Download Mitochondrial GFA2 Is Required for Synergid Cell

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 Death
in Arabidopsis
Cory A. Christensen,1 Steven W. Gorsich, Ryan H. Brown,2 Linda G. Jones, Jessica Brown,2 Janet M. Shaw,
and Gary N. Drews3
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, and
few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern
essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed
this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect
in 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 Saccharomyces
cerevisiae (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 GFA2
is 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 that
plays an integral role in the angiosperm life cycle. It develops within ovules and most often consists of an egg cell, a
central cell, two synergid cells, and three antipodal cells
(Figure 1). The FG is important for many aspects of the angiosperm reproductive process: in addition to giving rise to
the embryo and endosperm of the seed, the FG plays a role
in 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 seed
development (Ohad et al., 1996; Chaudhury et al., 1997;
Grossniklaus et al., 1998).
FG development requires many fundamental cellular processes, including mitosis, vacuole formation, cell wall formation, nuclear migration, nuclear fusion, and cell death
(Figure 1). Thus, mutations that affect these processes are
likely to affect the FG, exhibit reduced transmission through
the FG, and appear at reduced frequency in the sporophyte
1 Current
address: Paradigm Genetics, Inc., 108 Alexander Drive,
P.O. Box 14528, Research Triangle Park, NC 27709-4528.
address: Plant Biology Graduate Group, Section of Molecular and Cellular Biology, University of California, 1 Shields Avenue,
Davis, CA 95616.
3 To whom correspondence should be addressed. E-mail drews@biosci.
biology.utah.edu; fax 801-581-4668.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.002170.
2 Current
generation. Consequently, genetic analysis of these cellular
processes is likely to require gametophytic screens and
analysis of the gametophyte generation. An example of this
is cell death, which is important in the diploid sporophyte
generation for tissue development, senescence, and plant
defense (Martienssen, 1997; Buckner et al., 1998; Richberg
et al., 1998; Lam et al., 1999). However, because cell death
occurs during the gametophytic phase (Figure 1), cell death
mutants may not be identified readily in sporophyte-oriented
screens (Martienssen, 1997).
Little is known about the molecular and genetic processes that govern FG development and the FG’s reproductive functions. The number and identities of genes expressed in the FG, and the proportion of these that are
unique to the FG, are unknown. These genes presumably
regulate and mediate FG development and function. However, for all but a few of these genes, specific functions in
the FG are unknown. Also, little is known about FG physiology. The physiological pathways that exist in the FG, and
the extent to which FG physiology is independent from that
of the surrounding sporophytic tissue, are unknown. Thus, it
is not clear whether FG development and physiology are
governed primarily by the haploid or the diploid genome.
As a step toward addressing these issues, several groups
have performed screens for gametophytic mutants in Arabidopsis and maize, and a number of FG mutations have been
identified (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
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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 defines
stage 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 form
the homodiploid secondary nucleus (stage FG6). Finally, the antipodal cells degenerate, producing a mature FG (stage FG7). Early during the fertilization process, one of the synergid cells is induced to undergo cell death (data not shown). Category designations show the developmental
stages 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.
and Kermicle, 2001). However, relatively few of these mutations have been characterized at the phenotypic and molecular levels, and a specific function in the FG is known for
only few genes.
The present study had two main objectives. The first was
to determine the extent to which FG development and function are controlled/mediated by FG-expressed genes and to
identify specific genes required by these developmental/reproductive steps. To address these issues, we have identified and characterized a large collection of FG mutants. The
second objective was to initiate a molecular genetic analysis
of cell death during FG development. To this end, we identified and characterized a mutant, gfa2, that has a defect in
synergid cell death during the reproductive process. Our
analysis of the GFA2 gene suggests a role for mitochondria
in cell death in plants.
RESULTS
Analysis of 39 Female Gametophyte Mutants
To gain insight into the steps of FG development mediated
by FG-expressed genes, we identified and analyzed a large
collection of FG mutants. We identified 31 new FG mutants
(fem5 to fem38) from T-DNA– and transposon-mutagenized
lines (see Methods) and analyzed these along with the previously identified ctr1 mutant (Kieber and Ecker, 1994). These
mutants are summarized in Table 1. Because the FG is haploid, complementation tests could not be performed to determine whether any of the FG mutations are allelic. However, 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 to
date (22 of 39 mutants), the T-DNA/transposon insertion
sites fall within different genomic regions (C.A. Christensen,
R.H. Brown, and G.N. Drews, unpublished data), suggesting
that 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 to
transmit the mutant allele), we crossed heterozygous mutants as females to wild-type males and scored the number
of heterozygous (kanamycin-resistant) and homozygous
wild-type (kanamycin-sensitive) progeny (Table 2). To determine whether these mutations also affect the male gametophyte, we crossed heterozygous mutants as males to wild-type
females and scored the number of heterozygous (kanamycin-resistant) and homozygous wild-type (kanamycin-sensitive) progeny (Table 2). As shown in Table 2, of the new mutations, fem8, fem9, fem17, and fem20 appear to affect the
FG specifically.
To determine the steps of FG development affected by
the mutations, we analyzed the terminal phenotypes using
confocal laser scanning microscopy (CLSM) (Christensen et
al., 1997, 1998) and divided the mutants into phenotypic
categories. To comprehensively define the spectrum of possible phenotypes, we included seven previously analyzed
mutants (gfa2 to gfa5 and fem2 to fem4; Table 1) in our categorization scheme. Together, the 39 FG mutants fall into
five phenotypic categories (Table 2). These phenotypic categories along with wild-type development are summarized in
Figure 1. Confocal images of the FG mutants are shown in
Figure 2.
