INTRODUCTION
Embryogenesis generates the primary plant body of the
seedling which is fairly uniform among flowering plants
(Steeves and Sussex, 1989). How this body organisation is
established in the plant embryo has become a focus of genetic
analysis, using Arabidopsis as a model (Jürgens et al., 1991).
A large-scale screen for mutations affecting the seedling body
organisation identified genes that appear to play specific roles
in one of three different aspects: apical-basal pattern, radial
pattern or shape (Mayer et al., 1991). Mutations in those genes
were shown to alter embryogenesis before or at the heart stage
when the primordia of seedling structures become morphologically
recognisable.
In Arabidopsis and other crucifers, the embryo grows by
oriented cell divisions such that the seedling structures can be
traced back to groups of cells in the early embryo (Schulz and
Jensen, 1968; Tykarska, 1976, 1979; Mansfield and Briarty,
1991; Jürgens and Mayer, 1994). Such regularity of cell
division pattern is not common to all flowering plant embryos
(Natesh and Rau, 1984), and thus it is not obvious how it
relates to embryonic pattern formation. However, recent
studies of embryonic pattern mutants in Arabidopsis have
provided evidence that oriented cell divisions reflect processes
underlying pattern formation. Mutations in the GNOM gene
render the normally asymmetric division of the zygote nearly
symmetric, resulting in an enlarged apical daughter cell which
also divides abnormally; subsequent cell divisions are highly
irregular, yielding a seedling with a grossly perturbed apicalbasal
pattern (Mayer et al., 1993). Mutations in the
MONOPTEROS gene alter the cell division pattern of the basal
region of the embryo from the early-globular stage, producing
seedlings without hypocotyl and root (Berleth and Jürgens,
1993).
Mutations in the FASS (FS) gene were shown to drastically
change the shape of the seedling without altering its body
pattern; furthermore, fs embryos did not display the characteristic
cell shapes or cell arrangements by which the primordia
of seedling structures are normally recognised at the heart stage
(Mayer et al., 1991). These observations raised the possibility
that the seedling body is generated by two distinct processes
in the Arabidopsis embryo, pattern formation and morphogenesis,
and that the FS gene might only be involved in the latter
but not the former; the term ‘morphogenesis’ is used to cover
cell activities, such as oriented cell divisions and cell shape
changes, that generate body shape (for definition of the terms
see e.g. Slack, 1991). To test this idea, we have carried out a
detailed genetic and developmental analysis, and the results
indicate that the FS gene is continuously required for the elaboration
of cell shape and for correct orientation of cell walls
2967Development 120, 2967-2978 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
The pattern of cell division is very regular in Arabidopsis
embryogenesis, enabling seedling structures to be traced
back to groups of cells in the early embryo. Recessive
mutations in the FASS gene alter the pattern of cell division
from the zygote, without interfering with embryonic
pattern formation: although no primordia of seedling
structures can be recognised by morphological criteria at
the early-heart stage, all elements of the body pattern are
differentiated in the seedling. fass seedlings are strongly
compressed in the apical-basal axis and enlarged circumferentially,
notably in the hypocotyl. Depending on the
width of the hypocotyl, fass seedlings may have up to three
supernumerary cotyledons. fass mutants can develop into
tiny adult plants with all parts, including floral organs,
strongly compressed in their longitudinal axis. At the
cellular level, fass mutations affect cell elongation and orientation
of cell walls but do not interfere with cell polarity
as evidenced by the unequal division of the zygote. The
results suggest that the FASS gene is required for morphogenesis,
i.e. oriented cell divisions and positiondependent
cell shape changes generating body shape, but
not for cell polarity which seems essential for pattern
formation.
Key words: fass, embryo, Arabidopsis, morphogenesis, pattern
formation, polarity, shape, elongation
SUMMARY
Mutations in the FASS gene uncouple pattern formation and morphogenesis
in Arabidopsis development
Ramón A. Torres-Ruiz*,† and Gerd Jürgens‡
Institut für Genetik und Mikrobiologie, Lehrstuhl für Genetik, Universität München, Maria-Ward-Strasse 1a, D-80638 München,
FRG
*Author for correspondence
†Present address: Lehrstuhl für Gentechnik Höherer Pflanzen, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, FRG
‡Present address: Lehrstuhl für Entwicklungsgenetik, Universität Tübingen, Spemannstrasse 37-39, D-72076 Tübingen, FRG
2968
from very early stages of embryogenesis to the adult stage.
Thus, mutations in the FS gene uncouple pattern formation and
morphogenesis. Since cell polarity appears not to be affected
in fs embryos, we propose that polarity plays a pivotal role in
pattern formation.
MATERIALS AND METHODS
Plant strains
The wild-type strain used was the Landsberg erecta (Ler) ecotype.
The fass (fs) mutants were isolated on the basis of their seedling phenotypes
following EMS mutagenesis of Ler seeds (Mayer et al.,
1991). Mapping and complementation experiments involved the
marker strains W100 (an ap1; er py; hy2 gl1; bp cer2; ms1 tt3), W134
(ttg ga3) and ttg yi, which were kindly provided by M. Koornneef,
Agricultural University, Wageningen, The Netherlands (for description
of mutants, see Koornneef et al., 1983, 1987; Koornneef and
Hanhart, 1983).
Plant growth conditions and genetic crosses
Plants were grown as previously described (Mayer et al., 1993).
Flowers of plants to be used as female parents were emasculated with
foreceps and cross-pollinated on one of the following 3 days. The
anthers of male parents were either used directly after removal from
flowers or after storage at 4°C for up to 10 days.
Complementation tests
All lines had been isogenised by selfing at least four times. Plants to
be used as parents were tested for fs heterozygosity by phenotypic
analysis of embryos from selfed flowers. Younger flowers from heterozygous
plants were then test-crossed. Each complementation test
was repeated at least once. In total, more than 50, and often up to 400,
F1 seedlings from each combination were phenotyped on agar plates.
Recombination mapping
Pollen from fs heterozygous plants was used to pollinate flowers of
homozygous W100 and ttg yi plants. F1 plants were progeny-phenotyped
for fs heterozygosity. The F2 progeny were scored for W100
marker phenotypes and for segregation of fs seedlings (F3 generation).
