Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology Hong Wang 1, *, Yongming Zhou 1,2 , Susan Gilmer 2 , Steve Whitwill 1 and Larry C. Fowke 2 1 Saskatoon Research Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada, and 2 Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada Received 16 June 2000; revised 29 August 2000; accepted 14 September 2000. *For correspondence (fax +1 306 956 7247; [email protected]). Summary The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division. Keywords: Arabidopsis thaliana, CDK inhibitor, cell division, cell size, plant growth, plant development. Introduction Cell division has long been considered to play a significant role in plant growth and development based on analyses of organ ontogeny and cellular events that affect growth and development in plants and animals. The formation of a plant organ, and hence the plant itself, can be seen as structurally a result of cell multiplication through cell division and subsequent cell expansion. Cell migration, cell slippage and removal of cells through apoptosis do not contribute significantly to growth and morphogenesis in plants, as they do in animals (Meyerowitz, 1997). This characteristic, and the presence of physically rigid cell walls, make cell division the main means to achieve the proper organ size and shape of a plant species. Contrary to this traditional view, an alternative organis- mal view suggests that cell division is merely a ‘marker’ of plant growth, and it does not influence either plant growth or development (Kaplan and Hagemann, 1991). Results from some recent molecular and genetic studies (Hemerly et al., 1995; Smith et al., 1996; Torres-Ruiz and Jurgens, 1994; Traas et al., 1995) appear to lend support to the organismal theory. Therefore there is a paradox, with cell division being viewed as both important and unimportant for plant development (Meyerowitz, 1996). A number of developmental mutants have been identi- fied in which cell division is affected. Mutations in some developmental regulators, such as CLAVATA (CLV) (Clark et al., 1993; Clark et al., 1995) and PASTICCINO (PAS) (Faure et al., 1998), lead to increased cell divisions and often increased growth of certain tissues. On the other hand, loss of function in developmental regulators, such as CURLY LEAF (CLF) (Kim et al., 1998) and AINTEGUMENTA (ANT) (Mizukami and Fischer, 2000), resulted in a reduced number of cell divisions. Furthermore, mutations in genes such as FASS (Torres- Ruiz and Jurgens, 1994), TONNEAU (TON) (Traas et al., 1995) and TANGLED (TAN) (Smith et al., 1996) affected the The Plant Journal (2000) 24(5), 613–623 ª 2000 Blackwell Science Ltd 613
Transcript
Expression of the plant cyclin-dependent kinase inhibitorICK1 affects cell division, plant growth and morphology
Hong Wang1,*, Yongming Zhou1,2, Susan Gilmer2, Steve Whitwill1 and Larry C. Fowke2
1 Saskatoon Research Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada, and2 Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada
Received 16 June 2000; revised 29 August 2000; accepted 14 September 2000.*For correspondence (fax +1 306 956 7247; [email protected]).
Summary
The plant CDK inhibitor ICK1 was identi®ed previously from Arabidopis thaliana with its inhibitory
activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct
from known animal CDK inhibitors. Despite the initial characterization, there is no information on the
functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of
cell division during plant growth and development, transgenic plants were generated expressing ICK1
driven by the cauli¯ower mosaic virus 35S promoter. In comparison to control plants, growth was
signi®cantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type
plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modi®cations
in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that
35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses
showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells
in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These
results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by
ICK1 expression has profound effects on plant growth and development. They also suggest that
alterations of plant organ shape can be achieved by restriction of cell division.
ICK1. We present here the ®rst direct evidence that the
expression of ICK1 results in reduced CDK activity, reduced
cell number, and inhibition of growth. Signi®cantly, many
aspects of plant morphology and development were also
modi®ed in these 35S-ICK1 plants, indicating that cell
division is important not only for plant growth, but also for
plant development.
Results
Increased expression of ICK1 resulted in reduction of
CDK activity in plant cells
The ICK1 expression level increased signi®cantly in trans-
genic 35S-ICK1 Arabidopsis plants, as shown from several
independent analyses. Increased ICK1 expression was
observed in original T1 transformants (data not shown),
and was similarly observed in the progeny T2 plants
(Figure 1a), indicating that the increased level was due to
transgene integration. The increased expression was
detected in all tissues analysed (Figure 1b), as expected
since the 35S promoter activates gene expression in most
tissues. Expression of the CDK gene cdc2a did not
decrease, and perhaps showed a slight increase. For
comparison, a ubiquitin gene UBQ11 (Callis et al., 1993)
remained more consistent.