Category 1 and category 2 mutants had defects during
the nuclear division phase of megagametogenesis (stages
GFA2 Is Required for Cell Death
FG1 to early FG5) and failed to cellularize. With category 1
mutants (14 of 39 mutants), all or most ( 60%) of the mutant FGs arrested at stage FG1. Arrested FGs either persisted (Figure 2H) or degenerated (Figure 2G) during ovule
development. Mutant FGs that progressed beyond stage
FG1 exhibited category 2 defects. With category 2 mutants
(17 of 39 mutants), all or most (60%) of the mutant FGs
progressed beyond stage FG1. Category 2 defects included
abnormal 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 cellularized
and had defects during the later phases of megagametogenesis. Category 3 mutants (4 of 39 mutants) had defects
in cellular morphology, including abnormal nuclear positions
within cells, misshapen cells, and unusual cell features. With
fem4, the defects were quite pronounced and included irregular cell shapes and polarities of the egg and synergid
cells (Christensen et al., 1998). With the other three category
3 mutants, FG morphology was almost wild type: fem7 (Figure 2N) and fem9 (data not shown) FGs had slightly reduced
cytoplasm and slightly abnormal cell shape, and fem8 FGs
had many small vacuoles in the central cell cytoplasm (Figure 2O). Category 4 mutants (2 of 39 mutants) had defects in
fusion of the polar nuclei. With both mutants, the polar nuclei migrated properly, came to lie side by side, but failed to
fuse. 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)
female gametophyte2 (fem2)
female gametophyte3 (fem3)
female gametophyte4 (fem4)
female gametophyte5 (fem5)
female gametophyte6 (fem6)
female gametophyte7 (fem7)
female gametophyte8 (fem8)
female gametophyte9 (fem9)
female gametophyte10 (fem10)
female gametophyte11 (fem11)
female gametophyte12 (fem12)
female gametophyte13 (fem13)
female gametophyte14 (fem14)
female gametophyte15 (fem15)
female gametophyte16 (fem16)
female gametophyte17 (fem17)
female gametophyte18 (fem18)
female gametophyte19 (fem19)
female gametophyte20 (fem20)
female gametophyte21 (fem21)
female gametophyte22 (fem22)
female gametophyte23 (fem23)
female gametophyte24 (fem24)
female gametophyte25 (fem25)
female gametophyte26 (fem26)
female gametophyte29 (fem29)
female gametophyte30 (fem30)
female gametophyte31 (fem31)
female gametophyte33 (fem33)
female gametophyte34 (fem34)
female gametophyte35 (fem35)
female gametophyte36 (fem36)
female gametophyte37 (fem37)
female gametophyte38 (fem38)
gametophytic factor2 (gfa2)
gametophytic factor3 (gfa3)
gametophytic factor4 (gfa4)
gametophytic factor5 (gfa5)
–
2462-5
2465-35
TJ160
TJ13
TJ1335
TJ1350
TJ1507
TJ2111
TJ2118
a308
a466
B271
A839
A845
E160
TJ761
RB46
RB81
RB158
RB160
RB299
RB302
RB309
RB312
RB353
RB117
RB140
RB184
RB273
RB279
RB341
RB405
RB428
RB433
102
13/21
84
114
X-ray
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
Transposon
Transposon
Transposon
Transposon
Transposon
Transposon
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
T-DNA
L-er
Ws
Ws
Col
Col
Col
Col
Col
Col
Col
L-er
L-er
L-er
L-er
L-er
L-er
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Col
Ws
Ws
Ws
Ws
Kieber and Ecker, 1994
Christensen et al., 1998
Christensen et al., 1998
Christensen et al., 1998
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Feldmann et al., 1997
Feldmann et al., 1997
Feldmann et al., 1997
Feldmann et al., 1997
Col, Columbia; L-er, Landsberg erecta; Ws, Wassilewskija.
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Table 2. Penetrance, Female Gametophyte Specificity, and Phenotype
Mutant
Penetrance in FGa
Penetrance in MGa
Terminal Phenotypeb
ctr1-2
fem2
fem3
fem4
fem5
fem6
fem7
fem8
fem9
fem10
fem11
fem12
fem13
fem14
fem15
fem16
fem17
fem18
fem19
fem20
fem21
fem22
fem23
fem24
fem25
fem26
fem29
fem30
fem31
fem33
fem34
fem35
fem36
fem37
fem38
gfa2
gfa3
gfa4
gfa5
58%c
44%d
42%d
100%d
87% (685)
93% (455)
81% (831)
67% (441)
42% (431)
93% (1275)
54% (624)
73% (1111)
84% (406)
79% (105)
100% (99)
66% (206)
98% (66)
39% (305)
100% (306)
34% (154)
100% (108)
100% (710)
98% (826)
83% (504)
86% (342)
99% (260)
17% (387)
15% (183)
66% (506)
94% (174)
100% (300)
35% (295)
99% (315)
42% (328)
51% (330)
99% (571)
82% (281)
61% (1252)
79% (441)
0%c
0%d
44%d
42%d
72% (1799)
86% (811)
30% (614)
9%e (623)
12%e (857)
92% (1347)
100% (100)
100% (403)
100% (423)
100% (218)
64% (64)
71% (298)
10%e (133)
65% (694)
100% (459)
0% (413)
100% (376)
99% (601)
43% (1024)
16% (726)
99% (847)
17% (796)
71% (535)
97% (774)
88% (256)
70% (214)
98% (360)
31% (602)
92% (246)
16% (668)
53% (355)
86% (704)
99% (365)
100% (908)
84% (649)
Wild type (5)
Stage FG1d (1)
Stage FG1d (1)
Abnormal cellular morphology, stage FG5/6/7 (3)
Abnormal nuclear number, size, and position (2)
Abnormal cellular morphology (3)
Abnormal cellular morphology (3)
Abnormal cellular morphology (3)
Stage FG1 (1)
Abnormal nuclear number and position (2)
Stage FG1 to stage FG6 (2)
Stage FG1 (1)
Abnormal nuclear number, cellularization; twinning (2)
Abnormal nuclear number, position; collapse (2)
Abnormal nuclear number and cellularization (2)
No FG (1)
Wild type (5)
Stage FG1 to stage FG2 (1)
Abnormal nuclear number and position (2)
Stage FG1, stage FG5 (2)
Stage FG3 to stage FG5 (2)
Stage FG4 to stage FG5 (2)
Stage FG3 to stage FG5 (2)
Stage FG1, stage FG3 to stage FG5 (2)
Abnormal nuclear number, position; stage FG1 to FG2 (2)
Stage FG1 (1)
Stage FG1 (1)
Stage FG1 to stage FG5; abnormal nuclear number (2)
Stage FG1, stage FG3 (1)
Stage FG1, stage FG3 to stage FG4 (2)
Stage FG3 (2)
Stage FG1 (1)
Stage FG1, stage FG3 to stage FG4 (2)
Stage FG1 (1)
Stage FG1 (1)
Unfused polar nucleid (4)
Unfused polar nuclei, cellularization failured (4)
Stage FG1d (1)
Stage FG1d (1)
a FG,
female gametophyte; MG, male gametophyte. The number of progeny scored is in parentheses.
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.
b Phenotypic
gfa3 affected both nuclear fusion (predominantly) and cellularization (Christensen et al., 1998).
Category 5 mutants (2 of 39 mutants) appeared normal at
the terminal developmental stage (stage FG7), suggesting
that megagametogenesis was not affected. This finding, together with the reduced transmission of these mutations
through the FG (Table 2), suggests that category 5 mutations affect one of the FG’s reproductive functions (pollen
tube 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 that
the disrupted gene corresponds to the FIS2 gene (Luo et al.,
1999), which is required to repress endosperm development
before fertilization (Chaudhury et al., 1997).
To investigate the role of CTR1, we analyzed the postpollination phenotypes of ctr1. In ctr1/ctr1 siliques, 92% (45 of
49) of ovules contained a pollen tube in the micropyle (97%
in the wild-type control), indicating that pollen tube guidance
GFA2 Is Required for Cell Death
was not affected significantly. We next examined fertilized
ctr1 embryo sacs at 15 to 48 h after pollination (HAP) with
wild-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 contained a central nucleus and many small vacuoles; the endosperm contained two to four nuclei; and one synergid
was degenerated. These data suggest that ctr1 FGs become fertilized but arrest very early in seed development.
Together, these data suggest that most (95%) FGexpressed genes are required for megagametogenesis and
that 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 proportion (5%) of FG-expressed genes play no role in
megagametogenesis and instead are required for one of the
FG’s reproductive functions.
The gfa2 Mutation Affects Synergid Cell Death
In an effort to identify the genes involved in cell death during
FG development, we analyzed our mutant collection for
lines that display cell death defects. Cell death occurs three
times during FG development: first, immediately after meiosis, three of the megaspores undergo cell death; second,
late during megagametogenesis, the three antipodal cells
degenerate (Figure 1); third, in response to pollination, one
of the synergid cells undergoes cell death (Christensen et
al., 1997). As discussed above, the analysis of megagametogenesis in each of our FG mutants did not reveal mutants
with defects in megaspore or antipodal cell death.
