Download Large-scale histological analysis of leaf mutants using two simple

The Plant Journal (2006) 48, 638–644
doi: 10.1111/j.1365-313X.2006.02896.x
TECHNICAL ADVANCE
Large-scale histological analysis of leaf mutants using two
simple leaf observation methods: identification of novel
genetic pathways governing the size and shape of leaves
Gorou Horiguchi1,2,*, Ushio Fujikura2, Ali Ferjani1,†, Naoko Ishikawa1 and Hirokazu Tsukaya1,3
National Institute for Basic Biology, Okazaki Institute for Integrated Bioscience, Myodaiji-cho Nisigo Naka 38, Okazaki, Aichi
444-8585, Japan,
2
School of Life Sciences, Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan, and
3
Graduate School of Science, University of Tokyo, Science Building #2, 7-3-1 Hongo, Tokyo 113-0033, Japan
1
Received 5 April 2006; revised 23 July 2006; accepted 25 July 2006.
*For correspondence (fax þ81 564 55 7513; e-mail [email protected]).
†
Present address: Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan.
Summary
Observations of cellular organization are essential in understanding the mechanisms underlying leaf
morphogenesis. These observations require several preparative steps, such as fixation and clearing of organs,
and such procedures are time-consuming and labor-intensive for large-scale analyses. Thus, we have
developed simple methods for the observation of leaf epidermal and mesophyll cells. To visualize the
epidermis, a gel cast was made of the leaf surface, which was then observed under a light microscope. To
visualize the leaf mesophyll cells, leaves were immersed in a solution containing Triton X-100, briefly
centrifuged, and then viewed under a light microscope. These methods allowed us to conduct a histological
phenome analysis for a large number of known and newly isolated leaf-shape/size mutants of Arabidopsis
thaliana by measuring various parameters, including cell number, size, and distribution of cells within a leaf
blade. Mutants showed changes in leaf size caused by specific increases or decreases in the number and/or size
of cells. In addition, altered cell distributions in the leaf blade were observed, resulting from increases or
decreases in the number of cells along the proximo-distal or medio-lateral axis, or recruitment of cells along a
particular axis at the expense of other leaf parts. These results provide a phenomic view of the cellular behavior
involved in organ size control and leaf-shape patterning.
Keywords: Arabidopsis thaliana, cell number, cell size, leaf shape, leaf size, hormones.
Introduction
To understand plant morphogenesis, detailed observations
of the cellular organization of a given organ are essential.
Physical or optical sectioning of tissues and scanning electron microscopic observation are most often employed for
this purpose.
Several mutants of Arabidopsis thaliana have been
defined by various anatomical characteristics. In the case
of leaves of A. thaliana, cross-sectioning has been used to
analyze cellular phenotypes in multiple cell layers, consisting of the adaxial epidermal layer, palisade and spongy
mesophyll layers, and the abaxial epidermal layer (Tsuge
et al., 1996), and the organization of vascular tissues
638
(McConnell and Barton, 1998). Scanning electron microscopy and nail-polish imprints are often used to observe
structural features of the epidermis, such as the morphology
and distribution of trichomes, guard cells and pavement
cells (Berger and Altmann, 2000; Telfer et al., 1997; Yu et al.,
2005). To vizualize vascular networks and identify mesophyll
cells within the dermis, whole leaves must be fixed and
cleared (Hamada et al., 2000; Tsuge et al., 1996).
Mutants displaying dramatic morphological alterations in
leaf polarity, such as the disruption of bilateral symmetry
and the failure to establish adaxial or abaxial identity, have
been extensively analyzed histologically and have provided
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
Comprehensive analysis of leaf size/shape mutants 639
a wealth of knowledge regarding leaf morphogenesis (for
reviews, see Tsukaya, 2002, 2005). In contrast, numerous
reports have described mutant phenotypes, including alterations in leaf shape and size (e.g. Berná et al., 1999; PérezPérez et al., 2002), but detailed cellular observations of leaf
phenotypes have often not been performed. Nonetheless,
we believe that these histological mutant phenotypes are
valuable sources of information for understanding the
cellular behaviors that affect final leaf size and shape
(Tsukaya, 2006). Thus, the development of easy methods
for histological analysis is necessary for a comparative
analysis of various leaf-shape/size mutants.
