Cyclin-dependent kinase activity maintains the shoot apicalmeristem cells in an undifferentiated state

Tarik Gaamouche1,2,†, Carmem-Lara de O. Manes1,2,†,‡, Dorota Kwiatkowska3, Barbara Berckmans1,2, Rachel Koumproglou4,

Sara Maes1,2, Tom Beeckman1,2, Teva Vernoux5, John H. Doonan4, Jan Traas5, Dirk Inze1,2 and Lieven De Veylder1,2,*

1Department of Plant Systems Biology, VIB, 9052 Gent, Belgium,2Department of Plant Biotechnology and Genetics, Ghent University, 9052 Gent, Belgium,3Department of Biophysics and Morphogenesis of Plants, University of Silesia, 40-032 Katowice, Poland,4John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK, and5Laboratoire de Reproduction et Developement des Plantes, Institut National de la Recherche Agronomique,

69364 Lyon Cedex 07, France

Received 28 May 2010; accepted 6 July 2010.*For correspondence (fax +32 9 3313809; e-mail lieven.de.veylder@psb.vib-ugent.be).†These authors contributed equally to this work.‡Present address: Universite Pierre et Marie Curie, Unite Mixte de Recherche 7621, Oceanographie Microbienne, Observatoire Oceanologique, 66651 Banyuls sur

Mer, France.

SUMMARY

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration

between cell division rates and fate specification is essential for normal organ formation and plant

development. However, the inter-dependence of cell-cycle machinery and meristem-organizing genes is still

poorly understood. To investigate this mechanism, we specifically inhibited the cell-cycle machinery in the

shoot apex by expression of a dominant negative allele of the A-type cyclin-dependent kinase (CDK) CDKA;1 in

meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot

apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven

into the differentiation pathway, as indicated by premature cell expansion and onset of endo-reduplication.

Although the meristem structure and expression patterns of the meristem identity genes were maintained in

most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype

correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a

threshold model in which the effect of the cell-cycle machinery on meristem organization is determined by the

level of CDK activity.

Keywords: Arabidopsis thaliana, cell cycle, CDK, endo-reduplication, organogenesis, shoot apical meristem.

INTRODUCTION

An important attribute of growing plants is their ability to

coordinate the events of cell division and cell differentiation.

A prominent example is the shoot apex, in which cell divi-

sions occur in the shoot apical meristem (SAM) and lateral

organ primordia, whereas cell differentiation happens in

meristem-derived cells that will form stem and lateral organ

tissues. At the organizational level, the dome-shaped SAM is

divided into functionally distinct domains that are charac-

terized by different division rates. A central zone (CZ) at the

summit harbors slowly dividing cells that can be distin-

guished from the surrounding peripheral zone (PZ) and the

rib meristem (RM) located beneath, which both exhibit more

frequent cell divisions. The PZ contains cells that will be

incorporated into the lateral organs. The CZ consists of stem

cells that replenish themselves. During primordium forma-

tion, PZ cells become the founder cells of lateral organs,

after which these cells are replaced by dividing pluripotent

cells from the CZ. As a consequence, the SAM can maintain

itself as a stable self-sustaining structure (Carraro et al.,

2006).

The self-perpetuation of the SAM is controlled by a group

of well-characterized genes. The SHOOT MERISTEMLESS

(STM) gene is expressed throughout the CZ, PZ and RM, and

keeps them in the indeterminate state of cells within the

SAM (Long et al., 1996; Long and Barton, 1998). By contrast,

expression of the WUSCHEL (WUS) gene is confined to a

26 ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 64, 26–37 doi: 10.1111/j.1365-313X.2010.04317.x

few cells in the proximal part of the CZ (the organizing center),

and maintains the stem cell identity of neighbor cells (Mayer

et al., 1998). When ectopically expressed, the combined

action of STM and WUS is sufficient to trigger the formation

of ectopic SAMs and organogenesis (Gallois et al., 2002;

Lenhard et al., 2002). WUS expression depends on the

CLAVATA (CLV) signaling pathway, and is negatively con-

trolled by the CLV1, CLV2 and CLV3 genes, which are

expressed in overlapping domains of the CZ (CLV1 and

CLV3) and other SAM zones (CLV2) (Clark et al., 1997; Fletcher

et al., 1999). In clv mutants, enlargement of the SAM appears

to result from ectopic WUS expression in a proximal region

of the CZ (Schoof et al., 2000). Moreover, over-expression of

CLV3 inhibits WUS expression (Brand et al., 2000). These

findings have led to a model in which stem cell maintenance

is regulated by a negative feedback loop mediated by the

WUS and CLV3 genes, in which the organizing center triggers

the distally located neighbors to specialize as stem cells, that

in turn signal back to restrict the size of the organizing center

(Brand et al., 2000; Schoof et al., 2000; Ito et al., 2006; Kondo

et al., 2006).

Meristem regulators have been proposed to act partially

through direct regulation of cell division (Laufs et al., 1998;

Reddy et al., 2004; Reddy and Meyerowitz, 2005), a mech-

anism that is conserved among eukaryotes. Cell-cycle tran-

sitions are controlled by heterodimeric serine–threonine

protein kinases, consisting of a catalytic cyclin-dependent

kinase (CDK) and a regulatory cyclin (CYC) subunit.

Although mRNA in situ hybridization can detect expression

of most CDK and cyclin genes in the SAM (de Almeida Engler

et al., 2009), it is still unclear how the cell division machinery

is controlled by the meristem patterning genes and whether

the cell-cycle machinery itself has an impact on meristem

organization. On the one hand, manipulation of the cell

division rate by over-expression of CYCA3;2, CYCD3;1 or the

fission yeast gene cdc25 provokes additional cell divisions

within the SAM, but without disturbing its organization

(Wyrzykowska et al., 2002; Dewitte et al., 2003). On the other

hand, over-expression or down-regulation of the mitotis-

specific B2-type CDKs results in severe meristematic defects

and disorganization of the SAM (Andersen et al., 2008).

Here, we used an alternative approach to inhibit cell-cycle

activity at the SAM by means of a dominant negative allele

(CDKA;1.N146) of CDKA;1 that is the major cell division-

controlling CDK in Arabidopsis thaliana and is needed at

both the G1/S and G2/M transition points (Porceddu et al.,

2001; Joubes et al., 2004). Its requirement in cell division has

been demonstrated by a reduced cell division rate in plants

that have low CDKA activity and by an arrest of the second

mitotic division during male gametogenesis in null mutants

(Hemerly et al., 1995; Iwakawa et al., 2006; Nowack et al.,

2006). As CDKA;1.N146 expression reduces the overall CDK

activity through competition with the endogenous CDKs for

cyclin subunits (Hemerly et al., 1995; Joubes et al., 2004),

we assessed the effects of a reduced cell division rate

on meristem functioning by specifically expressing

CDKA;1.N146 at the SAM under the control of the STM

promoter. Inhibition of CDK activity caused premature

differentiation of some meristem cells and induced several

morphological alterations, but did not have a drastic impact

on the meristem organization. Only some of the plants

displayed altered apex morphology, visible by meristem

splitting. The size of this population was found to correlate

with the level of expression of CDKA;1.N146. We propose

that the meristematic organization is relatively stable

against changes in cell division rates and is affected only

when the CDK activity drops below a hypothetical threshold

level, thereby reconciling previous contradictory observa-

tions on the link between cell division and meristem

organization.