We also asked whether synergid cell death after pollination is affected in our FG mutants. We focused on those mutants in which the egg apparatus (egg and synergid cells) is
morphologically normal: fem17/fis2 (discussed above), ctr1
(Figures 2S and 2T), and gfa2 (Figure 2P). As discussed
above (Figures 2S and 2T) and by others (Chaudhury et al.,
1997), the fertilization process appears to be normal in the
ctr1 and fem17/fis2 mutants, indicating that synergid cell
death is not affected in these mutants.
To determine whether the gfa2 mutation affects synergid
cell death, we pollinated gfa2/GFA2 pistils with wild-type
pollen and examined gfa2 embryo sacs (those with unfused
polar nuclei) at 7 to 24 HAP. In the wild type (Wassilewskija
[Ws-2] ecotype), one of the synergid cells was degenerated
by 7 HAP (Figure 2C) in essentially all (40 of 41) ovules. In
contrast to the wild type, with most (44 of 55) gfa2 FGs,
both synergids were intact and showed no signs of degeneration (Figures 2Q and 2R). All gfa2 embryo sacs that failed
to undergo synergid cell death (i.e., those with two intact
synergids) showed no signs of fertilization (cf. Figures 2A to
2E with Figures 2Q and 2R).
The synergid-cell-death defect of gfa2 could be a secondary consequence of a failure to attract pollen tubes. To address this issue, we pollinated gfa2/GFA2 pistils with wild-
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type pollen and scored the number of ovules with pollen
tubes in the micropyle. In gfa2/GFA2 siliques, 99% (148 of
149) of ovules had a pollen tube in the micropyle (100% in
the wild-type control), indicating that the gfa2 mutation does
not affect pollen tube guidance. Together, these data indicate that the GFA2 gene product is required for synergid
cell death during the fertilization process.
GFA2 Encodes a Member of the DnaJ Protein Family
We used inverse PCR (Triglia et al., 1988) to identify the
T-DNA insertion site in gfa2 mutants. The insertion site corresponded to locus At5 g48030 from BAC clone MDN11 on
chromosome 5. Introducing a wild-type copy of this gene
into the gfa2 mutant restored transmission of the gfa2 allele
through the FG and rescued the nuclear fusion defect (data
not shown), indicating that locus At5 g48030 corresponds to
the GFA2 gene. To determine GFA2 gene structure, we isolated a GFA2 cDNA clone and compared its sequence with
that of the genomic sequence. As shown in Figure 3, the
GFA2 gene consists of 18 exons, has an open reading frame
of 1368 bp, and is predicted to encode a protein of 456
amino acids. The T-DNA insert in the gfa2 mutant fell within
the second exon, likely creating a null allele (Figure 3).
BLAST searches against the SWISS-PROT database using the predicted GFA2 protein sequence revealed homology with bacterial DnaJ (HSP40) proteins. As shown in Figure 4, within the region of homology (residues 90 to 456),
GFA2 is 37% identical and 53% similar to Escherichia coli
DnaJ. DnaJ proteins are defined by the presence of an
70–amino acid J-domain, which contains the highly conserved HPD tripeptide (Kelley, 1998). GFA2 has this domain
and, like many other DnaJ family members, also contains a
Gly/Phe-rich domain, a Cys-rich zinc-finger domain, and a less
well-conserved C-terminal domain (Figure 4). The J-domain
and the Gly/Phe-rich domain are thought to mediate the interactions between DnaJ and its cochaperone DnaK, and
the zinc-finger and C-terminal domains are thought to mediate 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 GFA2
gene is expressed within the plant. First, we used reverse
transcriptase–PCR to assay the presence of GFA2 RNA in
various plant organs. Total RNA purified from roots, rosette
leaves, stems, open flowers, and apical tips (including unopened flowers) was used as a template to make singlestranded cDNA by reverse transcription, and primers specific for GFA2 and actin were used to amplify cDNAs from
each of the RNA populations. As shown in Figure 5, PCR
bands representing the spliced transcripts of both genes
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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 a
single 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), one
degenerated 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 nucleus. 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 peripheral
endosperm 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.
GFA2 Is Required for Cell Death
were amplified from each RNA population, indicating that
GFA2 RNA is present in each organ.
Second, we transformed plants with a GFA2::-glucuronidase (GUS) reporter construct and analyzed GUS expression in seedlings and whole flowering plants. As shown
in Figures 6A and 6B, strong GUS activity was detected in
all organs. GUS activity also was detected in ovules during
FG development (Figure 6B). Together, these data indicate
that the GFA2 gene is expressed throughout the plant.
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Arnim et al., 1998). By contrast, in plants containing the
35S::GFA2-GFP construct, GFP fluorescence was detected
in multiple small intracellular compartments (Figure 6D). This
pattern was seen in all tissues examined and colocalized
with a mitochondrion-specific dye, Mitotracker (Figures 6E
to 6G). Together, these data indicate that the GFA2 protein
localizes to mitochondria.
GFA2 Partially Complements a Yeast mdj1 Deletion
GFA2 Is Similar to Yeast Mdj1p and Localizes
to Mitochondria
We compared GFA2 to the J-domain proteins from Saccharomyces cerevisiae and found that Mdj1p is the yeast protein most similar to GFA2 (data not shown). Mdj1p is a mitochondria-targeted DnaJ family member that functions as a
chaperone in the mitochondrial matrix (Rowley et al., 1994;
Westermann et al., 1996; Duchniewicz et al., 1999). This sequence similarity suggests that GFA2 might be a mitochondria-targeted protein. Furthermore, the GFA2 N terminus
has the characteristics of a mitochondrial targeting sequence (Figure 4) (Gavel et al., 1988; Roise, 1997).
To determine whether GFA2 is targeted to mitochondria,
we transformed plants with a GFA2–green fluorescent protein (GFP) translational fusion construct driven by the 35S
promoter of Cauliflower mosaic virus (35S::GFA2-GFP). As a
control, we also transformed plants with a GFP that lacks a
targeting sequence (35S::GFP) (von Arnim et al., 1998). With
both constructs, transgenic plants appeared normal. Control plants containing the 35S::GFP construct exhibited GFP
fluorescence in the cytoplasm and nucleus (Figure 6C), in
agreement with previous studies (Grebenok et al., 1997; von
The sequence similarity and mitochondrial localization suggest that GFA2 is the Arabidopsis ortholog of yeast MDJ1.
To test this possibility further, we determined whether GFA2
protein can substitute for Mdj1p in yeast by expressing
GFA2 in an mdj1 yeast strain. The yeast strains used in this
study are listed in Table 3. To generate an MDJ1 null allele,
we replaced the entire MDJ1 coding region with the HIS3
gene. We refer to this allele as mdj1::HIS3. As expected
from previous studies (Rowley et al., 1994; Duchniewicz et
al., 1999), mdj1::HIS3 cells at 25C failed to grow on the
nonfermentable carbon source, glycerol, but grew slowly on
the fermentable carbon source, dextrose. This petite phenotype is consistent with a defect in mitochondrial function. In
addition, at 37C, mdj1::HIS3 cells failed to grow on both
carbon sources, indicating that Mdj1p function also is required for survival at high temperature (Duchniewicz et al.,
1999).