Recombination frequencies (RF) were determined as RF = x/(2-x)
where x is the proportion of fs heterozygous plants among all F2 plants
showing the same marker phenotype. Map distances were calculated
from RF using Haldane´s mapping function.
Analysis of seedling phenotypes
Analysis of seedling phenotypes was carried out as described in
Mayer et al. (1993). Dark-grown seedlings were obtained by the
following modification: the Petri dishes were wrapped in aluminium
foil, stored at 4°C for 4 days and incubated at 25°C for up to 2 weeks.
Two batches of (strong) fs seedlings, grown on soil, were sprayed with
10−5 M gibberellin (90% GA3; Sigma) every 5 days during a period
of 3 weeks.
Length measurements of seedlings
For quantitative comparisons, conditions were kept as similar as
possible: seeds from parental lines and F1 seeds of trans-heterozygous
combinations were stored in paper bags for at least 3 weeks, sown on
agar plates, vernalised at 4°C for at least 5 days and transferred to
25°C under constant light. Due to the large number of crosses (and
resulting F1 seedlings), the data were collected from three series of
experiments as indicated by different symbols in Fig. 2.
Length and width of seedlings were measured after germination on
agar plates using an Olympus VM stereomicroscope equipped with
an eye-piece scale bar (10 mm, 100 divisions). The seedlings were
examined at 40× magnification (one division corresponding to 0.025
mm). The length of the seedling was measured from the tip of the
basal end to the cotyledonary node, i.e. where the cotyledons are
inserted into the axis. The width of the seedling was measured across
the cotyledonary node. The mean values of length measurements fell
into three different groups, which were designated ‘strong’, ‘intermediate’
and ‘weak’ (see Fig. 2). Trans-heterozygous combinations
were classified as ‘intermediate’ if their standard deviation values
(taken as sn-1) did not overlap with the mean value of their parents.
All combinations, except one, fulfilled this criterion and were classified
accordingly. The exceptional case was the trans-heterozygote
from the cross fs3-515 × fsU93-11 (field 55 in Table 1); classification as
‘intermediate’ was retained since the standard deviation overlapped
only slightly with the mean value of one parent.
Whole-mount preparations
In most cases embryos or seedlings were directly mounted in gum
arabic/chloral hydrate/glycerol (30 g/200 g/20 g in 50ml). Whole
mounts were used to study the phenotypic effects of fs mutations
during early and late development by comparing large numbers of
wild-type and mutant embryos. Several strong and weak fs alleles
were analysed with a Zeiss Axiophot using phase-contrast or
Nomarski (DIC) optics. Photographs were taken using an Agfa Pan
25 film.
For DAPI staining, young leaves were fixed in 3.7% formaldehyde
in PBT buffer for 4 hours (room temperature) or overnight (4°C),
washed 3× with PBT and stained with DAPI (1 µg/ml) in PBT buffer
for 1 hour. Excess stain was removed by washing 3× with PBT. The
leaves were mounted in glycerol and analysed with the Zeiss
Axiophot equipped with an epifluorescence lamp (filter no. 487901,
UV-H365).
Histological analysis
Ovules were removed from staged siliques, transferred to fixative
(3.7% formaldehyde, 5% acetic acid, 50% ethanol, 0.01% Triton X-
100) and fixed overnight at 4°C. Fixed ovules were processed for
embedding in Spurr’s resin (Spurr, 1969). Ovules were washed twice
for 20 minutes in 50% ethanol and then dehydrated in a series of
increasing concentrations of ethanol (60%, 70%, 80%, 90% and twice
in 100% for 20 minutes each). After the final 100% ethanol treatment
Spurr’s embedding medium was added in increasing concentrations
(5:1, 2:1, 1:1, 1:3 of ethanol : Spurr’s; each treatment for at least 30
minutes). Finally, two changes of Spurr´s embedding medium were
applied to remove remaining traces of ethanol. The embedding
medium was hardened by incubating overnight at 60°C. Sections 3-5
µm thick were made with a Reichert-Jung microtome, placed onto
poly-L-lysine coated microscope slides, stained with toluidine-blue
and mounted in Spurr’s medium. Photographs were taken with a Zeiss
Axiophot microscope-camera using Agfa-Pan (25 ASA), Kodak
Ektachrome (64 ASA) or Kodak Gold II (100 ASA) films.
RESULTS
Genetic characterisation of fass mutants
Twelve EMS-induced mutants were independently isolated on
the basis of their characteristic seedling phenotype (Mayer et
al., 1991). The seedlings from nine mutant lines were
extremely compressed in the apical-basal axis and enlarged in
diameter as compared to wild-type seedlings (Fig. 1A). The
three other mutants showed a similar but less drastic seedling
phenotype (see Fig. 1B). Crosses of two of the mutants with
the remaining mutants established a single complementation
group which was named FASS (FS) after the barrel shape of
mutant seedlings.
Subsequent complementation analysis of all possible pairR.
A. Torres-Ruiz and G. Jürgens
2969The effect of the FASS gene on morphogenesis
wise combinations yielded three classes of phenotype among
the F1 trans-heterozygous seedlings: strong, weak and intermediate
(Fig. 1B, Table 1). The strong or weak phenotypes
resulted from crossing any two strong or weak alleles, respectively.
Intermediate phenotypes were only observed in crosses
of any strong with any weak allele. Although visual inspection
of seedlings enabled classification of mutant phenotypes, there
was some variability within each class so we quantitated the
phenotypes by measuring the lengths of mutant seedlings (Fig.
2). Care was taken to compare F1 trans-heterozygous seedlings
with mutant seedlings from their parental lines under identical
conditions (for details, see Material and Methods). The nine
strong alleles and all their trans-heterozygous combinations
produced very short seedlings (0.5-0.75 mm long), which
showed little variability in length. The three weak alleles and
their trans-heterozygous combinations yielded fs seedlings that
were more variable in length, ranging from 1.25 mm to 2 mm.
Trans-heterozygous combinations of strong and weak alleles
gave seedlings of intermediate length, ranging from 0.75 to
1.25 mm. There was almost no overlap between the intermediate
and the other two phenotypes, suggesting that the former
reflected a distinct residual level of gene activity.
