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Genetic Analyses of the Pretty Few Seeds2 Locus on Ovule Development
Ovules are the developmental precursors of seed. The maternal tissues surrounding the growing embryo are
critical for its growth and development. A number of mutations affecting the development of these maternal
organs result in female sterility. This investigation describes a new genetic locus that is necessary to the
development of these maternal tissues and fertility. The pretty few seeds2 (pfs2) mutant exhibits specific defects
in gametophyte formation and integument morphogenesis. This locus is inherited as a maternal trait, which
indicates that the defects in gametophyte development are secondary to development of the maternal
organs. Examination of genetic interactions of the pfs2 locus with other well-characterized ovule loci has clarified
the role of this locus on ovule development. Interestingly, the double-mutant phenotypes of the PFS2 locus with
the inner no outer and strubbelig loci all had integuments that appear similar to telomes. Based on the
prehistoric fossil record, telomes are believed to be the evolutionary precursors of ovules. Based on these data,
we hypothesize that the PFS2 gene might have played an important role in integument evolution.
A small angiosperm, Arabidopsis thaliana, has been utilized as a model organism to study plant reproduction.
The growth of A. thaliana ovules serves as a general model for angiosperm ovule development. Numerous
studies have characterized the development of wild-type A. thaliana ovules (Modrusan et al., 1994; Robinson-
Beers et al., 1992; Schneitz et al., 1995). In addition, molecular and genetic studies have made steady progress
to identify and predict additional loci that regulate steps of Arabidopsis ovule development (for reviews see, Gasser
et al., 1998; Grossniklaus and Schneitz, 1998). For these reasons, it is practical to use this member of the
mustard family to define genetic steps of ovule development.
Herein, we describe a new locus that regulates ovule development in Arabidopsis thaliana. Through the
genetic analysis of the interactions between the PFS2 locus and other ovule loci, the function of gene in the
ovule developmental pathways will be examined. Identification of the function of the PFS2 gene on
ovule development could provide a tool to explore important aspects of plant reproduction.
Plant growth and genetics
Plants were grown on sterile soil (Fafard Mix #2, Conrad Fafard Inc., Agawam, MA) at 220C with a fluence rate
of ~100 pE/m2/s. The pfs2 mutant was crossed with the following ovule mutants: aintegumenta (ant), bell
(bell), fiddle faddle (ffd), inner no outer (/no), nozzle (nzz), and strubbelling (sub). These crosses were made
by fertilizing pfs2 pistils with pollen from other mutants. Segregating F2 plants were examined as described
below. The probabilities of observed segregation ratios in double-mutant populations were determined by chi
Ovule morphology and anatomy
An MZFL3 stereomicroscope was used to examine the morphology (Leica Microsystems,
Heidelberg, Germany) and determine phenotype of ovules in segregating double mutant populations.
The internal anatomy of cleared ovules was visualized using Normarsky optics, where differences in
the refractive index of the samples generated contrast. Ovules were cleared in BB4.5 (Herr, 1971).
Scanning electron microscopy (SEM)
Single and double mutants were examined using an SEM. Each pistil was dissected along the length of
a carpel, which exposed the ovules, and fixed in FAA (10% formalin, 5% acetic acid, 50%
ethanol). Specimens were dehydrated in a graded ethanol series and dried with a critical point
dryer. Dried specimens were mounted on stubs, dissected, sputter-coated with platinum, and
examined using a Hitachi S-4000 field emission scanning electron microscope (Tokyo,
Japan). Microscopic images were processed using Adobe Photoshop 5.5 (Adobe Systems, Inc., San
Ovule development in wild-type plants
The development of wild-type Arabidopsis ovules has been characterized (Robinson-Beer et al.,
1992). An ovule initiates from the growth of cells in the placenta. Ovule primordia differentiate into
three distinct zones: funiculus, chalaza, and nucellus (Fig. 1A). Within the nucellus, a megaspore
mother cell undergoes meiosis. A single meiotic product divides and differentiates into a seven-
celled gametophyte (Fig. 1C). Integument primoridia originate from the chalazal tissue and
differentiate into the inner and outer integuments (Fig. 1B). These integuments protect and nourish
the nucellus and developing embryo sac. Asymmetric growth of the outer integument leads to
the curvature of the distal end of the ovule, until it is adjacent to the funiculus (Fig. 1C).
A B C
Figure 1. A) Ovule primordia emerge from the placenta. These primordia differentiate into three
distinct zones: the nucellus (n), chalaza (c), and funiculus (f). B) Integument primordia emerge from
the chalaza region and develop into the inner integument (ii) and outer integument (oi). A cell within
the nucellus undergoes meiosis, forming three polar bodies and a single functional megaspore. C)
This megaspore divides three times to produce the embryo sac (es), which contains the
antipodals, synergids, egg, and central cell. The outer integument undergoes asymmetric growth,
causing the distal tip of the ovule to curve until it is adjacent to the funiculus.