We introduced an expression vector containing the GFA2
cDNA (pRS416-MET25-GFA2) into heterozygous yeast cells
(MDJ1/mdj1::HIS3). As a control, we transformed heterozygous cells (MDJ1/mdj1::HIS3) with the pRS416-MET25 vector alone. Transformed heterozygous diploids then were
sporulated, 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 projected 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 chalazal
end, 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 egg
cell). 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 degenerated 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 sections
taken at the terminal developmental stage or at the HAP indicated. In the CLSM images, cytoplasm is shown in gray, vacuoles are shown in
black, and nucleoli are shown in white. In the depictions, cytoplasm is shown in light gray, vacuoles are shown in black, nuclei are shown in dark
gray, 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, nucleus; 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.
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The Plant Cell
dextrose-containing plates and grown at 25, 30, and 37C.
As shown in Figure 7, mdj1::HIS3 cells containing the GFA2
expression vector grew slightly faster than mdj1::HIS3 cells
containing the empty vector at 25C (cf. rows 3 and 4) and
30C (cf. rows 7 and 8).
In addition, the presence of GFA2 rescued the lethality of
mdj1::HIS3 at 37C (cf. rows 11 and 12). However, the presence of the GFA2 expression vector did not restore the ability of mdj1::HIS3 cells to grow on glycerol (data not shown).
These data (summarized in Table 4) indicate that Arabidopsis GFA2 can partially substitute for Mdj1p in yeast. These
data, along with the sequence similarity and mitochondrial
localization, suggest very strongly that GFA2 is the ortholog
of yeast MDJ1 and that GFA2 protein functions as a chaperone in the mitochondrial matrix.
The gfa2 Mutation Does Not Affect Viability
Figure 3. GFA2 Gene Sequence.
Exons are indicated with uppercase letters. A 33-bp deletion associated with the site of the T-DNA insert is indicated by strikethrough.
The position of the T-DNA left border is indicated above the
strikethrough. Plant DNA on the other side of the T-DNA insert is adjacent to the internal T-DNA sequence. The predicted GFA2 protein
contains two in-frame ATG codons (marked in boldface and underlined) in its N terminus. The first Met codon is expected to initiate
translation because it satisfies the consensus sequence criteria for a
translation initiation codon (Kozak, 1991). Some of the exon-intron
boundaries derived from the cDNA sequence differ from the annotation of the At5 g48030 locus shown in the MIPS Arabidopsis thaliana
database (MATDB; http://mips.gsf.de/proj/thal/db/index.html). Miscalled exons and splice sites relative to the annotation for At5
g48030 are underlined. The underlined regions are indicated as exons or introns according the structure of the GFA2 cDNA.
The data presented above suggest that the gfa2 mutation
causes defects in mitochondrial function. Thus, the gfa2 defects could be a secondary consequence of decreased metabolic activity. To assess this possibility, we asked whether
gfa2 FGs express genes associated with central cell differentiation. We analyzed the expression of a FIE reporter gene
construct (FIE::GFP) whose expression is initiated in the
central cell at approximately the time of polar nuclei fusion
(Yadegari et al., 2000). We examined ovules from plants that
were heterozygous for the gfa2 mutation and homozygous
for FIE::GFP.
As expected, approximately half (39 of 70) of the ovules
contained a FG with unfused polar nuclei. All of the ovules
examined (n 98) expressed FIE::GFP in the central cell
(data not shown). We also examined ovules from plants heterozygous for the gfa2 mutation and containing a FIE-GFP
fusion protein construct (FIE::FIE-GFP). GFP expressed
from this construct is localized in the central cell nucleus
(Kinoshita et al., 2001). As shown in Figures 6H and 6I, the
FIE::FIE-GFP construct is localized to the nucleus and is expressed at the same level in mutant and wild-type FGs.
gfa2 FGs exhibited several other indicators of cell viability
and metabolic activity. First, the failure of polar nuclei fusion
was the only defect observed during megagametogenesis
(Figure 2P). In the wild type, many other energy-requiring
steps (e.g., cell wall formation) occurred at approximately
the same time as nuclear fusion. Yet, none of these steps
appeared to be affected in the gfa2 mutant (Figure 2P). Furthermore, the nuclei, vacuoles, and cell membranes in gfa2
FGs were intact and showed no signs of degradation (Figures 2P and data not shown). Second, as discussed above,
gfa2 FGs attracted pollen tubes. The presence of a functional 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 emasculated
flowers, gfa2 FGs persisted as long as wild-type FGs (data
GFA2 Is Required for Cell Death
Figure 4. Alignment of the Predicted Protein Sequences for Arabidopsis GFA2, Yeast Mdj1p, and E. coli DnaJ.
The J domain (residues 90 to 156) and the zinc-finger domain (residues 238 to 294) are double underlined. The Gly/Phe-rich domain
(residues 157 to 237) lies between these two domains. Sequence
similarity is indicated by shading. Relative to DnaJ, the predicted
GFA2 sequence contains an 89-residue extension at the N terminus
that has several characteristics of a mitochondrial targeting sequence: an overrepresentation of Arg, Ala, and Ser residues (25 of
89 residues); a paucity of Asp and Glu residues (3 of 89 residues);
and an ability to form an amphiphilic -helix (residues 7 to 16 may
form an amphiphilic -helix region) (Gavel et al., 1988; Roise, 1997).
not shown). Together, these data suggest that gfa2 FGs are
viable and metabolically active.
2223
to affect specific processes and can be ordered within the
FG developmental pathway (Figure 1). These data indicate
that megagametogenesis is controlled/mediated extensively
by FG-expressed genes and that FG-expressed genes control/mediate essentially every step of FG development. The
one developmental step for which no mutants were recovered is degeneration of the antipodal cells, possibly because mutations that affect this step occur infrequently or
do not affect FG viability.
In addition, we identified a mutant class that includes ctr1
and fem17, in which megagametogenesis is not affected
detectably. This, together with the fact that these mutations
exhibit reduced transmission through the FG, suggests that
the affected genes are required for one of the FG’s reproductive functions. Consistent with this notion, the T-DNA insertion in fem17 (C.A. Christensen, R.H. Brown, and G.N.
Drews, unpublished data) disrupts the previously identified
FIS2 gene (Luo et al., 1999), which plays a role in the induction of endosperm development (Chaudhury et al., 1997).
The CTR1 gene encodes a Raf Ser/Thr protein kinase that
is involved in ethylene signal transduction (Kieber et al.,
1993). Seed development becomes arrested soon after the
fertilization of ctr1 FGs with wild-type pollen (Figure 2S).
These data suggest either that maternal expression (i.e., FG
expression) of the CTR1 gene is required for embryo and/or
endosperm development after fertilization or that the CTR1
gene is imprinted paternally and required after fertilization
for embryo and/or endosperm development (Chaudhury and
Berger, 2001).