The p13Suc1-associated Cdc2-like histone H1 kinase
activity was analysed with the same source tissues that
were used in gene expression analyses. Results in Figure 1
show that, coinciding with increased ICK1 expression, the
Cdc2-like kinase activity decreased signi®cantly in com-
parison to control plants. This decrease was observed in
independent 35S-ICK1 plants (Figure 1a) and different
tissues (Figure 1b). As there was no decrease in the level of
expression for positive cell-cycle regulators such as cdc2a,
it is concluded that the decreased Cdc2 kinase activity is
directly due to inhibition by increased ICK1 expression in
these 35S-ICK1 plants.
Growth was signi®cantly inhibited by ICK1 expression
Among 50 independent transformants examined, the
majority displayed signi®cant growth and morphological
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changes. One striking change was the much smaller size of
transformants compared to control plants (Figure 2). The
smaller size of 35S-ICK1 plants persisted through all stages
of plant development, and was re¯ected by the size of
most organs including leaf, stem, root and ¯oral organs. It
was consistent from original T1 to subsequent generations
(e.g. T2 and T3), indicating that it is genetically stable.
There was a range in the extent of phenotypic alterations
among independent transformants. Consistent with this
observation, the level of ICK1 expression varied among
different transformants (data not shown). It is well estab-
lished that there is a wide range of variation in the level of
expression for a given gene introduced into independent
transgenic plants.
The inhibition of growth was initially assessed by fresh
weight of transgenic T2 and control seedlings grown in
petri plates. Results indicated that plants of independent
35S-ICK1 lines had lower fresh weight than wild-type
plants (data not shown). For quanti®cation of growth
inhibition under typical physiological conditions, 35S-ICK1
and control plants were grown in soil. The fresh weight (at
3 weeks) was signi®cantly lower in a number of independ-
ent 35S-ICK1 lines in comparison to plants without 35S-
ICK1 or plants harboring the 35S-GUS construct (Table 1).
Transgenic 35S-ICK1 plants were smaller than control
plants. In some lines, 35S-ICK1 plants were on average less
than one-tenth of the control plants that did not carry 35S-
ICK1. The ®nding that the smaller size (and lower fresh
weight) of 35S-ICK1 plants was consistent for plants grown
under different growth conditions suggests that the inhib-
ition of growth was not due to variations in growth
conditions. These data clearly show that growth was
signi®cantly inhibited by ICK1 overexpression.
Many aspects of plant morphology and development
were altered
The 35S-ICK1 plants showed profound changes in the
morphology of organs such as leaves. Depending on
transgenic lines, there was a range of changes in leaf
shape (Figure 3), in addition to a reduction in size. In some
lines, leaves were signi®cantly serrated (Figure 3b,c). In
wild-type plants, only slight serration occurred in adult
Figure 1. Analyses of RNA expression and p13Suc1-associated histone H1kinase activity.(a) Analysis of different transgenic lines. Floral samples were collectedfrom wild-type plants (WT), plants carrying 35S-GUS (GUS), andindependent 35S-ICK1 lines (lanes 3±9). Total RNA was isolated,separated by electrophoresis and transferred onto nylon membrane. Theblot was probed with 32P-labeled ICK1 (®rst row), cdc2a (second row),and UBQ11 (third row). Ethidium bromide staining of the RNA gel isshown (fourth row). Activity of histone H1 kinase was analysed (®fth row)using kinases puri®ed with p13Suc1 af®nity chromatograph from the sametissue sources. (b) Analysis of different plant tissues. Plants used were T3
plants of wild-type genotype (W) and transgenic genotype (T) that weresegregated from the same T2 line with a single copy of 35S-ICK1, thusallowing more precise comparisons. Pairwise comparison (W versus T)was made for various tissue types. Abbreviations above the data: Sh-2,2-week-old shoots (collected from seedlings grown in petri plates); Rt-2,2-week-old roots (collected from seedlings grown in petri plates); Lf-4, 4-week-old leaves; Fl-4, 4-week-old ¯owers; St-4, 4-week-old stems. As in(a), RNA blot was probed with ICK1 (®rst row), Cdc2a (second row), andUBQ11 (third row). Ethidium bromide staining and CDK-like histone H1kinase activity are also shown (fourth and ®fth rows, respectively). Notethat the low level of ICK1 expression in wild-type plants is not visible onthe blot under the exposure time used to detect ICK1 expression in35S-ICK1 plants.
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leaves. The expression of ICK1 resulted in much more
prominent serration of the leaves, and this characteristic
was observed in almost all leaves in 35S-ICK1 plants with
strong phenotype of growth inhibition. The smaller leaves
and shorter leaf petioles gave 35S-ICK1 plants a more
compact appearance (Figure 1). Root growth of 35S-ICK1
plants was similarly affected (data not shown).
