weidentified seven mutants (fem5 to fem10 and fem17) from 1228T-DNA–mutagenized lines generated with the pD991 vector (Campisiet al., 1999); in the second screen, we identified six mutants (fem11to fem16) from 819 transposon-mutagenized lines generated withthe DsG transposon (Sundaresan et al., 1995); and in the thirdscreen, we identified 18 mutants (fem18 to fem38) from 3426 T-DNA–mutagenized lines generated with the pCGN1547 vector (McBrideand Summerfelt, 1990). The mutant frequency from these threescreens was 0.6, 0.7, and 0.5%, respectively.

Phenotypic Analysis of Mutants

Confocal laser scanning microscopy was performed as describedpreviously (Christensen et al., 1997, 1998). For analysis of pollentube guidance, siliques at 24 h after pollination were fixed in 10%glacial acetic acid, 30% chloroform, and 60% absolute ethanol for�1.5 h; rinsed with tap water; soaked in 4 N NaOH at 65�C for 15 to20 min; rinsed with tap water; and then stained in 0.1% aniline blue(Smith and McCully, 1978) and 0.1 M K3PO4 overnight. Stained tissuewas mounted in a drop of glycerol, pressed with a cover slip, and ex-amined using a fluorescence microscope. In all cases, terminal phe-notypes were analyzed. To obtain ovules of the appropriate stage,we emasculated flowers of stage 12c, waited 24 to 48 h, and pro-cessed the tissue for confocal analysis (Christensen et al., 1997).

Sequence Analysis

We used GENSCANW (http://CCR-081.mit.edu/cgi-bin/genscanw.cgi) to identify genomic sequence features such as exon/intronboundaries and poly(A) addition sites (Vector NTI Suite version 5.3for Macintosh; Informax Inc., Bethesda, MD) for manipulation ofplasmid constructs, primer design, and analysis of protein se-quences. We used the AlignX module of Vector NTI suite with a BLO-SUM matrix and the neighbor-joining algorithm (Saitou and Nei,1987) for the alignment of protein sequences.

GFA2 Gene Cloning and gfa2 Complementation

We used inverse PCR to identify the plant sequence flanking theT-DNA border in the gfa2 mutant (Triglia et al., 1988). We used theHindIII restriction enzyme with the LB1 (5�-CATCTCATCGATGCT-TGGTAATAATTG-3�) and LB2 (5�-GAGCTATTGGCACACGAA-

GAATGGT-3�) primers. Approximately 600 bp was amplified thatcontained T-DNA left border sequence and 240 bp of plant DNA.

The construct for molecular complementation of the gfa2 mutationconsisted of the GFA2 promoter fused to the GFA2 cDNA (see be-low). A 3654-bp XhoI fragment from a partial genomic subclone ofBAC MDN11 (plasmid pBSII-C/P5�GFA2-8) was subcloned into theXhoI site in the pCRII-6/14GFA2c plasmid, resulting in plasmid pCR-GFA2c/g. To add a transcription termination sequence, the primers3�NOS-F (SacI; 5�-GCGCGAGCTCTGAATCCTGTTGCCGGTCTTG-3�)and 3�NOS-R (PstI-SacI; 5�-GCGCGAGCTCTGCAGCGATCTAGT-AACATAGATGACAC-3�) were used to amplify and introduce restric-tion sites into the 3� nopaline synthase (NOS) termination sequencefrom pGEM NOS-1.

This product was digested with SacI and ligated into the SacI siteof pCR-GFA2c/g. A 5513-bp SphI-PstI fragment corresponding tothe rescue construct (GFA2::GFA2-NOS) then was isolated from theresultant plasmid, pCR-GFA2c/g-NOS, blunted, and subcloned intothe unique PmeI restriction site of the T-DNA vector pCGNlox2b(Sieburth et al., 1998), resulting in transformation vector plox-GFA2c/g-NOS. Transgenic progeny from wild-type plants transformed withplox-GFA2c/g-NOS were selected and crossed with the gfa2 mutant.By crossing and screening with PCR, we generated lines that wereheterozygous for the gfa2 mutation and homozygous for the GFA2transgene. In such lines, FG morphology was analyzed, and trans-mission of the gfa2 allele through the FG and male gametophyte wastested.

Reverse Transcriptase PCR and cDNA Cloning

RNA was isolated using the Plant RNeasy purification kit (Qiagen,Valencia, CA) from ecotype Columbia. First-strand cDNA synthesiswas performed using the SuperScript Preamplification System forFirst Strand cDNA Synthesis (Gibco BRL). Subsequent amplificationof the first-strand cDNA was performed using Biolase polymerase(Bioline, Reno, NV), and all thermal cycling was performed on an MJResearch PTC-200 thermal cycler. For tissue-specific reverse tran-scriptase–PCR, we used primer sets gfa2-11 (5�-CAACCCTCACTG-GTGATGTTGTCGTGAA-3�) and gfa2-14 (5�-GTAGAATTAGTT-AAAAGTAAAACCCAGACA-3�) along with ActConF (5�-GATTTG-GCATCACACTTTCTACAATG-3�) and ActConR (5�-GTTCCACCACTG-AGCACAATG-3�) to assay the presence of spliced cDNAs from theGFA2 and actin genes, respectively.

