Cellular Parameters of the Shoot Apical Meristem in Arabidopsis

15 Pages·1998·0.5 MB·English

The Plant Cell, Vol. 10, 1375–1389, August 1998, www.plantcell.org © 1998 American Society of Plant Physiologists
Cellular Parameters of the Shoot Apical Meristem
in Arabidopsis
Patrick Laufs,a Olivier Grandjean,b Claudia Jonak,a,1 Kiên Kiêu,c and Jan Traasa,2
aLaboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026 Versailles
Cedex, France
bDépartement de Génétique et d’Amélioration des Plantes, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France
cUnité de Biométrie, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France
The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With
the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a de-
tailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija
ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a se-
ries of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within
the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was
four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with
the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the
CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, be-
cause the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone
of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results
suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the
CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic
analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased
size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis
showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation
rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem
function.
INTRODUCTION
Shoot apical meristems (SAMs) are small groups of dividing whereas L2 and L3 provide cells for the inner parts of the or-
cells that initiate all of the aerial parts of the plant. According gans (Satina et al., 1940; reviewed in Steeves and Sussex,
to a widely accepted model, SAMs are composed of func- 1989).
tionally distinct zones (see, e.g., Steeves and Sussex, 1989; Recently, genetic analysis with different model species
Medford, 1992; Clark, 1997; Kerstetter and Hake, 1997). In has led to the identification of a number of genes involved in
spermatophytes, the cells at the summit of the SAM, which meristem function. In Arabidopsis, the SHOOT MERISTEM-
form the central zone, are thought to function as a source of LESS (STM) gene, a member of the KNOTTED homeobox
cells for the peripheral zone, where organ primordia are initi- family, is necessary for setting up and maintaining the SAM
ated, and for the underlying rib zone, which gives rise to the (Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al.,
pith of the stem. Superimposed on this organization into 1996), whereas the receptor kinase CLAVATA1 (CLV1) is
zones is a partitioning into layers. In dicots, three layers can necessary for limiting meristem size (Clark et al., 1993,
be distinguished, and they are called L1, L2, and L3. Cell lin- 1997). A range of other mutations leading to abnormal mer-
eage studies have shown that a great majority of the cells istem size has been described. For instance, in wuschel, cell
derived from the meristematic L1 layer form the epidermis, proliferation within the meristem appears to be reduced,
whereas fasciata and mgoun (mgo), like clv, show meristem
overgrowth (Leyser and Furner, 1992; Laux et al., 1996;
1Current address: Institute of Microbiology and Genetics, Dr. Bohrgasse
Laufs et al., 1998b). Although the macroscopic modifica-
9, A-1030 Vienna, Austria.
2To whom correspondence should be addressed. E-mail traas@ tions observed in these and other mutants have been well
versailles.inra.fr; fax [phone]. characterized, it is often not obvious how the mutations affect
1376 The Plant Cell
development at the cellular level. For instance, one of the The morphology of 12 meristems of each ecotype was
proposed functions for the CLV1 and CLV3 genes is the lim- more precisely analyzed by determining the height of the
itation of cell division rates in the central zone (Clark et al., meristem in four directions (see Methods and Figure 2I). In
1993, 1995, 1996; Laux and Schoof, 1997), and more or less the two ecotypes, no significant differences were found in
direct roles in the coordination of cell division have been meristem shape along the four radii defined in Figure 2I.
proposed for a range of genes (Meyerowitz, 1997; Traas and Therefore, we considered the meristems to be symmetric
Laufs, 1998). However, there are no detailed studies de- dome-shaped structures. Meristem height at different dis-
scribing the spatial and temporal distribution of cell division tances from the central axis is represented in Figure 3. The
and expansion in any mutant. Indeed, at present, no refined meristem shape was also directly determined on longitudi-
description of the proliferation and growth characteristics of nal sections (Figure 1C), which confirmed the shape found
the meristematic cells in the widely used model plant Arabi- by reconstructions of serial transverse sections (Figure 3).
dopsis has been reported.
To define a frame of reference for future work on the con-
trol of cell proliferation and growth in the meristem, we have
Cell Size and Cell Number in the Inflorescence Meristem
quantified cell size and mitotic activity in the young inflores-
cence meristem of Arabidopsis by using confocal laser scan-
As shown in Figures 1 and 2, the L1, L2, and L3 layers could
ning microscopy and image analysis. Together, our results
be clearly distinguished. Based on the partitioning into lay-
lead to a cellular model of this meristem. We have used this
ers and the four concentric zones defined in Methods and
model to analyze division patterns in two mutants showing
Figure 2I, the meristem was divided into seven subdomains
meristem overgrowth. They are clv3-1 and mgo2.