Morphology of pfs2 ovules
The pfs2 mutants displayed defects in megaspore and gametophyte differentiation. The embryo sac
in pfs2 mutants was disorganized, often containing fewer cells than normal (Figure 2). In addition,
the growth of integuments was retarded and often displayed aberrant morphology (Figure 2). Only
a small number of ovules made anatomically normal embryo sacs; most had fewer than the
normal complement of seven cells.
stage 1-11 stage 2-111 stage 3-VI
Figure 2. The ovule anatomy of pfs2 and of wild-type were compared. Whole-mount preparations
were depicted with optical sections. A) At stage 1-11, ovule primordia began to differentiate into
the funiculus, chalaza, and nucellus regions. B) At stage of 2-III, the inner integument primordia
(iip) and outer integument primordia (oip) emerged from the chalaza region in the wild type. C) In
the pfs2 mutant, the size of the megaspore mother cell (mmc) was highly reduced compared to the
mmc of the wild type. Additional cells, marked with arrows, occupied the region where normally
there would be the megaspore mother cell. D) The embryo sac of a wild-type ovule contained polar
nuclei (pn), an egg cell (e), and synergids (s). (E) In the pfs2 mutant, a reduced number of cells
was detected in its embryo sac.
Effects of pfs2 locus on fecundity
The number of viable ovules in randomly selected pistils was counted in wild-type and pfs2
mutant plants. Carpels from the pfs2 mutants formed an average of 2.0 ï¿½1.2 viable seeds, while
wild-type carpels averaged 47.2 ï¿½4.5 ovules. Thus, the pfs2 mutation clearly caused a
significant reduction in fertility (P = 3.7 X 10-22).
The frequencies of ovule phenotypes in the F2 populations that were segregating for multiple mutations were tabulated.
Parent genotype Parent phenotype WT:pfs2:SM:DM Confidence level (X2) p-value
pfs2/+bel/+ Wild type 15:1:6:2 3.56 0.31
pfs2/+ffd/+ Wild type 19:5:3:1 1.97 0.58
pfs2/-Hno/+ Wild type 30:8:4:2 3.52 0.32
pfs2/+nzz/+ Wild type 21:6:9:2 0.74 0.86
pfs2/+sub/+ Wild type 23:12:4:3 4.14 0.25
The segregations of mutations closely correlated with the expected Mendelian ratio of 9:3:3:1. [WT: pfs2 : single mutant (SM): double mutant (DM)]. A chi-
squared test, which compared the observed frequencies with the expected frequencies, was used to determine the probability of these distributions (p-value).
The phenotypes of ovules from segregating F2 populations were determined using a
stereomicroscope (Table 1). The populations segregated for mutations closely correlate with
the expected Mendelian ratio of 9:3:3:1 (wild-type: single mutant A: single mutant
B: double mutant AB). We found the following results in the double-mutant analysis: (1) ant is
epistatic to pfs2 (Fig. 3); (2) bell and ffd exhibited additive genetic interaction with pfs2 (Fig. 4-5);
(3) synergistic genetic interactions existed between pfs2 and ino, nzz, and sub (Fig. 6-8).
Figure 3. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In ant
mutants, integuments failed to emerge from the chalaza. B) The pfs2/-ant/- double mutant was
not distinguishable from the ant single mutant. C) The megaspore mother cell was absent from
the nucellus (n) of ant mutants. D) In the internal anatomy of the pfs2/-ant/- double mutant, the
phenotypic characteristics of the ant single mutant were observed. This evidence indicated that
the ANT was epistatic to the PFS2.
Figure 4. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) The
bell single mutant failed to form either an inner or outer integument. Instead, the mutant
developed into an abnormal integument-like structure (ils). B) The embryo sac was absent in the
bell single mutant. C) The pfs2/-bell/- double mutant exhibited a bell-shaped integument
structure, which was a typical characteristic of the bell single mutant. In addition, the growth of
the integument-like structure was reduced. D) The pfs2/-bell/- double mutant failed to develop
an embryo sac.
Figure 5. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the
ffd single mutant, the orientation and shape of the integument cells were irregular, which led an
abnormal ovule morphology. In the ffd single mutant, the cells of the adjacent ovules sometimes
fused. B) The ffd mutant's outer integument (oi) did not fully develop, so did not encapsulate the
inner integument (ii). C) In addition to having the ffd single-mutant characteristics, the pfs2/-
ffd/- double mutant had pfs2 characteristics, such as aberrant integument expansion and
shortened integuments. D) No embryo sac developed in pfs2/-ffd/- double mutants.
PFS2 regulates ovule zonation
Examining genetic interactions is a valuable tool for determining the function of loci in
developmental pathways. There are three common outcomes of double mutant analysis:
additive, epistatic, and synergistic interactions.