The FS gene was mapped against morphological markers,
using the W100 and ttg yi strains (for details, see Material and
Methods). For most markers, the recombination frequencies
were close to 50%, suggesting independent segregation. By
contrast, only 9% (11/124) of all ttg plants tested produced fs
seedlings, which corresponds to a genetic distance of about 5
cM between the two genes. In the same region of chromosome
5 lies GA3, a gene required for the biosynthesis of gibberellic
acid, which mutates to produce a dwarf plant phenotype
(Koornneef et al., 1983). We tested for the possibility that ga3
and fs might be mutations in the same gene by crossing fs/+
with ga3 plants. All their F1 progeny were normal. To exclude
the possibility that ga3 and fs might be complementing alleles,
lines were established from F1 plants that produced both fs
seedling and ga3 plants, and normal-looking plants of the next
generation were progeny-tested. Some F2 plants were recombinant
since they did not produce fs seeds. In addition, fs
seedlings did not respond to gibberellic acid whereas ga3
plantlets do, by elongating their axis. Thus, the two phenotypes
are caused by mutations in different, though closely linked,
genes.
The fs seedling phenotype
Although fs seedlings have grossly abnormal shapes, they do
not lack any of the pattern elements that characterise normal
seedlings, and the overall body pattern is normal (Fig. 3). In
whole-mount preparations of wild-type seedlings, the
following elements of the apical-basal pattern can be recognised
by specific features: shoot meristem, cotyledons,
hypocotyl, root and root meristem (Fig. 3A). The cotyledons
have circles of vascular strands which are bisected by a central
strand. The root-hypocotyl boundary is marked by an abrupt
change in birefringence and by the presence of root hairs below
the boundary. The root meristem consists of compact cells
which appear bright in dark-field optics. By these criteria, the
same apical-basal pattern elements are present in fs seedlings
although extremely compressed in the strong phenotype and
less so in the weak phenotype (Fig. 3B,C,E). Specifically, the
vascular ramifications in the cotyledons are essentially as in
wild-type, indicating that the cotyledons are distally complete.
Root hairs are present in fs at the same relative position as in
wild type (Fig. 3B). Of the radial pattern elements, two appear
Fig. 1. (A) Seedling phenotype
of the strong allele fassR226-32
(right) showing extreme
compression of all body
elements; wild-type on the left.
Colours indicate orientation in
the mutant: the dark green at
the apical end represents the
cotyledons, the light-green
region corresponds to the
hypocotyl ending in a broad,
white root end (9X). (B)
Comparison of homozygous
strong (fsR226-32/fsR226-32),
trans-heterozygous
(fsR226-32/fsU93-11) and
homozygous weak (fsU93-11/
fsU93-11) fass seedlings.
(C) Cotyledon number
variation of fass seedlings
(allele fs R2-3, from left to right,
one to five cotyledons). Note
the π and pairwise arrangement
in seedlings with three or more
cotyledons respectively.
2970 R. A. Torres-Ruiz and G. Jürgens
Fig. 2. Length measurements of fass homozygous and trans-heterozygous seedlings resulting from the crosses shown in Table 1. Data are given
for measurements taken from three series of crosses. The corresponding mean values with standard deviations, sn-1, are indicated by three
different symbols: open circle for the first, filled circle for the second and open square for the third series (for details see Materials and Methods
and text). Values for homozygous mutant seedlings, from selfing of weak or strong alleles, are grouped by alleles (horizontal bars) as indicated.
Data for trans-heterozygous fass seedlings are grouped by combinations (see Table 1): strong/strong (No. 1-36), strong/weak (No. 37-63) and
weak/weak (No. 64-66) trans-heterozygotes. Dotted lines serve as orientation on the scale.
3-515 R2-3 R224-31 R226-32 R239-19 R325-23 S241-9 T277-14 U284-30 G211-31
3-515 X
S 1 S 2 S 4 S 7 S 11 S 16 S 22 S 29 IN 37
–/9 22/– –/23 16/19 28/– –/24 –/18 27/– 20/–
R2-3 X
S 3 S 5 S 8 S 12 S 17 S 23 S 30 IN 38
54/– 16/– 18/20 21/– 28/24 23/– 15/– 23/–
R224-31 X
S 6 S 9 S 13 S 18 S 24 S 31 IN 39
19/31 22/36 25/– –/23 –/26 4/21 28/–
R226-32 X
S 10 S 14 S 19 S 25 S 32 IN 40
–/20 19/24 –/22 24/– –/21 22/–
R239-19 X
S 15 S 20 S 26 S 33 IN 41
–/19 –/15 –/19 17/– 29/–
R325-23 X
S 21 S 27 S 34 IN 42
–/15 –/25 22/– 14/–
S241-9 X
S 28 S 35 IN 43
–/17 22/– 31/–
T277-14 X
S 36 IN 44
16/– 24/–
U284-30 X
IN 45
27/8
G211-31 X
R392-23
U93-11
R392-23 U93-11
IN 46 IN 55
20/18 29/–
IN 47 IN 56
19/– 25/–
IN 48 IN 57
24/– 30/–
IN 49 IN 58
–/17 24/19
IN 50 IN 59
28/– 21/–
IN 51 IN 60
29/– –/24
IN 52 IN 61
26/– –/20
IN 53 IN 62
–/24 –/18
IN 54 IN 63
–/24 17/20
W 64 W 65
25/31 30/–
X
W 66
–/26
X
Table 1. Complementation matrix of fass mutant
Parental alleles are given on the left and at the top. Strong and weak alleles are separated by double lines. For easy reference in the text every combination is
given a number in the upper right corner of the respective field (total 66 fields). The phenotypes of F1 seedlings were classified for each combination as strong
(S), intermediate (IN) or weak (W) (based on length measurements, see text and Fig. 2). Numbers indicate percentages of fass seedlings from each cross. Slashes
separate the results of reciprocal crosses (ca. 20% of the cases). Values to the left or right of the slash refer to the female parent shown on the left or at the top,
respectively.