We developed two very simple techniques for histological
observation of leaf tissues. Using these methods, we have
been able to analyze a large number of leaf mutants isolated
in our laboratory (Horiguchi et al., 2006) and by others,
including hormonal mutants. To obtain a histological ’phenome’ data set, we focused on the total number and final size
of leaf cells because they are the most important parameters
characterizing the quantitative aspect of the leaf as a determinate organ. In addition, parameters that reflect the
arrangement of cells within a leaf blade are informative of
changes in leaf shape. These data allowed us to postulate
diverse genetic pathways involved in leaf size and shape
control, and also provided an updated view of hormonal
action in leaf development. We thus established a comprehensive view of cellular behaviors affecting leaf size and
shape.
Results and discussion
Observation of the leaf epidermis
Because the epidermis is a transparent tissue, it is difficult to
trace accurately the jigsaw-shaped individual epidermal cells
using optical microscopes. In addition, the epidermal surface
is not completely flat at a microscopic level, making it difficult
to focus over a wide area of tissue, unless scanning electron
microscopy is used. To overcome these problems, we
developed a simple observation method, the dried-gel
method. A drop of 2% low-melt agarose containing 0.01%
bromophenol blue pre-warmed at 50C was placed on a glass
slide, and the leaf sample was immediately gently placed on
it. Once the gel solidified, the leaf material was carefully
peeled off, and the remaining gel cast was left to dry for about
10 min. The gel cast was then observed without a cover glass
under a differential phase contrast microscope (Figure 1).
Although this method cannot be applied to serial analysis of
epidermis development, it allows quicker acquisition of epidermal image than the nail polish imprinting method, which
requires two preparative steps, namely dental resin imprinting and subsequent copying of the resin surface by nail polish
(Berger and Altmann, 2000). The images obtained using the
dried-gel method are of high quality, and the contours of
Figure 1. Epidermal tissues observed using the dried-gel method.
(a) Adaxial epidermis (upper panel) and abaxial epidermis (lower panel) of
leaf blades.
(b) Adaxial epidermis of leaf petiole.
(c) Adaxial epidermis of wild-type (upper panel) and an3-4 mutant (lower
panel) petals.
Bars ¼ 100 lm.
individual cells, including guard cells (Figure 1a), can be
easily distinguished, thereby allowing rapid measurements
of cell size, shape (length, width and perimeter) and density,
and stomatal density. This method can also be used to
observe the epidermis of other organs, such as petioles
(Figure 1b) and petals (Figure 1c). Because petal cells are
conical in shape, they are quite difficult to observe under a
microscope without using this method. As an example, we
compared the adaxial epidermis of petals of the wild-type
(Figure 1c, upper panel) and the angustifolia3 (an3) mutant,
also known as grf interacting factor1 (gif1; Kim and Kende,
2004) (Figure 1c, lower panel). We previously showed that
the an3 mutant has fewer, but larger, leaf cells than the wildtype (Horiguchi et al., 2005). The petal epidermal cells of the
an3 mutant were also found to be larger than those of the
wild-type, demonstrating the efficacy of this method.
Observation of palisade cells
Hoyer’s solution, which contains chloral hydrate, and its
modified versions are often used in histological observations because of their tissue-clearing ability (Anderson,
1954). The optimal concentration of chloral hydrate may
differ depending on the tissues examined. Therefore, we
previously determined the optimal concentration of chloral
hydrate solution for leaves (Tsuge et al., 1996). Leaves were
fixed in formalin-acetic acid-alcohol (FAA) under a vacuum
and then cleared in chloral hydrate solution (200 g chloral
hydrate, 20 g glycerol, 50 ml H2O). This method reproducibly provides good images of cells (Figure 2a; note that the
size of mesophyll cells in the an3 mutant [right] is larger than
in the wild-type [left]).
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644
640 Gorou Horiguchi et al.
Figure 2. Observation of palisade cells in fully expanded first leaves.
(a) Palisade cells in leaves from wild-type (left panel) and the an3-4 mutant
(right panel) cleared using chloral hydrate solution.
(b) Palisade cells in leaves from wild-type (left panel) and the an3-4 mutant
(right panel) prepared using the centrifugation method.