RESULTS

Expression of a dominant negative CDKA;1 allele under the

control of the STM promoter perturbs post-embryonic

development

To investigate the role of CDK activity in cell fate determi-

nation within the SAM, we generated transgenic plants that

(a) (b) (c)

Figure 1. Molecular characterization of transgenic lines harboring the CDKA;1.N146 mutant allele under the control of the STM promoter.

(a) Expression of the STM promoter in the SAM as visualized by confocal microscopy of 1-week-old PSTM:YFP-histone H4 seedlings.

(b) Quantitative real-time expression analysis of CDKA;1.N146 in PSTM:CDKA;1.N146 lines B3-4 and B3-5, and Col-0 plants. The values are means of three

independent replicates. The ACTIN2, EF1-a and CYTOCHROME B5 expression levels were used as a reference.

(c) CDK kinase activity in 8-day-old wild-type (Col-0) versus PSTM:CDKA;1.N146 lines with histone H1 as the substrate. The kinase activity level of the control plants

was arbitrary set to 100.

CDK dose-dependent control of meristem activity 27

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

expressed the dominant negative CDKA;1.N146 allele under

the control of the SHOOT MERISTEMLESS (STM) promoter

(PSTM). The promoter used corresponded to the 4.5 kb 5’

untranslated region of the STM gene and mimicked the

expression pattern of the endogenous STM gene (Figure 1a).

Homozygous PSTM:CDKA;1.N146 lines were selected for

molecular and phenotypic analyzes. Expression of the

transgene in the plant apices was confirmed by RT-PCR for at

least six independent lines. For downstream analysis, we

focused on two independent lines homozygous for a single

T-DNA locus, CDKA;1.N146 B3-4 and CDKA;1.N146 B3-5

(Figure 1b). Although these two lines showed an almost

twofold difference in the level of CDKA;1.N146 expression,

the CDK activity was equally reduced by approximately 50%

in both lines (Figure 1c). As both lines show identical phe-

notypes (see below), corresponding to their equal inhibition

of CDK activity, we assumed that a proportion of the detected

CDKA;1.N146 transcripts were not translated in the

CDKA;1.N146 B3-4 line, perhaps because of the presence of a

truncated T-DNA locus.

Homozygous PSTM:CDKA;1.N146 transgenic lines from

the T3 generation were germinated on non-selective med-

ium and scored for phenotypes. During the vegetative phase

of development, various phenotypes were observed within a

single population. Approximately 2% of the plants had a

severe growth phenotype with malformed cotyledons and/

or fused leaves (Figure 2a,b), and showed early growth

arrest. However, the majority of the transgenic plants

presented less severe, but still pronounced, morphological

alterations, such as retarded growth and small and

mis-shapen leaves (Figure 2c–e), but the inflorescence shoot

apex was not altered during the reproductive developmental

phase, (Figure 2f,g). As observed for wild-type plants, flower

primordia were initiated in the typical Fibonacci spiral

pattern (Figure 3ai,aii), but the mutant flowers were smaller

than those of the wild-type and aberrantly shaped

(Figure 3b–e). In scanning electron micrographs, abnormal-

ities were observed during the early stages of flower

primordium development, in particular during sepal forma-

tion. Boundaries between sepal primordia did not develop

properly (Figure 3aiii), i.e. they appeared fused, and these

primordia grew slowly, such that exposure of the central

part of the flower primordia was longer than in wild-type

flowers (Figure 3aiv,av). Additionally, the overall carpel cell

size was increased, indicating a reduction in total cell

number (Figure 3f–i), as observed for leaf epidermal cells

(see below).

Cell-cycle inhibition in the STM domain affects cell division

in the meristem and the developing organs

To visualize the cellular morphology of the PSTM:CDKA;

1.N146 meristems, mature embryos and vegetative SAMs

from 2- and 8-day-old seedlings, respectively, were analyzed

by confocal and light microscopy. Embryonic meristems

were defective in shape and size. The embryonic SAM was

much smaller and more rounded than that of the wild type,

and the tunica layers (L1 and L2) were indistinct

(Figure 4a,b). Longitudinal sections from SAMs of 8-day-old

seedlings confirmed the altered meristem organization and

shape, although the two tunica layers (L1 and L2) were rec-

ognizable at this stage (Figure 4c,d). Quantitative analysis

showed that meristem size, assessed as the distance

between opposing primordia measured along the SAM

surface, was clearly reduced in the PSTM:CDKA;1.N146 lines

(Figure S1).

To analyze cell division rates and determine cell fate on

the shoot apex surface, the sequential replica method was

used (Dumais and Kwiatkowska, 2002). Replicas taken from

the inflorescence shoot apices were apparently non-uni-

form in the PSTM:CDKA;1.N146 transgenic plants. The size

of the transgenic meristems varied, as did the number of

cells on the SAM surface (Figure 4e–g), but the number of

cells was significantly smaller in the transgenic

PSTM:CDKA;1.N146 plants than that in wild-type plants

(123 � 25 versus 63 � 24, respectively; mean � SD, n = 8;

P value <0.002 by the non-parametric Kruskal–Wallis test).

(a) (b)

(d) (e) (f) (g)

(c) Figure 2. Phenotypic characterization of PSTM:

CDKA;1.N146 lines.

(a–c) Twelve-day-old seedlings of a wild-type

plant (a) and PSTM:CDKA;1.N146 plants with

severe (b) and weak (c) phenotypes.

(d,e) Three-week-old wild-type (d) and PSTM:

CDKA;1.N146 (e) plants.

(f,g) Six-week-old wild-type (f) and PSTM:

CDKA;1.N146 (g) plants.

28 Tarik Gaamouche et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

The cell division frequency, determined by comparison of

cell numbers in two consecutive sequential replicas, was

also variable, and was lower in transgenic than in wild-type

SAMs (Figure 4e–g). A striking feature of the

PSTM:CDKA;1.N146 inflorescence meristems was the non-

homogeneous size of the cells, i.e. some cells were

apparently larger than others (Figure 5a–c). These

cells did not divide, but expanded (Figure 5a–c; cells

labeled by asterisks and circles). Cells were elongated

along the meristem radius, unlike typical isodiametric

meristematic cells. This shape most probably resulted

from anisotropic growth that is specific to the meristem

periphery, i.e. faster growth along the meristem radius

than along its circumference. Atypical cells were also

found on the adaxial flower primordium boundary: these

cells were elongated along the periphery (i.e. along the

circumference), which often appeared wrinkled (Figure 5a–

c), in contrast to the wild-type apices (Figure 4e). The

wrinkled appearance was due to deep grooves (creases) in

the meristem surface that occur at anticlinal walls in

contact with superficial meristem cells. These deep

grooves are a distinctive feature of differentiating cells,

such as cells on the sepal surface (Figure 3aii).