2971The effect of the FASS gene on morphogenesis
Fig. 3. Whole-mount preparations of wild-type and fs seedlings showing internal structures. (A) Wild-type with a simply reticularised vascular
system in the cotyledons. The vascular strands run tightly together through the center of the hypocotyl into the root. The upper end of the root is
marked by root hairs and a more intense birefringence, which is also seen in the apical and especially the basal end (root meristem zone). The
wild-type is not shown in full length (interruptions indicated). (B-F) fs seedlings (same stage and same magnification as the wild type). The
main features of the wild type are also visible in fs seedlings (note the epidermal layer in the cotyledons). Vascular strands are clearly separated
from each other in the hypocotyl and the root (small arrows). (B-D) Strong fs phenotype. Dicotyledoneous seedlings in B (side view) and C
(top view): vascular system is sometimes interrupted. (D) fs seedling with three cotyledons: arrowheads point to the supernumerary vascular
strand and cotyledon respectively. (E,F) Weak fs phenotypes: compare with B and D. Dark-field optics.
2972
bright in dark-field optics: the epidermal layer and the centrally
located, closely spaced vascular strands (Fig. 3A). In fs
seedlings, the epidermal layer is best seen in the cotyledons
(Fig. 3B,E). The two vascular strands running
the length of the axis are separated in both
weak and strong phenotypes; in the latter, the
vascular strands are abnormally shaped and
often interrupted (Fig. 3B,C). Some mutant
seedlings have three cotyledons (see below); in
these cases, there are also three, rather than
two, vascular strands in the hypocotyl (Fig.
3D,F).
Histological sections of strong fs seedling
phenotypes revealed regional differences in
cell size and cell shape (Fig. 4A). The
hypocotyl and the embryonic root lack the
regular arrangement of cell files found in the
wild type and show irregularly enlarged cells
both in the epidermis and in the ground tissue.
Narrow cells in the centre may represent the
vascular tissue (Fig. 4B). Near the lower end of
the seedling axis, large surface cells form
bulges, which may correspond to incipient root
hairs (Fig. 4C). The enlarged root pole is
covered by layers of compressed cells, presumably
the central portion of the root cap,
which overlie the small, densely cytoplasmic
cells of the root meristem; no quiescent centre
can be recognised by anatomical criteria (Fig.
4D). The cotyledons consist, by and large, of
normal-sized cells. The epidermal layer is
clearly recognisable, and stomatal guard cells
are well-differentiated (Fig. 4E,F). Narrow
cells in the centre seem to correspond to the
vascular cells found in wild-type cotyledons
(Fig. 4F). The shoot meristem is represented by
a group of small, densely cytoplasmic cells
bulging between the bases of the cotyledons
(Fig. 4G). Thus, the various tissues and organs
are present in fs seedlings, although cell shapes
and arrangements are highly abnormal.
The abnormal shapes and arrangements of
cells are probably responsible for the compression
of the seedling axis. We tested whether
this defect could be reversed or mitigated by
growing fs seedlings in the dark since light is
known to have an inhibitory effect on stem
elongation. We compared the seedling phenotypes
of the three weak and two strong alleles
in the light and in the dark, by measuring their
lengths and their widths (Table 2). In the strong
alleles, the seedlings were almost as wide as
long in the light and they elongated very little
in the dark. By contrast, the weak-phenotype
seedlings elongated markedly in the dark
although much less than did wild-type
seedlings. Among the weak alleles, fsU93-11
was less responsive than the other two and gave
broader seedlings both in the light and in the
dark, suggesting that this allele is slightly
stronger (see also below).
Abnormal cotyledon numbers in fs seedlings
Mutant seedlings, in spite of their altered shape, essentially
showed the overall body pattern seen in wild-type seedlings,
R. A. Torres-Ruiz and G. Jürgens
Fig. 4. Histological sections of young fs seedlings. (A) Longitudinal section.
(B-G) Higher magnification: sections of different seedling regions, specific features
marked by arrowheads (not all taken from the same section as in A). (B) Hypocotyl:
elongated cells (vascular precursors) in the center. (C) Upper root region: putative root
hair cells. (D) Root end: root cap (darkly stained), cell walls abutting a group of densely
cytoplasmic cells, which include the root meristem. (E) Cotyledon: stomatal cells
(tangential section). (F) Cotyledon: elongated cells (including vascular precursors).
(G) Shoot apical meristem zone.
2973The effect of the FASS gene on morphogenesis
except for one recurrent deviation. Regardless of the fs allele,
cotyledon number was abnormal in a large proportion of
mutant seedlings (Table 3). The vast majority of exceptional
seedlings had three cotyledons, some had only one cotyledon
and others had four or even five cotyledons. Supernumerary
cotyledons correlated well with the widths of mutant seedlings.
The strong alleles produced 10-19% mutant seedlings with
three cotyledons as compared to 15% for the weak allele
fsU93-11 and 6-8% for the other two alleles (Table 3). The allele
fsU93-11 also caused a larger seedling width than the other weak
alleles did, suggesting that the increased number of cotyledons
is a secondary defect (see Table 2).
The supernumerary cotyledons were arranged in specific
ways (Fig. 1C). In tricotyledonous seedlings, two different
configurations were observed. Either all three cotyledons were
spaced evenly, forming a triangle; or, more often, two cotyledons
facing each other abutted the third at a right angle, thus
forming a π-shaped arrangement. Nearly all seedlings with
four cotyledons (and those with five) had the cotyledons
grouped in pairs which, in turn, were variably positioned
relative to each other.
Development of fs embryos
It had been shown previously that fs embryos exhibit abnormal
arrangements of cell walls at the heart stage (Mayer et al.,
1991). We analysed, in histological sections, fs embryos from
very early stages in order to determine when their development
starts to deviate from normal and how the extreme compression
of the seedling axis comes about. We compared mutant
with wild-type embryos of approximately the same stages (Fig.
5). Since the cellular features that are used for staging wildtype
embryos (Jürgens and Mayer, 1994) are lacking in fs
embryos, we tentatively identified equivalent stages by size,
approximate cell numbers and the corresponding developmental
stage of the endosperm (Mansfield and Briarty, 1990). To
facilitate the description of fs embryogenesis, we will first
briefly describe wild-type embryo development (for details, see
Mansfield and Briarty, 1991; Jürgens and Mayer, 1994).