The fully processed cDNA fragments amplified by these primersets were 385 bp for GFA2 and 650 bp for actin. To amplify a cDNAencompassing the entire open reading frame of GFA2, we usedprimer set gfa2-6 (5�-TAGGGTTTTAACTTTGGCTGCTCTGTCTCAA-3�) and gfa2-14. The cDNA was cloned into the pCRII-TOPO vectorusing the TOPO TA cloning kit (Invitrogen, Carlsbad, CA), resulting inplasmid pCRII-6/14GFA2c.

GFA2::�-Glucuronidase Reporter Construct

To make the �-glucuronidase (GUS) reporter plasmid, we startedwith pBSII-mGFP5-ter that was prepared by amplifying the mGFP5coding region and the transcription termination region from pAVA393(von Arnim et al., 1998) using mGFP5-F (5�-GGGGCTCGAGCC-ATGGGTAAAGGAGAACTTTTCACT-3�) and mGFP5-R (5�-GGG-GGGTACCAGGTCACTGGATTTTGGTTTTAGGAA-3�), which intro-duced XhoI and KpnI sites, respectively, and cloned this product in


pBluescript II KS�. The uidA coding sequence from transgenic GUS-expressing plants (transformed with the pD991 enhancer trap vector[Campisi et al., 1999]) was amplified using primers GUS-F (5�-GGGGCCATGGCATTACGTCCTGTAGAAACCCCAA-3�) and GUS-R(5�-GGGGTCTAGAGTTGTTTGCCTCCCTGCTGCGGTT-3�), whichintroduced a NcoI site at the uidA start codon and an XbaI site at theuidA stop codon.

The PCR product was digested with NcoI and XbaI and used to re-place the mGFP5 coding region in pBSII-mGFP5-ter, resulting inplasmid pBSII-GUS-ter. The GFA2 upstream regulatory sequencesand the first three exons were obtained by PCR amplification usingthe gfa2-17 (5�-GGGGCTGCAGCAGATCAGTCGACGTTCATGCCA-TCTCAT-3�) and gfa2-18 (5�-GGGGCCATGGCAAACGAAGAACCT-GTTCCATGAAAAGACCT-3�) primers to introduce PstI and NcoIsites, respectively. The resulting PCR product was cloned into pBSII-GUS-ter using the PstI and NcoI sites, resulting in plasmid pBSII-GFA2pro-GUS. The GFA2::GUS-ter cassette was liberated by BssHIIdigestion and blunting and subcloned into the blunted XbaI site ofpCGN1547, resulting in plasmid pCGN-GFA2pro-GUS. This con-struct includes 2993 bp of sequence upstream of the translationalstart codon and the first three exons of GFA2.

Progeny from plants transformed with pCGN-GFA2pro-GUS wereselected on 50 �g/mL kanamycin MS medium. After �10 days ofgrowth, seedlings were transplanted to Magenta boxes (Gibco BRL)containing only MS medium and allowed to develop until 5 to 10flowers were present. Plants were stained in 50-mL conical tubescontaining 25 mL of 1 mM 5-bromo-4-chloro-3-indolyl-�-glucuronicacid (U.S. Biological, Swampscott, MA), 1 mM potassium ferrocya-nide, 1 mM potassium ferricyanide, 0.1% Triton X-100, and 50 mMsodium phosphate buffer, pH 7.0. Holes were punched in the lid ofthe tube to allow air exchange.

A house vacuum was used for infiltration during the first 10 to 20min of staining. Tissue was incubated in stain at 37�C overnight andthen cleared in 70% ethanol. Flowers and siliques were stained in thesame way, except that they were removed from the plants and thecarpels were dissected using a 30-gauge syringe needle and stainedin 1.5-mL tubes. Images of GUS-stained whole plants were takenwith a hand-held Nikon Coolpix 990 digital camera (Tokyo, Japan).Images of flowers were taken with an Olympus SZx12 dissecting mi-croscope equipped with the same camera (Tokyo, Japan).

GFA2-mGFP5 Protein Localization Construct

We used the plasmid pAVA393 (von Arnim et al., 1998) to construct aC-terminal fusion of mGFP5 to the GFA2 protein. We amplified theGFA2 open reading frame from plasmid pCR-GFA2c/g using primersgfa2-19 (5�-GGGGCCATGGTCCCTTCCAATGGCGCAAAGGTT-3�)and gfa2-20 (5�-CCCCCCATGGCTCCGCCACCTCCGCCACCC-TGGGAAGATCCAGTTGCTGTGCGCT-3�), which introduce a NcoIsite at the 5� and 3� ends of the gene and introduce six Gly codons atthe 3� end of the gene. The gfa2-19/gfa2-20 PCR product was di-gested with NcoI and cloned into the NcoI site of pAVA393, resultingin plasmid pAVA393-GFA2-mGFP5(6G).