(Figure 4, A to G). The distance between the nuclei, which is
a direct measure for cell size, was determined in each zone
(Table 1). The results point to a number of minor differences
and similarities between the two ecotypes. Cell size in-
RESULTS creased from the outer layer to the inner parts of the mer-
istem. This difference in cell size was visible on the longitudinal
and transverse sections (see, e.g., Figures 1C, 1D, and 2G).
Wild-Type Inflorescence Meristems
In both ecotypes, cell size was uniform throughout L1, and
no central zone or peripheral zone distinction could be made
As described previously (Smyth et al., 1990), the Arabidop-
based on this criterion. L3 cells were larger than L1 cells in
sis inflorescence meristem produces floral primordia at its
both Ler and WS. The meristem shape curve (Figure 3) was
flank in regular, spiral, clockwise (Figure 1A), or counter-
used for calculating the area of the meristem surface. By us-
clockwise patterns (Figure 1B). The cellular parameters of
ing the mean L1 surface/cell ratio, we found that in both
the meristem were determined on transverse and longitudi-
ecotypes, the outer L1 layer of the average meristem is
nal optical sections of the apical dome (Figures 1C, 1D, and
composed of (cid:122)150 cells (Table 1).
2A to 2H). The zone analyzed extended from the top of the
meristem to the meristem base, which was defined as the
level at which the third visible primordium (P3) joins the mer-
istem (Figures 1A, 1B, 1E, and 2G; see also Methods). This Mitotic Index in the Inflorescence Meristem
primordium was at stage 2, as defined by Smyth et al.
(1990). A total of 55 Wassilewskija (WS) and 53 Landsberg The number of mitoses in individual Ler meristems varied
erecta (Ler) apices were optically sectioned and analyzed. from three to 17 (mean 9.3 (cid:54) 0.6; n (cid:53) 49); in WS, values be-
Note that the apices were growing rapidly because they tween two and 21 were found (mean 9.8 (cid:54) 0.6; n (cid:53) 50). The
were harvested when the most developed flower bud had mitotic index was determined in different subdomains of L1
just opened (stage 13, as defined by Smyth et al., 1990). (Figure 4, A and D), L2 (E and F), and L3 (subdomain G). For
Figures 1A and 1B show that the three-dimensional informa- this purpose, the number of cells in interphase and mitosis
tion obtained with the confocal laser scanning microscope in each subdomain was counted in at least 30 meristems of
correctly reflects the living structure. each ecotype (Table 2). In the central part of the L1 layer
The diameter and height of the meristems were measured (subdomain A), the mitotic index was lower than at the pe-
using transverse serial sections. In WS, the base of the mer- riphery (subdomain D). These differences are significant with
istematic dome had a mean diameter of 61.8 (cid:54) 1.2 (cid:109)m P values of 0.7 and 5.6% for Ler and WS, respectively. Sim-
(mean (cid:54) SE; n (cid:53) 50), whereas in Ler, meristems were found ilarly, the mitotic index in the center of the L2 layer (subdo-
to be wider, with a mean diameter of 69.5 (cid:54) 0.9 (cid:109)m (n (cid:53) 49; main E) was lower than in the periphery (subdomain F). P
see Table 1). The mean height of the WS meristems was values were 11 and 10% for Ler and WS, respectively. Al-
19.3 (cid:54) 0.8 (cid:109)m, which is slightly more than the 15.0 (cid:54) 0.5 though not all of these differences are significant at the level
(cid:109)m found for Ler. This shows that under our conditions, of 5%, these results suggest that in the L1 and L2 layers, an
meristems in Ler are flatter and wider than they are in WS. inner zone characterized by reduced mitotic activity can be
Cellular Parameters of the Arabidopsis Meristem 1377
Figure 1. Wild-Type Inflorescence Meristems.
(A) Scanning electron microscopy of a young inflorescence meristem (ecotype WS).
(B) Three-dimensional reconstruction of a young inflorescence meristem (ecotype WS). Fifty-one optical sections were used.
(C) Longitudinal optical section of a WS inflorescence meristem. Note the presence of distinct cell layers and the difference in cell size between
the outer and inner layers.
(D) Detail of a longitudinal optical section of a Ler inflorescence meristem. The difference in cell size in the three layers is clearly visible.
