Cellular Parameters of the Shoot Apical Meristem in

The Plant Cell, Vol. 10, 1375–1389, August 1998, www.plantcell.org © 1998 American Society of Plant Physiologists

Cellular Parameters of the Shoot Apical Meristemin Arabidopsis

Patrick Laufs,

a

Olivier Grandjean,

b

Claudia Jonak,

a,1

Kiên Kiêu,

c

and Jan Traas

a,2

a

Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026 Versailles Cedex, France

b

Département de Génétique et d’Amélioration des Plantes, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France

c

Unité 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. Withthe 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 Wassilewskijaecotypes. 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 withinthe inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that wasfour to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type withthe 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 zoneof slowly dividing cells in meristems of

clv3-1

seedlings. This zone was not detectable in the wild type. These resultssuggest 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 microscopicanalyses suggest that

mgo2

is impaired in the production of primordia, and we previously proposed that the increasedsize of the

mgo2

meristem could be due to an accumulation of cells at the periphery. Our morphometric analysisshowed that

mgo2

meristems, in contrast to those of

clv3-1

, have an enlarged periphery with high cell proliferationrates. This confirms that

clv3-1

and

mgo2

lead to meristem overgrowth by affecting different aspects of meristemfunction.

INTRODUCTION

Shoot apical meristems (SAMs) are small groups of dividingcells that initiate all of the aerial parts of the plant. Accordingto a widely accepted model, SAMs are composed of func-tionally distinct zones (see, e.g., Steeves and Sussex, 1989;Medford, 1992; Clark, 1997; Kerstetter and Hake, 1997). Inspermatophytes, the cells at the summit of the SAM, whichform the central zone, are thought to function as a source ofcells for the peripheral zone, where organ primordia are initi-ated, and for the underlying rib zone, which gives rise to thepith of the stem. Superimposed on this organization intozones is a partitioning into layers. In dicots, three layers canbe distinguished, and they are called L1, L2, and L3. Cell lin-eage studies have shown that a great majority of the cellsderived from the meristematic L1 layer form the epidermis,

whereas L2 and L3 provide cells for the inner parts of the or-gans (Satina et al., 1940; reviewed in Steeves and Sussex,1989).

Recently, genetic analysis with different model specieshas led to the identification of a number of genes involved inmeristem function. In Arabidopsis, the

SHOOT MERISTEM-LESS

(

STM

) gene, a member of the

KNOTTED

homeoboxfamily, is necessary for setting up and maintaining the SAM(Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al.,1996), whereas the receptor kinase CLAVATA1 (CLV1) isnecessary for limiting meristem size (Clark et al., 1993,1997). A range of other mutations leading to abnormal mer-istem size has been described. For instance, in

wuschel

, cellproliferation within the meristem appears to be reduced,whereas

fasciata

and

mgoun

(

mgo

), like

clv

, show meristemovergrowth (Leyser and Furner, 1992; Laux et al., 1996;Laufs et al., 1998b). Although the macroscopic modifica-tions observed in these and other mutants have been wellcharacterized, it is often not obvious how the mutations affect

1

Current address: Institute of Microbiology and Genetics, Dr. Bohrgasse9, A-1030 Vienna, Austria.

2

To whom correspondence should be addressed. E-mail [email protected]; fax 33-1-30-83-30-99.

1376 The Plant Cell

development at the cellular level. For instance, one of theproposed functions for the

CLV1

and

CLV3

genes is the lim-itation of cell division rates in the central zone (Clark et al.,1993, 1995, 1996; Laux and Schoof, 1997), and more or lessdirect roles in the coordination of cell division have beenproposed for a range of genes (Meyerowitz, 1997; Traas andLaufs, 1998). However, there are no detailed studies de-scribing the spatial and temporal distribution of cell divisionand expansion in any mutant. Indeed, at present, no refineddescription of the proliferation and growth characteristics ofthe meristematic cells in the widely used model plant Arabi-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 havequantified cell size and mitotic activity in the young inflores-cence meristem of Arabidopsis by using confocal laser scan-ning microscopy and image analysis. Together, our resultslead to a cellular model of this meristem. We have used thismodel to analyze division patterns in two mutants showingmeristem overgrowth. They are

clv3-1

and

mgo2.

RESULTS

Wild-Type Inflorescence Meristems

As described previously (Smyth et al., 1990), the Arabidop-sis inflorescence meristem produces floral primordia at itsflank in regular, spiral, clockwise (Figure 1A), or counter-clockwise patterns (Figure 1B). The cellular parameters ofthe meristem were determined on transverse and longitudi-nal optical sections of the apical dome (Figures 1C, 1D, and2A to 2H). The zone analyzed extended from the top of themeristem to the meristem base, which was defined as thelevel at which the third visible primordium (P3) joins the mer-istem (Figures 1A, 1B, 1E, and 2G; see also Methods). Thisprimordium was at stage 2, as defined by Smyth et al.(1990). A total of 55 Wassilewskija (WS) and 53 Landsberg

erecta

(L

er

) apices were optically sectioned and analyzed.Note that the apices were growing rapidly because theywere harvested when the most developed flower bud hadjust opened (stage 13, as defined by Smyth et al., 1990).Figures 1A and 1B show that the three-dimensional informa-tion obtained with the confocal laser scanning microscopecorrectly reflects the living structure.