In the three independent screens we performed, mutant
frequency was 0.5 to 0.7% among T-DNA/transposon lines
(see Methods). However, this number is an underestimate
because lines containing multiple independently segregating inserts were not considered in segregation distortion
screens. In our T-DNA screens, approximately one-third of
the lines contained multiple inserts; thus, the corrected frequency of FG mutants was 1.0%. Mutant frequencies similar to this have been reported by other groups (Moore et al.,
1997; Bonhomme et al., 1998; Howden et al., 1998). In a
saturation screen, 180,000 T-DNA insertions must be screened
DISCUSSION
FG-Expressed Genes are Required throughout
FG Development
One of the objectives of this study was to identify and characterize a large collection of mutants so that we could infer
the developmental and reproductive steps regulated and
mediated by FG-expressed genes. Our analysis has shown
that a large number of FG mutants can be recovered (discussed below) and that these mutants have defects
throughout development. Many of these mutations appear
Figure 5. Reverse Transcriptase–PCR Analysis of GFA2 Expression.
2224
The Plant Cell
to achieve a 95% probability of mutating any gene of median size (Krysan et al., 1999), and approximately three alleles per gene are isolated on average. Using these three
numbers, we estimate that 600 (0.01 180,000 0.33)
independent FG loci could be identified via mutation.
The gfa2 Mutation Affects Cell Death and
Nuclear Fusion
A second objective of this study was to initiate a genetic
analysis of cell death during FG development. Cell death
plays a prominent role in FG development: soon after meiosis, three of the meiotic products (megaspores) undergo cell
death; late during megagametogenesis, the three antidopdal cells degenerate; and soon after pollination, one of the
synergid cells undergoes cell death. Although we did not
identify mutants with defects in megaspore or antipodal cell
death, one mutant, gfa2, exhibited a defect in synergid cell
death. In the wild type, most FGs contain a degenerating
synergid cell at 7 HAP (Figure 2C), whereas in most gfa2
FGs, a degenerating synergid cell is not observed even at
24 HAP (Figure 2Q). Megaspore and antipodal cell death
both occur normally in gfa2 mutants, suggesting that synergid cell death may occur via a different mechanism than
megaspore and antipodal cell death.
The synergid cell death process has been described in a
number of species. An exact time course has not been reported; however, the process invariably involves a dramatic
decrease in cell volume, collapse of the vacuoles, and complete disintegration of the plasma membrane and most organelles (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 been
described in Arabidopsis. Thus, the extent to which synergid cell death in Arabidopsis resembles synergid cell death
in other species remains unknown. It also is not clear which
Table 3. Yeast Strains Used in This Study
Figure 6. GFA2::GUS Expression in the Wild Type, GFA2-GFP Protein Localization in the Wild Type, and FIE::FIE-sGFP Expression in
the gfa2 Mutant.
(A) and (B) Histochemical assay for the expression of the
GFA2::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 expressing 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.
Strain Name Genotype
JSY4037
JSY4042
JSY4046
JSY4048
JSY4058
JSY4059
MATa/ ura3-52/ura3-52 his3200/his3200
leu21/leu21 trp163/trp163 lys2202/lys2202
mdj1::HIS3/MDJ1 with pRS416-MET25-GFA2
MATa/ ura3-52/ura3-52 his3200/his3200
leu21/leu21 trp163/trp163 lys2202/lys2202
mdj1::HIS3/MDJ1 with pRS416-MET25
MATa ura3-52 his3200 leu21 trp163 lys2202
mdj1::HIS3 with pRS416-MET25-GFA2
MATa ura3-52 his3200 leu21 trp163 lys2202
MDJ1 with pRS416-MET25-GFA2
MATa ura3-52 his3200 leu21 trp163 lys2202
MDJ1 with pRS416-MET25
MATa ura3-52 his3200 leu21 trp163 lys2202
mdj1::HIS3 with pRS416-MET25
GFA2 Is Required for Cell Death
2225
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 pRS416MET25 were diluted serially (1:10) and grown at 25, 30, and 37C on dextrose.
step of synergid cell death is affected in gfa2 mutants. To
resolve these issues, we are analyzing synergid cell death in
wild-type and gfa2 FGs using transmission electron microscopy.
The gfa2 mutation also affects the fusion of the polar nuclei during megagametogenesis (Figure 2P). Fusion of the
polar nuclei begins with contact of the endoplasmic reticulum (ER) membranes that are continuous with the outer nuclear membranes of the two nuclei. Fusion of the ER membranes results in outer nuclear membranes that are
continuous. Finally, the inner nuclear membranes come into
contact and merge (Jensen, 1964; Schulz and Jensen,
1973; Sumner and Van Caeseele, 1990; Huang and Russell,
1992a). In gfa2 mutants, the outer nuclear membranes of
the polar nuclei come into contact but do not fuse (Figure
2P and data not shown), suggesting that the GFA2 gene
product is required for the membrane fusion step.
GFA2 Is a Mitochondrial DnaJ Protein
GFA2 encodes a member of the DnaJ protein family, which
function as chaperones alone or in association with DnaK
(HSP70) partners. Chaperone activity prevents the aggregation or misfolding of nascent polypeptides, assists in the as-
sembly/disassembly of protein complexes, prevents nonproductive interactions with other cellular components, and
prevents protein aggregation during stress (Hartl, 1996;
Bukau and Horwich, 1998; Kelley, 1998; Fink, 1999). Of the
yeast DnaJ family members, GFA2 is most similar to mitochondrially localized Mdj1p. GFA2 protein localized to mitochondria (Figures 6E to 6G), and the GFA2 gene partially
complemented a yeast mdj1 deletion (Figure 7, Table 4). Together, these data suggest very strongly that GFA2 is the
Arabidopsis ortholog of MDJ1.
Disruption of the MDJ1 gene leads to mitochondrial genome loss and reduced oxidative phosphorylation and ATP
production by mitochondria (Rowley et al., 1994; Prip-Buus
et al., 1996; Westermann et al., 1996; Duchniewicz et al.,
1999). The gfa2 mutation most likely causes defects in mitochondrial function similar to those caused by the mdj1 mutation (Rowley et al., 1994; Prip-Buus et al., 1996; Westermann
et al., 1996; Duchniewicz et al., 1999). Thus, the gfa2 defects
could be a secondary consequence of reduced metabolic
activity.
Several lines of evidence argue against this possibility.
First, the nuclear fusion defect is the only defect detected
during megagametogenesis (Figures 2P to 2R) (Christensen
et al., 1998). Nuclear division, nuclear migration, cell wall
formation, and cell differentiation all occur normally. If gfa2
2226
The Plant Cell
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
FGs were energy deficient, pleiotropic effects on these other
energy-requiring processes would be expected. Second,
gfa2 FGs do not appear degraded and persist within unfertilized ovules as long as wild-type FGs. Third, gfa2 FGs are
metabolically active: they attract pollen tubes and express
genes at the same level as wild-type cells (Figures 6H and
6I). Decreased metabolic activity would be expected if gfa2
FGs were energy deficient. Together, these observations
suggest that the cell death and nuclear fusion defects are
not attributable simply to a lack of respiratory activity and
reduced ATP levels.
These observations also suggest that the surrounding
sporophytic tissue provides metabolites to the FG. As discussed above, oxidative phosphorylation and ATP production most likely are reduced in gfa2 FGs. If this is the case,
then the apparently normal metabolic activity of gfa2 FGs
suggests that this energy deficiency is rescued by the provision 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 established
most clearly in mammals, in which the mitochondrial intermembrane space contains several cell death activators
(e.g., cytochrome c) and effectors (e.g., nucleases). In response to a variety of cell death stimuli, the cell death activators/effectors are released from mitochondria (Hengartner,
2000; Wang, 2001). The cell death activators promote
cell death by activating caspases that are largely responsible for the morphological changes associated with cell
death (Hengartner, 2000). Much less is known about cell
death in plants, and a role for mitochondria is not firmly established (Lam et al., 1999; Jones, 2000).