Bars ¼ 100 lm.
sense. For each mutant, we measured the area, length
and width of the leaf blade, determined the number and
area of palisade cells in the sub-epidermal layer, and
counted the number of palisade cells aligned along the
proximo–distal (P–D) and medio–lateral (M–L) axes. We
then calculated the leaf index (leaf blade length/width
ratio) and cell number index (ratio of the number of cells
in the P–D/M–L axes) (see Table S1).
The quantitative phenotypes of these plants were verified
using the leaf blade area and number and size of palisade
cells in the sub-epidermal layer. Most mutants examined
were small-leaf mutants; large-leaf mutants were relatively
rare. Changes in leaf area were associated with specific
changes in the number and/or size of cells (Figure 3).
Theoretically, there are nine possible classes of mutants,
according to changes in cell number and size (Horiguchi
et al., 2006). Mutants with normal cell number and size were
associated with changes in leaf shape, and this particular
class is discussed later. We previously found mutants of all
possible combinations except for an increase in both cell
number and size (Horiguchi et al., 2006). Here, we found that
ethylene insensitive3 (ein3), ethylene response1 (etr1) and
auxin response factor2 (arf2) mutants potentially belong to
this previously unidentified class (Table 1). Thus, by characterizing these known hormonal mutants we are able to
find mutants for all possible combinations of changes in leaf
cell number and size.
This method, however, requires several intricate steps,
such as the removal of intercellular air bubbles, fixation and
clearing. Thus, we developed a very simple alternative
method. Leaves were placed into a microcentrifuge tube and
immersed in 0.1% Triton X-100, followed by centrifugation at
10 000 g for 1 min at room temperature. This centrifugation
method not only removed air bubbles from intercellular
spaces, but also sedimented the chloroplasts, greatly facilitating counting the number of cells (Figure 2b). These
images are of sufficient quality for measuring the size of
individual palisade cells [compare palisade cells in the wildtype and the an3 mutant (Figure 2b)]. Thus, this method
could serve as an alternative for performing quick observations of leaf mesophyll cells.
Verification of leaf-shape/size mutants by the number and
size of leaf cells
We applied the centrifugation method to previously isolated mutants (Horiguchi et al., 2006) and new mutant
lines (the number of mutant lines has expanded from 147
to 205). We also examined the leaf phenotypes of several
hormonal mutants because most of these mutants are
reported to have distinct leaf phenotypes, although their
cellular organization has not been examined in a strict
Figure 3. Characterization of palisade cells in 205 mutant lines.
The number and size of palisade cells in each mutant were determined using
the centrifugation method. Data represent the average cell number and cell
size of each mutant line normalized to those of the wild-type (n ¼ 8). Open
triangles, squares and circles indicate that either cell size or cell number, or
both, are statistically different from corresponding wild-type values, respectively, while closed circles indicate that neither cell size nor cell number are
significantly different from the wild-type values (P < 0.05, Student’s t-test).