The diameter and height of the meristems were measuredusing transverse serial sections. In WS, the base of the mer-istematic dome had a mean diameter of 61.8

6

1.2

m

m(mean

6

SE

;

n

5

50), whereas in L

er

, meristems were foundto be wider, with a mean diameter of 69.5

6

0.9

m

m (

n

5

49;see Table 1). The mean height of the WS meristems was19.3

6

0.8

m

m, which is slightly more than the 15.0

6

0.5

m

m found for L

er.

This shows that under our conditions,meristems in L

er

are flatter and wider than they are in WS.

The morphology of 12 meristems of each ecotype wasmore precisely analyzed by determining the height of themeristem in four directions (see Methods and Figure 2I). Inthe two ecotypes, no significant differences were found inmeristem shape along the four radii defined in Figure 2I.Therefore, we considered the meristems to be symmetricdome-shaped structures. Meristem height at different dis-tances from the central axis is represented in Figure 3. Themeristem shape was also directly determined on longitudi-nal sections (Figure 1C), which confirmed the shape foundby reconstructions of serial transverse sections (Figure 3).

Cell Size and Cell Number in the Inflorescence Meristem

As shown in Figures 1 and 2, the L1, L2, and L3 layers couldbe clearly distinguished. Based on the partitioning into lay-ers and the four concentric zones defined in Methods andFigure 2I, the meristem was divided into seven subdomains(Figure 4, A to G). The distance between the nuclei, which isa direct measure for cell size, was determined in each zone(Table 1). The results point to a number of minor differencesand similarities between the two ecotypes. Cell size in-creased from the outer layer to the inner parts of the mer-istem. This difference in cell size was visible on the longitudinaland transverse sections (see, e.g., Figures 1C, 1D, and 2G).In both ecotypes, cell size was uniform throughout L1, andno central zone or peripheral zone distinction could be madebased on this criterion. L3 cells were larger than L1 cells inboth L

er

and WS. The meristem shape curve (Figure 3) wasused for calculating the area of the meristem surface. By us-ing the mean L1 surface/cell ratio, we found that in bothecotypes, the outer L1 layer of the average meristem iscomposed of

z

150 cells (Table 1).

Mitotic Index in the Inflorescence Meristem

The number of mitoses in individual L

er

meristems variedfrom three to 17 (mean 9.3

6

0.6;

n

5

49); in WS, values be-tween two and 21 were found (mean 9.8

6

0.6;

n

5

50). Themitotic index was determined in different subdomains of L1(Figure 4, A and D), L2 (E and F), and L3 (subdomain G). Forthis purpose, the number of cells in interphase and mitosisin each subdomain was counted in at least 30 meristems ofeach ecotype (Table 2). In the central part of the L1 layer(subdomain A), the mitotic index was lower than at the pe-riphery (subdomain D). These differences are significant withP values of 0.7 and 5.6% for L

er

and WS, respectively. Sim-ilarly, the mitotic index in the center of the L2 layer (subdo-main E) was lower than in the periphery (subdomain F). Pvalues were 11 and 10% for L

er

and WS, respectively. Al-though not all of these differences are significant at the levelof 5%, these results suggest that in the L1 and L2 layers, aninner zone characterized by reduced mitotic activity can be

Cellular Parameters of the Arabidopsis Meristem 1377

defined. The mitotic index in L3 subdomain G was found tobe similar to the one found in central L2 subdomain E.

Floral Primordia

Cell size and mitotic index were measured in the outer layerof young flora P3 and P4 meristems (which were at stage 2,as defined by Smyth et al., 1990; Tables 1 and 2). No signifi-cant differences were found between P3 and P4. Cell sizewas slightly increased when compared with the cell size inthe inflorescence meristem (Table 1). The mitotic index wascomparable in the primordia from both ecotypes. The values

are in the same range as in the periphery of the meristem(subdomain D).

Spatial Distribution of the Mitoses within the L1 Layer of the Inflorescence Meristem

To describe meristem organization more precisely, we ana-lyzed the spatial distribution of the mitoses in the L1 layer. In50 WS and 49 L

er

meristems, we found 309 and 402 mito-ses, respectively, in the L1 layer. The mean mitotic indexthroughout the L1 layer was 4.1

6

0.3% in WS and 5.6

6

0.4%in L

er.

The position of every mitotic cell was determined: aline joining the mitotic cell to the meristem axis was drawn

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 betweenthe 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, floralprimordia. Bars in (A) to (C) 5 100 mm; bar in (D) 5 10 mm.

1378 The Plant Cell

Figure 2. Optical Sections of a Wild-Type Inflorescence Meristem (Ler).

(A) to (H) Consecutive optical sections (at 2-mm intervals) through an inflorescence meristem. In (A) to (C), L1 cells are visible. In (D) and (E), theless bright L2 cells appear in the center. Mitotic cells are visible throughout the meristem (arrowheads). In (H), the three youngest primordia areindicated. The third primordium (P3) joins the meristem flank in the section presented in (G), which is considered as the basal section. Bars 525 mm.(I) Higher magnification of the basal section (shown in [G]) showing the measurement references. The meristem is included in an outer circle C1of radius R0; the reference axis (Ref) is drawn. Three inner circles C2, C3, and C4 are drawn. Two diameters that were used for determining themeristem shape are shown. Bar 5 25 mm.(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 toeach mitotic cell. The position is defined by the length of R and its angle (a) to the reference axis. Bar 5 25 mm.