Very early development of wild-type embryos is characterised
by a sequence of precise cell divisions, resulting in the
octant-stage embryo (Fig. 5A,C). The zygote divides asymmetrically
to give a small apical and a large basal cell. These
two cells and their progeny differ in staining intensity: the
apical cell, which is densely cytoplasmic, stains more darkly,
while the basal cell, which has a large vacuole stains more
lightly (Mansfield and Briarty, 1991). The apical cell undergoes
three rounds of cell division, two vertical and one horizontal,
to generate the proembryo of the octant stage, which is
Table 2. Width and length measurements of fass seedlings in the light and the dark*
Width of fass seedlings Length of fass-seedlings
Light(l) Dark(d) Light (l) Dark (d)
Allele (scale units†) (scale units†) l/d (scale units†) (scale units†) l/d
R224-31 24.0±4.4 23.1±3.6 1.04 31.8±5.3 36.7±9.0 0.87
R226-32 25.2±2.9 23.8±3.4 1.06 28.0±5.0 32.4±6.0 0.86
G211-31 17.2±1.8 15.4±1.1 1.12 60.5±5.6 120.2±21.6 0.50
R392-23 16.8±1.7 14.0±1.6 1.20 62.3±6.8 118.5±23.1 0.53
U93-11 20.8±2.6 17.8±1.6 1.17 64.9±9.4 98.5±17.3 0.66
Hypocotyl 68.2±7.1 367.6±64.0 0.19
Wt Root 410.4±46.0 210.4±40.4 1.95
Total 7.8±0.9 6.9±0.7 1.13 478.6±53.1 578.0±104.4 0.83
*Taken 5 days after germination.
†100 scale units=2.5 mm.
The double line separates strong fass alleles, weak fass alleles and wild type (wt) respectively.
Table 3. Number of cotyledons in fass plants
No. of Tricot.*
seedlings fs seedlings Monocot.* Dicot.* π-arrangement† Tetracot.*
Allele scored (%) % % % (%) %
3-515 1826 410 (22.5) <1 88 11 (58) <1
R2-3 1885 413 (21.9) <2 81 16 (57) <2‡
R224-21 1867 438 (23.5) <1 82 17 (73) <1
R226-32 2194 491 (22.4) <1 89 10 (49) n.d.
R239-19 1643 366 (22.3) <1 83 15 (73) <2
R325-23 1742 437 (25.1) <2 82 16 (61) <1
S241-9 1457 327 (22.4) <1 80 19 (61) <1
T277-14 1924 397 (20.6) <2 84 13 (62) <2
U284-30 1823 466 (25.6) <2 84 14 (66) <1
G211-31 2622 524 (20.0) 1 93 6 (59) n.d.
R392-23 2607 603 (23.1) <2 90 8 (38) <1
U93-11 2912 791 (27.2) <1 84 15 (61) <1
*Refers to fass seedlings.
†% of fass seedlings with three cotyledons, two arranged as a pair and the third positioned at 90° relative to the pair (see text and Fig. 1C).
‡Also rare pentacotyledonous seedlings observed (see Fig. 1C).
The double line separates strong and week alleles.
2974
composed of an upper and a lower tier, each of four cells. The
basal cell produces, by repeated horizontal divisions, a file of
7-9 cells of which all but the uppermost one form the suspensor
(Fig. 5E,G). The uppermost derivative of the basal cell, which
also stains lightly, becomes the hypophysis which joins the
proembryo. Region-specific changes transform the octant stage
into a globular embryo. Initially, tangential cell divisions
produce the primordium of the epidermis (protoderm) as an
R. A. Torres-Ruiz and G. Jürgens
Fig. 5. Embryo
development of fass in
comparison to wild type
(comparable stages, wildtype:
A, C, E, G, I, K, fs:
B, D, F, H, J, L). All
sections same
magnification except K
and L (approx. half the
magnification of A-J).
(A,B) One cell stage: the
apical cell is densely
cytoplasmic and darkly
stained. The first cell wall
(arrowhead) is horizontal
in A and oblique in B
(note the nuclei with
nucleoli; approx. 400×).
(C,D) Octant cell stage:
the cell wall separating
suspensor (s) and
proembryo is horizontal
(arrowheads, note highly
vacuolated suspensor
cells). (E,F) Early
globular stage: the
epidermis primordium
(protoderm, pt) and the
hypophysis (hy) are
recognisable in E but not
in F. A cell at the
equivalent position of the
hypophysis is marked in
F. (G,H) Late globular
stage: root primordium
(rp) and vascular
primordium (vp,
elongated cells).
Irregularly arranged cells
(arrow) in H may
correspond to the rp.
Irregular surface layer in
H (note cells extending
into the embryo,
arrowheads). (I,J) Heart
stage: cotyledon
primordia (co) and
cellular endosperm (ce)
visible in I; the latter has
been probably damaged in
J. (K,L) Torpedo stage:
cotyledon primordia (co)
are distinguishable in fs.
Note also cellular
endosperm (ce).
2975The effect of the FASS gene on morphogenesis
outer cell layer (Fig 5E). Slightly later, the innermost cells can
be recognised as the primordium of the vascular tissue (Fig.
5G). Oriented cell divisions of the lower-tier derivatives result
in elongation of the embryonic axis, and the hypophysis
generates, by a series of highly regular divisions, the incipient
root primordium. Within a short period of time, the symmetry
of the developing embryo changes from radial to bilateral.
Rapid cell divisions in the apical region of the late-globular
embryo initiate the two cotyledonary primordia, giving the
embryo a heart shape (Fig. 5G,I). At the basal pole, the derivatives
of the hypophysis are now arranged in three cell layers
(Fig. 5I). The late-heart stage embryo displays the primordia of
the main tissues and structures of the seedling. The endosperm,
which has been nuclear, cellularises during this stage (Fig. 5I).
During the torpedo stages, the embryo mainly grows by
elongation of both the cotyledons and the hypocotyl which now
consist of several layers of cell files (Fig. 5K). Bending of the
cotyledons, which eventually reach the level of the root pole,
and enlargement of the shoot meristem and the root primordium
complete the growth of the embryo.
Very early embryos from fs/+ plants were inspected for
deviation from the regular pattern of cell division (Fig. 5B,D).
As in the wild type, fs zygotes divided asymmetrically, giving
two daughter cells of different sizes and staining intensity.
Occasionally, the cell wall separating the apical from the basal
daughter cell was oriented obliquely, instead of perpendicular
to the axis, suggesting that the FS gene normally acts before
the division of the zygote (compare Fig. 5A and B). However,
the majority of mutant embryos could not be recognised by the
abnormal orientation of the first cell wall, neither at the onecell
stage nor at later stages when other features enabled recognition
of fs embryos. The apical cell also tended to divide irregularly,
and by the octant-stage, fs embryos looked abnormal.