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644
Comprehensive analysis of leaf size/shape mutants 641
Table 1 Characterization of leaf organization in hormonal mutants
Mesophyll cells
2
2
Pavement cells
Genotype
Leaf area (mm )
Area (lm )
Number
Area (lm2)
Number
Stomatal index
Wild-type
aba2-1
aba3-1
axr1-3
axr1-12
arf2-1
arf7-1
arf7-1 arf19-1
ctr1-12
ein3-1
eto1-1
etr1-1
jar1-1
48.0 3.6 (100)
20.6 2.7* (43)
33.4 3.8* (70)
28.8 2.4*(60)
22.5 2.7* (47)
69.7 5.0* (145)
40.8 1.8* (85)
21.4 3.4* (45)
5.5 0.9* (12)
57.5 5.5* (120)
25.0 4.0* (52)
65.1 6.7* (136)
36.6 5.6* (76)
4186 444 (100)
3093 300* (74)
3388 393* (81)
3116 454* (75)
2540 369* (61)
4595 666 (110)
3390 389* (81)
1824 100* (44)
2085 238* (50)
4432 421 (106)
2856 465* (68)
5067 612* (121)
3545 310* (85)
11510 1194 (100)
6524 919* (57)
9450 1149* (82)
9636 884* (84)
9343 1229* (81)
14672 1715* (128)
12522 1145 (109)
12054 2298 (105)
2975 286* (26)
12993 1471* (113)
8494 1410* (74)
12828 1304* (112)
10111 1088* (88)
3603 208 (100)
2285 170* (63)
3096 309* (86)
2666 376* (74)
2033 402* (56)
4541 247* (126)
3444 292 (96)
1752 162.3* (49)
1631 173* (45)
3847 230 (107)
2686 323* (75)
4885 722* (134)
2865 312* (80)
22762 (100)
16603 (73)
19737 (87)
18368 (81)
17965 (79)
28026 (123)
19996 (88)
19955 (88)
6037 (27)
25051 (110)
12760 (56)
21597 (94.9)
22422 (99)
17.7 0.7
19.4 0.4*
19.1 0.4*
17.7 0.6
16.9 0.4*
18.9 0.4*
17.5 0.6
17.5 0.6
18.9 1.1*
17.3 0.5
17.4 0.9
16.5 0.7*
18.3 0.7
Leaves from 25-day-old plants were used. Data are means SD (n ¼ 8 for each line); relative values compared with wild-type are shown in
parentheses. For each leaf, the average cell area was determined by measuring 20 palisade cells or all epidermal cells within a 0.4 lm2 area. The
total number of palisade cells was determined by dividing the leaf area by the palisade cell density for each leaf. The total number of epidermal cells
was estimated by dividing the mean leaf area by the mean epidermal cell size (pavement plus guard cells). Stomatal index (SI) is determined by the
following formula: SI ¼ [S/(E þ S)] · 100 where S is the number of stomata per unit area and E is the number of epidermal cells per unit area
(Mishra, 1997). Asterisks indicate significant differences from the wild-type (Student’s t-test, P < 0.05).
Genetic pathways controlling leaf proportions via cell
proliferation
Measurements of the number of palisade cells along two
leaf axes allowed us to classify mutants according to axisdependent changes in cell number. We then examined
whether such changes were correlated with changes in leaf
shape. The leaf index roughly represents the overall proportions of the leaf blade. The first leaves of the wild-type are
almost circular (i.e. a leaf index of approximately 1.0), thus
most leaf-shape mutants have narrower/longer or shorter/
wider leaf blades than the wild-type (Figure 4a,c). We compared the leaf index and cell number index within individual
mutant lines. Changes in the leaf index were correlated with
changes in the cell number index (Figure 4a), suggesting
that cell proliferation rather than polar cell expansion is
affected in these mutants.
Leaf-shape mutants can be further sub-classified according to changes in the number of palisade cells along leaf
axes. Mutants in which the cell number index differed by
more than 10% from that of the wild-type are shown in
Figure 4(b). In many cases, the changes were caused by a
greater decrease in the number of palisade cells along one
axis than the other. For example, an3/gif1 produces narrow
leaves because of a severe decrease in cell number along the
M–L axis compared to the P–D axis (Horiguchi et al., 2005;
Kim and Kende, 2004). Mutants that are associated with
specific changes in cell number along one particular leaf axis
have narrow, long, wide or short leaves (Figure 4c). Palisade
cell numbers along the P–D or M–L axis in these mutants
ranged from 75 to 125% of cell numbers in the wild-type
(Figure 4b), suggesting the occurrence of positive and
negative genetic pathways for leaf-shape control involving
cell proliferation. The only characterized mutant in this subclass is rot4-1D, which is associated with a specific decrease
in cell number along the P–D axis (Narita et al., 2004)
(Figure 4c). An alternative interpretation of axis-specific
phenotypes is that these mutants are impaired in both
appropriate patterning and cell proliferation; i.e. the altered
cell number along a specific leaf axis is just a coincidence
because of the weak phenotypes. These possibilities should
be examined in future investigations of mutant alleles and
their corresponding gene function.
Two other sub-classes had less obvious phenotypes in
relation to the total number of palisade cells. Interestingly,
these sub-classes were associated with an increase in the
number of palisade cells along one axis at the expense of
cells along the other axis. Thus, these mutations affected the
direction of growth in proliferative tissues, rather than
proliferating activity itself. Although a long-range signal
that orients growth has been proposed in petals of
Antirrhinum majus (Rolland-Lagan et al., 2003), how the
direction of growth is guided in leaf primordia is not yet
understood. Thus, these mutants may be a desirable starting
material for the investigation of this issue.
