Copyright  2000 by the Genetics Society of America
SHORT INTEGUMENTS 2 Promotes Growth During Arabidopsis
Reproductive Development
Jean Broadhvest, Shawn C. Baker1 and Charles S. Gasser
Section of Molecular and Cellular Biology, University of California, Davis, California 95616
Manuscript received November 30, 1999
Accepted for publication March 2, 2000
ABSTRACT
The short integuments 2 (sin2 ) mutation arrests cell division during integument development of the
Arabidopsis ovule and also has subtle pleiotropic effects on both sepal and pistil morphology. Genetic
interactions between sin2 and other ovule mutations show that cell division, directionality of growth, and
cell expansion represent at least partially independent processes during integument development. Doublemutant analyses also reveal that SIN2 shares functional redundancy with HUELLENLOS in ovule primordium outgrowth and proximal-distal patterning and with TSO1 in promotion of normal morphological
development of the four whorls of primary floral organs. All of these observations are consistent with
SIN2 being a promoter of growth and cell division during reproductive development, with a primary role
in these processes during integument development. On the basis of the floral pleiotropic effects observed
in a majority of ovule mutants, including sin2, we postulate a relationship between ovule genes and the
evolutionary origin of some processes regulating flower morphology.
P
LANT morphogenesis is dependent on tight integration of cell division and cell expansion. Morphogenesis often involves coordinated growth among different cell lineages to form single structures, implying
regulation through intercellular communication and
non-cell-autonomous developmental signals. In recent
years, genetic and molecular approaches have led to
significant new insights into the regulation of some aspects of morphogenesis, including the control of floral
organ identity and the maintenance of the shoot apex
(for review see Meyerowitz 1997). Despite these advances, we have little information on the specifics of
regulation of directional growth and cell division during
formation of individual plant organs (Meyerowitz
1997; Schneitz et al. 1998b).
The bitegmic Arabidopsis ovule is being used as a
morphogenetic model to help understand the regulation of growth and organogenesis, and a number of
genes regulating ovule development have been identified through genetic mutant screens (reviewed in Angenent and Colombo 1996; Gasser et al. 1998; Schneitz
1999). These genes can be separated in two different
classes on the basis of their effects on growth. The first
class encompasses genes that promote or suppress
growth, mostly through regulation of cell division or
cell expansion. huellenlos (hll) and aintegumenta (ant)
mutations arrest integument growth prior to or during
Corresponding author: Charles S. Gasser, Section of Molecular and
Cellular Biology, University of California, 1 Shields Ave., Davis, CA
95616. E-mail: [email protected]
1
Present address: Illumina, Inc., Suite 200, 9390 Towne Centre Dr.,
San Diego, CA 92121.
Genetics 155: 899–907 ( June 2000)
initiation (Elliott et al. 1996; Klucher et al. 1996;
Baker et al. 1997; Schneitz et al. 1998a). ANT and
HLL were also shown to be functionally redundant in
promoting ovule primordia growth and patterning of
the ovule (Schneitz et al. 1998a). SUPERMAN (SUP)
and INNER NO OUTER (INO) act as growth suppressor
and promoter of the outer integument, respectively,
and both genes are needed for development of the
asymmetric form of this structure (Gaiser et al. 1995;
Sakai et al. 1995; Villanueva et al. 1999). The short
integuments 1 (sin1) mutations suppress growth of the
integuments by impeding cellular expansion in these
structures (Robinson-Beers et al. 1992; Ray et al.
1996a). The second group of ovule growth loci help
determine the shape or identity of the growing tissue.
Weak tso1 mutations affect directional control of cell
growth and division, resulting in relatively disorganized
integument tissues (Hauser et al. 1998). In bell1 (bel1)
mutants, an integument-like structure grows in place of
the integuments, apparently due to loss of integument
identity (Robinson-Beers et al. 1992; Modrusan et al.
1994; Ray et al. 1994). The discovery of novel genes
involved in ovule development shows the complexity
and redundancy of growth regulation in a relatively
simple structure and adds to our knowledge of regulation of plant morphogenesis.