Striking changes also occurred in ¯oral organs. In wild-
type plants, the fully opened ¯owers were spread and the
top of ¯owers often exceeded the in¯orescence apex. In
35S-ICK1 plants, ¯owers stayed closer and usually were at
the same level or below the in¯orescence apex (Figure 3d
versus Figure 3e). On the in¯orescence of 35S-ICK1 plants,
the reduced distance between ¯owers was probably due to
reduced growth of the in¯orescence stem and ¯ower
pedicels. Thus the ¯owers appeared as a compact cluster
when viewed from the top (Figure 3f versus Figure 3g).
Changes in size and morphology were also evident in
individual ¯owers. The ¯owers of 35S-ICK1 plants had
smaller or shorter sepals, petals and stamens (Figure 3h,j
versus 3i,k). Mature petals of normal Arabidopsis ¯owers
bent at the halfway point horizontally above sepals, while
those of 35S-ICK1 plants point straight upwards. Petals of
35S-ICK1 plants were also narrower with serration along
the top edge (Figure 3i,k). These changes were so
profound that transgenic 35S-ICK1 plants bore little
resemblance to the wild-type Arabidopsis plants.
Most of the above changes could theoretically be
attributed to reduction of cell division and thus organ
growth. However, there were also developmental changes
beyond this simple explanation. (1) 35S-ICK1 plants
¯owered earlier and had signi®cantly fewer leaves than
control plants at ¯owering time (Table 1); precocious
¯owering of 35S-ICK1 plants was similarly observed
Figure 2. Growth and morphology oftransgenic 35S-ICK1 Arabidopsis plants.(a) Plants at 3 weeks of wild type (top leftpot), 35S-GUS (bottom left pot), and fourindependent 35S-ICK1 lines (other fourpots). (b) Comparison of a representativewild-type Arabidopsis plant (left) and a 35S-ICK1 plant (right) at 3.5 weeks. The 35S-ICK1plant was smaller and showed majormorphological alteration (inset).
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Table 1. Growth and development of transgenic 35S-ICK1 Arabidopsis plants. 35S-ICK1 and control plants were grown in growthchambers. For growth evaluation, shoot (above-ground tissues) FW of 21-day-old plants was determined. For development evaluation,¯owering time and leaf number (rosette plus in¯orescence leaves on the primary axis) were obtained
aSD, standard deviation.**Signi®cance (t-test) from the Wt control plants at P < 0.001;*Signi®cance at P < 0.05 level.
Figure 3. Modi®cations of organ morphology in transgenic 35S-ICK1 plants.(a±c) Changes in leaf size and morphology. Leaves were placed in order from oldest (left) to youngest (right).(a) Leaves of a wild-type (Wt) Arabidopsis plant (top row), a 35S-GUS plant (bottom row), and four independent 35S-ICK1 plants (middle four rows). (b)Enlargement of leaves of 35S-ICK1 plants from (a) showing changes in shape and serration. (c) Enlargement of leaves from the ®rst and second rows in(b). (d±k). Changes in size and morphology of ¯oral organs. (d,e) Side view of in¯orescence of Wt (d) and 35S-ICK1 (e) plants. (f,g) Top view ofin¯orescence of Wt (f) and 35S-ICK1 (g) plants. (h,i) Single ¯ower of Wt (h) and 35S-ICK1 (i) plants. Note smaller ¯owers of 35S-ICK1 plant with alteredorgans, especially sepals and petals. (j,k) Top view of ¯owers of Wt (j) and 35S-ICK1 (k) plants showing narrower serrated petals on the 35S-ICK1 plant.
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inance which was evident in two ways: reduced promin-
ence of the primary in¯orescence, and a large number of
lateral branches.
Cell division, not cell expansion, was inhibited
Two factors could contribute to the smaller organ and
plant size observed. The 35S-ICK1 plants might have fewer
cells due to inhibition of cell division or smaller cells due to
inhibition of cell growth. To address these questions, the
structure and cell size of 35S-ICK1 and control plants were
examined by SEM and light microscopy. It was consist-
ently observed that the cells of 35S-ICK1 plants in all
tissues examined (leaves, hypocotyl, root and ¯ower
organs) were on average slightly larger than the corres-
ponding cells in control plants (Figure 4).