The aberrant leaf phenotype of the PSTM:CDKA;1.N146

transgenic plants suggested that inhibition of cell division

within the STM domain of the SAM has a dramatic effect on

the cell cycle during plant development. To test this

hypothesis, we analyzed the leaves in more detail. The first

true mature leaves showed a decrease in the surface area of

the leaf lamina of 24 and 32% in the CDKA;1.N146 B3-4

and B3-5 lines, respectively, compared to control plants

(Figure 6a). To investigate whether this reduction in leaf size

was caused by inhibition of cell division or cell expansion,

the mean cell area of abaxial epidermal cells was determined

from drawing-tubus images, and the total cell number in the

leaf epidermis was calculated from the ratio of the leaf

lamina surface area to the mean cell area. Significant

differences were observed between wild-type and trans-

genic lines (Figure 6b,c). Both CDKA;1.N146 lines displayed

a statistically significant reduction (43 and 37%, respectively)

(a)

(f) (g) (h) (i)

(b) (c)

(d) (e)(i)

(iii) (iv) (v)

(ii)

Figure 3. Floral and inflorescence phenotypes of PSTM:CDKA;1.N146 lines.

(ai,aii) Arrangement of flower primordia and aberrations in primordium development in PSTM:CDKA;1.N146 lines. Top views of inflorescence shoot apices of wild-

type (ai) and transgenic (aii) plants surrounded by flower primordia (labeled P1–P9, numbered from the youngest primordium to the oldest). (aiii–av) Close-up views

of abnormally developed flower primordia representing consecutive stages of early sepal formation. (aiii) Six-plastochron-old primordium. No clear boundaries

(asterisks) are visible between sepals of two pairs (S1 and S2). (aiv) Six-plastochron-old primordium with apparent sepals of two pairs (S1 and S2) with a developed

boundary. (av) Seven-plastochron-old primordium with developed sepals of two pairs (S1 and S2), but exhibiting retarded growth. At this stage in the wild-type, the

sepals start to or completely overtop the central portion of the flower primordium [compare with P7 in (ai)].

(b,d) Wild-type flowers.

(c,e) Mutant flowers of small size and abnormal shape.

(f–i) Scanning electron microscopy of carpels. (f) Wild-type carpel. (h) Mutant carpel. Epidermal cells of wild-type (g) and mutant (i) carpels.

Scale bars = 30 lm (ai,aii), 10 lm (aiii–av), 3 mm (f,h) and 800 lm (g,i).

CDK dose-dependent control of meristem activity 29

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

in the epidermal cell number and an increase in cell size (30

and 46%, respectively), in accordance with the microscopic

analysis of leaf epidermal cells (Figure 6d,e). Similarly,

enlargement of the palisade mesophyll cells was observed

(Figure 6f,g). From these results, it can be inferred that the

smaller leaves from the PSTM:CDKA;1.N146 plants are a

consequence of reduced cell proliferation rates that cannot

be fully compensated for by increased cell size.

Inhibition of CDK activity at the shoot apex results in

premature differentiation of SAM cells

The results described above indicate that cells within the

shoot apex of PSTM:CDKA;1.N146 lines entered cell differen-

tiation prematurely. Because of the relationship between cell

size, differentiation and endo-reduplication, the DNA ploidy

distribution pattern within the shoot apex of wild-type and

PSTM:CDKA;1.N146 plants was compared. 4¢,6-diamidino-2-

phenylindole (DAPI) staining of longitudinal sections

through the inflorescence SAM showed the presence of a

proportion of cells with strongly fluorescent nuclei in trans-

genic plants, indicating that they might have undergone

endo-reduplication (Figure 5d,e). In agreement, measure-

ments of the DNA ploidy level by flow cytometry on dissected

vegetative apices showed a clear increase in the 4C and 8C

DNA content in transgenic samples (Figure 5f,g). In addition,

an extra 16C peak was observed. Endo-reduplication-linked

S-phase progression was confirmed by mRNA in situ

hybridization, with very similar expression patterns for

the S-phase marker gene HISTONE H4 in wild-type and

PSTM:CDKA;1.N146 SAMs (Figure 5h,i). Thus, although

CDKA;1.N146 expression inhibited the mitotic cell cycle, it

did not affect the endo-reduplication cycle.

Inter-dependency of meristem organization and the level

of cell-cycle inhibition

To study the effects of a reduced cell-division rate on the

meristem organization, STM, WUS, CLV3 and ASYMMETRIC

LEAVES 1 (AS1) transcripts were analyzed by mRNA in situ

hybridization (Figure S2). The expression patterns of these

genes did not differ significantly between the transgenic and

wild-type plants, in either the CZ, PZ or RM, indicating no

significant change in meristem organization. However,

phenotypic analysis of 3-week-old PSTM:CDKA;1.N146 plants

showed that 30% of a total population of 200 plants exam-

ined had a bushy appearance with multiple leaf rosettes, and

thus an increased total number of leaves (Figure 7a–e). A

more detailed analysis of these plants revealed that sec-

ondary vegetative rosettes developed side-by-side with the

primary rosette (Figure 7a,b). Tissue sectioning of 8-day-old

seedlings revealed that the vegetative SAM in these plants

(a)

(e) (f) (g)

(b) (c) (d)

Figure 4. Meristem organization and cell division analysis of PSTM:CDKA;1.N146 lines.

(a,b) Confocal micrographs of embryonic SAMs from wild-type (a) and PSTM:CDKA;1.N146 (b) plants.

(c,d) Longitudinal sections through the SAM visualized by optical microscopy of wild-type (c) and PSTM:CDKA;1.N146 transgenic (d) seedlings. For confocal and light

microscopy, plant material was stained with 4¢,6-diamidino-2-phenylindole (DAPI) and 0.1% toluidine blue, respectively.

(e–g) Distribution of anticlinal divisions of superficial apex cells (outer tunica layer) shown on scanning electron micrographs of top views of wild-type and

transgenic inflorescence shoot apices. Cells that had divided within the 24 h time frame between replicas are labeled by dots. The apex portion, which was

recognized in the replicas is outlined. (e) Wild-type apex. Forty-six of 104 cells had divided. (f) Transgenic apex. Nineteen of 105 cells in the outlined region had

divided. (g) Transgenic apex with clearly reduced meristem size. Seven of 38 cells had divided.

Scale bars = 20 lm (c,d) and 10 lm (e–g).