(E) Graphic representation of the spatial references used.
Arrowheads indicate mitotic cells. P1 to P7 are floral primordia numbered from the youngest to the oldest. IM, inflorescence meristem; P, floral
primordia. Bars in (A) to (C) (cid:53) 100 (cid:109)m; bar in (D) (cid:53) 10 (cid:109)m.
defined. The mitotic index in L3 subdomain G was found to are in the same range as in the periphery of the meristem
be similar to the one found in central L2 subdomain E. (subdomain D).
Spatial Distribution of the Mitoses within the L1 Layer of
Floral Primordia the Inflorescence Meristem
Cell size and mitotic index were measured in the outer layer To describe meristem organization more precisely, we ana-
of young flora P3 and P4 meristems (which were at stage 2, lyzed the spatial distribution of the mitoses in the L1 layer. In
as defined by Smyth et al., 1990; Tables 1 and 2). No signifi- 50 WS and 49 Ler meristems, we found 309 and 402 mito-
cant differences were found between P3 and P4. Cell size ses, respectively, in the L1 layer. The mean mitotic index
was slightly increased when compared with the cell size in throughout the L1 layer was 4.1 (cid:54) 0.3% in WS and 5.6 (cid:54) 0.4%
the inflorescence meristem (Table 1). The mitotic index was in Ler. The position of every mitotic cell was determined: a
comparable in the primordia from both ecotypes. The values line joining the mitotic cell to the meristem axis was drawn
1378 The Plant Cell
Figure 2. Optical Sections of a Wild-Type Inflorescence Meristem (Ler).
(A) to (H) Consecutive optical sections (at 2-(cid:109)m intervals) through an inflorescence meristem. In (A) to (C), L1 cells are visible. In (D) and (E), the
less bright L2 cells appear in the center. Mitotic cells are visible throughout the meristem (arrowheads). In (H), the three youngest primordia are
indicated. The third primordium (P3) joins the meristem flank in the section presented in (G), which is considered as the basal section. Bars (cid:53)
25 (cid:109)m.
(I) Higher magnification of the basal section (shown in [G]) showing the measurement references. The meristem is included in an outer circle C1
of radius R; the reference axis (Ref) is drawn. Three inner circles C2, C3, and C4 are drawn. Two diameters that were used for determining the
0
meristem shape are shown. Bar (cid:53) 25 (cid:109)m.
(J) Higher magnification of the basal section (G) showing how the position of mitotic cells is determined. A line (R) is drawn from the center to
each mitotic cell. The position is defined by the length of R and its angle ((cid:97)) to the reference axis. Bar (cid:53) 25 (cid:109)m.
Cellular Parameters of the Arabidopsis Meristem 1379
and projected onto the meristem base. The position of mi-
totic cells was determined by two parameters: R, the dis-
tance to the meristem axis; and (cid:97), the angle between the
projected line and a reference axis (see Figure 2J and Meth-
ods). The length R was normalized for the meristem radius
R . Figures 5A and 5B show the position of each mitotic cell
0
projected onto the meristem base.
The advantage of recording the spatial position of every
mitotic cell before a division into areas is that one set of data
can be used to compare mitotic rates in a whole range of
subdivisions, which can be chosen freely a posteriori. For Figure 3. Wild-Type Meristem Shape Curve.
analyzing the radial distribution of the mitoses, the meristem
The meristem height at five reference points is shown. This shows
L1 layer was divided into three to 10 concentric zones, and
that the meristem of Ler is slightly flatter and wider than that of WS.
Horizontal bars, standard error for meristem diameter; vertical bars,
maximum standard error for meristem height; 12 meristems analyzed.
Table 1. Morphological and Cellular Parameters of Wild-Type
Inflorescence Meristemsa
the mitotic index was determined in each zone. Because all
Meristem Zones Cell Sizeb ((cid:109)m)
of the subdivisions led to similar results, the subdivision into
WSc five concentric zones is shown as an example in Figure 6A.