Interestingly, mutations in a majority of the genes
regulating ovule development also cause floral aberrations. For example, ap2 mutations affect ovule integuments and the identity of some floral organs (Bowman
et al. 1989; Kunst et al. 1989; Jofuku et al. 1994; Modrusan et al. 1994). SUP appears to control expression of
the floral class B genes and the ovule gene INO through
900
J. Broadhvest, S. C. Baker and C. S. Gasser
possibly similar non-cell-autonomous mechanisms
(Sakai et al. 1995; Villanueva et al. 1999). These observations suggest a molecular relationship between floral
and ovule morphogenic pathways.
We report here the characterization of short integuments 2 (sin2), a novel mutation arresting cell division
in both integuments of Arabidopsis ovules. Genetic interactions between sin2 and other ovule mutations
show that directional regulation, cell expansion, and
cell division are partially independent processes governing integument development. SIN2 shares functional
redundancies with at least two different genes regulating
flower and ovule growth. Floral pleiotropic effects of
sin2 and other ovule mutants lead us to postulate a
connection between some genes regulating ovule development and the evolution of floral organs.
MATERIALS AND METHODS
Plant material: sin2 was isolated from ethyl methanesulfonate-mutagenized Landsberg erecta (Ler) ecotype as described
previously for other ovule mutants (Robinson-Beers et al.
1992). Mutants were backcrossed at least three times to wildtype Ler plants prior to further analysis. Plants were grown as
described previously (Kranz and Kirchheim 1987; Robinson-Beers et al. 1992). Pistil measurements and ovule counts
were performed under a Zeiss (Oberkochen, Germany) SV8
stereomicroscope. Epidermal cell counts were done from scanning electron micrographs. Because of the three-dimensional
nature of the ovules, counts were compiled from multiple
images and represent a best estimate of the true cell counts.
Genetic mapping of SIN2: A mapping population (F2 progeny) was generated by crossing sin2 and Co-3 (Columbia) wildtype plants. Using DNA samples (Edwards et al. 1991) from
34 sin2 plants in the mapping population, sin2 was mapped
to chromosome II at a position 2.5 cM south of the cleaved
amplified polymorphic sequence (CAPS) m429 marker
(Konieczny and Ausubel 1993) and 3.5 cM north of the
simple sequence length polymorphisms (SSLP) AthBIO2
(Bell and Ecker 1994). Further analysis was done by genotyping 918 plants from the mapping population for both m429
and AthBIO2 loci, which allowed for unambiguous determination of the genotype at the SIN2 locus.
Scanning electron microscopy: Samples were prepared as
described previously (Hauser et al. 1998) and were examined
with a Hitachi (Tokyo) S3500N scanning electron microscope
at an accelerating voltage of 5 or 10 kV. Images were acquired
digitally and were edited for publication in Photoshop 4.0 for
Macintosh (Adobe Systems, Inc., San Jose).
Confocal laser scanning microscopy: Arabidopsis inflorescences were fixed and stained with fluorescent periodic acidSchiff reagent and were examined under a Zeiss LSM410 laser
scanning confocal microscope as described previously (Baker
et al. 1997).
Double-mutant analyses: Pollen from plants homozygous
for a specific mutation was used to fertilize emasculated flowers
of sin2 heterozygous plants. Seeds were collected from these
crosses and sowed as described (Kranz and Kirchheim 1987;
Robinson-Beers et al. 1992). All F1 plants were phenotypically
wild type. F2 families, showing segregation for both sin2 and
the mutation under study, were further analyzed. When possible or in doubt, a putative double-mutant plant was backcrossed to a wild-type Ler plant to confirm the presence of
both mutations by observation of their segregation in the
backcross F2⬘ progeny.
In situ hybridizations: Sections of wild-type or sin2 inflorescences 8- to 10-␮m thick were prepared and hybridized with
digoxigenin-UTP-labeled probes as described previously
(Vielle-Calzada et al. 1999). BEL1 probes were generated
from pLR115 (gift from Linda Margossian and Robert L.
Fischer, University of California, Berkeley, CA) as described
(Reiser et al. 1995). ANT probes were generated from pcDNA5
(gift from David Smyth, Monash University, Melbourne) as
described (Elliott et al. 1996).
RESULTS
Wild-type ovule development: Detailed descriptions
of ovule development in Arabidopsis have been presented previously (Robinson-Beers et al. 1992;
Schneitz et al. 1995). A summary is presented here.
The Arabidopsis ovule arises as a cylindrical primordium
from the placental tissues found adjacent to both sides
of the septum inside the pistil (Figure 1A). Initiation
of both integuments occurs in the midzone of the ovule
primordium and defines this zone as the chalaza (Figure
1C). The inner integument initiates as a ring of cells
around the circumference of the ovule primordium and
exhibits symmetrical cylindrical growth until it surrounds and encases the nucellus (Figure 1, C and E).