Re-evaluation of hormonal mutants
The development of these simple, easy observation methods prompted us to re-characterize known hormonal mutants because they are known to affect growth and
development, but lack quantitative descriptions of leaf
morphology. We measured the leaf area, and the area and
number of sub-epidermal palisade and adaxial epidermal
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644
642 Gorou Horiguchi et al.
(a)
(b)
(c)
Figure 4. Geometric distribution of palisade cells in leaf blades.
(a) Comparison of the leaf index (leaf blade length/width ratio) and cell
number index (ration of number of cells in the proximo–distal [P–D] and
medio–lateral [M–L] axes) within 205 mutant lines (n ¼ 8 for each mutant line).
(b) Comparison of palisade cell numbers along the P–D and M–L axes. Leafshape mutants with differences of >10% (squares, 51 mutant lines) and <10%
(circles, 29 mutant lines) in cell number index compared to the wild-type are
shown (n ¼ 8 for each mutant line). Closed squares and circles indicate the
mutantlinesshownin(c).Thepositionofthewild-typeisindicatedbyadiamond.
(c) Appearance of leaf-shape mutants. Fully expanded first leaves taken from
25-day-old plants are shown. From left to right, line 1027 (narrow leaf),
line 2063 (long leaf), wild-type, line 2023 (wide leaf) and rot4-1D (short leaf).
Bar ¼ 5 mm.
pavement cells, and determined the stomatal index
(Table 1).
We found several results that have not been previously
reported. An early report of auxin resistant 1 (axr1) alleles
suggested that axr1 mutations mainly affect cell number in
relation to leaf development (Lincoln et al., 1990). However,
we found that both cell number and size were reduced in
axr1-3 and axr1-12 leaves, irrespective of cell type (Table 1).
In contrast, when we examined the arf7 arf19 double mutant
(Okushima et al., 2005b), we found a specific effect of these
mutations on cell size. In the double mutant, cell proliferation was not affected at a statistically significant level, but
cell size and leaf blade area were reduced to a similar extent
to each other in comparison to the wild-type, as reported by
Wilmoth et al. (2005). In contrast, the large-leaf phenotype of
the arf2 mutant (Okushima et al., 2005a) was associated with
an increase in the number and size of leaf cells (Table 1).
These results indicate specific roles of particular members of
the ARF gene family in promoting leaf cell expansion. The
effects of mutations in other members of the ARF gene
family on leaf cell proliferation and expansion should be
examined.
We also found an overlooked phenotype in ethylenerelated mutants. Although the constitutive triple response1
(ctr1) mutant is defective in cell expansion (Kieber et al.,
1993), we found that cell proliferation was inhibited to an
even greater extent than cell expansion in this mutant. The
ctr1-12 mutant contained only 26% of the number of leaf
cells found in the wild-type, which is one of the lowest cell
numbers among all the mutants we examined, whereas its
cell size was reduced to about 50% of that of the wild-type
(Table 1). Likewise, the ethylene overproducer 1 (eto1)
mutations (Guzmán and Ecker, 1990) also inhibited both cell
expansion and proliferation (Table 1). Although the relationship between ethylene and cell division has been
characterized by only a few studies (Kazama et al., 2004),
the strong defect in cell proliferation in ctr1 indicates the
importance of normal ethylene signaling during the proliferative phase of leaf development.
ABA and ethylene have opposing effects on leaf blade
expansion (LeNoble et al., 2004; León and Sheen, 2003).
Whereas etr1 and ein3 mutants produced larger leaves,aba
deficient2 (aba2) and aba3 mutants produced smaller leaves
than the wild-type. These differences were correlated with
increases in both cell number and size for the etr1 and ein3
mutants, and decreases in both cell number and size for the
aba2 and aba3 mutants (Table 1). We also examined the
effect of the jasmonate resistant1 (jar1) mutation (Staswick
et al., 2002) on leaf development; this mutation caused
marginal decreases in cell number and size (Table 1). Thus,
both cell proliferation and expansion were affected in most
hormonal mutants examined, suggesting that these hormones play a role in the growth of whole organs. However,
as demonstrated by the specific defect of arf7 arf19 mutants
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644
Comprehensive analysis of leaf size/shape mutants 643
in leaf cell expansion, it is tempting to speculate that genes
downstream of these hormone-related genes play a specific
role in either cell proliferation or expansion. How hormonal
perception and subsequent signal transduction are linked to
the processes of cell proliferation and expansion remains to
be determined.
