Robert Et Al. J Plant Physiol. 2007

ARTICLE IN PRESS
Journal of Plant Physiology ] (]]]]) ]]]]]]
1
3
www.elsevier.de/jplph
5
7
9
11
13
15
17
Morphological analysis of seed shape in Arabidopsis

thaliana reveals altered polarity in mutants of the
ethylene signaling pathway
Ce
line Roberta, Arturo Noriegaa, Angel Tocinob, Emilio Cervantesa,
a
23
Departamento de Produccio
n Vegetal, IRNASA-CSIC, Salamanca, Apartado 257, Salamanca, Spain
Departamento de Matematicas, Universidad de Salamanca, Plaza de la Merced, 1, 37008 Salamanca, Spain
21
19
Received 24 April 2007; received in revised form 3 July 2007; accepted 1 October 2007
33
35
31
37
39
41
43
53
55
47
51
57
45
49
The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid.
The accuracy of this approximation is discussed. Considering its limitations, it allows
a geometric approximation to the analysis of changes occurring in seed shape during
imbibition prior to seed germination as well as the differences in shape between
genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2,
etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other
mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show
different response to imbibition than the wild type. Imbibed seeds of the wild type
increase their asymmetry compared with the dry seeds. This is detected by the
relative changes in the curvature values in both poles. Thus, during imbibition of the
wild-type seeds, the reduction in curvature values observed in the basal pole gives
them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in
curvature values occurs in both basal and apical poles, and its shape remains as a
prolate spheroid. Our data indicate that the ethylene signaling pathway is involved,
in general, in the complex regulation of seed shape and, in particular, in the
establishment of polarity in seeds, controlling curvature values in the seed poles.
& 2007 Elsevier GmbH. All rights reserved.
TE
29
Summary
KEYWORDS
Arabidopsis;
Curvature;
Ellipsoid of revolution;
Ethylene;
Ovoid;
Polarity;
Q1 Seed
EC
27
PR
25
Corresponding
author.
Tel.:
+34 92321 9606;
+34 92321 9609.
E-mail address: ecervant@usal.es (E. Cervantes).
fax:
Introduction
59
Recent analysis by molecular techniques has

revealed many genes involved in the control of
organ shape as well as general architecture in the
model plant Arabidopsis thaliana. For example,
61
0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2007.10.005
Please cite this article as: Robert C, et al. Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in
mutants of the ethylene signaling.... J Plant Physiol (2007), doi:10.1016/j.jplph.2007.10.005
XML:ver:5:0:1
6 jena
JPLPH : 50519
Prod:Type:FTP
pp:110col:fig::1;3;4
ED:SathishSN
PAGN:Sakthi SCAN:
63
65
67
ARTICLE IN PRESS
2
17
19
21
23
25
27
29
31
33
35
37
69
71
73
75
77
79
81
83
85
87
89
91
93
103
49
65
101
45
63
99
43
61
97
41
59
95
39
47
15
Seeds of A. thaliana var. Columbia (col) and its

mutants ctr1-1 (Kieber et al., 1993), eto1-1 (Chae et
al., 2003; Woeste et al., 1999), etr1-1 (Chang et al.,
1993), ein2-1 (Guzma
n and Ecker, 1990), as well as the
triple mutant ein2-1, ers1-2, etr1-7 (Hall and Bleecker,
2003) were used.
Q2
Three different seed treatments were carried out in
the analysis. The rst involved dry seeds. For the second,
seeds were imbibed for 3 h, and for the third treatment,
seeds were imbibed for 24 h in distilled water. For the
second and third treatments, seeds were previously
sterilized (2 min ethanol 70%, 7 min 2% sodium hypochlorite, 3 washed in distilled water). After sterilization, every seed was placed over a piece of graph paper
on a medium containing wateragar (1%).
The visual inspection of seeds of A. thaliana revealed a
number of symmetries and the existence of a longitudinal
axis. In addition, the longitudinal sections resemble
ellipses and the transversal sections are similar to circles
(Figure 1). The seed surface may thus be adjusted by a
prolate spheroid. A prolate spheroid is the ellipsoid of
revolution obtained by rotating an ellipse about its major
axis (Figure 2). It then has two semi-axes of the same
length as the minor semi-axis of the ellipse (here denoted
by b); the third semi-axis of the ellipsoid coincides with
the major semi-axis of the ellipse (here denoted by a).
The seeds were observed, one by one, with a Nikon
SMZ-2T stereo microscope. Considering the seeds as
13
57
67
Materials and methods
11
PR
values in both poles, we described the changes

that occur during imbibition. Further, new phenotypic traits for mutants in the ethylene signal
transduction pathway are described. Our results
suggest that the ethylene signaling pathway is
involved, in general, in the complex regulation of
seed shape and, in particular, in the establishment
of polarity in seeds.
TE
EC
Dinneny et al. (2006) described genes involved in