After inner integument initiation, the outer integument
initiates on the abaxial (toward the base of the carpel)
side of the ovule primordium and has an asymmetric
growth pattern (Figure 1, C and E). Parallel with the
development of both integuments from the chalaza, the
distal portion of the primordium differentiates to form
the nucellus while the proximal portion will constitute
the funiculus. Concomitant but opposite asymmetric
growth of the funiculus and the outer integument give
the ovule its final configuration (Figure 1G).
sin2 ovule phenotype and ontogeny: sin2 is a singlelocus recessive mutation that produced complete female
sterility in homozygous plants. The frequency of mutant
plants was lower than the expected 3:1 ratio in segregating populations [229 wt : 54 sin2 (ratio 4 : 1, ␹2 ⫽ 0.15,
P ⫽ 0.70 )]. This altered segregation ratio indicated
either incomplete penetrance or reduced viability of
sin2 plants. Using flanking markers, we determined the
genotype at the SIN2 locus in 918 plants from a segregating mapping population (see materials and methods). All 165 homozygous sin2 plants exhibited the mutant phenotype, demonstrating complete penetrance.
Ovules of sin2 plants developed as wild type up to the
point when both integuments initiated (compare Figure
1, A and C, and Figure 1, B and D). The growth of
both integuments arrested shortly after initiation, and
at anthesis sin2 ovules had two short integuments comprising fewer cells than wild-type integuments (Figure
1F). The epidermis of wild-type outer integument is
composed of ⵑ200 cells arranged in 9–10 files whereas
sin2 outer integument comprised only ⵑ10–30 cells.
SIN2 Promotes Reproductive Growth
Figure 1.—Scanning electron micrographs of wild-type (A,
C, E, and G) and sin2 (B, D, F, and H) ovules. Stages of ovule
development according to Schneitz et al. (1995). (A) Stage
1-I, wild-type ovules and (B) stage 1-II, sin2 ovules arise as
cylindrical primordia from placental tissue. (C and D) Stage
2-III, both integuments have initiated. (E) Stage 2-V, wild-type
integuments grow toward the nucellus apex. (F) Stage 2-V,
sin2 integument growth arrests before the nucelli are covered.
(G) Stage 3-VI (anthesis), asymmetric growth of the outer
integument and the funiculus results in amphitropous wildtype ovules. (H) Stage 3-VI (anthesis) sin2 integuments are
short and do not encase the nucellus; f, funiculus; ii, inner
integument; n, nucellus; oi, outer integument; p, primordia.
Bars, 25 ␮m.
Slight variations in integument length were observed
even among ovules from a single sin2 pistil, but both
integuments were always substantially shorter than wild
type, leaving the nucelli fully exposed (Figure 1H). On
the basis of these observations, SIN2 appears necessary
for progression of integument growth following initiation of these structures during Arabidopsis ovule development. Confocal laser scanning microscopy observations of sin2 nucelli showed that megasporogenesis was
901
Figure 2.—sin2 floral effects. Stages of floral development
according to Bowman (1994). (A) At anthesis (stage 13), a
wild-type Arabidopsis pistil is composed of a stigma, a short
style, and two valves joined by the replum. (B) Stage 13 sin2
pistil bearing an outgrowth on one valve (arrowhead). (C)
Stage 16 sin2 pistil showing postpollination characteristics of
wild-type pistils (elongated valve cells and interspersed stomata; Bowman 1994) but with the style/stigma split in the axis
of one of the valves (arrow) and an outgrowth (arrowhead)
present on the valve in the axis of the cleft stigma. (D) Stage
12 wild-type floral bud. Sepal tips have a smooth edge made
up of small uniform cells. (E) Stage 11 sin2 floral bud. Sepals
have jagged tips made up of cells of variable size and shape.
p, petal; r, replum; se, sepal; sg, stigma; st, style, v, valve. Bars,
250 ␮m.
arrested before formation of the megaspore mother cell
(data not shown).