Finally, we are planning to donate mutants isolated by us
and described in this study to public seed banks after the
accomplishment of more detailed characterization of several
mutants. We hope that our mutant collection serves the
Arabidopsis research community as a useful tool for developmental biology.
Conclusions
Experimental procedures
We developed two simple methods for the histological
observation of leaves that greatly facilitated the characterization of leaf mutants. Cellular-phenotype-based classification of several leaf size/shape mutants largely
elucidated the genetic architecture of leaf expansion.
We showed a good correlation between leaf index and cell
number index among our mutant lines. This suggests that
although leaf shape in wild-type is very simple (almost
circular in the case of the first leaves), the final leaf shape
could be considered as the integrated output of several
developmental regulatory pathways influencing morphogenesis. Two types of regulation would account for altered
leaf shapes in these mutants. First, in every round of cell
division, the orientation of the cell division plane relative to
the leaf axes must be decided. If such a decision is affected
by a mutation, leaf shape may be altered accordingly. The
second possibility is that there are unknown sub-domains in
leaf primordia where cell proliferation is locally promoted or
inhibited. In this case, locally altered cell number is merely a
consequence of mis-specification of a particular pattern
rather than the cause of altered leaf shape. These possibilities are not mutually exclusive, and whether such regulations are operative in leaf morphogenesis or not is unclear.
This important issue will be addressed in future work using
our mutant lines.
For organ-size control, we have been able to classify
specific mutants positively or negatively affecting either cell
proliferation or expansion. These mutants will serve as
valuable tools to examine the details of developmental
processes in comparison with hormonal mutations, which
have pleiotropic effects on cell proliferation and expansion
in most cases. In terms of the cellular patterning involved in
two-dimensional leaf expansion, we identified new polaritydependent cell-proliferation mutants. Our mutant analysis
classified mutants by similar cellular phenotypes. Mutations
from the same or opposite classes may be involved in the
same developmental pathway. Furthermore, mutants in the
same class may be sub-classified by comparing their leaf
development kinetics. Together with our earlier work on
polarity-dependent cell expansion, revealed by the characterization of the an and rot3 mutants (Kim et al., 1998, 1999,
2002; Tsuge et al., 1996), the in-depth analysis of each
mutant, as well as the examination of genetic interactions
between different mutant classes, will reveal the organization of the gene network for leaf blade expansion.
Plant materials
Wild-type plants and all mutants were of the Col-0 background.
Mutants with a line number below 2000 were isolated from X-ray or
gamma-ray-irradiated M2 populations, and those with a number
2000 and over were from T-DNA-tagged T2 populations. Seeds were
sown on rock wool (Nittobo, Tokyo, Japan), and seedlings were
grown at 22C under a 16 h light/8 h dark cycle at a light intensity of
40 lmol m)2 sec)1, and were watered daily with 0.5 g l)1 Hyponex
solution (Hyponex Japan, Osaka, Japan). The seeds of aba2-1, aba31, axr1-3, axr1-12, arf2-1, arf7-1,arf7-1arf19-1, ctr1-12, ein3-1, eto1-1,
etr1-1 and jar1-1 mutants were obtained from the Arabidopsis
Biological Resource Center (ABRC, Columbus, OH, USA).