In this study, we show that the GFA2 gene is required for
synergid cell death and that the absence of GFA2 protein
most likely compromises mitochondrial function. These data
suggest very strongly that mitochondria play a role in cell
death in plants. Several other studies support this conclusion.
First, in several experimental systems, the release of cytochrome c from mitochondria has been shown to precede
cell death (Balk et al., 1999; Stein and Hansen, 1999; Sun et
al., 1999; Balk and Leaver, 2001).
Second, Bax, a cell death–promoting member of the Bcl-2
protein family, triggers cell death when expressed in plant
cells. Deletion of the C-terminal hydrophobic tail (transmembrane domain) eliminates this activity, and fusion of the
transmembrane domain to GFP targets GFP to mitochondria, suggesting that mitochondrial targeting of Bax is necessary for its death-promoting activity in plants (Lacomme
and Santa Cruz, 1999).
Third, victorin, a fungal toxin that binds mitochondrial Gly
decarboxylase, induces cell death in treated tissue (Navarre
and Wolpert, 1999).
Finally, mutants or experimental manipulations that lead
to increased levels of reactive oxygen species, which are
produced primarily in the mitochondrion, can induce cell
death 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 mitochondrial matrix, it is unlikely to play a direct role in cell
death. More likely, the gfa2 mutation affects cell death indirectly by compromising the folding of proteins that are involved directly in the cell death process. For example,
GFA2-deficient mitochondria may fail to undergo the morphological and biochemical changes required to release cytochrome c in response to cell death stimuli. To investigate
these issues further, we are analyzing mitochondrial morphology and cytochrome c localization during synergid cell
death in wild-type and gfa2 FGs.
Role of GFA2 in Nuclear Fusion
Essentially nothing is known about the molecular aspects of
nuclear fusion in plants. However, genetic and biochemical
studies in yeast have identified a number of proteins required
for nuclear fusion during karyogamy, which occurs via a process similar to nuclear fusion in plants (Rose, 1996). All of the
identified proteins localize to the ER, and most of them are
components of the translocon (Rose, 1996; Brizzio et al.,
1999). Although a direct requirement for these translocon components in membrane fusion has been demonstrated, a specific role of the translocon in nuclear fusion remains unclear.
The mitochondrial localization of GFA2 raises the question of what role this protein plays in nuclear fusion. As discussed above, the nuclear fusion defect does not appear to
be attributable simply to a lack of respiratory activity. One
possibility is that functional mitochondria are required for
GFA2 Is Required for Cell Death
nuclear fusion in plants for reasons other than energy production. For example, nuclear membrane fusion may require
diffusible factors derived from one of the mitochondrion’s
other metabolic activities, including hemes, cytochromes,
carbon skeletons, and thymidylates (Mackenzie and McIntosh,
1999; Kushnir et al., 2001).
Alternatively, mitochondria may provide factors via physical
contact with the outer nuclear membrane (Lichtscheidl et al.,
1990). For example, lipid composition, which is important for
membrane trafficking within the secretory pathway (Burger,
2000; Huijbregts et al., 2000; Huttner and Schmidt, 2000), may
be an important factor in membrane fusion, and mitochondrialnuclear contact may be required to shuttle lipids between the
two compartments (Staehelin, 1997). The analysis of additional
nuclear fusion mutants as well as mitochondrial mutants in Arabidopsis and yeast should help to clarify the possible role of
the mitochondrion in nuclear fusion in these two organisms.
Synergid Cell Death in Arabidopsis
The timing and induction of synergid cell death appears to be
variable among species. In some species, synergid cell death
requires pollination and does not occur if pollination is prevented (Christensen et al., 1997; Huang and Russell, 1992b;
Jensen et al., 1983), which suggests that synergid degeneration
is not a feature of the megagametogenesis developmental program in these species. By contrast, in other species, synergid
cell death occurs before pollination and clearly is not dependent on pollination, which suggests that synergid degeneration
is an integral aspect of the megagametogenesis developmental program in these species (Huang and Russell, 1992b). Thus,
different species may use different mechanisms to induce synergid cell death.
In Arabidopsis, synergid cell death does not occur in the
absence of pollination (Christensen et al., 1997), and a degenerating synergid cell is apparent soon after pollination
(within 7 HAP; Figure 2C). These observations suggest very
strongly that synergid cell death is induced by pollination
or the presence of pollen tubes within the female tissue
(Christensen et al., 1997). At present, it is not known
whether synergid degeneration in Arabidopsis occurs before
or upon pollen tube arrival at the FG. Thus, it is unclear
whether synergid cell death is triggered by a long-range signal or upon FG–pollen tube contact.
In some species, the synergid degenerates only after pollen tube arrival, and degeneration is thought to occur via a
mechanical process: the release of pollen tube contents into
the synergid cell may cause a massive increase in volume
and pressure, resulting in a bursting of the synergid membrane (van Went and Willemse, 1984; Willemse and van
Went, 1984; Russell, 1992; Higashiyama et al., 2000). The
gfa2 mutation should not affect a mechanical process such
as this, suggesting that synergid degeneration in Arabidopsis entails a physiological response by the synergid cell.
The gfa2 phenotype suggests that synergid degeneration
2227
is not required for pollen tube attraction. The synergid cells
are the source of a pollen tube attractant (Higashiyama et
al., 2001), and synergid cell death could, in principle, be important 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 and
ovules containing gfa2 FGs attracted pollen tubes, suggesting that the attractant was released by intact synergid cells.
These data are consistent with those showing that at least
one intact synergid is required for pollen tube attraction
(Higashiyama et al., 2001).
Summary
We have shown that screens for FG mutants can identify
genes important for many developmental and cellular processes, including cellularization, nuclear fusion, cell death,
and the induction of seed development. One example of this
is GFA2, which is required for cell death of the synergid cell.
We have shown that GFA2 protein localized to mitochondria
and that the gfa2 mutation most likely affected mitochondrial function. These data suggest very strongly that mitochondria are involved in cell death in plants. Thus, the role
of mitochondria as important regulators of cell death appears to be conserved evolutionarily in plants, mammals
(Green and Reed, 1998), and Caenorhabditis elegans (Chen
et al., 2000; Parrish et al., 2001).
METHODS
Plant Material and Growth Conditions
Arabidopsis thaliana growth conditions were as described previously, except that plants were grown in Scott’s Terra-lite soil mix
(Maysville, OH) (Christensen et al., 1997). We obtained seeds from
transgenic plants for two FIE reporter construct lines. Line 24 contains 4.9 kb of FIE promoter fused to sGFP (FIE::sGFP) (Yadegari et
al., 2000), and line 11 contains 1.6 kb of FIE promoter fused to the
FIE cDNA and sGFP (FIE::FIE-sGFP) (Kinoshita et al., 2001).