Sin2 floral phenotypes: Besides having effects on ovule
development, subtle morphological aberrations were
also observed in the gynoecia (pistils) and sepals of
sin2 flowers. At anthesis, a wild-type Arabidopsis pistil
comprises an apical stigma, a short style, and two basal
valves separated by a replum (Figure 2A). Most sin2
pistils had a cleft stigma and/or style (Figure 2C). This
cleft was always in the axis of one of the valves and not
in the plane of the replum. The valve on the cleft side
of the stigma sometimes bore an outgrowth (Figure 2,
B and C) and, less frequently, both valves of a pistil bore
outgrowths (Table 1). The tissue forming the outgrowths had the appearance of valve tissue, except at
the tip, where it did not resemble any floral tissue. These
aberrant pistil phenotypes were not observed in all
902
J. Broadhvest, S. C. Baker and C. S. Gasser
TABLE 1
Effects of sin2 on mature pistil morphology
Lera
sin2b
a
b
Average
length
(mm)
% of pistils with
cleft stigma/style
1 outgrowth
2 outgrowths
Average ovule
number/pistil
2.9 ⫾ 0.5
2.0 ⫾ 0.4
0
60
0
30
0
14
56 ⫾ 4
33 ⫾ 7 (n ⫽ 71)
% of pistils bearing:
Mature pistils from emasculated wild-type flowers, n ⫽ 10.
n ⫽ 100 unless otherwise specified.
flowers of sin2 plants (Table 1) and the cleft and outgrowth varied in size (Figure 2, B and C). At anthesis,
sin2 pistils were also shorter and bore fewer ovules than
pistils from emasculated wild-type flowers (Table 1).
Distribution of the ovules along the placenta was also
affected in sin2 pistils with the distance between ovules
being generally greater than in wild type (not shown).
Sepals were also affected in sin2 flowers. Wild-type
sepal margins are smooth and consist of very small
rounded cells (Figure 2D; Bowman 1994). The sepals
of all sin2 flowers had fewer such cells on their margins,
especially toward the tips. The absence of margin tissue
made the tips of sin2 sepals jagged (Figure 2E). While
sin2 plants reached the same final size as wild-type
plants, they exhibited a slightly slower growth rate. No
other vegetative effects of sin2 were observed.
Genetic interactions: Double mutants were generated
to investigate the interactions between SIN2 and other
genes regulating ovule growth and development. The
observed segregation ratios were as expected for each
genetic interaction examined (Table 2) and no partial
dominance was observed for any of the segregating mutations. Except as noted otherwise, flowers of doublemutant plants had phenotypes that were consistent with
simple addition of the floral effects of the two single
mutations (data not shown).
sin2 ant-5: ANT encodes a putative transcription factor
containing two AP2 domains (Elliott et al. 1996;
Klucher et al. 1996) and has recently been shown to
promote growth of all Arabidopsis lateral organs (Mizukami and Fischer 2000). During floral morphogenesis,
ant mutations affect the expansion and the number of
primary floral organs, but have more severe effects on
integument development (Elliott et al. 1996; Klucher
et al. 1996; Baker et al. 1997; Krizek 1999). Ovules
of putative null ant-5 mutants (Gln227 to stop codon,
eliminating the C-terminal half of the ANT protein including both AP2 domains; B. A. Krizek, personal communication) fail to develop integuments, forming at
most a small integumentary ridge from the chalazal
region (Figure 3A; Baker et al. 1997). ANT is thought
to be necessary for promotion of integument primordia
initiation and growth (Elliott et al. 1996; Klucher et
al. 1996; Baker et al. 1997; Schneitz et al. 1998a). sin2
ant-5 double-mutant ovules were not different in appearance from those of ant-5 single mutants (Figure 3B),
indicating that ant is epistatic to sin2 with respect to
ovule development.
sin2 ino-1: ino mutations are specific to ovules and
affect only outer integument development. In the strong
ino-1 allele, ovules fail to initiate outer integuments but
development of the inner integuments is not impaired
and is similar to wild type (Figure 3C; Baker et al. 1997).
sin2 ino double mutants displayed additivity as their
ovules lacked outer integuments and bore short inner
integuments at anthesis (Figure 3D).