Histological analysis
Whole leaves and leaf cells were observed under a stereoscopic
microscope (MZ16a) and a Nomarski differential interference contrast microscope (DMRX E) (both from Leica Microsystems, Tokyo,
Japan), respectively. Palisade cells in the sub-epidermal layer and
adaxial epidermal cells in the center of the leaf blade between the
mid-vein and the leaf margin were examined. The density of palisade cells per unit area in this region (0.4 lm2) was determined, and
the area of the leaf blade was divided by this value to calculate the
total number of palisade cells in the sub-epidermal layer. For estimation of the total number of epidermal cells, the mean leaf blade
area was divided by the mean epidermal cell size per unit area
(0.4 lm2). To determine the cell area, 20 palisade cells from each
leaf were measured. To determine the cell number index, palisade
cells were counted along the P–D axis about five-cell distance away
from the mid-vein, and along the M–L axis at the widest region of
the leaf blade. The stomatal index (SI) was determined according to
the formula, SI ¼ [S/(E þ S)] · 100, where S is the number of stomata per unit area and E is the number of epidermal cells per unit
leaf area (Mishra, 1997). All measurements were performed using
Image J software (http://rsb.info.nih.gov/ij/).
Acknowledgements
We thank Dr H. Fukaki (Nara Institute of Science and Technology,
Nara, Japan), Dr T. Shikanai (Kyushu University, Fukuoka, Japan)
and Dr K. Torii (University of Washington, WA, USA), for seeds of
the sgr1-1, paa1-4 and er-102 mutants, respectively. We thank Ms
C. Yamaguchi and Ms M. Nagura for daily care of the plants. This
work was supported by a Grant-in-Aid for Scientific Research on
Priority Areas and for Young Scientists (B) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, by
a Grant-in-Aid for Creative Scientific Research from the Japan
Society for the Promotion of Science, and by grants from the BioDesign Program of the Ministry of Agriculture, Forestry and Fishes
of Japan, the Toray Science Foundation, and the Sumitomo
Foundation.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644
644 Gorou Horiguchi et al.
Supplementary Material
The following supplementary material is available for this article
online:
Table S1. Histological characterization of leaf-shape/size mutants
This material is available as part of the online article from http://
www.blackwell-synergy.com.
References
Anderson, L.E. (1954) Hoyer’s solution as a rapid permanent
mounting medium for bryophytes. Bryologist, 57, 242–244.
Berger, D. and Altmann, T. (2000) A subtilisin-like serine protease
involved in the regulation of stomatal density and distribution in
Arabidopsis thaliana. Genes Dev. 14, 1119–1131.
Berná, G., Robles, P. and Micol, J.L. (1999) A mutational analysis of
leaf morphogenesis in Arabidopsis thaliana. Genetics, 152, 729–
742.
Guzmán, P. and Ecker, J.R. (1990) Exploiting the triple response of
Arabidopsis to identify ethylene-related mutants. Plant Cell, 2,
513–523.
Hamada, S., Onouchi, H., Tanaka, H., Kudo, M., Liu, Y.-G., Shibata,
D., Machida, C. and Machida, Y. (2000) Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of
shoots without juvenile leaves. Plant J. 24, 91–101.
Horiguchi, G., Kim, G.-T. and Tsukaya, H. (2005) The transcription
factor AtGRF5 and the transcription coactivator AN3 regulate cell
proliferation in leaf primordia of Arabidopsis thaliana. Plant J. 43,
68–78.
Horiguchi, G., Ferjani, A., Fujikura, U. and Tsukaya, H. (2006)
Coordination of cell proliferation and cell expansion in the control
of leaf size in Arabidopsis thaliana. J. Plant Res. 119, 37–42.
Kazama, H., Dan, H., Imaseki, H. and Wasteneys, G.O. (2004) Transient exposure to ethylene stimulates cell division and alters the
fate and polarity of hypocotyl epidermal cells. Plant Physiol. 134,
1614–1623.
Kieber, J.J., Rothernberg, M., Roman, G., Feldmann, K.A. and Ecker,
J.R. (1993) CTR1, a negative regulator of the ethylene response
pathway in Arabidopsis, encodes a member of the Raf family
protein kinases. Cell, 72, 427–441.
Kim, J.H. and Kende, H. (2004) A transcriptional coactivator, AtGIF1,
is involved in regulating leaf growth and morphology in Arabidopsis. Proc. Natl Acad. Sci. USA, 101, 13374–13379.
Kim, G.-T., Tsukaya, H. and Uchimiya, H. (1998) The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the
cytochrome P-450 family that is required for the regulated polar
elongation of leaf cells. Genes Dev. 12, 2381–2391.