L1 layer
These zones, when projected onto the meristem base, have
Zone A 4.4 (0.2)
an equal width of 0.2 R . The number of mitoses in each
Zone B 4.7 (0.1) 0
zone was determined. Based on the surface of each zone
Zone C 4.7 (0.1)
and the mean L1 cell size, the cell number in each ring was
Zone D 4.7 (0.1)
Zones A(cid:49)B(cid:49)C(cid:49)D 4.6 (0.2) calculated and used for estimating the mitotic index, which
L2 layer is shown in Figure 6A. The results obtained by using this
Zone E 4.7 (0.2) method were reliable because the values found at the mer-
Zone F 4.5 (0.1) istem center and border were similar to those found by di-
L3 layer rect counting (Table 3, subdomains A and D). In WS and Ler,
Zone G 5.3 (0.2) the mitotic index increased from the inner zone to the outer
Primordium 3 5.5 (0.2)
zone of L1. The Hotelling’s T2 test (see Mardia et al., 1979)
Primordium 4 5.4 (0.2)
showed that differences among zones 1 to 5 were significant
Lerd
at the 5% level (P values are 0.2% for WS and 3% for Ler).
L1 layer
The spatial distribution of the mitotic indices suggested
Zone A 5.0 (0.2)
Zone B 5.1 (0.1) the presence of two distinct zones. These two zones could
Zone C 4.9 (0.1) correspond to the central and peripheral zones proposed in
Zone D 5.2 (0.3) the meristem organization model. For this reason, we looked
Zones A(cid:49)B(cid:49)C(cid:49)D 5.0 (0.2) for optimal partitioning into two zones. For WS, the optimal
L2 layer division into two concentric zones is obtained when the
Zone E 5.8 (0.3) boundary lies at 0.25 R from the meristem axis. This de-
0
Zone F 5.0 (0.1)
fined an inner zone of approximately eight cells and an outer
L3 layer zone of (cid:122)140 cells in the average meristem. The mitotic in-
Zone G 5.7 (0.3)
dex in the outer zone was approximately three times higher
Primordium 3 5.3 (0.2)
than in the inner zone (Figure 6B). Note that for all partitions
Primordium 4 5.7 (0.1)
with a boundary lying between 0.2 and 0.7 R , the difference
0
aThe number of L1 cells (150 for WS and 147 for Ler) was deduced
between the two local mitotic indices was significant at the
from the calculated surface of the average meristem (Figure 3) and
5% level. For Ler, the optimal partitioning into two concen-
the mean L1 cell size.
tric zones was obtained when the boundary lay at 0.4 R
bCell size was determined by measuring the distance between the 0
from the meristem axis. The inner and outer zones con-
nuclei in the subdomains (Figure 4). Mean values and standard devi-
ations for 12 WS and 12 Ler meristems are shown. (Numbers within tained (cid:122)20 and 130 cells, respectively, and the mitotic index
parentheses indicate standard errors.) showed a 50% increase in the outer zone (Figure 6C). Again
cThe meristem diameter for WS is 61.8 (cid:109)m (1.2), and the meristem the determination of the optimal partitioning was subject to
height is 19.3 (cid:109)m (0.8) (50 meristems analyzed). imprecision: for all partitions with a boundary lying between
dThe meristem diameter for Ler is 69.5 (cid:109)m (0.9), and the meristem 0.25 and 0.75 R , the difference between the two local mi-
0
height is 15.0 (cid:109)m (0.5) (49 meristems analyzed). totic indices was significant at the 5% level.
1380 The Plant Cell
primordium) and the sector in which a primordium would de-
velop after P0 (sector P-1; see Figures 7C and 7D). Note that
these sectors are morphologically similar and that there is
no sign of primordia development. The mitotic index in the
P0 sector was higher than in P-1 (Figures 7C and 7D). This
difference was significant, with a P value of 1.8 and 0.7% for
Ler and WS, respectively, as shown by the Student’s t test.
Analysis of clv3-1 and mgo2 Meristems
Inflorescence Meristem Morphology and Cell Size
Meristem sizes of clv3-1 and mgo2 mutants are increased
(Clark et al., 1995; Laufs et al., 1998b; Figures 8 and 9).
Figure 4. Definition of Meristem Subdomains.
Flower primordia are initiated at the meristem flanks. In con-
Seven subdomains were defined within the meristem. The L1 layer trast with the wild type, no regular initiation pattern could be
was subdivided into four concentric subdomains (A to D), which,
recognized in the mutants (Figures 8 and 9). In clv3-1 and
when projected onto the meristem base, correspond to the four cir-
mgo2, the meristem base was defined as the level at which
cular domains defined in Figure 2I (each is 0.25 R in width). The L2
0 the first stage 2 flower joins the meristem flank (Figures 8A,
cells beneath the subdomain A form the subdomain E. On the mer-
8B, and 9). Meristem shapes of clv3-1 and mgo2 were vari-
istem base, L2 and L3 cells form, respectively, subdomains F and G.