sin2 bel1-6: Mature bel1-6 ovules do not have integuments but bear an integument-like structure (ILS) in
TABLE 2
Double-mutant analysis with sin2
F2 segregation
Observed
Expecteda
␹2
P
12:3:2:2 (WT : ant : sin2 : sin2 ant)
21:6:4:2 (WT: ino : sin2 : sin2 ino)
20:7:5:1 (WT: bel1 : sin2 : sin2 bel1)
124:29:32:5 (WT: sin1 : sin2 : sin2 sin1)
46:22:8:4 (WT: hll : sin2 : sin2 hll)
37:11:8:2 (WT: sup : sin2 : sin2 sup)
63:19:10:4 (WT: tso1 : sin2 : sin2 tso1)
12:4:3:1
12:4:3:1
12:4:3:1
16:4:4:1c
12:4:3:1
12:4:3:1
12:4:3:1
1.61
0.38
0.28
1.09
3.70
1.4
1.7
0.65
0.94
0.96
0.78
0.30
0.70
0.63
Mutant
ant-5
ino-1
bel1-6
sin1-2
hll-1
sup-5
tso1-3
b
sin2 mutants segregate ⵑ4:1.
Wild type.
c
sin1 mutants segregate ⵑ4:1 (Robinson-Beers et al. 1992).
a
b
SIN2 Promotes Reproductive Growth
903
Figure 3.—Scanning electron micrographs of single- and double-mutant ovules at anthesis (stage 3-VI; Schneitz et al. 1995).
(A) ant-5 ovules have integumentary ridges in place of integuments. (B) sin2 ant-5 ovules are similar to ant-5 ovules. (C) ino-1
ovules fail to initiate outer integuments but development of the inner integuments is not impaired. (D) sin2 ino-1 ovules lack
outer integuments and bear short inner integuments. (E) bel1-6 ovules have ILS in place of integuments. Protuberances (arrowheads) formed from the ILS. The funiculi consist of more cells than those of wild type. (F) sin2 bel1-6 ovules had smaller ILS
than bel-6 ovules and protuberances were present. The funiculi are shorter than bel1-6 ovules and display an increase in diameter
relative to sin2 ovules. (G) sin1-1 integuments are made up of the same numbers of cells as wild type, but are short due to
impeded cell expansion. (H) sin2 sin1-1 integument cell numbers are as in sin2 ovules but with reduced cell expansion. (I) hll-1
ovules have short funiculi and nucelli, and only limited integument initiation from the chalaza is sometimes observed. Occurrence
of collapsed cells in the distal portion can be observed. (J) sin2 hll-1 pistils bear only abortive ovule primordia where cell collapse
is observed. (K) sup-5 outer integuments are more radially symmetrical than wild type. (L) sin2 sup-5 ovules are similar to sin2
ovules. f, funiculus; ii, inner integument; ils, integument-like structure; n, nucellus; oi, outer integument. Bars, 50 ␮m.
the chalazal region (Figure 3E; Robinson-Beers et al.
1992; Modrusan et al. 1994; Ray et al. 1994). At anthesis,
protuberances are often observed on the distal surface
of the ILS that may take on nucellar or carpel identity
later during its development (Robinson-Beers et al.
1992; Modrusan et al. 1994; Ray et al. 1994; Herr 1995;
Gasser et al. 1998). bel1 funiculi contain more cells and
can be longer than those of wild-type ovules (Figure 3F;
Robinson-Beers et al. 1992; Schneitz et al. 1997). The
BEL1 homeodomain protein, a putative transcription
factor (Reiser et al. 1995), appears to be necessary for
establishment of integument identity and for cessation
of cell division in the funiculus (Robinson-Beers et al.
1992; Modrusan et al. 1994; Ray et al. 1994). At anthesis,
the ovules of sin2 bel1-6 plants bore smaller ILS than
bel-6 ovules. The funiculi did not show the abnormal
elongation observed in bel1 single mutants, but did show
a marked increase in diameter relative to sin2 single
mutants (Figure 3F). Protuberances were present on
the ILS of the sin2 bel1-6 ovules at anthesis (Figure 3F)
and some of the ovules became carpelloid (not shown).
sin2 sin1-2: In the Ler ecotype, integuments of sin1-2
ovules do not fully elongate, and the ovules superficially
resemble those of sin2. But in contrast to sin2 mutants,
integuments of sin1-2 ovules have the same number of
cells as wild type and their short length results from
a reduction in cell elongation (Figure 3G; RobinsonBeers et al. 1992; Lang et al. 1994; Ray et al. 1996a,b).