30 Tarik Gaamouche et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

was split, resulting in formation of shoot apices with two

fused domes (Figure 7f). During ontogenesis, the flower

organs were small and the siliques were extremely short

with few seeds inside (data not shown). Also, the epidermis

of the carpels consisted of enlarged, irregularly shaped cells

(Figure 7g,h), an indication of strongly inhibited cell divi-

sion. As the carpel cell division phenotype was more severe

in plants with a split meristem than in plants with a single

(a) (d) (e)

(f)

(h) (i)

(g)(b)

(c)

Figure 5. DNA replication and cell expansion in

PSTM:CDKA;1.N146 SAMs.

(a–c) Developmental sequences of a transgenic

inflorescence shoot apex. Primordia (P1–P5) are

numbered from the youngest primordium to the

oldest. Three stages (replicas) are shown, taken

at 24 h intervals. Asterisks and circles indicate

cells that appeared normal in the first (a) or the

second stage (b), but did not divide within the

next 24 h, although their size increased consid-

erably (c). Deep wrinkles were apparent between

some cells at the primordium P3 boundary

(indicated by arrowheads).

(d,e) DAPI-stained longitudinal sections of

7-week-old inflorescence shoot meristems from

wild-type (d) and PSTM:CDKA;1.N146 plants.

Arrows point to enlarged nuclei (e).

(f,g) DNA ploidy distribution for nuclei isolated

from 8-day-old seedling shoot meristems of

wild-type (f) and PSTM:CDKA;1.N146 transgenic

plants (g).

(h,i) Histone H4 expression pattern in wild-type

(h) and PSTM:CDKA;1.N146 transgenic plants (i).

Scale bar = 10 lm (a–c) and 30 lm (d,e).

(a)

(d) (e) (f) (g)

(b) (c)

Figure 6. Cell and leaf lamina size analysis of mature PSTM:CDKA;1.N146 leaves compared to those of the wild-type.

(a–c) Leaf surface area (a), cell number (b) and abaxial epidermis cell size (c). All measurements were obtained on 3-week-old first leaves. Values are means for 6–10

plants (*P = 0.06, **P = 0.01, ***P = 0.001, ****P = 0.0001, *****P = 0.00001, Student’s t test, for comparison with wild-type).

(d–g) Microscopy analysis of adaxial epidermis cells and palisade mesophyll cells from wild-type (d,f) and a transgenic line (e,g). Scale bar = 25 lm.

CDK dose-dependent control of meristem activity 31

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meristem, we hypothesized that the shoot apex phenotype

correlated with the level of PSTM:CDKA;1.N146 activity. To

test this hypothesis, we compared the number of plants with

at least two SAMs under long-day and continuous light

conditions. In both the wild-type and transgenic plants, an

increase in day length did not significantly affect the

expression level of endogenous CDKA;1 (Figure 7i), but

triggered an increase in the level of expression of STM

(Figure 7j), as reported previously (Geier et al., 2008).

Accordingly, the transcription level of the CDKA;1.N146

transgene was increased under continuous light conditions

in the PSTM:CDKA;1.N146 plants (Figure 7k). Additionally,

under continuous light conditions, the number of plants

with multiple rosettes increased by approximately 20%

(a)

(c) (f)

(i) (j)

(k) (l)

(b) (d)

(e)

(g) (h)

Figure 7. Phenotypic analysis of PSTM:CDKA;1.N146 plants exhibiting a severe phenotype.

(a) Three-week-old plant with two rosettes developing side by side.

(b) Twelve-day-old seedling with two young rosettes.

(c) Six-week-old plant with a bushy appearance.

(d,e) Dissected rosette leaves (cut to flatten the leaf lamina) of wild-type (d) and PSTM:CDKA;1.N146 transgenic plants (e).

(f) Longitudinal section through the vegetative SAM of a PSTM:CDKA;1.N146 transgenic line. The SAM had two domes.

(g,h) Scanning electron micrographs of a PSTM:CDKA;1.N146 carpel (g) and its epidermal cells (h). Scale bars = 3 mm (g) and 800 lm (h).

(i–k) Quantitative real-time expression analysis of CDKA;1 (i), STM (j) and CDKA;1.N146 (k) in 1-week-old wild-type and PSTM:CDKA;1N146 seedlings grown either

under long-day (LD; 16 h light) or continuous light (CL) conditions. The values are the means of three independent replicates. The ACTIN2, EF1-a and CYTOCHROME

B5 expression levels were used as a reference. Expression levels observed in Col-0 (LD) (i,j) or CDKA;1.N146 (LD) (k) were arbitrary set to 1.

(l) Percentage of transgenic plants exhibiting double rosettes after 3 weeks of growth under LD and CL photoperiods (n = 300).

**P = 0.05 and ***P = 0.007 (Student’s t test, LD versus CL).

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(Figure 7l), indirectly implying a correlation between the

appearance of multiple apices and the level of cell-cycle

inhibition.

As independent proof that the CDKA;1.N146 expression

level affects meristem organization, the number of plants

with multiple meristems was compared in homozygous

versus hemizygous PSTM:CDKA;1.N146 populations. Hemi-

zygous lines were obtained by pollinating wild-type plants

with pollen from the PSTM:CDKA;1.N146 homozygous

plants. Genotyping the offspring confirmed a high success

rate (>90%) for cross-pollination. The hemizygous

PSTM:CDKA;1.N146 plants had lost the typical malformed

leaf phenotype of the homozygous plants (Figure 8a,b),

corresponding with a decrease in the CDKA;1.N146 expres-

sion level (Figure 8c), and the number of plants with two or

more rosettes was significantly reduced (Figure 8d). The

reduction in the number of plants with multiple meristems

was accompanied by a decrease in the CDKA;1.N146 tran-

script level of >80%, rather than the theoretically expected

50% in hemizygous plants. This observation is probably due

to a reduction in the relative abundance of STM-expressing

cells as a consequence of the decrease in SAM number in the

hemizygous lines.