able. Most of the early, rapidly growing clv3-1 inflorescence
meristems adopted a symmetric dome shape (Figures 8C
and 8D). However, oval, single-domed (Figure 8E), or fasci-
The angular distribution of mitoses was analyzed. For this ated meristems could be distinguished (Figure 8F). Like clv3,
purpose, the meristem was divided into three, four, or five young mgo2 meristems formed a symmetric dome (Figures
sectors, and the mitotic index in each sector was deter- 9A and 9B). Fasciated meristems were also observed (Fig-
mined. Figures 7A and 7B show the results obtained when ure 9C). In contrast to those of clv3-1, these meristems were
the meristem was subdivided into five sectors of 72(cid:56). The composed of several juxtapositioned units.
Hotelling’s T2 test showed that the dividing cells were not To compare the mutants with the wild type, we analyzed
evenly distributed over the sectors. We tested the hypothe- only the symmetric, dome-shaped meristems. The morpho-
sis of a local variation of mitotic activity associated with pri- logical and cellular parameters of these clv3-1 and mgo2
mordia initiation. For this purpose, two 40(cid:56) sectors were meristems are described in Table 3. The meristems are
defined that centered on either P0 (position of next incipient wider and higher than the corresponding Ler and WS wild-
Table 2. Mitotic Index in Wild-Type Inflorescence Meristems
WSa Lera
Zones nb Cell Number Mitosis Number Mitotic Index nb Cell Number Mitosis Number Mitotic Index
Meristem L1 Layer
Zone A 50 516 8 1.5% (0.6) 49 579 19 3.3% (0.8)
Zone D 30 1290 58 4.5% (0.5) 30 1716 95 5.5% (0.5)
Meristem L2 Layer
Zone E 50 545 9 1.6% (0.7) 49 459 11 2.4% (0.7)
Zone F 30 1183 44 3.7% (0.6) 30 1459 59 4.0% (0.5)
Meristem L3 Layer
Zone G 30 1652 35 2.1% (0.4) 30 1037 25 2.4% (0.4)
Primordium 3 32 1308 63 4.8% (0.5) 32 1410 68 4.8% (0.4)
Primordium 4 24 1157 57 4.9% (0.6) 21 801 40 5.0% (0.7)
aThe total number of cells and the number of mitoses in each subdomain were counted and used for calculating the mitotic index (standard er-
rors appear within parentheses).
bBecause the number of cells per subdomain was relatively low (sometimes as low as five cells in subdomains A and E), the cells from all of the
meristems were pooled. n, number of meristems.
Cellular Parameters of the Arabidopsis Meristem 1381
Mitosis in L1 and L3 of clv3-1 Inflorescence Meristems
In the L1 layer of 34 symmetric clv3-1 meristems, 420 mito-
ses were found. The mean mitotic index in this layer was
found to be 2.8 (cid:54) 0.2%, which is comparable to the value
found for the inner zone in Ler. The index in the primordia
(4.6%) was comparable to that of the wild type. After re-
cording the position of all mitotic cells, we divided the L1
surface into five concentric zones and calculated the mitotic
index in each zone as described for the wild type (Figure
10A). The distribution of mitotic cells was not homogeneous,
as shown by the Hotelling’s T2 test, but no significant differ-
ences between the meristem center and periphery were
found. As a consequence, it was not possible to divide the
L1 layer into two zones with a different mitotic index. The
mitotic indices in the four outer clv3-1 zones were signifi-
cantly different from those of the Ler outer zone value but
not from the Ler inner zone defined in Figure 6C. We could
not find any significant difference between the inner clv3-1
zone and the inner or outer Ler zone. This is due to the high
variability of the mitotic index in the meristem center of clv3-1.
Because the CLV1 gene is known to be expressed in the L3
area of the meristem, it is possible that all clv mutants are
primarily perturbed in this area. Therefore, we also deter-
mined the mitotic index in L3 of the mutant. For this pur-
pose, the percentage of dividing cells was determined in L3
of 32 clv3-1 meristems. In a total population of 3782 cells,
we identified 49 mitotic cells. The mean mitotic index calcu-
lated from the values of all individual meristems was 1.3 (cid:54)
0.2%, which is lower than the values found in wild-type mer-
istems (Table 2).