The basal cell often produced fewer derivatives which were
separated by horizontal or oblique cell walls (Fig. 5B,D). The
shorter suspensor frequently failed to push the developing
embryo into the cavity of the embryo sac.
In fs embryos, the octant cells did not produce a distinct
outer cell layer (Fig. 5F), and the innermost cells of the midglobular
embryo did not show the morphological features of
the vascular primordium (Fig. 5H). The derivatives of the
‘lower tier’ divided more or less at random, and the positional
equivalent of the hypophysis produced a cell group that, by
morphological criteria, did not resemble the incipient root primordium
(Fig. 5H). The suspensor cells, which were recognisable
by their light staining, had irregularly oriented cell
walls (Fig. 5H). The growing embryo merely increased in size
but did not change its shape and thus, by the time the wildtype
embryo had turned heart-shaped, the mutant embryo
resembled an oversized globular embryo.
The continuing irregular cell divisions of fs embryos made
the cells at the surface form a sloppy layer; although discernable
as a layer, the surface cells varied in size and shape (Fig.
5J). The internal cells as well remained disorganised with
respect to cell wall orientation. The cells in the centre did not
show morphological features of vascular precursors except that
in some sections, they appeared narrower than the surrounding
cells of the putative ground tissue. From the late-heart stage,
bulges representing cotyledonary primordia developed from the
apical surface of the embryo (Fig. 5L). The cells between their
bases as well as cells at the basal pole of the embryo exhibited
features of meristematic cells: they were small, rich in
cytoplasm and less vacuolated than other cells. Some heartstage
embryos had a basally located cell group that was marked
off against the main cell mass. This cell group may have been
derived from the hypophysis (Fig. 5F,H); by size and vacuolation,
these cells resembled suspensor cells. In contrast to the
wild type where the derivatives of the hypophysis are arranged
in a highly regular manner, the basal cell group of fs embryos
showed variable cell wall orientations, sometimes horizontal, in
other cases oblique or vertical (Fig. 5F,H,J). This variability
made it difficult to recognise the basal cell group unambiguously
as the incipient root primordium. There were fewer
suspensor cells than in wild-type and they differed from
embryonic cells by staining lightly (Fig. 5H,J,L). The
endosperm of developing fs seeds was normal. Nuclear
endosperm was associated with the embryo until the heart-stage
and then replaced by cellular endosperm (compare Fig. 5J with
L). Continuing irregular cell divisions resulted in a mature
embryo that was extremely compressed in the apical-basal axis
but considerably enlarged radially. Although the cotyledons did
not bend, the embryo filled up the entire seed at maturity.
Postembryonic development of fs mutants
Mutant fs seedlings, though short and thick, were not lethal and
developed into adult plants that were extremely dwarfed, even
in comparison to ga3 plants (Fig. 6A). Weak fs alleles
completed postembryonic development in a regular manner
whereas strong alleles rarely did. Initially, a thick root emerged
from the root pole of the seedling, and the shoot meristem
produced primary leaves which appeared in the region between
the stunted cotyledons (Fig. 6B). Subsequent activity of the
shoot meristem generated organs that were compressed in their
proximal-distal axis. The extent to which organ shape was
altered reflected the relative strength of the fs allele. For
example, the leaf blade seemed to be immediately attached to
the stem in strong alleles while a thick petiole was recognisable
in weak alleles (compare Fig. 6C with G,H). In some fs
plants, different leaf arrangements were observed, which may
be correlated with the different spatial relations of the cotyledons
described above (Fig. 6G,H).
The fs phenotype was also expressed at the cellular level,
which was most clearly seen in the leaf epidermis. Epidermal
cells, which are characteristically interdigitated in wild type,
appeared roundish in fs plants (compare Fig. 6K with L). Furthermore,
leaf trichomes, which normally have three branches
positioned in a very specific way (Fig. 6I; Hülskamp et al.,
1994) were abolished in strong fs alleles; weak alleles were
associated with unbranched trichomes (Fig. 6J).
The flowers of adult fs plants showed the normal number
and position of organs (compare Fig. 6D with E). However,
flower organs were compressed in their proximal-distal axis
(Fig. 6F). This effect was particularly pronounced for stamens
and pistils, and the pistils contained thickened ovules. Regardless
of the allele strength, the fs plants examined did not set
seed. These results suggest that the FS gene is required during
post-embryonic development in a similar manner as in embryo-
genesis.
DISCUSSION
The analysis of pattern formation in the Arabidopsis embryo
2976 R. A. Torres-Ruiz and G. Jürgens
Fig. 6. Postembryonic development of fs. (A) Comparison of adult wild-type (wt), dwarf mutant (ga-3) and fs plants (fs, marked by a white
line). (B) Strong-phenotype seedling with emerging root (arrowhead) and primary leaves (arrows). (C) Adult plant with flowers (strong allele).
(D,E) Comparison of wild-type (D) and fs flowers (E, weak allele). (F) Comparison of wild-type (upper row) and fs (lower row) flower organs
(from left to right: pistil, stamen, petal and sepal). (G,H) Tricotyledoneous fs plants (weak allele); G: cotyledons evenly spaced (arrowheads),
and three staggered primary leaves; H: cotyledons irregularly spaced, two primary leaves. (I,J) Leaf trichome: wild type (I), fs trichome (J;
weak allele); DAPI-stain (note the densely stained nuclei). (K,L) Epidermal cell layer: wild-type (K) and fs (L; weak allele).
2977The effect of the FASS gene on morphogenesis
is facilitated by the regularity of cell divisions that characterise
the early stages of embryogenesis (Mansfield and Briarty,
1991; Jürgens and Mayer, 1994). The primordia of seedling
structures which become recognisable in the heart-stage
embryo can thus be traced back to cell groups in the very early
embryo. Oriented cell divisions and changes in cell shape are
associated with the formation of seedling primordia as well as
with their elaboration during later stages of embryogenesis.