DISCUSSION

Although it is generally accepted that CDK/CYC complexes

perform fundamental control of cell division in plants, the

manner by which the cell-cycle machinery is integrated into

the meristem organization is still poorly understood. Here,

we have confirmed a link between cell-cycle control and

meristem function by specifically inhibiting cell divisions in

the SAM. Cell divisions in the vegetative SAM are necessary

for SAM self-perpetuation, a process in which the general

shape and size of the SAM are maintained despite the mer-

istem continuously providing cells that are incorporated into

leaf primordia. Strikingly, although the cell cycle was aimed

at inhibition in the SAM exclusively, plants expressing

PSTM:CDKA;1.N146 showed clear morphological alterations,

such as mis-shapen leaves. The phenotypic changes were

accompanied by a reduction in cell number and an increase

in cell size. Developmental defects because of mis-expres-

sion of cell-cycle regulatory genes have been described

previously (Riou-Khamlichi et al., 1999; De Veylder et al.,

2001; Weingartner et al., 2004), but, because of the use of

ubiquitously expressed promoters, the influence of the

malfunctioning meristem on the overall plant phenotype

could not be determined. In other cases, the effects on

meristem organization were so strong that they hampered

post-meristematic development, as observed upon over-

expression or knockout of B2-type CDKs (Andersen et al.,

2008). Although the possibility cannot be totally excluded

that a residual amount of CDKA;1.N146 protein may remain

in the cells after they have left the meristem (because of

protein stability), and thus contribute to the reduced leaf cell

number, the data presented indicate that a reduced cell

division rate within the meristem exerts a severe negative

effect on cell divisions outside the meristem. As noted pre-

viously, early stages of leaf primordium development can

take place without any cell division (Foard and Haber, 1961;

(a)

(c)

(d)

(b)

Figure 8. Phenotypic comparison of PSTM:CDKA;1N146 plants.

(a,b) Three-week-old homozygous (a) and hemizygous (b) PSTM:CDKA;1.N146

plants.

(c) CDKA;1.N146 expression levels measured by quantitative real-time PCR.

The values are means of three independent replicates. The ACTIN2, EF1-a and

CYTOCHROME B5 expression levels were used as a reference. Expression

levels observed in Col-0 were arbitrarily set to 1.

(d) Percentage of transgenic plants exhibiting double rosettes after 3 weeks of

growth (n = 200, ****P < 0.0001, Student’s t test, for comparison with

hemizygous plants).

CDK dose-dependent control of meristem activity 33

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

Haber, 1962), supporting the ‘organismal’ theory, which

states that the frequency and pattern of cell divisions do not

drive organogenesis, but are secondary to an intrinsic

developmental plan. Nevertheless, because the cell number

increases exponentially during the initial stages of leaf

development (De Veylder et al., 2001; Boudolf et al., 2004),

primordia consisting initially of a strongly reduced number

of cells are expected to eventually develop in leaves made

of fewer cells.

Alternatively, the reduction in cell number in leaves might

be due to the decrease in CDK activity when the cells leave

the meristem. In Arabidopsis, leaf cells exit the cell cycle in

an apex-to-base direction (Donnelly et al., 1999). If this

gradient coincides with a basipetal gradient in CDK activity,

cells will probably exit the cell cycle when the level of CDK

activity drops below a critical threshold level required to

drive mitosis. We propose that primordia with a lower initial

CDK level leave the cell cycle before those with a higher

level, eventually resulting in a reduction in leaf cell number

in PSTM:CDKA;1.N146 plants. Earlier cell-cycle exit and

differentiation are substantiated by an increase in the overall

DNA ploidy level of PSTM:CDKA;1.N146 leaf cells (data not

shown), because the onset of endo-reduplication correlates

with exit from the mitotic cell cycle (Boudolf et al., 2004;

Verkest et al., 2005; Vlieghe et al., 2005; Dewitte et al., 2007).

A similar increase in the DNA ploidy level was observed in

the case of meristem-specific over-expression of the CDKA;1

inhibitor-encoding gene ICK2/KRP2 (Verkest et al., 2005).

The expression of ICK2/KRP2 has been postulated to reduce

the level of CDKA;1 activity below a threshold level neces-

sary for mitotic cell divisions, but that is still high enough to

enable repeated rounds of DNA replication. Similarly,

expression of the dominant negative CDKA;1 allele probably

has a profound effect on the mitotic cell cycle, but not on the

endo-reduplication cycle, triggering an early mitosis-to-

endocycle transition, and eventually a higher DNA ploidy

level. This process may take place at the SAM, because the

enlarged cells at the meristem correlate with an increase in

the number of cells with an enlarged nucleus. As endo-

reduplicating cells rarely divide, premature differentiation of

cells within the meristem might also have a significant

negative effect on the final cell number within the organism.

With regard to the abnormal leaf morphology in

PSTM:CDKA;1.N146 plants, some of the SAM cells that

entered the endo-reduplication cycle prematurely might be

assumed to be incorporated into the leaf primordium.

Together the general reduction in the cell number of

primordia and the endocycling of some primordium cells

might obstruct leaf organogenesis.

Despite the clear defects in plant development and the

cellular phenotypes within the meristem, the majority of the

PSTM:CDKA;1.N146 plants had no clearly altered meristem

organization. In situ hybridization and expression analysis of

SAM regulatory genes revealed no difference between

transgenic and wild-type plants. The absence of visible

meristem organization phenotype appears to contradict

other published data. Local over-expression of the RETINO-

BLASTOMA-RELATED 1 (RBR1) gene at the SAM triggered

differentiation of meristematic cells together with loss of

expression of meristem identity markers (Wyrzykowska

et al., 2006). Analogously, treatment of meristems with

microtubule-depolymerizing drugs caused meristem cells

to differentiate (Grandjean et al., 2004), while loss of

expression or over-expression of B2-type CDKs resulted in

a total loss of meristem integrity (Andersen et al., 2008). In

contrast, local stimulation of cell division rates by over-

expression of CYCA3;2 and expression of fission yeast

cdc25, or inhibition of cell proliferation by knock-out of

D3-type cyclin, had none or only mild effects on meristem

organization (Wyrzykowska et al., 2002; Dewitte et al., 2007).

Surprisingly, upon over-expression of CDKA;1.N146 in the

STM domain, a sub-population of plants had a split SAM in

the vegetative phase, indicating some meristematic defects

in this sub-population of plants. This phenotype resembles

that obtained by surgical manipulations of the SAM in which

destruction of the CZ resulted in regeneration of new SAMs

from the PZ (Reinhardt et al., 2003). The split-meristem

phenotype was correlated with the PSTM:CDKA;1.N146

expression level, suggesting a dose-dependent relationship

between inhibition of cell division and the effect on meri-

stem organization. Although the product of the dominant

negative CDKA;1.N146 allele probably specifically targets

the A-type CDKs, the reduced cell proliferation rate caused

by a decrease in CDKA;1 activity will affect other CDKs. Thus,

rather than attributing the meristem defects solely to

CDKA;1, we assume that the overall effect on CDK activity

is responsible for the observed phenotypes. We propose a

CDK activity threshold model in which weak CDK inhibition

suppresses the cell division activity without affecting mer-

istem organization, whereas strong inactivation of CDK

leads to a loss of meristem identity (Figure 9), which may

be followed by new meristem formation. This hypothesis

reconciles the apparently conflicting data available in the

literature, in which the presence or absence of clear meri-

stem organization phenotypes might be correlated with the

strength of cell-cycle inhibition. The loss-of-meristem orga-

nization phenotype of B2-type CDK mutants might be

a consequence of the severe inhibition of cell division

(Andersen et al., 2008). By contrast, plants lacking the

D3-type cyclin show only a mildly defective cell cycle

without clear meristem patterning alterations (Dewitte et al.,

2007). The model implies that high CDK activity within the

meristem is necessary to maintain cells in an undifferenti-

ated state, as supported by the induction of differentiation of

root stem cells by co-suppression of an F-type CDK-activat-

ing kinase (Umeda et al., 2000). Similarly, in a leaf callus

culture, continuous CDK activity is important to maintain

cells in an undifferentiated state (Yamaguchi et al., 2003).