Plant Transformation
T-DNA constructs were introduced into Agrobacterium tumefaciens
strain ASE by freeze-thaw transformation (Chen et al., 1994). Arabidopsis plants were transformed by the floral dip method (Clough and
Bent, 1998). Transformed progeny were selected by sowing surfacesterilized 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, and
screened for reduced seed set or the presence of abnormal seeds
2228
The Plant Cell
(white or brown seeds). Selfed seeds were collected from plants exhibiting reduced seed set, and the ratio of kanamycin-resistant to
kanamycin-sensitive progeny was determined. With lines exhibiting a
resistant-to-sensitive ratio of 1.5:1, 20 individuals were transferred to soil and screened to determine if reduced seed set cosegregated with kanamycin resistance. Two individuals were used in reciprocal crosses with the wild type, and lines exhibiting significantly
reduced transmission of kanamycin through the female gametophyte
(FG) were selected for further analysis. Backcrossed individuals were
transplanted to soil for repeated reciprocal crosses (2 individuals),
repeated cosegregation analysis (50 individuals), and analysis of the
FG terminal phenotype (2 individuals). The theoretical aspects of this
screen have been described previously (Christensen et al., 1998;
Drews et al., 1998).
We performed three independent screens. In the first screen, we
identified seven mutants (fem5 to fem10 and fem17) from 1228
T-DNA–mutagenized lines generated with the pD991 vector (Campisi
et al., 1999); in the second screen, we identified six mutants (fem11
to fem16) from 819 transposon-mutagenized lines generated with
the DsG transposon (Sundaresan et al., 1995); and in the third
screen, we identified 18 mutants (fem18 to fem38) from 3426 T-DNA–
mutagenized lines generated with the pCGN1547 vector (McBride
and Summerfelt, 1990). The mutant frequency from these three
screens was 0.6, 0.7, and 0.5%, respectively.
GAATGGT-3) primers. Approximately 600 bp was amplified that
contained T-DNA left border sequence and 240 bp of plant DNA.
The construct for molecular complementation of the gfa2 mutation
consisted of the GFA2 promoter fused to the GFA2 cDNA (see below). A 3654-bp XhoI fragment from a partial genomic subclone of
BAC MDN11 (plasmid pBSII-C/P5GFA2-8) was subcloned into the
XhoI site in the pCRII-6/14GFA2c plasmid, resulting in plasmid pCRGFA2c/g. To add a transcription termination sequence, the primers
3NOS-F (SacI; 5-GCGCGAGCTCTGAATCCTGTTGCCGGTCTTG-3)
and 3NOS-R (PstI-SacI; 5-GCGCGAGCTCTGCAGCGATCTAGTAACATAGATGACAC-3) were used to amplify and introduce restriction sites into the 3 nopaline synthase (NOS) termination sequence
from pGEM NOS-1.
This product was digested with SacI and ligated into the SacI site
of pCR-GFA2c/g. A 5513-bp SphI-PstI fragment corresponding to
the rescue construct (GFA2::GFA2-NOS) then was isolated from the
resultant plasmid, pCR-GFA2c/g-NOS, blunted, and subcloned into
the unique PmeI restriction site of the T-DNA vector pCGNlox2b
(Sieburth et al., 1998), resulting in transformation vector plox-GFA2c/gNOS. Transgenic progeny from wild-type plants transformed with
plox-GFA2c/g-NOS were selected and crossed with the gfa2 mutant.
By crossing and screening with PCR, we generated lines that were
heterozygous for the gfa2 mutation and homozygous for the GFA2
transgene. In such lines, FG morphology was analyzed, and transmission of the gfa2 allele through the FG and male gametophyte was
tested.
Phenotypic Analysis of Mutants
Confocal laser scanning microscopy was performed as described
previously (Christensen et al., 1997, 1998). For analysis of pollen
tube 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 65C for 15 to
20 min; rinsed with tap water; and then stained in 0.1% aniline blue
(Smith and McCully, 1978) and 0.1 M K3PO4 overnight. Stained tissue
was mounted in a drop of glycerol, pressed with a cover slip, and examined using a fluorescence microscope. In all cases, terminal phenotypes were analyzed. To obtain ovules of the appropriate stage,
we emasculated flowers of stage 12c, waited 24 to 48 h, and processed 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/intron
boundaries and poly(A) addition sites (Vector NTI Suite version 5.3
for Macintosh; Informax Inc., Bethesda, MD) for manipulation of
plasmid constructs, primer design, and analysis of protein sequences. We used the AlignX module of Vector NTI suite with a BLOSUM 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 the
T-DNA border in the gfa2 mutant (Triglia et al., 1988). We used the
HindIII restriction enzyme with the LB1 (5-CATCTCATCGATGCTTGGTAATAATTG-3) and LB2 (5-GAGCTATTGGCACACGAA-
Reverse Transcriptase PCR and cDNA Cloning
RNA was isolated using the Plant RNeasy purification kit (Qiagen,
Valencia, CA) from ecotype Columbia. First-strand cDNA synthesis
was performed using the SuperScript Preamplification System for
First Strand cDNA Synthesis (Gibco BRL). Subsequent amplification
of the first-strand cDNA was performed using Biolase polymerase
(Bioline, Reno, NV), and all thermal cycling was performed on an MJ
Research PTC-200 thermal cycler. For tissue-specific reverse transcriptase–PCR, we used primer sets gfa2-11 (5-CAACCCTCACTGGTGATGTTGTCGTGAA-3) and gfa2-14 (5-GTAGAATTAGTTAAAAGTAAAACCCAGACA-3) along with ActConF (5-GATTTGGCATCACACTTTCTACAATG-3) and ActConR (5-GTTCCACCACTGAGCACAATG-3) to assay the presence of spliced cDNAs from the
GFA2 and actin genes, respectively.
The fully processed cDNA fragments amplified by these primer
sets were 385 bp for GFA2 and 650 bp for actin. To amplify a cDNA
encompassing the entire open reading frame of GFA2, we used
primer set gfa2-6 (5-TAGGGTTTTAACTTTGGCTGCTCTGTCTCAA3) and gfa2-14. The cDNA was cloned into the pCRII-TOPO vector
using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA), resulting in
plasmid pCRII-6/14GFA2c.
GFA2::-Glucuronidase Reporter Construct
To make the -glucuronidase (GUS) reporter plasmid, we started
with pBSII-mGFP5-ter that was prepared by amplifying the mGFP5
coding region and the transcription termination region from pAVA393
(von Arnim et al., 1998) using mGFP5-F (5-GGGGCTCGAGCCATGGGTAAAGGAGAACTTTTCACT-3) and mGFP5-R (5-GGGGGGTACCAGGTCACTGGATTTTGGTTTTAGGAA-3), which introduced XhoI and KpnI sites, respectively, and cloned this product in
GFA2 Is Required for Cell Death
pBluescript II KS. The uidA coding sequence from transgenic GUSexpressing plants (transformed with the pD991 enhancer trap vector
[Campisi et al., 1999]) was amplified using primers GUS-F (5GGGGCCATGGCATTACGTCCTGTAGAAACCCCAA-3) and GUS-R
(5-GGGGTCTAGAGTTGTTTGCCTCCCTGCTGCGGTT-3), which
introduced a NcoI site at the uidA start codon and an XbaI site at the
uidA stop codon.