the control of shape in stamens and carpels, while
other genes were described that regulate the at
shape of leaves (summarized by Tsukaya, 2005).
The shape of organs is the result of the integration
of multiple processes, including cell cycling,
cellular elongation and communication and the
direction of cell proliferation, and thus all sets of
genes active in these processes may also be
involved in shape determination. The multiplicity
of factors that affect organ and plant size and
shape indicate complex regulation; thus, global
regulatory mechanisms must also exist that may be
able to compensate or buffer sudden environmental changes that, otherwise, would have deleterious effects. The cytoskeleton organization and
microtubules, in particular, are very important in
cell size and shape determination. Indeed, they are
involved in the arrangement of the deposition of
the wall microbrils (Hepler and Palevitz, 1974). All
these aspects are known to be under the control of
the ethylene sensing and signaling pathway. Thus,
Apelbaum and Burg (1971) showed that ethylene
altered the orientation of microbrils in Pisum
sativum stems.
Seed shape is also the visible result of processes
submitted to complex regulatory mechanisms.
Leon-Kloosterziel et al. (1994) have described
mutations that have an effect in integument
development with a direct consequence in the
Arabidopsis seed shape.
In this work, our objective was to investigate in
detail the effects that diverse mutants in the
ethylene signal-transduction exert on seed shape.
For this, we rst need to adjust the seed shape to a
geometric form. After adjusting seed shape to a
prolate spheroid and calculating the curvature
C. Robert et al.
105
51
107
53
109
55
111
Figure 1. Photograph of Arabidopsis thaliana cv. Columbia in longitudinal (left) and polar (right) views.
JPLPH : 50519
ARTICLE IN PRESS
Analysis of seed shape in Arabidopsis thaliana: Altered polarity in ethylene mutants
11
13
15
17
19
21
23
29
31
4parea
perimeter2
To understand changes that occurred in the seed

shape, a volume approximation was made. The volume
of an ellipsoid of revolution was calculated from the
values of the longitudinal views area A and the short
35
EC
33
b
b
37
39
49
Results
71
Geometric description of seed shape in dry

seeds of Arabidopsis thaliana cv. Columbia
73
The visual inspection of seed images suggests

their adjustment with an ellipsoid of revolution. To
conrm this, two tests were developed. One was
based on comparison of areas, the other utilized
curvature analysis in both poles.
The values given by the program analysis for the
area of the gures corresponding to longitudinal
and polar views of seeds were compared with those
corresponding to an ellipse (longitudinal view) or to
a circle (for the polar view). No differences were
found between the calculated and the observed
values (Table 1).
Circularity indices for the images observed in
polar views of wild-type Columbia dry seeds yielded
an average value of 0.88 (Table 2).
The comparison of curvature values between the
two seed poles provides information about whether
there are differences in shape between them. In
the case of dry seeds, there was no difference
between maximum curvature values obtained for
both poles (data not shown).
These results led us to accept the idea that the
shape of the dry seed is approximately an ellipsoid
of revolution.
Figure 2. Prolate spheroid (a and b are the long and

short semi-axes, respectively).
Longitudinal view
Polar view
79
81
83
85
87
89
91
93
95
97
99
105
107
Mean
Standard error
Mean
Standard error
186,554.59
193,493.75
29,877.4113
31,431.0973
142,716.233
148,642.588
24,534.4761
25,811.4664
109
53
55
77
103
Table 1. Statistical analysis (t-test) for the comparison between calculated areas and software given areas values for
longitudinal and polar views of dry seeds of Arabidopsis cv. Columbia
51
75
101
47
P2
65
69
45
41
43
P1
63
67
Bezier curves corresponding to the seed poles in the

images were obtained and their curvature values
calculated according to Cervantes and Tocino (2005).
Curvature is expressed in radians per micron (rad/m) and
provides an idea of sharpness or roundness in curves.
TE
61
57
59
4
V Ab.
3
27
semi-axis b obtained from Analysis according to the