34 Tarik Gaamouche et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

Hence, probably only substantial changes in the cell division

rates or the proliferation status of meristem cells will result

in a change in meristem organization. We postulate that, in

the case of the SAM of the PSTM:CDKA;1.N146 transgenic

line, the CDK level is always close to the putative threshold,

and subtle differences below or above this threshold are

sufficient to trigger apparently different phenotypes.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

The STM promoter (PSTM) was amplified from the F24O1 BAC cloneas a 4.5 kb fragment, as described previously (Verkest et al., 2005),and cloned into the pBIN+ vector, into which a cassette containingthe CDKA;1.N146 allele (Hemerly et al., 1995) together with a 3’ NOSterminator was introduced. Arabidopsis thaliana (L.) Heyhn. plants(ecotype Columbia, Col-0) were grown at 22 � 2�C under a 16 h/8 hlight/dark photoperiod (65 lE m)2 sec)1) on agar-solidified culturemedium (half-strength Murashige and Skoog medium, 0.5 g/L MES,10 g/L sucrose, 0.8% plant tissue culture agar), except where indi-cated. Agrobacterium tumefaciens-mediated transformation wasperformed by floral dip as described previously (Clough and Bent,1998). Transgenic lines were selected on kanamycin-containingmedium.

Sequential replicas of inflorescence meristems

To obtain sequential replicas, plants were grown under short-dayconditions (11 h/13 h light/dark) for 13 weeks, followed by 4 weeksof long-day conditions (16 h/8 h light/dark). Replicas were takenfrom inflorescence apices of main shoots or axillary shoots (30–150 mm long) as described previously (Williams and Green, 1988;Kwiatkowska, 2004). Sequences were taken every 24 h for 2–4 daysfrom inflorescence apices of transgenic plants. Previously obtained

sequential replicas of Col-0 (Kwiatkowska, 2004, 2006) were used forcomparison. All the electron micrographs for cell identification andcounting were improved by means of the MATLAB image pro-cessing toolbox (http://www.mathworks.com/products/matlab/), toenable more precise recognition of the anticlinal walls of surfacecells.

In situ hybridization

The RNA of 12-day-old seedlings from wild-type and mutant linesfixed in FAA (10% formaldehyde, 50% EtOH, 5% glacial acetic acid)was hybridized in situ. Full-length riboprobes were synthesized forthe STM (At1G62360), WUS (At2G17950), CLV3 (At2G27250), AS1(At2G37630), H4 (At1G07820) and CDKB1 (At3G54180) genes by PCRamplification with universal primers specific to the gene-containingvectors. Samples were processed as described previously (Dreaet al., 2005), with the following modifications: the slides were pro-cessed using the automated slide processor InsituPro VSi (http://www.intavis.com) from the hybridization step to signal detection,hybridization and post-hybridization washes were performed at55�C, samples were incubated in 1:1250 diluted anti-digoxigeninantibody conjugated to alkaline phosphatase (http://www.roche.com) for 1.5 h, and the signal was developed using Western Bluestabilized substrate (http://www.promega.com). Sections werephotographed using a Nikon E800 microscope with a digital camera(http://www.nikoninstruments.com).

Real-time quantitative PCR analysis

For real-time quantitative PCR, total RNA was isolated from a pool of200 plantlets at 7 days after germination using an RNeasy plant minikit (http://www.qiagen.com). For each sample, 1 lg was reverse-transcribed into cDNA using the SuperScript first-strand synthesissystem for RT-PCR (http://www.invitrogen.com). QuantitativeRT-PCR amplification of the cDNA was performed using a LightCy-clerTM 480 (http://www.roche.com), with the gene-specific primers5¢-ATTGCGTATTGCCACTCTCATAGG-3¢ and 3¢-TCCTGACAGGGATACCGAATGC-5¢ for CDKA;1 (At3G48750), 5¢-CGGAGGACATGCAGTTTGTA-3¢ and 3¢-AAAGCATGGTGGAGGAGATG-5¢ for STM,and 5¢-AAGCTTTTGAGATCAGTTTCTTG-3¢ and 3¢-ACTTTATTGCCAAATGTTTGAA-5¢ for the CDKA;1.N146 transgene. Differences inthe amount of target cDNA used as input were normalized againstthree reference genes amplified using primers 5¢-GGCTCCTCTTAACCCAAAGGC-3¢ and 3¢-CACACCATCACCAGAATCCAGC-5¢for ACTIN2 (At3G46520), 5¢-CTGGAGGTTTTGAGGCTGGTAT-3¢ and3¢-CCAAGGGTGAAAGCAAGAAGA-5¢ for EF1-a (At5G60390), and5¢-CGACACTGCAAGGGACATGA-3¢ and 3¢-ACGTATGTCCTAGTTGCTGGAACA-5¢ for CYTOCHROME B5 (AT5G53560).

Kinase assay

Eight-day-old plants were harvested for total protein extraction andkinase activity tests. CDK-like proteins were purified from totalprotein extracts on p10CKS1 Sepharose beads (De Veylder et al.,1999), and their ability to phosphorylate histone H1 was determinedas described previously (Azzi et al., 1994; Corellou et al., 2000).Radioactive histone H1 was quantified using a STORM Phospho-Imager and ImageQuant software (http://www.gehealthcare.com).

Flow cytometric analysis

Frozen plant material was chopped with a razor blade in 800 ll ofnucleus extraction solution (http://www.partec.com), supplementedwith 200 ll of staining solution (http://www.partec.com). Thehomogenate was filtered through a 30 lm mesh. The nuclei were

Figure 9. CDK activity threshold model for meristem organization.

High CDK activity is required to maintain cells within the SAM in an

undifferentiated state. A decrease in CDK activity has no effect on meristem

organization unless it drops below a hypothetical threshold level, below

which the meristematic cells exit the cell cycle affecting meristem identity and

organization.

CDK dose-dependent control of meristem activity 35

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

analyzed using a CyFlow cytometer and FloMax software (http://www.partec.com).