Mitotic Index in Vegetative Meristems of clv3-1 and Ler
Figure 5. Spatial Distribution of the Mitoses in the L1 Layer of Wild-
Type Meristems. To confirm the phenotype observed in the inflorescence, we
The positions of the mitotic cells are projected onto the basal sec- also determined the mitotic index in vegetative meristems of
tion. 7-day-old clv3-1 and Ler seedlings. For this purpose, trans-
(A) WS ecotype. Positions of mitoses in 50 meristems are repre- verse sections of 54 wild-type plants and 50 mutants were
sented. analyzed (Table 4). Vegetative meristems of Ler had a mean
(B) Ler ecotype. Positions of mitoses in 49 meristems are repre- width of 53.2 (cid:54) 0.43 (cid:109)m and a mean height of 13.3 (cid:54) 0.36
sented. (cid:109)m. clv3-1 meristems had a diameter of 81.2 (cid:54) 0.6 (cid:109)m and
P1, P2, P3, and P0 are positions of the three youngest primordia a height of 31.2 (cid:54) 0.7 (cid:109)m. Note that here the base of the
and of the next developing primordium, respectively. Bars indicate
meristem was defined as the point at which the meristem
the mean cell size, which is 4.6 (cid:109)m for WS and 5.0 (cid:109)m for Ler.
joins the primordia or young leaves. Wild-type plants tended
to have a lower mitotic index (3.2%) in the central part rather
than at the periphery (4.8%), but the statistical test did not
allow us to conclude that these differences were significant.
type meristems, respectively. Cell size was measured in Therefore, statistically, the wild-type meristem had to be con-
seven subdomains, as described for wild-type meristems sidered as a unit for comparison to the mutant. In clv3-1 mu-
(Table 3). In clv3-1, L1 cells were smaller than in wild-type Ler. tants, the inner two zones had a significantly lower mitotic
In mgo2, L1 cells are larger than in wild-type WS. In addi- index (2.1 and 2.0%) than the two outer zones (4.1 and 5.0%),
tion, variations in cell size were observed between individual as shown by Hotelling’s T2 test. When compared with the
meristems. The mean cell number in the L1 layer was in- wild type, the mitotic index in the two inner zones of clv3-1
creased in both mutants when compared with the wild type. was significantly lower, albeit at a P value of 8%. In contrast,
1382 The Plant Cell
Figure 6. Mitotic Index in Different Parts of the Meristem and in the Floral Primordia of the Wild Type.
Subdivisions in five (A) and two ([B] and [C]) concentric zones are shown. Bars show mean and standard error.
(A) Subdivision of the L1 into five concentric zones. The mitotic index in each zone and in the floral primordia has been calculated and is repre-
sented for WS (white bars) and Ler (black bars).
(B) Mitotic index in the inner zone (I.Z.) and outer zone (O.Z.) of WS meristems. The boundary between the two zones lies at 0.25 R.
0
(C) Mitotic index in the I.Z. and O.Z. of Ler meristems. The boundary between the two zones lies at 0.4 R.
0
no significant difference was found between the two outer DISCUSSION
zones of clv3-1 and the wild-type meristem.
On the whole, these data as well as those obtained on the
The initiation of organs in specific patterns requires stimula-
inflorescence meristem show that the enlargement of the
tion or inhibition of growth in specific regions within the mer-
clv3-1 meristem is not due to an increased mitotic index. In
istem. To initiate pattern formation, the cells within these
fact, the observation that the mitotic index remains low in a
areas must be instructed to grow and divide at different
large part of the mutant meristem is in line with the hypothe-
rates. How and at what level growth is regulated are still
sis that the central part has increased in size dramatically.
matters for debate. It has been proposed that morphogene-
sis is controlled at the level of the whole organism and that
cell division merely follows local growth rates (e.g., Kaplan
Spatial Distribution of the Mitoses in the L1 Layer and Hagemann, 1991; Hagemann, 1992). This is opposed to
of mgo2 the view that growth is driven by a direct activation of the
cellular mechanisms coordinating cell division and growth.