Kim, G.-T., Tsukaya, H., Saito, Y. and Uchimiya, H. (1999) Changes in
the shapes of leaves and flowers upon overexpression of
cytochrome P450 in Arabidopsis. Proc. Natl Acad. Sci. USA, 96,
9433–9437.
Kim, G.-T., Shoda, K., Tsuge, T., Cho, K.-H., Uchimiya, H., Yokoyama,
R., Nishitani, K. and Tsukaya, H. (2002) The ANGUSTIFOLIA gene
of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion,
the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell wall formation. EMBO J. 21, 1267–
1279.
LeNoble, M.E., Spollen, W.G. and Sharp, R.E. (2004) Maintenance of
shoot growth by endogenous ABA: genetic assessment of the
involvement of ethylene suppression. J. Exp. Bot. 55, 237–245.
León, P. and Sheen, J. (2003) Sugar and hormone connections.
Trends Plant Sci. 8, 110–116.
Lincoln, C., Britton, J.H. and Estelle, M. (1990) Growth and development of the axr1 mutants of Arabidopsis. Plant Cell, 2, 1071–
1080.
McConnell, J.R. and Barton, M.K. (1998) Leaf polarity and meristem
formation in Arabidopsis. Development, 125, 2935–2942.
Mishra, M.K. (1997) Stomatal characteristics at different ploidy
levels in Coffea L. Ann. Bot. 80, 689–692.
Narita, N.N., Moore, S., Horiguchi, G., Kubo, M., Demura, T.,
Fukuda, H., Goodrich, J. and Tsukaya, H. (2004) Overexpression
of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana. Plant J. 38,
699–713.
Okushima, Y., Mitina, I., Quach, H.L. and Theologis, A. (2005a)
AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator. Plant J. 43, 29–46.
Okushima, Y., Overvoorde, P.J., Arima, K. et al. (2005b) Functional
genomic analysis of the AUXIN RESPONSE FACTOR gene family
members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell, 17, 444–463.
Pérez-Pérez, J.M., Serrano-Cartagena, J. and Micol, J.L. (2002)
Genetic analysis of natural variations in the architecture of Arabidopsis thaliana vegetative leaves. Genetics, 162, 893–915.
Rolland-Lagan, A.-G., Bangham, J.A. and Coen, E. (2003) Growth
dynamics underlying petal shape and asymmetry. Nature, 422,
161–163.
Staswick, P.E., Tiryaki, I. and Rowe, M.L. (2002) Jasmonate res\ponse locus JAR1 and several related Arabidopsis gene encode
enzymes of the firefly luciferase superfamily that show activity on
jasmonic, salicylic, and indole-3-acetic acids in an assay for
adenylation. Plant Cell, 14, 1405–1415.
Telfer, A., Bollman, K.M. and Poethig, S. (1997) Phase change and
the regulation of trichome distribution in Arabidopsis thaliana.
Development, 124, 645–654.
Tsuge, T., Tsukaya, H. and Uchimiya, H. (1996) Two independent
and polarized processes of cell elongation regulate leaf blade
expansion in Arabidopsis thaliana (L.) Heynh. Development, 122,
1589–1600.
Tsukaya, H. (2002) Leaf development. In The Arabidopsis Book
(Somerville, C.R. and Meyerowitz, E.M., eds). Rockville, MD:
American Society of Plant Biologists (doi/10.1199/tab.0072, http://
www.aspb.org/publications/arabidopsis/).
Tsukaya, H. (2005) Leaf shape: genetic controls and environmental
factors. Int. J. Dev. Biol. 49, 547–555.
Tsukaya, H. (2006) Mechanism of leaf shape determination. Annu.
Rev. Plant Biol. 57, 477–496.
Wilmoth, J.C., Wang, S., Tiwari, S.B., Joshi, A.D., Hagen, G.,
Guilfoyle, T.J., Alonso, J.M., Ecker, J.R. and Reed, J.W. (2005)
NPH4/ARF7 and ARF19 promote leaf expansion and auxininduced lateral root formation. Plant J. 43, 118–130.
Yu, L., Yu, X., Shen, R. and He, Y. (2005) HYL1 gene maintains
venation and polarity of leaves. Planta, 221, 231–242.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 638–644