Microscopic analysis

Plastic embedding with Technovit 7100 resin (http://www.kulzer-technik.de) and sectioning were performed according to the man-ufacturer’s protocol. Serial sections (5 lm) were stained with 0.1%toluidine blue and mounted using Depex-mounting medium (http://uk.vwr.com). Sections were photographed under a Diaplan lightmicroscope (http://www.leica-microsystems.com). The nuclearDNA was visualized as described previously (Bemis and Torii, 2007).Slides were observed under a 40· objective after 4¢,6-diamidino-2-phenylindole (DAPI) staining and using an appropriate filter on anAxioImager M1 equipped with an AxioCam HRm camera (http://www.zeiss.com). Digital images were acquired and analyzed usingAxioVision 4.6 software. Final images were obtained using Photo-shop 6.0 (http://www.adobe.com/). Dissected embryos of seedlings2 and 7 days after germination were analyzed by confocal micros-copy as described previously (Autran et al., 2002).

ACKNOWLEDGEMENTS

We thank all members of the Cell Cycle Group and the SY-STEMtraining network for useful discussions and suggestions, and Mar-tine De Cock for help in preparing the manuscript. This work wassupported by grants from the Interuniversity Attraction PolesProgram (IUAP VI/33), initiated by the Belgian State Science PolicyOffice, and the European Union SY-STEM (MRTN-GT-2004-005336).L.D.V. is a postdoctoral fellow of the Fonds voor WetenschappelijkOnderzoek – Vlaanderen (FWO).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Distance between opposing leaf primordia measuredalong the meristem surface.Figure S2. Spatial expression pattern analysis of SAM patterninggenes.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

de Almeida Engler, J., De Veylder, L., De Groodt, R. et al. (2009) Systematic

analysis of cell cycle gene expression during Arabidopsis development.

Plant J. 59, 645–660.

Andersen, S.U., Buechel, S., Zhao, Z., Ljung, K., Novak, O., Busch, W.,

Schuster, C. and Lohmann, J.U. (2008) Requirement of B2-type cyclin-

dependent kinases for meristem integrity in Arabidopsis thaliana. Plant

Cell, 20, 88–100.

Autran, D., Jonak, C., Belcram, K., Beemster, G.T.S., Kronenberger, J.,

Grandjean, O., Inze, D. and Traas, J. (2002) Cell numbers and leaf devel-

opment in Arabidopsis. A functional analysis of the STRUWWELPETER

gene. EMBO J. 21, 6036–6049.

Azzi, L., Meijer, L., Ostvold, A.-C., Lew, J. and Wang, J.H. (1994) Purification of

a 15-kDa cdk4- and cdk5-binding protein. J. Biol. Chem. 269, 13279–13288.

Bemis, S.M. and Torii, K.U. (2007) Autonomy of cell proliferation and devel-

opmental programs during Arabidopsis aboveground organ morphogen-

esis. Dev. Biol. 304, 367–381.

Boudolf, V., Barroco, R., de Almeida Engler, J., Verkest, A., Beeckman, T.,

Naudts, M., Inze, D. and De Veylder, L. (2004) B1-type cyclin-dependent

kinases are essential for the formation of stomatal complexes in Arabid-

opsis thaliana. Plant Cell, 16, 945–955.

Brand, U., Fletcher, J.C., Hobe, M., Meyerowitz, E.M. and Simon, R. (2000)

Dependence of stem cell fate in Arabidopsis on a feedback loop regulated

by CLV3 activity. Science, 289, 617–619.

Carraro, N., Peaucelle, A., Laufs, P. and Traas, J. (2006) Cell differentiation

and organ initiation at the shoot apical meristem. Plant Mol. Biol. 60,

811–826.

Clark, S.E., Williams, R.W. and Meyerowitz, E.M. (1997) The CLAVATA1 gene

encodes a putative receptor kinase that controls shoot and floral meristem

size in Arabidopsis. Cell, 89, 575–585.

Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agro-

bacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,

735–743.

Corellou, F., Bisgrove, S.R., Kropf, D.L., Meijer, L., Kloareg, B. and Bouget,

F.-Y. (2000) A S/M DNA replication checkpoint prevents nuclear and cyto-

plasmic events of cell division including centrosomal axis alignment and

inhibits activation of cyclin-dependent kinase-like proteins in fucoid

zygotes. Development, 127, 1651–1660.

De Veylder, L., De Almeida Engler, J., Burssens, S., Manevski, A., Lescure, B.,

Van Montagu, M., Engler, G. and Inze, D. (1999) A new D-type cyclin of

Arabidopsis thaliana expressed during lateral root primordia formation.

Planta, 208, 453–462.

De Veylder, L., Beeckman, T., Beemster, G.T.S., Krols, L., Terras, F., Landrieu,

I., Van Der Schueren, E., Maes, S., Naudts, M. and Inze, D. (2001) Functional

analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13,

1653–1667.

Dewitte, W., Riou-Khamlichi, C., Scofield, S., Healy, J.M.S., Jacqmard, A.,

Kilby, N.J. and Murray, J.A.H. (2003) Altered cell cycle distribution,

hyperplasia, and inhibited differentiation in Arabidopsis caused by the

D-type cyclin CYCD3. Plant Cell, 15, 79–92.

Dewitte, W., Scofield, S., Alcasabas, A.A. et al. (2007) Arabidopsis

CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-

limiting for cytokinin responses. Proc. Natl. Acad. Sci. USA, 104, 14537–

14542.

Donnelly, P.M., Bonetta, D., Tsukaya, H., Dengler, R.E. and Dengler, N.G.

(1999) Cell cycling and cell enlargement in developing leaves of Arabid-

opsis. Dev. Biol. 215, 407–419.

Drea, S., Corsar, J., Crawford, B., Shaw, P., Dolan, L. and Doonan, J.H. (2005)

A streamlined method for systematic, high resolution in situ analysis of

mRNA distribution in plants. Plant Methods, 1, 8.1–8.11.

Dumais, J. and Kwiatkowska, D. (2002) Analysis of surface growth in shoot

apices. Plant J. 31, 229–241.

Fletcher, J.C., Brand, U., Running, M.P., Simon, R. and Meyerowitz, E.M.

(1999) Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot

systems. Science, 283, 1911–1914.

Foard, D.E. and Haber, A.H. (1961) Anatomic studies of gamma-irradiated

wheat growing without cell division. Am. J. Bot. 48, 438–446.

Gallois, J.-L., Woodward, C., Reddy, G.V. and Sablowski, R. (2002) Combined

SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in

Arabidopsis. Development, 129, 3207–3217.

Geier, F., Lohmann, J.U., Gerstung, M., Maier, A.T., Timmer, J. and Fleck, C.

(2008) A quantitative and dynamic model for plant stem cell regulation.

PLoS ONE, 3, e3553.1–e3553.10.

Grandjean, O., Vernoux, T., Laufs, P., Belcram, K., Mizukami, Y. and Traas, J.

(2004) In vivo analysis of cell division, cell growth, and differentiation at the

shoot apical meristem in Arabidopsis. Plant Cell, 16, 74–87.

Haber, A.H. (1962) Nonessentiality of concurrent cell divisons for degree of

polarization of leaf growth. I. Studies with radiation-induced mitotic inhi-

bition. Am. J. Bot. 49, 583–589.