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

a

, the angle between theprojected line and a reference axis (see Figure 2J and Meth-ods). The length

R

was normalized for the meristem radius

R

0

. Figures 5A and 5B show the position of each mitotic cellprojected onto the meristem base.

The advantage of recording the spatial position of everymitotic cell before a division into areas is that one set of datacan be used to compare mitotic rates in a whole range ofsubdivisions, which can be chosen freely a posteriori. Foranalyzing the radial distribution of the mitoses, the meristemL1 layer was divided into three to 10 concentric zones, and

the mitotic index was determined in each zone. Because allof the subdivisions led to similar results, the subdivision intofive concentric zones is shown as an example in Figure 6A.These zones, when projected onto the meristem base, havean equal width of 0.2

R

0

. The number of mitoses in eachzone was determined. Based on the surface of each zoneand the mean L1 cell size, the cell number in each ring wascalculated and used for estimating the mitotic index, whichis shown in Figure 6A. The results obtained by using thismethod were reliable because the values found at the mer-istem center and border were similar to those found by di-rect counting (Table 3, subdomains A and D). In WS and L

er

,the mitotic index increased from the inner zone to the outerzone of L1. The Hotelling’s T2 test (see Mardia et al., 1979)showed that differences among zones 1 to 5 were significantat the 5% level (P values are 0.2% for WS and 3% for L

er

).The spatial distribution of the mitotic indices suggested

the presence of two distinct zones. These two zones couldcorrespond to the central and peripheral zones proposed inthe meristem organization model. For this reason, we lookedfor optimal partitioning into two zones. For WS, the optimaldivision into two concentric zones is obtained when theboundary lies at 0.25 R0 from the meristem axis. This de-fined an inner zone of approximately eight cells and an outerzone of z140 cells in the average meristem. The mitotic in-dex in the outer zone was approximately three times higherthan in the inner zone (Figure 6B). Note that for all partitionswith a boundary lying between 0.2 and 0.7 R0, the differencebetween the two local mitotic indices was significant at the5% level. For Ler, the optimal partitioning into two concen-tric zones was obtained when the boundary lay at 0.4 R0

from the meristem axis. The inner and outer zones con-tained z20 and 130 cells, respectively, and the mitotic indexshowed a 50% increase in the outer zone (Figure 6C). Againthe determination of the optimal partitioning was subject toimprecision: for all partitions with a boundary lying between0.25 and 0.75 R0, the difference between the two local mi-totic indices was significant at the 5% level.

Table 1. Morphological and Cellular Parameters of Wild-Type Inflorescence Meristemsa

Meristem Zones Cell Sizeb (mm)

WSc

L1 layerZone A 4.4 (0.2)Zone B 4.7 (0.1)Zone C 4.7 (0.1)Zone D 4.7 (0.1)Zones A1B1C1D 4.6 (0.2)

L2 layerZone E 4.7 (0.2)Zone F 4.5 (0.1)

L3 layerZone G 5.3 (0.2)

Primordium 3 5.5 (0.2)Primordium 4 5.4 (0.2)

Lerd




Primordium 3 5.3 (0.2)Primordium 4 5.7 (0.1)

a The number of L1 cells (150 for WS and 147 for Ler) was deducedfrom the calculated surface of the average meristem (Figure 3) andthe mean L1 cell size.b Cell size was determined by measuring the distance between thenuclei in the subdomains (Figure 4). Mean values and standard devi-ations for 12 WS and 12 Ler meristems are shown. (Numbers withinparentheses indicate standard errors.)c The meristem diameter for WS is 61.8 mm (1.2), and the meristemheight is 19.3 mm (0.8) (50 meristems analyzed).d The meristem diameter for Ler is 69.5 mm (0.9), and the meristemheight is 15.0 mm (0.5) (49 meristems analyzed).

Figure 3. Wild-Type Meristem Shape Curve.

The meristem height at five reference points is shown. This showsthat 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.

1380 The Plant Cell

The angular distribution of mitoses was analyzed. For thispurpose, the meristem was divided into three, four, or fivesectors, and the mitotic index in each sector was deter-mined. Figures 7A and 7B show the results obtained whenthe meristem was subdivided into five sectors of 72 8. TheHotelling’s T2 test showed that the dividing cells were notevenly distributed over the sectors. We tested the hypothe-sis of a local variation of mitotic activity associated with pri-mordia initiation. For this purpose, two 408 sectors weredefined that centered on either P0 (position of next incipient

primordium) and the sector in which a primordium would de-velop after P0 (sector P-1; see Figures 7C and 7D). Note thatthese sectors are morphologically similar and that there isno sign of primordia development. The mitotic index in theP0 sector was higher than in P-1 (Figures 7C and 7D). Thisdifference was significant, with a P value of 1.8 and 0.7% forLer 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).Flower primordia are initiated at the meristem flanks. In con-trast with the wild type, no regular initiation pattern could berecognized in the mutants (Figures 8 and 9). In clv3-1 andmgo2, the meristem base was defined as the level at whichthe first stage 2 flower joins the meristem flank (Figures 8A,8B, and 9). Meristem shapes of clv3-1 and mgo2 were vari-able. Most of the early, rapidly growing clv3-1 inflorescencemeristems adopted a symmetric dome shape (Figures 8Cand 8D). However, oval, single-domed (Figure 8E), or fasci-ated meristems could be distinguished (Figure 8F). Like clv3,young mgo2 meristems formed a symmetric dome (Figures9A and 9B). Fasciated meristems were also observed (Fig-ure 9C). In contrast to those of clv3-1, these meristems werecomposed of several juxtapositioned units.