formula:
PR
25
ellipsoids of revolution, photographs of the polar and

longitudinal views were taken with a Nikon Coolpix 950
digital camera according to the diagram shown in Figure
2 (polar from P1 and longitudinal views from P2). For
Columbia dry seeds, 20 photographs of the longitudinal
view and 18 of its transversal view were taken. Regarding
other treatments and genotypes, 10 photographs of every
view were used. Photographs were analyzed with the
software AnalySISr (Soft Imaging Systems), specialized in
image processing. Long and short diameters were directly
obtained from the photographs. In this process, graph
paper allowed us to convert pixels into mm.
Two independent measures for the areas of every view
were obtained: one obtained directly by the image
analysis program and the other calculated using the
diameter length in the area formula for circles (polar
view), or the axis length for ellipses (longitudinal view).
The statistical comparison between both values allows us
to accept or reject whether the gures are indeed circles
or ellipses.
In addition, to conrm that the polar view coincides
with a circle, a circularity index was calculated (Schwarz,
1980). Circularity index, denoted by I, is a measure that
establishes the degree of roundness of a geometrical
gure in a plane and for a circumference equals to 1. It
was calculated with the values of area and perimeter
given by the software analysis according to the formula:
Calculated area
Analysis area
Probability (0.05)
0.4786
0.4976
JPLPH : 50519
111
ARTICLE IN PRESS
4
1
C. Robert et al.
Table 2.
study
Mean and standard error of circularity index for the polar views of dry seeds in the different genotypes under
59
3
5
Mean
Std. error
7
9
col
ctr1-1
ein2-1
eto1-1
etr1-1
triple
0.87817394
0.0065203
0.85808874
0.01867804
0.85197882
0.02369746
0.86379887
0.0377162
0.85877989
0.02287831
0.84418956
0.03137029
The shape of dry seeds in mutants in the

ethylene signaling pathway
35.00%
30.00%
11
3h imbibition
20.00%
15.00%
10.00%
5.00%
55
53
51
lu
A comparison of changes occurring in the

shape of wild-type and mutant seeds after
imbibition
TE
EC
Q3
Figure 3 graphically represents the increase in

major parameters at 3 and 24 h, which showed a
10% increase after 3 h of imbibition, showing no
further change up to 24 h after imbibition. The long
diameter also increased in the rst 3 h, though to a
lesser extent (5%), and kept growing until 24 h.
Area, perimeter and volume values corresponding
to the gures of longitudinal views of seeds
increased proportionally after 3 h of imbibition,
showing very little increase between 3 and 24 h of
imbibition.
In the course of imbibition, maximum curvature
values decreased in the basal pole but not in the
apical pole (Table 3). This suggests that, while dry
seeds are similar to ellipsoids of revolution, after
24 h imbibition, wild-type Columbia seeds resemble
the shape of an ovoid (roughly speaking, an egg).
The term ovoid refers to a three-dimensional gure
obtained by the revolution of a curve (also called
ovoid) similar to an ellipse but only with one axis of
symmetry. Although ovoids and ellipsoids are
similar gures and have many characteristics in
common, there exists an important difference
between them: the lack of symmetry of ovoids
across the equatorial plane. This property is a
reection of the polarity observed in seed shape.
49
vo
47
er
45
et
43
er
31
41
et
er
Change in the shape of seeds following

imbibition
29
39
et
27
37
rim
ia
am
Figure 3. Diagram showing percent of increase in the

dimensions of Arabidopsis thaliana wild-type, dry seeds
and after 3 and 24 h of imbibition.
25
35
pe
td
di
23
ea
ar
or
ng
sh
lo
21
0.00%
19
Seed volume was larger in the wild-type Columbia and in the triple mutant (Table 4) than in the
mutants eto1-1 and ctr1-1; these genotypes presented shorter lengths in both axes.
In general, the shape of the wild-type Columbia
seeds is more homogeneous than the shape of the
mutants. This is conrmed by the comparison of
standard error of the mean values of circularity
index for the polar view (Table 2).
Seeds of the mutant genotypes deviated in a
number of aspects from the wild type. For
example, eto1-1 and the triple mutant genotypes
have more elongated seeds. This is supported by
the ratios long/short axis length as shown in Table
5. Seeds of etr1-1 and the triple mutant have very
irregular morphologies (Figure 4).
17
25.00%
PR
% of dry seed
15
61
63
65
67
24h imbibition
13
33
57
As a consequence of imbibition, a general size