The integuments of sin2 sin1-2 ovules were more reduced in size than in either of the single mutants (Figure
3H). The number of cells in both integuments appeared
similar to those of sin2 single mutants, but most of these
cells were smaller than in those of sin2 single mutants
(Figure 3H). These results show that some expansion
must occur in the integument cells of sin2 ovules.
sin2 hll-1: Strong hll alleles (e.g., hll-1) arrest ovule
development at an early stage. At anthesis, ovules of
hll-1 plants have short funiculi and nucelli, and only
904
J. Broadhvest, S. C. Baker and C. S. Gasser
limited integument initiation from the chalaza is sometimes observed (Figure 3I). A striking phenotype of the
hll-1 allele is the occurrence of collapsed cells in the
distal portion of the ovules (Figure 3I; Schneitz et al.
1998a). HLL appears to have a role in the early steps
of ovule development and in regulation of cell death
in these structures (Schneitz et al. 1998a). The combination of sin2 and hll-1 had an even greater affect on
ovule development than either single mutant. At anthesis, sin2 hll-1 pistils bore only rudimentary ovule primordia that appeared to have arrested following only very
limited growth, and cell death was observed in the entire
abortive primordia (Figure 3J). This synergistic phenotype indicates a role for SIN2 in promotion of the earliest
stages of ovule primordia growth (Figure 3J).
sin2 sup-5: Arabidopsis sup flowers have supernumerary stamens, reduced carpels, and aberrant ovules
(Schultz et al. 1991; Bowman et al. 1992; Gaiser et al.
1995; Sakai et al. 1995). Compared to wild type, sup
ovules have greater growth of the outer integument on
their adaxial side, leading to a more symmetrical outer
integument (Figure 3K; Gaiser et al. 1995; Sakai et al.
1995). SUP has been proposed to regulate cell division
on the adaxial side of the outer integument during
ovule development (Gaiser et al. 1995; Sakai et al.
1995), possibly through negative regulation of INO (Villanueva et al. 1999). sin2 sup-5 ovules were indistinguishable from those of sin2 single mutants, indicating
that sin2 is epistatic to sup during ovule morphogenesis
(Figure 3L).
sin2 tso1-3: While strong tso1 mutants develop highly
reduced, aberrant organs in the three inner floral
whorls (Liu et al. 1997), the weaker tso1-3 allele produces
relatively normal flowers with aberrant ovule integuments (Figure 4, A and C; Hauser et al. 1998). In tso1-3
ovules, cells of both integuments are misshapen and
are not organized in files due to an apparent loss of
directional regulation of cell elongation and division
(Figure 4A; Hauser et al. 1998). Plants homozygous for
both sin2 and tso1-3 had flowers exhibiting morphological defects in all the primary floral organs. In the firstformed sin2 tso1-3 flowers, the margins of organs in the
first three whorls were jagged and the pistils had split
styles/stigmas. Later flowers had reduced pistils and
more severely affected floral organs (Figure 4D). This
suggests that SIN2 plays a larger role in floral organ
development than indicated by the single-mutant phenotype and that SIN2 and TSO1 are genetically partially
redundant during flower development. Because of the
strong acropetal effect, only the first sin2 tso1-3 flowers
developed pistils bearing ovules. These ovules had short
integuments with slight evidence of cell disorganization
(Figure 4B). This result suggests that sin2 is additive
with tso1-3 during ovule development.
Expression of BEL1 and ANT in sin2 ovules: To learn
more about the basis of the phenotypic effects of the
sin2 mutation, we investigated the patterns of expression
Figure 4.—Scanning electron micrographs of interactions
between tso1-3 and sin2. (A) tso1-3 ovules have disorganized
integument tissues. (B) sin2 tso1-3 ovules have short integuments with evidence of tissue disorganization. (C) tso1-3 flowers have jagged sepal tips and may display slight pistil aberrations. (D) sin2 tso1-3 flowers have reduced pistils and severe
floral organ aberrations. f, funiculus; ii, inner integument; oi,
outer integument; n, nucellus; p, petal; se, sepal; sg, stigma;
sm, stamen; v, valve. Bars, 50 ␮m (A and B) and 250 ␮m (C
and D).
of ANT and BEL1 during sin2 ovule development
through in situ hybridization. In wild-type ovules, both
ANT and BEL1 have been shown to be initially expressed
throughout the ovule primordia, but in later stages of
development expression is restricted to the chalazal region and the emerging integuments (Reiser et al. 1995;
Elliott et al. 1996; Klucher et al. 1996). Comparisons
between wild-type and sin2 ovules were performed on
emerging ovule primordia (Figure 5, A, B, E, and F)
and on ovules where both integuments had initiated
(corresponding to the final sin2 ovule phenotype; Figure 5, C, D, G, and H). For both stages of development,
no differences were observed for the patterns of ANT
or BEL1 mRNA accumulation (Figure 5) between wildtype and sin2 ovules.