In the L1 layer of 38 symmetric mgo2 meristems, 252 mito- Whatever the hierarchical relationship between the basic
ses were found. The mean mitotic index in this layer was processes regulating cell morphogenesis and the mecha-
found to be 3.8 (cid:54) 0.3%, which is close to the wild-type nisms that drive growth of whole organs and multicellular
value. The method used to calculate the mitotic index in the domains, it is obvious that both levels of control are con-
clv3-1 inflorescence meristem requires a uniform cell size nected to each other (for discussion, see Jacobs, 1997). The
throughout the L1 layer. Because this is not the case in nature of this link is not understood at all, and determining
mgo2, we divided L1 into five concentric zones, and the mi- how the basic cellular processes of cell expansion and divi-
totic and total cell numbers in each zone were counted af- sion are integrated in the growing plant is becoming a cen-
terward for each individual meristem. The mitotic indices are tral issue in research into plant development.
represented in Figure 10B. The Hotelling’s T2 test did not al- At this stage, significant progress has been made in two
low us to detect a heterogenicity within the mutant SAM. It areas. First, a number of genes have been isolated in maize,
must be noted that, as shown in Figure 10B, the standard petunia, Antirrhinum, and Arabidopsis that are thought to
error was high, especially in the two inner zones. This vari- coordinate SAM function (reviewed in, e.g., Weigel and Clark,
ability could mask subtle differences within the meristem, 1996; Laufs et al., 1998a). Second, our knowledge regarding
which can only be detected by increased sampling. For the the basic mechanism of cell proliferation is rapidly increas-
statistical analysis, we considered the mgo2 meristem as a ing because major cell cycle regulators such as cyclins and
unit that was compared, on the whole, with the inner and cyclin-dependent kinases have now been identified, and their
outer zones of the WS wild type. This showed that mitotic expression patterns have been partially characterized in root
activity in the mutant was comparable to that in the outer and shoot meristems (reviewed in Francis et al., 1998). How
zone of the wild type (Figure 6B, O.Z.). are genes such as STM or CLV linked to the basic regulators
Cellular Parameters of the Arabidopsis Meristem 1383
of cell proliferation? Recently, several attempts were made
to address this issue. Meyerowitz (1997) as well as Traas
and Laufs (1998) classified a series of mutants in terms of
their cell division patterns (see also Clark and Schiefelbein,
1997; Laux and Schoof, 1997; Laufs et al., 1998a). Although
these studies provided an essential first step, they were
hampered by our lack of knowledge regarding meristem fine
structure in model systems. One can only guess at the effect
of certain mutations on cellular behavior. In this context, clv1
and clv3, which show an increased size of the SAM com-
pared with the wild type, provide a typical example. One of
the main hypotheses for CLV function proposes that the
genes could limit cell proliferation in the meristem. Until
Table 3. Morphological and Cellular Parameters of Circular clv3-1a
and mgo2a Inflorescence Meristems
Meristem Zones Cell Sizeb ((cid:109)m)
Figure 7. Mitotic Index in Meristem Sectors.
clv3-1c
L1 layer (A) and (B) Subdivision into five arbitrary sectors. (A) shows results
Zone A 4.3 (0.2) with WS; (B) shows results with Ler.
Zone B 4.3 (0.2) (C) and (D) Subdivision into two 40(cid:56) sectors centered on the position
Zone C 4.3 (0.2) at which a primordium will develop during the next plastochron (P0)
Zone D 4.2 (0.2) and on the zone to produce a primordia after P0 (P-1). (C) repre-
Zones A(cid:49)B(cid:49)C(cid:49)D 4.3 (0.2) sents WS; (D) represents Ler.
L2 layer The number of mitoses falling in the different sectors was deter-
Zone E 5.2 (0.2) mined. This result and the estimated cell number in each sector
Zone F 5.2 (0.3) were used for calculating the mitotic index. P1, P2, and P3 are posi-
L3 layer tions of the three youngest primordia.
Zone G 6.8 (0.3)
Primordium 4.7 (0.2)
mgo-2d
L1 layer
now, however, it was not possible to test this because no
Zone A 6.4 (0.4)
Zone B 6.0 (0.3) data regarding cellular proliferation rates were available.
Zone C 6.2 (0.3) The results presented here allow us to draw a three-dimen-
Zone D 6.2 (0.3) sional view of the Arabidopsis inflorescence meristem. In both
Zones A(cid:49)B(cid:49)C(cid:49)D 6.2 (0.3) Ler and WS, it can be defined as a flat dome composed of
L2 layer three clearly recognizable layers. In the outer layer, L1, cells
Zone E 6.3 (0.3) are of a relatively uniform size; however, moving toward L3,
Zone F 6.2 (03)
cell dimensions increase. We found that a subdivision into
L3 layer
zones based on differences in L1 cell size was not possible.