increase is expected. The increase was notably
greater in all mutant genotypes than in wild-type
seeds (Table 6).
With respect to mean curvature values at the
poles in dry seeds, no differences were detected
among genotypes (Figure 5). In the basal pole,
curvature values oscillated between 0.0076 for col
and 0.0093 for eto, and in the apical pole, between
0.0085 for col and 0.0122 for eto. For each
genotype there were no differences between
curvature values in both poles in the dry seeds.
After 24 h of imbibition, curvature values (Figure
5) decreased in both poles for all genotypes, except
in the apical pole of the wild-type Columbia seeds.
The greatest change in curvature was in the triple
mutant, and was more pronounced in the basal
pole.
Thus, differences in curvature between poles
were detected only in the wild type after 24 h of
imbibition. These seeds had smaller curvature
values in the basal pole and larger curvature values
in the apical pole. Only in this case can we conclude
JPLPH : 50519
69
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
101
103
105
107
109
111
ARTICLE IN PRESS
5
57
Table 3.
Treatments
Basal pole
Apical pole
T-student (po0.05)
59
Dry seeds
24 h imbibed seeds
0.0085
0.0077
0.0076
0.0066
0.230
0.001
61
t-Test to compare curvature values in the basal and apical poles
Curvature values were obtained for both poles from images of dry seeds and seeds after 24 h of imbibition of the wild-type Arabidopsis
thaliana var. Columbia.
65
9
Table 4a. Main parameters (obtained from Analysis, except volume) corresponding to seeds from different genotypes
of Arabidopsis thaliana
17
ctr1-1
19
21
Ein2-1
eto1-1
etr1-1
23
ein2-1, ers1-2, etr1-7
25
27
Area (mm2)
Perimeter (mm)
Volume (mm3)
69
599.85
49.58
535.73
38.12
591.85
40.93
533.98
36.46
544.90
60.24
633.76
73.53
394.34
36.45
340.61
54.77
370.52
42.40
309.99
31.29
365.42
33.99
375.78
31.45
1.93 105
3.14 104
1.48 105
3.06 104
1.78 105
2.94 104
1.30 105
2.25 104
1.66 105
2.77 104
1.88 105
2.44 104
1519.33
140.09
1517.79
148.78
1660.24
129.74
1448.17
115.87
1604.92
134.81
1741.16
130.08
5.15 107
1.33 107
3.46 107
1.26 107
4.45 107
1.26 107
2.73 107
7.35 106
4.09 107
1.01 107
4.74 107
8.47 106
71
In bold types, mean values. In normal types (below each mean), standard deviations. Obtained from a sample of 20 seeds for Columbia
and 10 seeds for other genotypes.
29
Genotypes
37
39
41
1
eto1-1
ctr1-1
etr1-1
ein2-1
triple
col
Sig.
10
10
10
10
10
20
2.73 107
3.46 107
3.46 107
4.09 107
4.45 107
35
Alfa 0.05
EC
33
of all genotypes, the approximation by ellipsoids of

revolution is correct. After 24 h of imbibition, the
shape of seeds of all other genotypes, except for
the wild type, is modeled to an ellipsoid of
revolution.
ANOVA
0.13891324
TE
Table 4b.
31
0.05750903
4.09 107
4.45 107
4.74 107
5.15 107
0.05012817
Comparison of seed volumes among genotypes.
Table 5. Mean values and standard deviations for the

ratio between long and short diameters in the dry seeds
47
43
45
51
53
55
col
49
col
Short diam (mm)
15
ctr1-1 ein2-1 eto1-1 etr1-1 triple
Mean
1.52 1.60
Std. dev. 0.12 0.19
67
Long diam (mm)
13
PR
11
63
1.61
0.12
1.73
0.15
1.50
0.17
1.70
0.24
that the approximation of the seed shape to the

ellipsoid of revolution was not accurate. Wild-type
seeds after 24 h of imbibition look more like ovoids
than like ellipsoids of revolution. For the dry seeds
73
75
77
79
81
83
85
87
89
91
Discussion
93
The shape of organs is a property integrated in

the architecture of organisms. Because these result
from adaptation to environmental conditions
through generations, both are programmed in the
genome and must be accurately regulated. Changes
in shape are not the result of the action of one, nor
a single group of genes or proteins, but need to be
thoroughly regulated by the interaction of global
pathways able to integrate signals from different
environmental stimuli. The ethylene signal transduction pathway is one such global integrative
pathway regulating plant architecture (Dolan,
1997), and as such it is well described in the model
plant Arabidopsis thaliana (Stepanova and Alonso,
2005). Our objective in this work was to investigate
whether mutations in diverse genes encoding
proteins of the ethylene transduction pathway
could affect seed shape, and to investigate how.
95
JPLPH : 50519
97
99
101
103
105
107
109
111
ARTICLE IN PRESS
6
1
C. Robert et al.
Genotype
col 2005
Longitudinal view
Transversal view
Genotype
eto1-1 2000
Longitudinal view
57
Transversal view
59
61
63
7
9
ctr1-1
2005
etr1-1 2005
65
67
11
13
ein2-1
2005
69
triple 2004
(ein2-1, ers12, etr1-7)
15
71
73
17
23
Table 6.
21
Figure 4. Photographs of Arabidopsis thaliana cv Columbia in longitudinal and polar views corresponding to dry seeds
of all the mutants used in this study.
Increase of size (percent) of the six genotypes under study after 24 h of imbibition
col (%)
ctr1-1 (%)
ein2-1(%)
7.90
10.80
20.06
8.84
32.24
22.26
45.52
77.56
28.85
153.76
13.11
29.63
49.43
19.50
96.40
29
0.0180
33
41
0.0080
77
81
83
85
87
89
91
93
39
0.0120
0.0100
17.78
52.45
82.66
28.34
180.66
95
Curvature poles
0.0140
37
25.82
34.94
63.00
24.79
118.87
EC
0.0160
35
14.30
46.11
67.67
23.88
142.67
TE
31
27
Long diameter
Short diameter
Area (long. view)
Perimeter (long. view)
Volume
triple (%)
PR
25
etr1-1(%)
eto1-1 (%)
75
79
19
0.0060
97
0.0040
43
51
C
U
49
101
0.0000
col
eto1-1
ctr1-1
45
47
99
0.0020
etr
ein2-1
ein2-1etr17ers1-2
apical pole dry seed