DISCUSSION
SIN2 regulates growth throughout reproductive development: All of the mutant phenotypes described herein
support a role for SIN2 as a primary promoter of integument growth and as a secondary growth promoter during other aspects of reproductive morphogenesis. In all
combinations with mutations producing at least one
integumentary structure, sin2 led to reduction in the
number of cells in such structures. This resulted in additive interactions with sin1, bel1, ino, and tso1. Such interactions imply that SIN2 regulates early stages of integument growth and suggest that elongation (SIN1),
SIN2 Promotes Reproductive Growth
Figure 5.—Comparison of ANT and BEL mRNA accumulation in wild-type (A, C, E, and G) and sin2 (B, D, F, and H)
ovules by in situ hybridization. In young flowers of both wild
type (A) and sin2 (B), ANT is expressed in the emerging ovule
primordia. In stage 2-III wild-type (C) and sin2 (D) ovules,
ANT is expressed at highest levels in the chalaza and emerging
integument primordia. BEL mRNA was found throughout
stage 1-I wild-type (E) and sin2 (F) ovule primordia. In stage
2-III wild-type (G) and sin2 (H) ovules, BEL1 mRNA was at
highest levels in the developing integuments and throughout
the chalaza. f, funiculus; ii, inner integument; n, nucellus; oi,
outer integument; ovp, ovule primordia. Bars, 50 ␮m (A and
B), 25 ␮m (C, D, G, and H), and 10 ␮m (E and F).
directionality of growth (TSO1), and cell division (SIN2)
are at least partially parallel processes that must interact
closely to generate appropriate morphogenesis of both
integuments. The epistasis of sin2 over sup (Figure 3L)
was consistent with sup affecting only later stages of
integument growth that never occur in sin2 mutants.
The strong synergism between sin2 and hll, which
nearly eliminates ovule development (Figure 3J), could
not have been predicted from either single-mutant phenotype. A similar phenotype was described for ant hll
905
double mutants (Schneitz et al. 1998a), suggesting that
SIN2, ANT, and HLL have redundant functions in ovule
primordia growth and the proximal-distal patterning of
ovules. A role for SIN2 in funiculus growth is further
indicated by the shorter length of the funiculi of sin2
bel1 mutants relative to bel1 single mutants. Thus, at
least three genes appear to promote ovule primordium
outgrowth and funiculus elongation. HLL must be a
key regulator of these processes as reduced growth is
observed in hll single mutants (Figure 3I). Because ant,
sin2, and ant sin2 ovules had wild-type funiculi, both
ANT and SIN2 must have secondary roles in these processes that become apparent only in the hll background.
The similar synergistic effects of either ant or sin2 with
hll may be an indication that ANT and SIN2 act in a
common secondary pathway. ANT could act first in this
secondary pathway and be a positive regulator of SIN2
activity. This is supported by the observation that transcriptional regulation of ANT is not affected in sin2
ovules (Figure 5, B and D). We also hypothesize that a
low level of SIN2 activity is still present in ant mutants
to explain the slightly stronger effects on ovule development observed in sin2 hll compared to ant hll plants
(Schneitz et al. 1998a) and the fact that no floral effects
were reported in ant tso1-3 double-mutant plants
(Hauser et al. 1998; see below).
An obvious explanation for the sterility of sin2 ovules
is the arrest in megaspore mother cell development.
While it is possible that SIN2 plays a direct role in the
regulation of megagasporogenesis, it was observed previously that mutations leaving exposed nucelli also display arrested megagametogenesis as a secondary effect
of the absence of integuments (Baker et al. 1997; Gasser et al. 1998). A similar hypothesis can be made for
the sin2 ovules.