To compare the mutants with the wild type, we analyzedonly the symmetric, dome-shaped meristems. The morpho-logical and cellular parameters of these clv3-1 and mgo2meristems are described in Table 3. The meristems arewider and higher than the corresponding Ler and WS wild-

Figure 4. Definition of Meristem Subdomains.

Seven subdomains were defined within the meristem. The L1 layerwas subdivided into four concentric subdomains (A to D), which,when projected onto the meristem base, correspond to the four cir-cular domains defined in Figure 2I (each is 0.25 R0 in width). The L2cells beneath the subdomain A form the subdomain E. On the mer-istem base, L2 and L3 cells form, respectively, subdomains F and G.

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 LayerZone 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 LayerZone 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 LayerZone 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)

a The 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).b Because 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 themeristems were pooled. n, number of meristems.


type meristems, respectively. Cell size was measured inseven subdomains, as described for wild-type meristems(Table 3). In clv3-1, L1 cells were smaller than in wild-type Ler.In mgo2, L1 cells are larger than in wild-type WS. In addi-tion, variations in cell size were observed between individualmeristems. The mean cell number in the L1 layer was in-creased in both mutants when compared with the wild type.

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 wasfound to be 2.8 6 0.2%, which is comparable to the valuefound 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 L1surface into five concentric zones and calculated the mitoticindex in each zone as described for the wild type (Figure10A). 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 werefound. As a consequence, it was not possible to divide theL1 layer into two zones with a different mitotic index. Themitotic indices in the four outer clv3-1 zones were signifi-cantly different from those of the Ler outer zone value butnot from the Ler inner zone defined in Figure 6C. We couldnot find any significant difference between the inner clv3-1zone and the inner or outer Ler zone. This is due to the highvariability of the mitotic index in the meristem center of clv3-1.Because the CLV1 gene is known to be expressed in the L3area of the meristem, it is possible that all clv mutants areprimarily 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 L3of 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 6

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

To confirm the phenotype observed in the inflorescence, wealso determined the mitotic index in vegetative meristems of7-day-old clv3-1 and Ler seedlings. For this purpose, trans-verse sections of 54 wild-type plants and 50 mutants wereanalyzed (Table 4). Vegetative meristems of Ler had a meanwidth of 53.2 6 0.43 mm and a mean height of 13.3 6 0.36mm. clv3-1 meristems had a diameter of 81.2 6 0.6 mm anda height of 31.2 6 0.7 mm. Note that here the base of themeristem was defined as the point at which the meristemjoins the primordia or young leaves. Wild-type plants tendedto have a lower mitotic index (3.2%) in the central part ratherthan at the periphery (4.8%), but the statistical test did notallow us to conclude that these differences were significant.Therefore, statistically, the wild-type meristem had to be con-sidered as a unit for comparison to the mutant. In clv3-1 mu-tants, the inner two zones had a significantly lower mitoticindex (2.1 and 2.0%) than the two outer zones (4.1 and 5.0%),as shown by Hotelling’s T2 test. When compared with thewild type, the mitotic index in the two inner zones of clv3-1was significantly lower, albeit at a P value of 8%. In contrast,

Figure 5. Spatial Distribution of the Mitoses in the L1 Layer of Wild-Type Meristems.

The positions of the mitotic cells are projected onto the basal sec-tion.(A) WS ecotype. Positions of mitoses in 50 meristems are repre-sented.(B) Ler ecotype. Positions of mitoses in 49 meristems are repre-sented.P1, P2, P3, and P0 are positions of the three youngest primordiaand of the next developing primordium, respectively. Bars indicatethe mean cell size, which is 4.6 mm for WS and 5.0 mm for Ler.

1382 The Plant Cell

no significant difference was found between the two outerzones of clv3-1 and the wild-type meristem.

On the whole, these data as well as those obtained on theinflorescence meristem show that the enlargement of theclv3-1 meristem is not due to an increased mitotic index. Infact, the observation that the mitotic index remains low in alarge part of the mutant meristem is in line with the hypothe-sis that the central part has increased in size dramatically.

Spatial Distribution of the Mitoses in the L1 Layerof mgo2

In the L1 layer of 38 symmetric mgo2 meristems, 252 mito-ses were found. The mean mitotic index in this layer wasfound to be 3.8 6 0.3%, which is close to the wild-typevalue. The method used to calculate the mitotic index in theclv3-1 inflorescence meristem requires a uniform cell sizethroughout the L1 layer. Because this is not the case inmgo2, we divided L1 into five concentric zones, and the mi-totic and total cell numbers in each zone were counted af-terward for each individual meristem. The mitotic indices arerepresented in Figure 10B. The Hotelling’s T2 test did not al-low us to detect a heterogenicity within the mutant SAM. Itmust be noted that, as shown in Figure 10B, the standarderror was high, especially in the two inner zones. This vari-ability could mask subtle differences within the meristem,which can only be detected by increased sampling. For thestatistical analysis, we considered the mgo2 meristem as aunit that was compared, on the whole, with the inner andouter zones of the WS wild type. This showed that mitoticactivity in the mutant was comparable to that in the outerzone of the wild type (Figure 6B, O.Z.).