The PCR product was digested with NcoI and XbaI and used to replace the mGFP5 coding region in pBSII-mGFP5-ter, resulting in
plasmid pBSII-GUS-ter. The GFA2 upstream regulatory sequences
and the first three exons were obtained by PCR amplification using
the gfa2-17 (5-GGGGCTGCAGCAGATCAGTCGACGTTCATGCCATCTCAT-3) and gfa2-18 (5-GGGGCCATGGCAAACGAAGAACCTGTTCCATGAAAAGACCT-3) primers to introduce PstI and NcoI
sites, respectively. The resulting PCR product was cloned into pBSIIGUS-ter using the PstI and NcoI sites, resulting in plasmid pBSIIGFA2pro-GUS. The GFA2::GUS-ter cassette was liberated by BssHII
digestion and blunting and subcloned into the blunted XbaI site of
pCGN1547, resulting in plasmid pCGN-GFA2pro-GUS. This construct includes 2993 bp of sequence upstream of the translational
start codon and the first three exons of GFA2.
Progeny from plants transformed with pCGN-GFA2pro-GUS were
selected on 50 g/mL kanamycin MS medium. After 10 days of
growth, seedlings were transplanted to Magenta boxes (Gibco BRL)
containing only MS medium and allowed to develop until 5 to 10
flowers were present. Plants were stained in 50-mL conical tubes
containing 25 mL of 1 mM 5-bromo-4-chloro-3-indolyl--glucuronic
acid (U.S. Biological, Swampscott, MA), 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, 0.1% Triton X-100, and 50 mM
sodium phosphate buffer, pH 7.0. Holes were punched in the lid of
the tube to allow air exchange.
A house vacuum was used for infiltration during the first 10 to 20
min of staining. Tissue was incubated in stain at 37C overnight and
then cleared in 70% ethanol. Flowers and siliques were stained in the
same way, except that they were removed from the plants and the
carpels were dissected using a 30-gauge syringe needle and stained
in 1.5-mL tubes. Images of GUS-stained whole plants were taken
with a hand-held Nikon Coolpix 990 digital camera (Tokyo, Japan).
Images of flowers were taken with an Olympus SZx12 dissecting microscope equipped with the same camera (Tokyo, Japan).
2229
Progeny from pCGN-35S::GFA2-mGFP5(6G) and pCGN-35S::
mGFP5 transformants were selected on 50 g/mL kanamycin MS
medium. After 5 to 10 days of growth, seedlings were harvested into
a freshly made staining solution of 500 nM CM-H2XRos (Mitotracker
Red; Molecular Probes, Eugene, OR) in a 1 MS salts solution
(11117-058; Gibco BRL) and allowed to stain for 15 min at room temperature. After staining, the seedlings were washed three times in
MS salts solution for 10 min (Hedtke et al., 1999).
Roots were mounted under a cover slip in a drop of 1% low-melting-point agarose stored at 37C (Gibco BRL). Images of mGFP5
fluorescence and Mitotracker fluorescence were captured using a
Leica LSM510 confocal laser scanning microscope (Wetzlar, Germany). The two channels were excited separately by 488-nm
(mGFP5) and 543-nm (Mitotracker) laser lines, and fluorescent emissions were gathered using the rhodamine/fluorescein isothiocyanate
filter set. Images of trichomes from plants transformed with pCGN35S::GFA2-mGFP5(6G) and pCGN-35S::mGFP5 were taken in the
same manner, except that whole seedlings were mounted in 1% low
melting point agarose.
Analysis of FIE::sGFP Expression in the gfa2 Mutant
These lines were crossed with gfa2, and progeny that carried both
the GFP construct and the gfa2 mutation were selected by GFP fluorescence and a PCR assay for the gfa2 mutation. We emasculated
flowers from these plants, waited 2 days, and then fixed half of them
for phenotypic examination and mounted the other half for GFP expression analysis. Before mounting the pistil in 1% low-melting-point
agarose (Gibco BRL), sepals, petals, and anthers were removed and
the carpel walls were dissected using a 30-gauge syringe needle. Images of gfa2 mutants expressing FIE::FIE-sGFP were taken as described above for GFA2-mGFP5 protein localization.
Yeast Growth
Standard methods were used for the growth, transformation, and genetic 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).
GFA2-mGFP5 Protein Localization Construct
MDJ1 Disruption and Complementation by GFA2
We used the plasmid pAVA393 (von Arnim et al., 1998) to construct a
C-terminal fusion of mGFP5 to the GFA2 protein. We amplified the
GFA2 open reading frame from plasmid pCR-GFA2c/g using primers
gfa2-19 (5-GGGGCCATGGTCCCTTCCAATGGCGCAAAGGTT-3)
and gfa2-20 (5-CCCCCCATGGCTCCGCCACCTCCGCCACCCTGGGAAGATCCAGTTGCTGTGCGCT-3), which introduce a NcoI
site at the 5 and 3 ends of the gene and introduce six Gly codons at
the 3 end of the gene. The gfa2-19/gfa2-20 PCR product was digested with NcoI and cloned into the NcoI site of pAVA393, resulting
in plasmid pAVA393-GFA2-mGFP5(6G).
The 35S Cauliflower mosaic virus::GFA2-6G-mGFP5 fusion protein construct in this plasmid was moved into pCGN1547 using
BamHI and HindIII restriction sites, resulting in plasmid pCGN35S::GFA2-mGFP5(6G). As a control for protein localization, the 35S
Cauliflower mosaic virus::mGFP5 cassette from pAVA393 also was
cloned into pCGN1547 using the BamHI and HindIII restriction sites,
resulting in plasmid pCGN-35S::mGFP5.
An mdj1::HIS3 disruption that replaced the MDJ1 coding region was
generated by gene replacement in a diploid strain as described
(Baudin et al., 1993). The resulting heterozygous diploid was sporulated, and the tetrads were dissected to obtain the mdj1::HIS3 haploid strains. To make the GFA2 complementation construct, a 1.716kb BamHI-XbaI fragment from pCRII-6/14GFA2c that contained the
GFA2 open reading frame was cloned into the BamHI-XbaI sites in
the polylinker of the yeast expression vector pRS416-MET25, resulting in plasmid pRS416-MET25-GFA2. The pRS416-MET25 and
pRS416-MET25-GFA2 vectors were transformed into the heterozygous diploid mdj1::HIS3/MDJ1 before sporulation and dissection.
Upon request, all novel materials described in this article will be
made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any
materials described in this article that would limit their use for noncommercial research purposes.
2230
The Plant Cell
Accession Numbers
The GenBank accession number for the GFA2 cDNA sequence is
AY103490. The GFA2 gene corresponds to locus At5 g48030 from
BAC clone MDN11 on chromosome 5 (GenBank accession number
AB017064).
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 California, Berkeley) for seeds from transgenic plants expressing
FIE::sGFP and FIE::FIE-sGFP; and F.B. Pickett for GUS staining suggestions. We also thank Ramin Yadegari and John Harada for critical
review of the manuscript. This work was funded in part by a National
Institutes of Health Developmental Biology Training Grant appointment 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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Mitochondrial GFA2 Is Required for Synergid Cell Death in Arabidopsis
Cory A. Christensen, Steven W. Gorsich, Ryan H. Brown, Linda G. Jones, Jessica Brown, Janet M.
Shaw and Gary N. Drews
Plant Cell 2002;14;2215-2232
DOI 10.1105/tpc.002170
This information is current as of June 14, 2017
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