Journal of Undergraduate Research
Volume 4, Issue 8 - April 2003
Genetic Analyses of the Pretty Few Seeds2 Locus on Ovule Development
Soon Hwang
ABSTRACT
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.
INTRODUCTION
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.
METHODS
Plant growth and genetics
Plants were grown on sterile soil (Fafard Mix #2, Conrad Fafard Inc.,
Agawam, MA) at 22°C with a fluence rate of ~100 µE/m2/s.
The pfs2 mutant was crossed with the following ovule mutants:
aintegumenta (ant), bel1 (bel1),
fiddle faddle (ffd), inner no outer (ino),
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 squared analyses.
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).
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 Jose, California).
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). 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.
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. 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-II, 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.
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 ±1.2 viable seeds, while wild-type
carpels averaged 47.2 ±4.5 ovules. Thus, the pfs2 mutation clearly
caused a significant reduction in fertility (P = 3.7 X 10-22).
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 bel1 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 bel1 single mutant. C) The pfs2/-bel1/-
double mutant exhibited a bell-shaped integument structure, which
was a typical characteristic of the bel1 single mutant. In addition,
the growth of the integument-like structure was reduced. D) The pfs2/-bel1/-
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.
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. 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. Baker, S. C., Robinson-Beers, K., Villanueva, J. M.,
Gaiser, J. C. and Gasser, C. S. (1997). Interactions among genes regulating
ovule development in Arabidopsis thaliana. Genetics 145, 1109-1124. Back to the Journal of Undergraduate
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Ovule morphology and anatomy
Scanning electron microscopy (SEM)
RESULTS
Ovule development in wild-type plants
Morphology of pfs2 ovules
Effects of pfs2 locus on fecundity
Table 1
The frequencies of ovule phenotypes in the F2 populations that were
segregating for multiple mutations were tabulated.
Parent genotype
WT:pfs2:SM:DM
pfs2/+bel/+
pfs2/+ffd/+
pfs2/+ino/+
pfs2/+nzz/+
pfs2/+sub/+
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).
Double-mutant analysis
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) bel1 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).
DISCUSSION
PFS2 regulates ovule zonation
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.
Ovule evolution
CONCLUSION
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