DISCUSSION

The initiation of organs in specific patterns requires stimula-tion or inhibition of growth in specific regions within the mer-istem. To initiate pattern formation, the cells within theseareas must be instructed to grow and divide at differentrates. How and at what level growth is regulated are stillmatters for debate. It has been proposed that morphogene-sis is controlled at the level of the whole organism and thatcell division merely follows local growth rates (e.g., Kaplanand Hagemann, 1991; Hagemann, 1992). This is opposed tothe view that growth is driven by a direct activation of thecellular mechanisms coordinating cell division and growth.Whatever the hierarchical relationship between the basicprocesses regulating cell morphogenesis and the mecha-nisms that drive growth of whole organs and multicellulardomains, it is obvious that both levels of control are con-nected to each other (for discussion, see Jacobs, 1997). Thenature of this link is not understood at all, and determininghow the basic cellular processes of cell expansion and divi-sion are integrated in the growing plant is becoming a cen-tral issue in research into plant development.

At this stage, significant progress has been made in twoareas. First, a number of genes have been isolated in maize,petunia, Antirrhinum, and Arabidopsis that are thought tocoordinate SAM function (reviewed in, e.g., Weigel and Clark,1996; Laufs et al., 1998a). Second, our knowledge regardingthe basic mechanism of cell proliferation is rapidly increas-ing because major cell cycle regulators such as cyclins andcyclin-dependent kinases have now been identified, and theirexpression patterns have been partially characterized in rootand shoot meristems (reviewed in Francis et al., 1998). Howare genes such as STM or CLV linked to the basic regulators

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 R0.(C) Mitotic index in the I.Z. and O.Z. of Ler meristems. The boundary between the two zones lies at 0.4 R0.


of cell proliferation? Recently, several attempts were madeto address this issue. Meyerowitz (1997) as well as Traasand Laufs (1998) classified a series of mutants in terms oftheir cell division patterns (see also Clark and Schiefelbein,1997; Laux and Schoof, 1997; Laufs et al., 1998a). Althoughthese studies provided an essential first step, they werehampered by our lack of knowledge regarding meristem finestructure in model systems. One can only guess at the effectof certain mutations on cellular behavior. In this context, clv1and clv3, which show an increased size of the SAM com-pared with the wild type, provide a typical example. One ofthe main hypotheses for CLV function proposes that thegenes could limit cell proliferation in the meristem. Until

now, however, it was not possible to test this because nodata regarding cellular proliferation rates were available.

The results presented here allow us to draw a three-dimen-sional view of the Arabidopsis inflorescence meristem. In bothLer and WS, it can be defined as a flat dome composed ofthree clearly recognizable layers. In the outer layer, L1, cellsare of a relatively uniform size; however, moving toward L3,cell dimensions increase. We found that a subdivision intozones based on differences in L1 cell size was not possible.

We demonstrate that superimposed on this pattern of cellsizes is a pattern of cell proliferation. It appears that the in-florescence SAM contains at least two populations of cells:one located at the summit of the dome and containingslowly dividing cells, and one at the flank and showing ahigher mitotic index. This pattern could correspond to theclassic division into central and peripheral zones proposedfor many other species. Until now, it has not been possibleto determine the exact size of these zones in Arabidopsis.Medford et al. (1992) suggested that the central zone couldbe just one to two cells wide in the vegetative meristem.Vaughan (1955), using cytohistology, proposed that the cen-tral zone is three to five cells wide. Brown et al. (1964)

Table 3. Morphological and Cellular Parameters of Circular clv3-1a and mgo2a Inflorescence Meristems

Meristem Zones Cell Sizeb (mm)

clv3-1c




Primordium 4.7 (0.2)mgo-2d


L2 layerZone E 6.3 (0.3)Zone F 6.2 (03)


Primordium 5.9 (0.3)

a The number of L1 cells for clv3-1 (on average 439 per meristem)was deduced from the calculated surface of the average meristemand the mean L1 cell size. The number of L1 cells in mgo2 (on aver-age 202 per meristem) was counted directly.b Cell size was determined by measuring the distance between thenuclei in the subdomains (Figure 4). Mean values and standard devi-ations for 12 clv3-1 meristems and 12 mgo2 meristems are shown.(Numbers within parentheses indicate standard errors.)c The meristem diameter for clv3-1 is 85.5 mm (1.2), and the mer-istem height is 43.0 mm (1.4) (34 meristems analyzed).d The meristem diameter for mgo2 is 83.9 mm (1.7), and the mer-istem height is 34.3 mm (1.4) (38 meristems analyzed).

Figure 7. Mitotic Index in Meristem Sectors.

(A) and (B) Subdivision into five arbitrary sectors. (A) shows resultswith WS; (B) shows results with Ler.(C) and (D) Subdivision into two 408 sectors centered on the positionat which a primordium will develop during the next plastochron (P0)and on the zone to produce a primordia after P0 (P-1). (C) repre-sents WS; (D) represents Ler.The number of mitoses falling in the different sectors was deter-mined. This result and the estimated cell number in each sectorwere used for calculating the mitotic index. P1, P2, and P3 are posi-tions of the three youngest primordia.