An epistatic interaction results when one mutation masks the phenotypic expression of another
mutation. Data revealed that the ant pfs2 double mutant was indistinguishable from the ant
single mutant (Fig. 3); ant was epistatic to pfs2. Reduction in cell division in the integument primordia
of ant mutants resulted in premature termination of ovule development (Baker et al., 1997). For
epistatic interactions, it is generally concluded that one mutation precedes another on a
developmental pathway. These data indicated that ant acts before pfs2 in ovule development.
When two mutations regulate independent and/or parallel pathways, double mutants are termed
additive phenotypes. This type of interaction was observed in pfs2 mutants in the bel 1 and ffd
mutant backgrounds. The bel 1 mutant is involved in determining ovule identity (Modrusan et al.,
1994; Robinson-Beers et al., 1992). Regulating a pathway independent from the bel 1 mutant, the
ffd mutant is involved in epidermal differentiation of ovules. The observed additive interactions
indicated that BEL 1 and FFD act independently of PFS.
Synergistic interactions were observed between pfs2 and other mutations that affect ovule
patterning (Figs. 6-8). When two loci perform similar functions, the double mutant, when compared
to the single mutants, exhibits a synergistic phenotype. Differentiation of the chalaza, integuments,
and nucellus is regulated by patterning genes. The phenotype of pfs2 indicated that PFS2
regulates differentiation of the integuments and nucellus (Fig. 2). The nzz locus encodes a novel
protein that is reported to be essential for nucellus zonation and sporocyte formation (Schiefthaler et
al., 1999; Yang et al., 1999). The SUB locus encodes a leucine-rich repeat kinase. The Sub-
phenotype and gene indicate this locus is involved in communication during the morphogenesis of
outer integuments. The INO locus is required for zonation and differentiation of the outer
integuments (Villanueva et al., 1999). The SUB, INO, and NZZ loci all regulate ovule patterning
and morphogenesis. The synergistic interaction of pfs2 with these three mutants indicates
PFS2 regulates similar processes. Thus, our analyses suggest that pfs2 orchestrates ovule patterning
of the integuments and nucellus in conjunction with SUB, INO, and NZZ.
Figure 6. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the
ino mutant, the outer integument (oi) primordia failed to develop. B) An embryo sac (es) formed
in the ino mutant. C) The pfs2/-ino/- double mutant had reduced growth of the outer integument,
thus resembling the development of the ino single mutant. In addition, the inner integument (ii)
was bifurcated. D) The double mutant lacked an embryo sac. In addition, the inner integument
bifurcated into finger-like projections. These projections appeared similar to telomes, which were
the evolutionary progenitors of the inner integument.
Figure 7. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the
nzz mutants, the integument morphology was similar to that of wild-type, although, the ovules
were reduced in size. B) In the nzz mutants, the embryo sac failed to form. C&D) In the pfs2/-
nzz/- double mutant, the integument primordia failed to develop. The reduction in size of the
inner and outer integument (ii, oi, respectively) led to the nucellus being exposed.
Figure 8. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) The
integument morphogenesis was disrupted in the sub mutant. In the sub single mutant, the
orientation of the integument cells was abnormal. B) In the sub single mutant, the megaspore
mother cell failed to differentiate into an embryo sac. C) The pfs2/-sub/- double mutant had
finger-like structures, which appear analogous to telomes. D) The embryo sac was absent in the
pfs2/-sub/- double mutant.
Integuments protect and nourish developing gametes and seeds. Paleobotanists have proposed that
the inner integument originated from the fusion of the sterilized sporangia (for a review see, Kenrick
and Crane, 1997). In a number of extinct taxa, the fossils had sterilized sporangia (termed telomes)
that partially or completely surrounded a fertile sporangium (Kenrick and Crane, 1997). As
female reproductive structures evolved, telomes fused together along their entire length, producing
an integument. In our phenotypic analyses of pfs2 double mutants, involving the ino, and sub loci,
ovules had telome-like structures. This ovule morphology is believed to be the evolutionary precursors
of ovules. The mimicking of telomes indicates that the radially symmetrical sporangia would have
to organize their growth around the centrally located fertile sporangia. Each of these genes was
involved in ovule patterning and acted as a communication system during the growth of
sterilized sporangia around fertile sporangium. Thus, it is probable that PFS2, SUB, and INO loci took
on roles in ovule patterning as telomes evolved into an integument.
Genetic models of ovule development demonstrate a close interplay between the development of
the gametophyte and integuments (Gasser et al., 1998; Grossniklaus and Schneitz, 1998). Our
present investigation of the pfs2 mutant phenotype revealed that this locus is necessary for
appropriate integument growth and megaspore mother cell differentiation. Our morphological
and histological analyses of the double mutant of pfs2 with other ovule mutants provided evidence
that PFS2 gene plays a vital role in the morphogensis of the ovule. Further work will be done
to determine if PFS2 gene specifies identity along the proximal-distal axis during ovule development
or by regulates cell polarity in a developmental field.
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