These cell activities are also responsible for generating
seedling body shape and may thus be covered by the term
‘morphogenesis’. Although in wild-type development, pattern
formation and morphogenesis are intimately coupled,
mutations in the FASS gene affect morphogenesis but not
pattern formation, suggesting that the regularity of cell division
is not instrumental in pattern formation in the Arabidopsis
embryo. In the following, we address two questions: (1) what
is the primary effect of the FASS gene in development, and (2)
what can be inferred from the fass phenotype about pattern
formation in the Arabidopsis embryo?
The primary effect of the FASS gene in development
Mutations in the FS gene cause a strong compression of the
longitudinal axes of the plant body and its organs throughout
development. By phenotypic criteria, the mutant alleles form
two distinct classes, strong and weak. Any trans-heterozygous
combination of weak with strong alleles gave a novel, intermediate,
phenotype which did not overlap the other two
classes. This result suggests that the strong phenotype reflects
the lack of function of the gene and that the intermediate
phenotype results from half the residual gene activity that is
associated with the weak alleles. A likely interpretation is that
the FS gene normally acts in a quantitative manner, with the
amount of functional product directly determining the biological
effect. If the expression of the fs phenotype reflects
residual gene activity, why did we not isolate alleles that would
produce intermediate phenotypes on their own? The answer
might be that the weak fs alleles represent a particular class of
mutations which make less product, rather than less active
product. This idea is consistent with the lack of partial complementation
between weak alleles, which stands in contrast to
the interaction between specific classes of gnom alleles (Mayer
et al., 1993).
That the strong alleles probably lack FS gene activity is also
supported by other observations. All strong alleles show essentially
the same extreme phenotype which varies little. Furthermore,
the fs alleles were isolated in a mutagenesis screen that
achieved saturation by statistical criteria and the number of fs
alleles was above the average allele frequency (Jürgens et al.,
1991; Mayer et al., 1991). We therefore consider it unlikely
that stronger fs alleles might mutate to embryonic lethality and
would thus not have been found. In summary, the strong alleles
probably lack gene activity but direct evidence will only come
from the molecular analysis of the FS gene.
The FS gene is probably required throughout development
and in all cell types. Lack of active FS gene product causes
different cellular phenotypes, depending on the tissue as well
as the developmental stage. In the fs embryo, cell walls are
oriented abnormally, and continuing cell divisions result in
grossly abnormal cell shapes and arrangements. At the seedling
stage, the postmitotic cells of the hypocotyl do not elongate
properly, even if challenged by the absence of light. In the leaf,
the epidermal cell layer does not acquire the characteristically
interdigitated pattern of lobed cells, and the trichome cells,
which normally undergo extensive morphogenesis (Hülskamp
et al., 1994), fail to grow out of the leaf surface in plants with
strong alleles or fail to branch if FS activity is reduced. All
these cellular processes, orderly division, elongation and morphogenesis,
require specific changes in the organisation of the
cell. Such changes may be brought about by the reorientation
of the cytoskeletal microtubules and the cellulose microfibrils
in the cell wall (for review see Staiger and Lloyd, 1991). For
example, the arrangement of microtubules changes during
elongation of the Arabidopsis zygote before the asymmetric
division occurs (Webb and Gunning, 1991), and similar observations
have been made on elongating cells of the pea
hypocotyl (Duckett and Lloyd, 1994). Cell shape is also influenced
by light and plant hormones such as auxin, ABA and
GA3 which alter the orientation of cortical microtubules
(Seagull, 1989; Sachs, 1991; Sakiyama-Sogo and Shibaoka,
1993). The strong fs phenotype is not markedly influenced by
light or by GA3, suggesting that the primary defect resides
within the cells. This is supported by the rare occurrence of
twin embryos of which one was mutant and the other was wildtype
(our unpublished observations). Considering the variety
of abnormal cell activities in fs mutants, it is tempting to
speculate that the primary function of the FS gene product may
be required for the proper organisation of the cell, either for its
maintenance or for mediating changes in response to spatial
cues.
Pattern formation and morphogenesis in
embryogenesis
Unlike many other plant species (Johri et al., 1992), Arabidopsis
and other crucifers display a regular pattern of cell
division in early embryogenesis (Schulz and Jensen, 1968;
Tykarska, 1976, 1979; Mansfield and Briarty, 1991; Jürgens
and Mayer, 1994). This regularity enabled primordia of
seedling structures to be recognised from early embryogenesis
and facilitated the identification of precursors for the meristems
of the root and the shoot (Dolan et al., 1993; Barton and
Poethig, 1993). Moreover, the mutant seedling phenotypes of
two genes involved in pattern formation in the Arabidopsis
embryo, GNOM and MONOPTEROS, were shown to correlate
topographically with abnormal patterns of cell division in the
early embryo (Mayer et al., 1993; Berleth and Jürgens, 1993).
These results supported the view that oriented cell divisions
are associated with pattern formation in the Arabidopsis
embryo. The fs phenotype, however, suggests that oriented cell
divisions are not instrumental for pattern formation: although
the primordia of seedling structures cannot be recognised by
morphological criteria at the heart stage, the body pattern of
the seedling is essentially normal (we interpret the occurrence
of supernumerary cotyledons as a secondary effect which is
caused by an enlarged hypocotyl). Thus, fs embryos mimic the
situation found in plant species such as cotton: the pattern of
cell division in the embryo is not regular and yet the seedling
has the same body pattern as in crucifers (Pollock and Jensen,
1964). It is therefore appropriate to view the regularity of cell
division in the Arabidopsis embryo as a special case: cells at
specific positions respond to pattern formation by changing cell
shape or by undergoing oriented cell division. In this view,
morphogenesis would be the consequence of pattern formation.
2978
The fs phenotype indicates that pattern formation is not
dependent on morphogenesis. Are the two processes independent
of each other or is morphogenesis a consequence of
pattern formation? The additive phenotype of the fass gnom
double mutant suggests that the two processes are independent
(Mayer, 1993), which raises the question of how pattern
formation and morphogenesis are normally integrated to
produce the wild-type seedling.
In order to identify factors influencing pattern formation in
the Arabidopsis embryo, one might thus ask what is not
changed in fs embryos. Although the division of the zygote
produces a variably oriented cell wall and subsequent divisions
of both its daughter cells can be abnormal, cell polarity is not
affected since the apical and the basal cell differ in staining
intensity as they do in wild-type. This is consistent with the
zygote being polarised in most plant species (Rutishauser,
1969; Natesh and Rau, 1984). The fs seedling itself demonstrates
that polarity is maintained throughout development
since it represents a polarised structure. Thus, cell polarity as
set up in the zygote might be essential for cells to acquire information
about their relative positions which may underlie
pattern formation. In some plant species, cell fate is then
presaged by very regular patterns of cell division and cell
arrangements, if the FS gene (or its homologue) is functional.