apical pole 24hr in water
basal pole dry seed

basal pole 24hr in water
Figure 5. Representation of the change in curvature values (rad/micron) in both poles, basal and apical, for all
genotypes. Bars represent curvature values for dry seeds and after 24 h of imbibition.
105
107
109
53
55
103
Genotypes
Previous results from our laboratory showed that

ethylene-insensitive mutants had reduced curvature values in their root apex during early root
development (Cervantes and Tocino, 2005) and also

in the embryonic root (Noriega et al., 2007).
Because the seed contains the embryo, of which
JPLPH : 50519
111
ARTICLE IN PRESS
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
23
21
PR
19
17
water distribution in seeds and ethylene has been

reported previously (Fountain et al., 1998).
Water uptake during imbibition is triphasic, and
the rst phase is mainly the consequence of matric
forces (Bewley and Black, 1994). Thus, our results
suggest that matric forces are restricted in the
wild-type seeds when compared with mutants.
Seed swelling during imbibition is due primarily to
an increase in the length of the short axis that is
associated with increased seed volume very early
upon imbibition, and followed by a slight elongation (increase in the long axis; Figure 3). The
number of cells remains constant before seed
germination and divisions occur only after radicle
protrusion (Barroco et al., 2005). Thus, the
increase in volume inside the seed may be
attributed to a rapid hydration of intercellular
spaces as well as swelling of the cells. As a
consequence, the seed becomes more rounded,
and the curvature in the basal pole decreases. The
increase in volume is also related to the circularity
index in the polar view. The lowest circularity index
in the polar view occurs in mutant seeds, and in
particular in the triple mutant. This is associated
with a greater variation (i.e. higher standard error
values, Table 2) and, thus, with increased irregularity in seed shape that may allow a greater
increase in volume than in the case of more regular
seeds, with higher circularity index, as in the wildtype seeds.
Water uptake through the seed is more uniform in
seeds of mutants in the ethylene signal transduction pathway (in particular the triple mutant) than
in wild-type seeds. In the mutants, there is a
reduction in curvature in both poles, whereas in the
wild type, there is not such a reduction in curvature
in the apical pole. During imbibition, the wild-type
seeds must have a mechanism that creates polarity,
i.e. that maintains the initial curvature values in
the apical pole and generates differences in
curvature values between both poles with increased values (curves more pronounced) in the
apical pole. This may be related with the reduced
increase in volume observed in the wild type, and,
in general, with the control of seed shape. In the
ethylene signal transduction pathway mutants, the
process of establishment of polarity is disrupted,
and in the triple mutant, the shape of the seeds is
very irregular and distorted. Changes in polar
curvature values during imbibition are maximal in
this genotype. Thus, our results show the relation
between the creation of polarity in the seeds
(altered in all the ethylene mutants) and the
overall maintenance of a regular shape in the seeds
(altered in the triple mutant with high variation in
the shape and aberrant seeds).
TE
15
EC
13
11
the radicle is an important part, and also because

curvature in the poles of the seed is an important
parameter dening seed shape, we reasoned that
the ethylene mutants could also present either
alterations in the curvature values of the seed in
the poles, or in their polarity. To analyze such
differences in polarity, or in general variations in
shape, we rst adjusted seed shape to a geometric
form.
The shape of A. thaliana seeds has been dened
here as prolate spheroid. A prolate spheroid is the
ellipsoid of revolution obtained by rotating an
ellipse about its major axis. In a general sense,
polarity refers to differences between the poles,
which, in the seed, may be due to the asymmetrical
distribution of the embryo, endosperm and other
cell types, tissues or structures inside the seed
cover. But in the geometric analysis presented
here, we prefer using the term polarity in a strict
sense, such that it reects only the differences in
curvature values between the two poles. In this
strict, geometrical sense, the A. thaliana var.
Columbia wild-type seeds acquired their polarity
in the course of imbibition. Only after 24 h of
imbibition was a difference between curvature
values in both poles observed in the wild-type
seeds. During imbibition, swelling of the cells
leads, in general, to increased roundness through
the surface of the seed, but in the wild-type seeds,
swelling is restricted to the basal pole (BP), and
does not occur in the apical pole (AP). Curvature
values in the BP decrease (i.e. the extremes
became more rounded), whereas in the AP, curvature maintains the values corresponding to the dry
seed (Figure 5). Thus, the wild-type Columbia dry
seeds are ellipsoids of revolution that, as a
consequence of the increase in polarity that occurs
in the course of imbibition, become ovoids. In
contrast, all mutant seeds analyzed in this work are
ellipsoids of revolution that increase their volumes
and remain being ellipsoids of revolution after
imbibition. Polarity is due to the differential
swelling of both poles; it occurs only in the wild
type after 24 h imbibition and not in any of the
ethylene signal transduction mutants tested. Thus,
the regulation of polarity in seeds is under the
control of the ethylene signal transduction pathway.
In the course of imbibition, the seeds swell,
resulting in an increase in volume. This increase in
volume is greater in the ethylene mutants, and in
particular in the triple mutant ein2-1, ers1-2, etr17 (Table 4) and is in agreement with higher water
uptake rates during seed imbibition in the mutants
(unpublished results). The relationship between
JPLPH : 50519
57
59
61
63
65
67
69
Q4
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
101
103
105
107
109
111
ARTICLE IN PRESS
8
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
23
21
PR
19
17
inhibitors. It has been much more difcult to nd