1384 The Plant Cell

reported 3H-thymidine incorporation experiments, but thecentral and peripheral zones could not be defined preciselybecause of the small number of meristems analyzed. It is ofnote that none of these estimations were based on quantita-tive analysis and that some of these studies included theanalysis of as few as one plant.

Based on the analyses of mitotic index, we found that theoptimal division into two zones lies at 0.4 and 0.25 times theradius of the Ler and WS meristems, respectively, whichwould correspond to central zones with a diameter of on av-erage four and six L1 cells. Other divisions, although lesslikely, are also possible, and the central part could be aswide as 0.75 times the radius. This imprecision can be ex-plained in several ways. It could be that there is no sharpchange in mitotic activity marking the limit of the two zones.This would be comparable to the situation in sunflower mer-istems, in which a gradual increase in mitotic activity fromthe center to the border of the central zone was reported(Davis et al., 1979). It must be kept in mind, however, that inthe Arabidopsis meristem, such a gradient would at beststretch over one or two cells. Alternatively, it could be that asharp boundary between the two zones exists but that thesize of the central part varies from meristem to meristem.

We also analyzed the mitotic patterns in the vegetativemeristem. Although the results suggest that the cells divideat a slower rate in the center than in other zones, these dif-

ferences were not found to be significant, probably becauseof the variability between the meristems. It could be, there-fore, that young wild-type meristems do not have a well-defined zone of low mitotic activity. Alternatively, becausesuch a zone might be as small as one cell, it could go unde-tected by our methods, especially if its position variesslightly from one meristem to another.

The angular distribution of mitoses in relation to the posi-tion of primordia was also studied. The mitoses are not dis-tributed evenly over the different sectors. In both wild-typeecotypes, we found a significant local increase associatedwith the position of P0, the primordium developing duringthe next plastochron, and low mitotic activity in P-1 (thezone in which no primordia will form within the next twoplastochrons, with plastochron being defined as the timeseparating the initiation of two successive organs). This re-sult shows that an increase in mitotic index precedes or atleast accompanies primordium outgrowth.

Precise data on the spatial distribution of mitotic activityare scarce. In pea, a modest increase in the mitotic indexhas been reported for the zone in which the next leaf primor-dium will form (Lyndon, 1970). This rate of cell division is notsufficient to account for the increase in cell number of thedeveloping primordium, suggesting that cells from adjacentzones continue to be recruited by the primordium (Lyndon,1970). Therefore, we propose that the relatively high mitotic

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) 5 100 mm.


activity observed in a large part of the L1 layer supportsgrowth of the young developing primordia (P0 to P3).

The usefulness of quantitative, structural analysis is dem-onstrated by our analysis of the clv3 and mgo2 inflores-cence meristems. In clv1 and clv3, the size of the meristemis increased during all phases of development. CLV1 andCLV3 have been proposed to act in the same pathway(Clark et al., 1995). Two models for CLV1 and CLV3 actionhave been proposed and discussed extensively in severalreviews (Clark et al., 1995, 1996; for discussion, see Liljegrenand Yanofsky, 1996; Clark and Schiefelbein, 1997; Laux andSchoof, 1997; Meyerowitz, 1997; Traas and Laufs, 1998;Laufs et al., 1998a). In the first model, CLV1 and CLV3 pro-mote the transition of cells from the central to the peripheralzones. In mutants, organ formation occurs at an increaseddistance from the meristem summit. According to this model,the meristem overgrows because of the accumulation ofcells that wait to be recruited by the developing organs. Inthe second model, CLV genes negatively regulate the prolif-eration rate of central zone cells. According to this hypothe-sis, cells accumulate because of an increased cell divisionrate in the meristem center of the mutants, which leads to an

increase in meristem size. The first model would lead to anincreased size of the central zone and hence to a reducedmean mitotic index in the whole meristem. In the secondscenario, the mitotic index in the central zone and thus inthe whole meristem would be increased.

Our analysis of clv3-1 shows that cell division ratesthroughout the enlarged inflorescence dome of the mutantare reduced when compared with the mean value found inthe wild type. The value found in clv3-1 is comparable to theone found in the inner zone of the wild type, suggesting thatthis area is increased in size in the mutant. Because we sec-tioned mutant meristems only to the first visible stage 2 pri-mordium, it could be argued that we might have missed apopulation of more rapidly dividing cells at the periphery. In-deed, our results show that the vegetative meristem of clv3-1has a large central area with a mitotic index that is lowerthan that in the periphery. Together, these observations al-low us to reject the hypothesis that the phenotype is due toan increased mitotic activity in the central zone. In contrast,the low cell proliferation rate is in line with the idea that inclv3-1, the central zone is increased in size and that CLV3 isinvolved in promoting the cell transition from the centralzone to the peripheral zone.

The production of more organs and hence more cells froman overall reduction of mitotic index appears contradictoryat first. This can be explained, however, if we consider theexisting model (see Introduction; Steeves and Sussex,1989), which supposes two discrete steps in the transitionfrom the central zone to the peripheral zone and finally intothe primordium. If the first step is inhibited, then centralzone cells accumulate, which indirectly increases the size ofthe peripheral zone. The extra cells needed for the increasein meristem size can come either from an increased mitoticactivity in the periphery or from a reduced incorporation ofcells into the primordia and hence a reduction in primordiumproduction. It is therefore possible that initially, primordiumproduction is reduced slightly in the mutant. Such a delaycould be difficult to detect because it could occur as early asduring embryogenesis. However, because the meristem in-creases in size continually, the total number of cell divisionsincreases also, which in turn leads to the production of extraorgans.