In essence, the fs phenotype suggests that pattern formation
and morphogenesis are two distinct processes in establishing
the body organisation of the seedling.
We thank F. Assaad, T. Berleth, Th. Fischer, G. Haberer, T. Laux,
W. Lukowitz, U. Mayer and S. Ploense for helpful suggestions and
critical reading of the manuscript, and U. Mayer for an introduction
to the semi-thin section method, T. Singer for technical assistance,
Maarten Koornneef for providing seeds from marker lines and H.
Jäckle for generous support during the initial phase of the work. This
work was supported by grants To 134/1-1, Ju 179/2-1 and Ju 179/2-
2 from the Deutsche Forschungsgemeinschaft.
REFERENCES
Barton, M. K. and Poethig, R. S. (1993). Formation of the shoot apical
meristem in Arabidopsis thaliana: an anlysis of development in the wild-type
and in the shoot meristemless mutant. Development 119, 823-831.
Berleth, T. and Jürgens, G. (1993).The role of the monopteros gene in
organising the basal body region of the Arabidopsis embryo. Development
118, 575-587.
Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts,
K., Scheres, B. (1993). Cellular organisation of the Arabidopsis thaliana
root. Development 119,71-84.
Duckett, C. M. and Lloyd, C. W. (1994). Gibberellic acid-induced
microtubule reorientation in dwarf peas is accompanied by rapid
modification of an alpha-tubulin isotype. The Plant J. 5, 363-372.
Hülskamp, M., Miséra, S. and Jürgens, G. (1994). Genetic dissection of
trichome cell development. Cell 76, 555-566.
Johri, B. M., Ambegaokar, K. B. and Srivastava, P. S. (1992). Comparative
Embryology of Angiosperms. Berlin: Springer.
Jürgens, G. and Mayer, U. (1994). Arabidopsis. In: EMBRYOS. Colour Atlas
of Development (ed. J. Bard), pp. 7-21. London: Wolfe Publ.
Jürgens, G., Mayer, U., Torres Ruiz, R. A., Berleth, T. and Miséra, S.
(1991). Genetic analysis of pattern formation in the Arabidopsis embryo.
Development Supplement1, 27-38.
Koornneef, M. and Hanhart, C. J. (1983). Linkage marker stocks of
Arabidopsis thaliana. Arabid. Inform. Serv. 20, 89-92.
Koornneef, M., Hanhart, C. J., van Loenen Martinet, E. P. and van der
Veen, J. H. (1987). A marker line that allows the detection of linkage on all
Arabidopsis chromosomes. Arabid. Inform. Serv. 23, 46-50.
Koornneef, M., van Eden, J., Hanhart, C. J., Stam, P., Braaksma, F. J. and
Feenstra, W. J. (1983). Linkage map of Arabidopsis thaliana. J. Hered. 74,
265-272.
Mansfield, S. G. and Briarty, L. G. (1990). Endosperm cellularization in
Arabidopsis thaliana L. Arabid.Inform. Serv. 27, 65-72.
Mansfield, S. G. and Briarty, L. G. (1991). Early embryogenesis in
Arabidopsis thaliana. II. The developing embryo. Can. J. Bot. 69, 461-476.
Mayer, U. (1993). Entwichlungsgenetische Untersuchungen zur
Musterbildung im Embryo der Blütenpflanze Arabidopsis thaliana. Thesis,
Tübingen.
Mayer, U., Büttner, G. and Jürgens, G. (1993). Apical-basal pattern
formation in the Arabidopsis embryo: studies on the role of the gnom gene.
Development 117, 149-162.
Mayer, U., Torres Ruiz, R. A., Berleth, T., Miséra, S. and Jürgens, G.
(1991). Mutations affecting body organization in the Arabidopsis embryo.
Nature 353, 402-407.
Natesh, S. and Rau, M. A. (1984). The embryo. In Embryology of
Angiosperms (ed. B. M. Johri), pp.377-443. Berlin: Springer.
Pollock, E. G. and Jensen, W. A. (1964). Cell development during early
embryogenesis in Capsella and Gossypium. Am. J. Bot. 51, 915-921.
Rutishauser, A. (1969). Embryologie und Fortpflanzungsbiologie der
Angiospermen. New York, Wien: Springer.
Sachs, T. (1991). Cell polarity and tissue patterning in plants. Development
Supplement 1, 83-93.
Sakiyama-Sogo, M. and Shibaoka, H. (1993). Gibberellin A3 and abscisic
acid cause the reorientation of the cortical microtubules in epicotyl cells of
the decapitated dwarf pea. Pl. Cell Physiol. 34, 431-437.
Schulz, R. and Jensen, W. A. (1968). Capsella embryogenesis: The egg,
zygote, and young embryo. Am. J. Bot. 55, 807-819.
Seagull, R. W. (1989). The plant cytoskeleton. CRC Critical Reviews in Plant
Science 8, 131-167.
Slack, J. M. W. (1991). From Egg to Embryo. Cambridge: Cambridge
University Press.
Spurr, A. P. (1969). A low-viscosity epoxy resin embedding medium for
electron microscopy. J. Ultrastrct. Res. 26, 31-43.
Staiger, C. J. and Lloyd, C. W. (1991). The plant cytoskeleton. Current
Opinion in Cell Biology 3, 33-42.
Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development.
Cambridge: Cambridge University Press.
Tykarska, T. (1976). Rape embryogenesis. I. The proembryo development.
Acta Soc. Bot. Pol. 45, 3-15.
Tykarska, T. (1979). Rape embryogenesis. II. Development of the embryo
proper. Acta Soc. Bot. Pol. 48, 391-421.
Webb, M. C. and Gunning, B. E. S. (1991). The microtubular cytoskeleton
during development of the zygote, proembryo, and free-nuclear endosperm
in Arabidopsis thaliana (L.) Heynh. Planta 184, 187-195.
(Accepted 30 June 1994
R. A. Torres-Ruiz and G. Jürgens