phenotypes for null mutants in the etr1 gene such
as etr1-7, because as predicted in the model, the
effect of this mutant will be complemented by the
action of genes encoding homologous proteins such
as etr2, ers1, ers2 or ein4 (Qu et al., 2007).
Similarly, if all the receptors actions converge in
EIN2, as predicted in the model, then it would be
impossible to nd an effect for the triple mutant
ein2-1, ers1-2, etr1-7 presenting additional traits
other than those described for the null mutant
ein2-1. In fact, developmental defects found in this
triple mutant, including altered cell size, were
found to be ein2-dependent (Hall and Bleecker,
2003). Nevertheless, further analysis demonstrated
that the triple mutant also presents growth
responses that are not present in ein2-1 null
mutants (Binder et al., 2004), thus demonstrating
that not all the activities of the ethylene receptors
are mediated through the action of EIN2 protein. In
this work, we have reported developmental alterations in the triple mutant. They are (1) irregularities in seed shape, (2) abnormal increase in seed
volume during germination and (3) abnormal
reduction of curvature values in both poles of the
seed and lack of polarity. Of these traits, 1 is
exclusive in the triple mutant and is absent in ein21 mutants, whereas 2 and 3 are more notable in the
triple mutant but are also present in ein2-1. This
result indicates that null mutants in both receptors
ETR1 and ERS1 have additional effects other than
those mediated via EIN2 protein, and present new
traits suitable for further analysis of the physiological action of the proteins. Interestingly, the role
for ETR1 receptor protein in hydrogen peroxide
signaling in stomatal guard cells has been recently
shown (Desikan et al., 2005), suggesting that the
ethylene receptors may be involved not only in
ethylene binding and signal transduction but also as
transducers of hydrogen peroxide. Ethylene receptors may be sensors of the redox state in the cells,
involved in general regulatory circuits beyond
ethylene sensing. This highlights an important
aspect of broad interest in genetics concerning
nomenclature. When we accept the name of a
gene, we must be aware that the name indicates
one function, but never the only function. Ethylene
mutants are altered not only in ethylene perception or signaling but also in other processes.
A common effect reported here for all the
ethylene mutants consists in the alteration of
polarity. This indicates that polarity may be viewed
as the result of a balanced situation in which,
alterations in any of two opposite directions would
result in a reduced curvature resulting in the lack
of polarity. This is in contrast with the known
TE
15
EC
13
11
Two signaling mechanisms, depending on auxins