The plasticity of integument growth observed among
sin2 ovules might result from variation in SIN2 activity,
suggesting that our only sin2 isolate might not represent
a null allele. sin2 was isolated through a genetic screen
based on female sterility and searches for additional
sin2 alleles have not yet been fruitful. Since SIN2 could
be expressed throughout the flower, putative null alleles
might generate a stronger floral phenotype and would
not have been identified as being putative alleles of
this mutation. Ongoing efforts to find other alleles in
populations arising from mutagenized heterozygous
SIN2/sin2 seeds are underway. On the basis of the latest
classical genetic map of chromosome 2 (http://www.
arabidopsis.org/chromosomes/), other characterized
mutations can be found in the vicinity of the SIN2 locus
(65 cM), including some that cause embryo lethality.
Because the correlation between physical and genetic
maps is imprecise, the potential allelism between sin2
and these other mutations must be tested on an individual basis.
We observed complete penetrance of sin2, showing
that the unexpectedly low segregation ratio of this muta-
906
J. Broadhvest, S. C. Baker and C. S. Gasser
tion (closer to 4:1 than the expected 3:1 for a recessive
trait) was due to a reduced frequency of homozygous
sin2 plants in the mature segregating population. The
deficiency in homozygous mutants could result from
reduced production, viability, or vigor of either sin2
embryo sacs or pollen. It could also be due to reduced
germination efficiency or increased seedling mortality
of sin2 plants. Experiments to differentiate among these
possibilities have thus far been inconclusive, but are still
in progress.
SIN2 roles in primary floral organ formation: The
synergistic effects on floral development observed in
sin2 tso1-3 double mutants (Figure 4D) suggest that SIN2
might be expressed throughout flowers, consistent with
the partially aberrant sepals and pistils observed in sin2
single-mutant flowers. The double-mutant phenotype
also suggests that directional cell expansion and cell
division are at least partially compensatory processes in
floral organ formation. A compensatory mechanism has
been proposed to be responsible for the relatively normal shape of maize leaves in the tangled-1 mutant, where
cell division planes are highly aberrant (Smith et al.
1996). Small perturbations in either cell division, as in
sin2, or directional expansion, as in tso1-3 (Hauser et
al. 1998), lead to slight organ deformities, but a combination of these two defects appears to prevent compensation, resulting in malformed organs. An alternative
explanation of our results implicates TSO1 as a negative
regulator of SIN2 expression in the flower. The tso1
mutation would lead to a higher ectopic expression
of altered SIN2 protein activity throughout the flower,
leading to the observed tso1 sin2 floral phenotype.
Pleiotropic floral effects are common among ovule
mutants: A majority of ovule mutants described to date
also have pleiotropic effects on other aspects of flower
development. These mutants include sup (Schultz et
al. 1991; Bowman et al. 1992; Gaiser et al. 1995; Sakai
et al. 1995), tso1 (Liu et al. 1997; Hauser et al. 1998),
leunig (Liu and Meyerowitz 1995), apetala2 (Bowman
et al. 1989; Kunst et al. 1989; Jofuku et al. 1994; Modrusan et al. 1994), and sin2 (this study). The pleiotropic
roles of these genes may be an indication of the evolutionary origin of some of the genes regulating flower
development. Ovules preceded flowers in the evolution
of seed plants. The lateral floral organs represent structures that must have been added to, or modified within,
the reproductive axis below the “ancestral” ovule
(Doyle 1994). Pathways for regulation of formation of
the lateral floral organs would have to derive from genes
in preexisting developmental pathways. Genes regulating ancestral ovule morphogenesis may have taken on
additional roles in regulation of aspects of floral development, while maintaining their roles in ovule development, and would thus exhibit pleiotropic mutant phenotypes like those described above. A comparison of
whole-genome expression analyses between each floral
organ and ovules might help to identify and differentiate genes with dual roles and those that are exclusive
to either floral or ovule development. Genes involved
in both developmental processes might have their origin
in the ovule morphogenetic pathway while genes specific for lateral floral organs would have been recruited
from other pathways. Such analyses would help clarify
the sources of genes that contributed to the evolution
of floral structures.
We thank David Smyth, and Linda Margossian and Robert L. Fischer
for the ANT and BEL1 cDNA clones, respectively. We also thank Beth
A. Krizek for sharing unpublished data and we thank reviewers for
constructive comments on the manuscript. We are grateful to the
members of the Bowman lab for exchanging ideas and help with the
in situ hybridization experiments and Rick Harris for help with the
scanning electron microscopy. Thanks to past and present members
of the Gasser lab for discussions. This work was supported by a grant
from the National Science Foundation (IBN98-08395).
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Communicating editor: V. L. Chandler