Our previous genetic and structural analyses indicated thatclv3 and mgo2 affect different processes, although bothmutations lead to an increase in meristem size. mgo2, incontrast to clv3, produces fewer leaves and flower organs(Laufs et al., 1998b). Therefore, we proposed that MGO2 isrequired for proper primordia initiation and may act by stim-ulating cells to leave the meristem and to be incorporatedinto the primordia. The quantitative data further support theidea that mgo2 and clv3 lead to an increase in meristem sizevia different pathways. We found that in mgo2 meristems,the mean mitotic activity is comparable to the one found inthe periphery of the wild type. Therefore, in contrast to that ofclv3-1, the population of slowly dividing cells is not in-creased in mgo2, but rather, the mutant appears to have an

Figure 9. mgo2 Inflorescence Meristems.

(A) Scanning electron microscopy of an mgo2 inflorescence mer-istem.(B) Symmetric dome-shaped mgo2 meristem.(C) Fasciated mgo2 meristem showing several subunits, as indi-cated by arrowheads.(B) and (C) are projections of serial transverse sections. Bars in (A)to (C) 5 100 mm.

1386 The Plant Cell

enlarged outer zone. These results are in agreement with theproposed role of MGO2 (Laufs et al., 1998b).

METHODS

Plant Strains and Growth Conditions

Wild-type Arabidopsis thaliana lines (ecotypes Wassilewskija [WS]and Landsberg erecta [Ler]) were generously provided by the Arabi-dopsis Biological Resource Center (Ohio State University, Colum-bus), and clavata3-1 (clv3-1; Ler background) was obtained from S.Clark (University of Michigan, Ann Arbor). Wild-type, clv3-1, and

mgoun2 (mgo2; WS background; Laufs et al., 1998b) plants weregrown in soil in a greenhouse under regulated conditions. Tempera-ture variations were relatively constant: the minimum night tempera-ture was 16.3 6 0.78C (mean value 6SD), and the maximum daytemperature was 26.6 6 1.88C. Apices of the main inflorescencestem were harvested when the most developed flower bud was atstage 13 (as defined by Smyth et al., 1990), that is, when it had justopened. All inflorescences used in this study were harvested on thesame day between 10 and 11 AM.

Confocal Laser Scanning Microscopy

Apices were fixed and stained with propidium iodide to visualizeDNA, as described by Clark et al. (1995). The presence of the

Figure 10. Mitotic Index in Different Parts of the Meristem and in the Floral Primordia of clv3-1, mgo2, and the Wild Type.

(A) Subdivision of the L1 layer into five concentric zones. The mean mitotic indices in each zone and in the floral primordia have been calculatedand are represented for clv3-1 (black bars) and the corresponding wild-type Ler (white bars). The extensions indicate standard errors.(B) Subdivision of the L1 layer into five concentric zones. The mean mitotic indices in each zone and in the floral primordia were calculated andare represented for mgo2 (black bars) and the corresponding wild-type WS (white bars). The extensions indicate standard errors.


strongly stained flower buds obscured the shoot apical inflorescencemeristem. Therefore, the more mature flower buds were removed un-der the dissecting microscope. The inflorescence was then verticallymounted in a drop of low melting point agarose (0.8% in water) andviewed directly in a Leica TCSNT confocal laser scanning micro-scope (Leica, Heidelberg, Germany) equipped with an argon/kryptonlaser (Omnichrome, Chino, CA). To visualize the propidium iodide,we used a BP568 band pass filter for excitation in combination witha long pass filter (LP590). All inflorescences were first scanned at alow magnification (316 objective, electronic zoom 31.5). Series of25 to 35 optical sections made at 3-mm intervals were projected as astereo pair to check the proper orientation of each sample. If re-quired, the apices were reoriented using glass needles to align thelongitudinal axis of the inflorescence as closely as possible to thevertical position. Subsequently, optical sections at 2-mm intervalsand a pinhole of 75 mm were made using a 340 lens, combined withan electronic zoom of 32 and a resolution of 512 3 512 pixels.

Each inflorescence shoot apical meristem (SAM) was opticallysectioned from the top down to the groove that separates the thirdvisible primordium (P3, which in all meristems was at stage 2; Smythet al., 1990) from the meristem (Figures 1 and 2). This level was arbi-trarily termed the meristem “base,” and the section through the basewas termed the “last” section of a series (Figure 1E). clv3-1 andmgo2 meristems do not show an obvious phyllotaxy, and flora mer-istems are not produced in a classic spiral pattern. Therefore, clv3-1and mgo2 apices were sectioned down to the groove that separatesthe first stage 2 primordium from the meristem. This level was termedthe base of the meristems.

Vegetative meristems were fixed and stained in propidium iodide,as described above. Subsequently, they were mounted horizontallybetween slide and coverslip and sectioned longitudinally using con-focal scanning microscopy.

Morphometric Analysis

All images were analyzed with Optimas 5.2 (Optimas Corporation,Bothell, WA) software. For every inflorescence meristem, a circle en-closing the meristem base was drawn on the last section. Subse-quently, a radius between the center of this circle and the point atwhich P3 joins the meristem was drawn on the same basal section.