The 35S Cauliflower mosaic virus::GFA2-6G-mGFP5 fusion pro-tein construct in this plasmid was moved into pCGN1547 usingBamHI and HindIII restriction sites, resulting in plasmid pCGN-35S::GFA2-mGFP5(6G). As a control for protein localization, the 35SCauliflower mosaic virus::mGFP5 cassette from pAVA393 also wascloned into pCGN1547 using the BamHI and HindIII restriction sites,resulting in plasmid pCGN-35S::mGFP5.

Progeny from pCGN-35S::GFA2-mGFP5(6G) and pCGN-35S::mGFP5 transformants were selected on 50 �g/mL kanamycin MSmedium. After 5 to 10 days of growth, seedlings were harvested intoa freshly made staining solution of 500 nM CM-H2XRos (MitotrackerRed; Molecular Probes, Eugene, OR) in a 1 � MS salts solution(11117-058; Gibco BRL) and allowed to stain for 15 min at room tem-perature. After staining, the seedlings were washed three times inMS salts solution for �10 min (Hedtke et al., 1999).

Roots were mounted under a cover slip in a drop of 1% low-melt-ing-point agarose stored at 37�C (Gibco BRL). Images of mGFP5fluorescence and Mitotracker fluorescence were captured using aLeica LSM510 confocal laser scanning microscope (Wetzlar, Ger-many). The two channels were excited separately by 488-nm(mGFP5) and 543-nm (Mitotracker) laser lines, and fluorescent emis-sions were gathered using the rhodamine/fluorescein isothiocyanatefilter set. Images of trichomes from plants transformed with pCGN-35S::GFA2-mGFP5(6G) and pCGN-35S::mGFP5 were taken in thesame manner, except that whole seedlings were mounted in 1% lowmelting point agarose.

Analysis of FIE::sGFP Expression in the gfa2 Mutant

These lines were crossed with gfa2, and progeny that carried boththe GFP construct and the gfa2 mutation were selected by GFP fluo-rescence and a PCR assay for the gfa2 mutation. We emasculatedflowers from these plants, waited 2 days, and then fixed half of themfor phenotypic examination and mounted the other half for GFP ex-pression analysis. Before mounting the pistil in 1% low-melting-pointagarose (Gibco BRL), sepals, petals, and anthers were removed andthe carpel walls were dissected using a 30-gauge syringe needle. Im-ages of gfa2 mutants expressing FIE::FIE-sGFP were taken as de-scribed above for GFA2-mGFP5 protein localization.

Yeast Growth

Standard methods were used for the growth, transformation, and ge-netic manipulation of Saccharomyces cerevisiae in rich medium(yeast extract plus peptone containing either dextrose or glycerol)and synthetic medium (synthetic dextrose) as described (Sherman,1986).

MDJ1 Disruption and Complementation by GFA2

An mdj1::HIS3 disruption that replaced the MDJ1 coding region wasgenerated by gene replacement in a diploid strain as described(Baudin et al., 1993). The resulting heterozygous diploid was sporu-lated, and the tetrads were dissected to obtain the mdj1::HIS3 hap-loid strains. To make the GFA2 complementation construct, a 1.716-kb BamHI-XbaI fragment from pCRII-6/14GFA2c that contained theGFA2 open reading frame was cloned into the BamHI-XbaI sites inthe polylinker of the yeast expression vector pRS416-MET25, result-ing in plasmid pRS416-MET25-GFA2. The pRS416-MET25 andpRS416-MET25-GFA2 vectors were transformed into the heterozy-gous diploid mdj1::HIS3/MDJ1 before sporulation and dissection.

Upon request, all novel materials described in this article will bemade available in a timely manner for noncommercial research pur-poses. No restrictions or conditions will be placed on the use of anymaterials described in this article that would limit their use for non-commercial research purposes.

2230 The Plant Cell

Accession Numbers

The GenBank accession number for the GFA2 cDNA sequence isAY103490. The GFA2 gene corresponds to locus At5 g48030 fromBAC clone MDN11 on chromosome 5 (GenBank accession numberAB017064).

ACKNOWLEDGMENTS

We thank the ABRC for the MDN11 BAC clones; Albrecht von Arnim(University of Tennessee) for the pAVA393 (35S::mGFP5) plasmid;Ramin Yadegari, Tetsu Kinoshita, and Bob Fischer (University of Cal-ifornia, Berkeley) for seeds from transgenic plants expressingFIE::sGFP and FIE::FIE-sGFP; and F.B. Pickett for GUS staining sug-gestions. We also thank Ramin Yadegari and John Harada for criticalreview of the manuscript. This work was funded in part by a NationalInstitutes of Health Developmental Biology Training Grant appoint-ment to C.A.C. and by grants from the National Science Foundation(IBN-96-30371) and Ceres, Inc., to G.N.D.

Received February 4, 2002; accepted May 19, 2002.

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DOI 10.1105/tpc.002170 2002;14;2215-2232Plant Cell

Shaw and Gary N. DrewsCory A. Christensen, Steven W. Gorsich, Ryan H. Brown, Linda G. Jones, Jessica Brown, Janet M.

Mitochondrial GFA2 Is Required for Synergid Cell Death in Arabidopsis

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