Zone G 6.2 (0.3)
We demonstrate that superimposed on this pattern of cell
Primordium 5.9 (0.3)
sizes is a pattern of cell proliferation. It appears that the in-
aThe number of L1 cells for clv3-1 (on average 439 per meristem)
florescence SAM contains at least two populations of cells:
was deduced from the calculated surface of the average meristem
one located at the summit of the dome and containing
and the mean L1 cell size. The number of L1 cells in mgo2 (on aver-
slowly dividing cells, and one at the flank and showing a
age 202 per meristem) was counted directly.
higher mitotic index. This pattern could correspond to the
bCell size was determined by measuring the distance between the
nuclei in the subdomains (Figure 4). Mean values and standard devi- classic division into central and peripheral zones proposed
ations for 12 clv3-1 meristems and 12 mgo2 meristems are shown. for many other species. Until now, it has not been possible
(Numbers within parentheses indicate standard errors.) to determine the exact size of these zones in Arabidopsis.
cThe meristem diameter for clv3-1 is 85.5 (cid:109)m (1.2), and the mer- Medford et al. (1992) suggested that the central zone could
istem height is 43.0 (cid:109)m (1.4) (34 meristems analyzed). be just one to two cells wide in the vegetative meristem.
dThe meristem diameter for mgo2 is 83.9 (cid:109)m (1.7), and the mer- Vaughan (1955), using cytohistology, proposed that the cen-
istem height is 34.3 (cid:109)m (1.4) (38 meristems analyzed).
tral zone is three to five cells wide. Brown et al. (1964)
1384 The Plant Cell
Figure 8. clv3-1 Inflorescence Meristems.
(A) Scanning electron microscopy of a clv3-1 inflorescence meristem. No regular phyllotactic pattern is visible.
(B) Scanning electron microscopy of a clv3-1 inflorescence meristem. A young stage 2 floral primordium is indicated by an arrowhead. The junc-
tion between the meristem and this primordium defines the base of the meristem.
(C) and (D) Symmetric dome-shaped clv3-1 meristems.
(E) Oval dome-shaped clv3-1 meristem.
(F) Fasciated clv3-1 meristem.
(C) to (F) are projections of serial transverse sections. Bars in (A) to (F) (cid:53) 100 (cid:109)m.
reported 3H-thymidine incorporation experiments, but the ferences were not found to be significant, probably because
central and peripheral zones could not be defined precisely of the variability between the meristems. It could be, there-
because of the small number of meristems analyzed. It is of fore, that young wild-type meristems do not have a well-
note that none of these estimations were based on quantita- defined zone of low mitotic activity. Alternatively, because
tive analysis and that some of these studies included the such a zone might be as small as one cell, it could go unde-
analysis of as few as one plant. tected by our methods, especially if its position varies
Based on the analyses of mitotic index, we found that the slightly from one meristem to another.
optimal division into two zones lies at 0.4 and 0.25 times the The angular distribution of mitoses in relation to the posi-
radius of the Ler and WS meristems, respectively, which tion of primordia was also studied. The mitoses are not dis-
would correspond to central zones with a diameter of on av- tributed evenly over the different sectors. In both wild-type
erage four and six L1 cells. Other divisions, although less ecotypes, we found a significant local increase associated
likely, are also possible, and the central part could be as with the position of P0, the primordium developing during
wide as 0.75 times the radius. This imprecision can be ex- the next plastochron, and low mitotic activity in P-1 (the
plained in several ways. It could be that there is no sharp zone in which no primordia will form within the next two
change in mitotic activity marking the limit of the two zones. plastochrons, with plastochron being defined as the time
This would be comparable to the situation in sunflower mer- separating the initiation of two successive organs). This re-
istems, in which a gradual increase in mitotic activity from sult shows that an increase in mitotic index precedes or at
the center to the border of the central zone was reported least accompanies primordium outgrowth.
(Davis et al., 1979). It must be kept in mind, however, that in Precise data on the spatial distribution of mitotic activity
the Arabidopsis meristem, such a gradient would at best are scarce. In pea, a modest increase in the mitotic index
stretch over one or two cells. Alternatively, it could be that a has been reported for the zone in which the next leaf primor-
sharp boundary between the two zones exists but that the dium will form (Lyndon, 1970). This rate of cell division is not
size of the central part varies from meristem to meristem. sufficient to account for the increase in cell number of the
We also analyzed the mitotic patterns in the vegetative developing primordium, suggesting that cells from adjacent
meristem. Although the results suggest that the cells divide zones continue to be recruited by the primordium (Lyndon,
at a slower rate in the center than in other zones, these dif- 1970). Therefore, we propose that the relatively high mitotic