This circle was used as a reference for further work and termed C1with radius R0. The center of this circle was defined as the center ofthe meristem base. From this center, a vertical line through the mer-istem summit defines the meristem axis (Figure 1E).

For the analysis of vegetative meristems, only the six median lon-gitudinal sections were taken. The meristems were subsequently di-vided into four “concentric” zones.

Meristem Size and Shape

Meristem height was determined by multiplying the total number ofsections by the distance between them (2 mm). Meristem width wasdetermined by measuring the length of R0. To determine meristemshape, three additional circles (C2, C3, and C4) with the same centeras C1 and radii of 0.75, 0.5, and 0.25 R0 were drawn. In addition, twoperpendicular diameters were drawn on C1 (see Figure 2I). The inter-sections between these diameters and the four circles defined 16reference points. Meristem height was determined at each of thesereference points. Alternatively, meristem shape was determined byexamining longitudinal optical sections.

Cell Size

Cell size was determined by measuring the mean distance betweenthe nuclei in each of the seven meristem subdomains described inFigure 3.

Mitotic Index

The numbers of interphase and mitotic (from late prophase to earlycytokinesis) cells were counted in at least 30 inflorescence mer-istems, in the subdomains described in Figure 3. For all mitotic indi-ces, standard errors were calculated according to the standardapproximation formula as given, for example, in Cochran (1977). Forvegetative meristems, only cells of the six median longitudinal sec-tions were counted in every zone.

Table 4. Mitotic Index in Wild-Type and clv3-1 7-Day-Old Vegetative Meristems

Lera,b clv3-1a,c

Meristem Zones nd Cell Number Mitosis Number Mitotic Index nd Cell Number Mitosis Number Mitotic Index

Meristem L1 LayerZone A 54 280 9 3.2% (1.0) 50 434 9 2.1% (0.7)Zone B 54 329 12 3.6% (1.0) 50 553 11 2.0% (0.6)Zone C 54 377 17 4.5% (1.1) 50 664 27 4.1% (0.8)Zone D 54 373 18 4.8% (1.0) 50 638 32 5.0% (0.8)

a The total number of cells and number of mitoses in each subdomain were counted and used for calculating the mitotic index (standard errorsappear within parentheses).b The meristem diameter for Ler is 53.1 mm (0.9), and the meristem height is 13.3 mm (0.4).c The meristem diameter for clv3-1 is 81.6 mm (1.3), and the meristem height is 31.2 mm (0.7).d n, number of meristems. For each meristem, six median longitudinal sections were analyzed.

1388 The Plant Cell

Spatial Distribution of Mitoses in L1 of Inflorescence Meristems

To determine the spatial distribution of mitotic activity in L1, the po-sition of every mitotic cell was recorded in the wild-type and clv3-1meristems. For this purpose, a line from the meristem axis to everymitotic cell in each section was drawn and projected onto the basalsection. The position of these mitoses was determined by measuringthe length of the projected line R and the angle a between the R andthe reference R0. Thus, every mitosis was characterized by two coor-dinates: the angle a and the distance R. As in many other species,the Arabidopsis meristem can initiate successive organs in either aclockwise ([1] meristem) or counterclockwise ([2] meristem) spiral.To make a direct comparison between these two possible types ofmeristems, we directly converted the values obtained for (2) mer-istems into the equivalent (1) meristem values.

For each meristem, the L1 layer was divided into five concentriczones. The first zone lies ,0.2 R0 from the meristem axis. The sec-ond zone lies between 0.2 and 0.4 R0 from the meristem axis, and soon. The average number of mitoses in each zone was determinedfrom the data described above. Furthermore, the mean surface areaof each zone was calculated using the curves shown in Figure 3.From the mean surface areas and the mean total number of cells inL1, the mean cell numbers were obtained for each zone. Thus, by di-viding the average number of mitoses by the mean number of cells,the average local mitotic indices were obtained for each zone.

Different divisions of the L1 layer into two concentric zones werealso investigated. The boundary between the two zones was chosenat 0.05 R0 intervals from 0.1 to 0.9 R0 from the meristem axis. Foreach division into two zones, the homogeneity was tested using theT2 procedure. The optimal division was defined as the division forwhich the difference between the two mean local mitotic indices isthe most significant.

Because L1 cell size in the mgo2 mutant is variable, mitotic celldistribution could not be analyzed as given. The L1 surface was di-vided into five concentric zones, and the mitotic and total cell num-bers in each zone were counted for each individual meristem.

The homogeneity hypothesis that “the mean local mitotic indicesare equal” was tested using the Hotelling’s T2 procedure (see, e.g.,Mardia et al., 1979).

ACKNOWLEDGMENTS

We thank Dr. Heather McKhann, Dr. Jan Dockx, and Dr. HermanHöfte for critical reading of the manuscript. P.L. was financed by agrant from the Ministère de l’Enseignement Supérieur et de la Re-cherche, and C.J. was financed by a fellowship from the EuropeanMolecular Biology Organization. We thank Joel Talbotec and JacquesGoujeaud for help in the greenhouse.

Received February 26, 1998; accepted June 23, 1998.

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DOI 10.1105/tpc.10.8.1375 1998;10;1375-1389Plant Cell

Patrick Laufs, Olivier Grandjean, Claudia Jonak, Kiên Kiêu and Jan TraasCellular Parameters of the Shoot Apical Meristem in Arabidopsis

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