Hemerly, A., de Almeida Engler, J., Bergounioux, C., Van Montagu, M.,

Engler, G., Inze, D. and Ferreira, P. (1995) Dominant negative mutants of

the Cdc2 kinase uncouple cell division from iterative plant development.

EMBO J. 14, 3925–3936.

Ito, Y., Nakanomyo, I., Motose, H., Iwamoto, K., Sawa, S., Dohmae, N. and

Fukuda, H. (2006) Dodeca-CLE peptides as suppressors of plant stem cell

differentiation. Science, 313, 842–845.

Iwakawa, H., Shinmyo, A. and Sekine, M. (2006) Arabidopsis CDKA;1, a cdc2

homologue, controls proliferation of generative cells in male gametogen-

esis. Plant J. 45, 819–831.

Joubes, J., De Schutter, K., Verkest, A., Inze, D. and De Veylder, L. (2004)

Conditional, recombinase-mediated expression of genes in plant cell cul-

tures. Plant J. 37, 889–896.

36 Tarik Gaamouche et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37

Kondo, T., Sawa, S., Kinoshita, A., Mizuno, S., Kakimoto, T., Fukuda, H. and

Sakagami, Y. (2006) A plant peptide encoded by CLV3 identified by in situ

MALDI-TOF MS analysis. Science, 313, 845–848.

Kwiatkowska, D. (2004) Surface growth at the reproductive shoot apex of

Arabidopsis thaliana pin-formed 1 and wild type. J. Exp. Bot. 55, 1021–

1032.

Kwiatkowska, D. (2006) Flower primordium formation at the Arabidopsis

shoot apex: quantitative analysis of surface geometry and growth. J. Exp.

Bot. 57, 571–580.

Laufs, P., Grandjean, O., Jonak, C., Kieu, K. and Traas, J. (1998) Cellular

parameters of the shoot apical meristem in Arabidopsis. Plant Cell, 10,

1375–1389.

Lenhard, M., Jurgens, G. and Laux, T. (2002) The WUSCHEL and SHOOT-

MERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot

meristem regulation. Development, 129, 3195–3206.

Long, J.A. and Barton, M.K. (1998) The development of apical embryonic

pattern in Arabidopsis. Development, 125, 3027–3035.

Long, J.A., Moan, E.I., Medford, J.I. and Barton, M.K. (1996) A member of the

KNOTTED class of homeodomain proteins encoded by the STM gene of

Arabidopsis. Nature, 379, 66–69.

Mayer, K.F.X., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G. and Laux, T.

(1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis

shoot meristem. Cell, 95, 805–815.

Nowack, M.K., Grini, P.E., Jakoby, M.J., Lafos, M., Koncz, C. and Schnittger,

A. (2006) A positive signal from the fertilization of the egg cell sets off

endosperm proliferation in angiosperm embryogenesis. Nat. Genet. 38,

63–67.

Porceddu, A., Stals, H., Reichheld, J.-P., Segers, G., De Veylder, L., De Pinho

Barroco, R., Casteels, P., Van Montagu, M., Inze, D. and Mironov, V. (2001)

A plant-specific cyclin-dependent kinase is involved in the control of G2/M

progression in plants. J. Biol. Chem. 276, 36354–36360.

Reddy, G.V. and Meyerowitz, E.M. (2005) Stem-cell homeostasis and growth

dynamics can be uncoupled in the Arabidopsis shoot apex. Science, 310,

663–667.

Reddy, G.V., Heisler, M.G., Ehrhardt, D.W. and Meyerowitz, E.M. (2004) Real-

time lineage analysis reveals oriented cell divisions associated with mor-

phogenesis at the shoot apex of Arabidopsis thaliana. Development, 131,

4225–4237.

Reinhardt, D., Pesce, E.-R., Stieger, P., Mandel, T., Baltensperger, K., Bennett,

M., Traas, J., Friml, J. and Kuhlemeier, C. (2003) Regulation of phyllotaxis

by polar auxin transport. Nature, 426, 255–260.

Riou-Khamlichi, C., Huntley, R., Jacqmard, A. and Murray, J.A.H. (1999)

Cytokinin activation of Arabidopsis cell division through a D-type cyclin.

Science, 283, 1541–1544.

Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F.X., Jurgens, G. and Laux, T.

(2000) The stem cell population of Arabidopsis shoot meristem is main-

tained by a regulatory loop between the CLAVATA and WUSCHEL genes.

Cell, 100, 635–644.

Umeda, M., Umeda-Hara, C. and Uchimiya, H. (2000) A cyclin-dependent

kinase-activating kinase regulates differentiation of root initial cells in

Arabidopsis. Proc. Natl. Acad. Sci. USA, 97, 13396–13400.

Verkest, A., de O. Manes, C.-L., Vercruysse, S., Maes, S., Van Der Schueren, E.,

Beeckman, T., Genschik, P., Kuiper, M., Inze, D. and De Veylder, L. (2005)

The cyclin-dependent kinase inhibitor KRP2 controls the onset of the

endoreduplication cycle during Arabidopsis leaf development through

inhibition of mitotic CDKA;1 kinase complexes. Plant Cell, 17, 1723–1736.

Vlieghe, K., Boudolf, V., Beemster, G.T.S., Maes, S., Magyar, Z., Atanassova,

A., de Almeida Engler, J., De Groodt, R., Inze, D. and De Veylder, L. (2005)

The DP-E2F-like DEL1 gene controls the endocycle in Arabidopsis thaliana.

Curr. Biol. 15, 59–63.

Weingartner, M., Criqui, M.-C., Meszaros, T., Binarova, P., Schmit, A.-C.,

Helfer, A., Derevier, A., Erhardt, M., Bogre, L. and Genschik, P. (2004)

Expression of a nondegradable cyclin B1 affects plant development and

leads to endomitosis by inhibiting the formation of phragmoplast. Plant

Cell, 16, 643–657.

Williams, M.H. and Green, P.B. (1988) Sequential scanning electron micro-

scopy of a growing plant meristem. Protoplasma, 147, 77–79.

Wyrzykowska, J., Pien, S., Shen, W.H. and Fleming, A.J. (2002) Manipulation

of leaf shape by modulation of cell division. Development, 129, 957–964.

Wyrzykowska, J., Schorderet, M., Pien, S., Gruissem, W. and Fleming, A.J.

(2006) Induction of differentiation in the shoot apical meristem by transient

overexpression of a retinoblastoma-related protein. Plant Physiol. 141,

1338–1348.

Yamaguchi, M., Kato, H., Yoshida, S., Yamamura, S., Uchimiya, H. and

Umeda, M. (2003) Control of in vitro organogenesis by cyclin-dependent

kinase activities in plants. Proc. Natl. Acad. Sci. USA, 100, 8019–8023.

CDK dose-dependent control of meristem activity 37

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 26–37