and cell wall components, respectively, are important in the establishment and maintenance of
polarity in plant cells (Souter and Lindsey, 2001).
Ethylene signal pathways interact closely with
auxin action (Rahman et al., 2002; Stepanova et
al., 2005), but also it has been long known that
ethylene may have an effect in the cytoskeleton
(Hepler and Palevitz, 1974). Auxins also interact
with the cytoskeleton (Sun et al., 2004). Thus, cell
shape and cell wall structure may be affected by
the direct action of ethylene signaling pathway on
cell wall components, or indirectly, via changes in
regulatory proteins, alterations in the sensibility to
hormones, calcium or other secondary messengers
or affecting general physiological aspects like the
redox status.
Geometric polarity is the result of anatomical
polarity and may be accurately measured by
curvature values in both apical and basal poles. In
the course of seed imbibition in the wild-type
seeds, geometric polarity, i.e. the differences
between maximum curvature values in both poles,
increase. Our results show that an intact ethylene
signal transduction pathway is required for the
establishment of polarity upon seed imbibition.
Wild-type seeds show this polarity at 24 h of
imbibition, which is apparent as differences between curvature values in both poles. Mutants in
any of the genes encoding proteins of the ethylene
signal transduction pathway resulted in a lack of
polarity after 24 h of imbibition.
The current model representing the ethylene
signal transduction pathway has been built on the
basis of diverse experimental evidence from different sources that include the analysis of mutant
genotypes, genetic analysis of epistasis, yeast
transformation, two-hybrid interaction, etc. (Stepanova and Alonso, 2005). The genetic analysis is
based generally in an all or nothing, lineal effect of
mutants, where all mutations tend to be grouped
into two classes: rst, those turning the mechanism
off, and second, those maintaining the system in an
active status resulting in the transcriptional activation of ethylene-regulated transcription factors and
genes. In the rst group, phenotypes resemble
wild-type plants treated with ethylene inhibitors
(either of action or synthesis). The second group of
mutants are characterized by phenotypes that
resemble wild-type plants treated with ethylene.
In this model, it is difcult to nd new phenotypes
for elements redundant in the model. For example,
etr1-1 mutant has a dominant constitutive effect.
Its phenotype is, in many aspects, similar to that of
the mutant ein2-1. Both mutants yield plants that
resemble wild-type plants treated with ethylene
C. Robert et al.
JPLPH : 50519
57
59
61
63
65
67
69
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
101
103
105
107
109
111
ARTICLE IN PRESS
phenotypes in ethylene signaling pathways, resulting in all or nothing responses where two contrasting effects are always observed similar to the
presence or absence of the phytohormone. On the
contrary, the response consisting in the lack of
polarity resembles more a non-linear process, in
which polarity is observed when the conditions
reach an equilibrium but disappears in case the
equilibrium is disrupted. Further disruption results
in severe shape alterations. It may be interesting to
investigate the specic effects that redox status
and free radicals may have in polarity.
7
9
11
13
15
Q5
Uncited reference
17
Kim et al. (1999).
21
19
Acknowledgments
23
25
We thank the late Dr. Anthony Bleecker and Dr.

Brad Binder for kindly providing us with seeds of the
triple mutant ein2-1, ers1-2, etr1-7.
27
41
43
45
47
49
51
53
55
TE
EC
39
37
35
33
Apelbaum A, Burg SP. Altered microbrillar orientation in

ethylene treated Pisum sativum stems. Plant Physiol
1971;48:64852.
Barroco RM, van Poucke C, Bergevoet JHW, de Veylder L,
Groot SPC, Inze D, et al. The role of the cell cycle
machinery in resumption of posembryonic development. Plant Physiol 2005;137:12740.
Bewley JD, Black M. Seeds. Physiology of development
and germination, 2nd ed. New York: Plenum Press;
1994.
Binder BM, OMalley RC, Wang W, Moore JM, Parks BM,
Spalding EP, et al. Arabidopsis seedling growth
response and recovery to etylene. A kinetic analysis.
Plant Physiol 2004;136:291320.
Cervantes E, Tocino A. Geometric analysis of Arabidopsis
root apex reveals a new aspect of the ethylene signal
transduction pathway in development. J Plant Physiol
2005;162:103845.
Chae HS, Faure F, Kieber JJ. The eto1, eto2, and eto3
mutations and cytokinin treatment increase ethylene
biosynthesis in Arabidopsis by increasing the stability
of ACS protein. Plant Cell 2003;15:54564.
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM.
Arabidopsis ethylene-response gene ETR1: similarity
of product to two component regulators. Science
1993;262:53941.
Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley
R, et al. A role for ETR1 in hydrogen peroxide signaling
31
References
29
PR
in
stomatal
guard
cells.
Plant
Physiol
2005;137(3):8314.
Dinneny JR, Weigel D, Yanofsky MF. NUBBIN and JAGGED
dene stamen and carpel shape in Arabidopsis.
Development 2006;133(9):164555.
Dolan L. The role of ethylene in the development of plant
form. J Exp Bot 1997;48(307):20110.
Fountain DW, Forde LC, Smith ES, Owens KR, Bailey DG,
Callaghan PT. Seed development in Phaseolus vulgaris
L. cv Seminole. 3. NMR imaging of embryos during
ethylene-induced precocious germination. Seed Sci
Res 1998;8:35765.
Guzma
n P, Ecker JR. Exploiting the triple response of
Arabidopsis to identify ethylene-related mutants.
Plant Cell 1990;2:51323.
Hall AE, Bleecker AB. Analysis of combinatorial loss-offunction mutants in the Arabidopsis ethylene receptors reveals that ers1 etr1 double mutant has severe
developmental defects that are EIN2 dependent. Plant
Cell 2003;15:203241.
Hepler PK, Palevitz BA. Microtubules and microlaments.
Annu Rev Plant Physiol 1974;25:30962.
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker
JR. CTR1, a negative regulator of the ethylene
response pathway in Arabidopsis, encodes a member
of the Raf family of protein kinases. Cell
1993;72:42741.
Kim G-T, Tsukaya H, Saito Y, Uchimiya H. Changes in the
shapes of leaves and owers upon overexpression of
cytochrome P450 in Arabidopsis. Proc Natl Acad Sci
USA 1999;96(16):94337.
Leon-Kloosterziel KM, Keijzer CJ, Koornneef M. A seed
shape mutant of Arabidopsis that is affected in
integument
development.
Plant
Cell
1994;6(3):38592.
Noriega A, Cervantes E, Tocino A. Ethylene responses in
Arabidopsis seedlings include the reduction of curvature values in the root cap. J Plant Physiol, 2007 [in
press].
Q6
Qu X, Hall BP, Gao Z, Schaller GE. A strong constitutive
ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene
receptors ETR1 and ERS1. BMC Plant Biol 2007;15 7:3.
Rahman A, Oshokawa S, Oono Y, Amakawa T, Goto N,
Tsurumi S. Auxin and ethylene response interactions
during Arabidopsis root hair development dissected by
auxin
inux
modulators.
Plant
Physiol
2002;130:190817.
Souter M, Lindsey K. Polarity and signalling in plant
embryogenesis. J Exp Bot 2001;51:97183.
Schwarz H. Two-dimensional feature-shape indices. Mikroskopie (Wien) 1980;37(Suppl):647.
Stepanova A, Alonso JM. Ethylene signalling and response
pathway: a unique signalling cascade with a multitude
of
inputs
and
outputs.
Physiol
Plant
2005;123:195206.
Stepanova AN, Hoyt JM, Hamilton AA, Alonso JM. A link
between ethylene and auxin uncovered by the
characterization of two root-specic ethylene-insen-
JPLPH : 50519
57
59
61
63
65
67
69
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
101
103
105
107
109
111
ARTICLE IN PRESS
10
F
O
O
PR
D
TE
EC
R
R
O
Tsukaya H. Leaf shape: genetic controls and environmental factors. Int J Dev Biol 2005;49:54755.
Woeste KE, Ye C, Kieber JJ. Two Arabidopsis mutants that
overproduce ethylene are affected in the posttranscriptionnal regulation of 1-1-aminocyclopropane-1carboxylic acid synthase. Plant Physiol 1999;119:
5218.
sitive mutants in arabidopsis. Plant Cell 2005;17:

223042.
Sun H, Basu S, Brady S R, Luciano RL, Muday GK.
Interactions between Auxin transport and the actin
cytoskeleton in developmental polarity of Fucus
distichus embryos in response to light and gravity.
Plant Physiol 2004;135:26678.
C. Robert et al.
JPLPH : 50519
9
11
13
15

Robert Et Al. J Plant Physiol. 2007

Uploaded by

Copyright:

Available Formats

You might also like

Robert Et Al. J Plant Physiol. 2007

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Robert Et Al. J Plant Physiol. 2007

Uploaded by

Copyright:

Available Formats

ARTICLE IN PRESS

Journal of Plant Physiology ] (]]]]) ]]]]]]

Morphological analysis of seed shape in Arabidopsis

Recent analysis by molecular techniques has

Seeds of A. thaliana var. Columbia (col) and its

Materials and methods

values in both poles, we described the changes

Dinneny et al. (2006) described genes involved in

To understand changes that occurred in the seed

Geometric description of seed shape in dry

The visual inspection of seed images suggests

Figure 2. Prolate spheroid (a and b are the long and

Bezier curves corresponding to the seed poles in the

semi-axis b obtained from Analysis according to the

ellipsoids of revolution, photographs of the polar and

The shape of dry seeds in mutants in the

A comparison of changes occurring in the

Figure 3 graphically represents the increase in

Change in the shape of seeds following

Figure 3. Diagram showing percent of increase in the

As a consequence of imbibition, a general size

t-Test to compare curvature values in the basal and apical poles

of all genotypes, the approximation by ellipsoids of

Comparison of seed volumes among genotypes.

Table 5. Mean values and standard deviations for the

Short diam (mm)

ctr1-1 ein2-1 eto1-1 etr1-1 triple

Long diam (mm)

that the approximation of the seed shape to the

The shape of organs is a property integrated in

apical pole dry seed

basal pole dry seed

Previous results from our laboratory showed that

development (Cervantes and Tocino, 2005) and also

water distribution in seeds and ethylene has been

the radicle is an important part, and also because

inhibitors. It has been much more difcult to nd

Two signaling mechanisms, depending on auxins

We thank the late Dr. Anthony Bleecker and Dr.

Apelbaum A, Burg SP. Altered microbrillar orientation in

sitive mutants in arabidopsis. Plant Cell 2005;17:

You might also like