An initial quantitative analysis of cell size was made
using pavement cells of the ®rst pair of true leaves on 15-
day-old plants growing in petri plates. Data showed that
cells of 35S-ICK1 plants were clearly larger than control
plants (data not shown). To better quantify the cell size
difference, fully expanded leaves (5th to 8th leaves) of 30-
day-old plants grown in soil were used for determining
leaf and epidermal cell size. The average leaf size of the
35S-ICK1 plants (lines) used was between 3.4 and 57.1% of
the leaf size of the wild-type plants (data not shown).
Pavement cells on the adaxial surface in similar areas of
leaves were measured. Cells in leaves of different 35S-ICK1
lines were 1.7±2.7 times larger than the cells of control
plants (Figure 5). The cell size was similar for the four
different leaves of each type of plant surveyed, indicating
that cells were at their mature size in these leaves. In
addition, there appeared to be a slightly wider variation in
cell size in 35S-ICK1 plants (Figure 4 and Figure 5).
From these analyses, two conclusions can be drawn. (1)
There was no inhibition of cell expansion, but there was a
strong inhibition of cell division due to ICK1 expression. (2)
There was some compensation for reduced cell number in
35S-ICK1 plants by increasing the size of cells. However, the
increase in cell size could not counter the more dominant
effect of growth inhibition by reduced cell number in 35S-
ICK1 plants. The smaller size of 35S-ICK1 plants was thus
entirely due to the reduction in cell number.
Discussion
ICK1 is able to inhibit CDK activity in plants
Mammalian CDK inhibitors play signi®cant roles in cell-
cycle regulation and animal development (Harper and
Elledge, 1996; Sherr and Roberts, 1995). Recent results
illustrate that their molecular functions are not limited to
simply inhibiting CDK activity (Sherr and Roberts, 1999).
The fact that Arabidopsis ICK1 can interact with a plant
CDK and inhibit the kinase activity, as observed with
mammalian inhibitors, suggests a similar role for ICK1
in vivo. On the other hand, ICK1 shares only limited
sequence similarity with animal CDK inhibitors and it has
distinct structural and functional properties (Wang et al.,
1997; Wang et al., 1998). The signi®cant divergence of ICK1
from animal counterparts, which is not known for other
major plant cell-cycle regulators, makes it dif®cult to
extrapolate results from animal inhibitors to the role of
ICK1 in cell division, growth and differentiation of plants. It
is therefore important to verify that the inhibitory activity
of ICK1 characterized in vitro is retained in vivo, and to
determine the possible effects on cell division.
Figure 4. Structural characterization of cells.Structural characterization of cells in wild type (Wt) (a,c,e,g) andtransgenic (Tr) 35S-ICK1 plants (b,d,f,h) by SEM (a,b,e,f,g,h) and lightmicroscopy (c,d). Note the larger size of cells in transgenic plants.(a,b) Fully expanded leaves from 30-day-old Wt (a) and Tr (b) plants(adaxial surface); (c,d) Leaf transverse sections of 15-day-old Wt (c) andTr (d) plants; (e,f) Styles of 30-day-old Wt (e) and Tr (f) plants(arrowheads indicate papillae on the stigma); (g,h) hypocotyls of 15-day-old Wt (g) and Tr (h) plants. All scale bars = 100 mm.
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The present study for the ®rst time shows that expres-
sion of the plant CDK inhibitor gene ICK1 is suf®cient to
inhibit Cdc2 kinase activity in planta. Such results were
obtained from different analyses using independent
transformants, plants of different generations, and differ-
ent tissues (Figure 1; data not shown), suggesting that CDK
inhibitors such as ICK1 are critical factors in regulating the
activity of plant CDKs. There was no corresponding
decrease in gene expression of positive cell-cycle regula-
tors such as Cdc2a with which ICK1 interacts (Wang et al.,
1998). Therefore the observed reduction of CDK was
probably due to direct inhibition of CDK by increased
levels of ICK1. The present results, together with previous
studies (Wang et al., 1997; Wang et al., 1998), clearly
establish the role of ICK1 as a plant CDK inhibitor.
Considering that the 35S promoter activates strong gene
expression in most plant tissues, it is surprising that most
transformants were able to set seeds, although in severe
cases the plants could not set seeds, or seedlings could not
develop normally. One explanation for the milder effect on
seed development is relatively weak gene expression
conferred by the 35S promoter during early embryogen-
esis and perhaps gametogenesis (Custers et al., 1999).
Despite high levels of ICK1 expression in the tissues
examined, different organs appeared to function normally.
These observations suggest that differentiated cells can
tolerate ICK1 much above the expression levels that are
normally present in these tissues.
Cell division, cell growth and plant growth
As plant organ growth is determined by the number of cell
divisions and the size of cells (Meyerowitz, 1997), it seems
self-evident that cell division and cell growth should be co-
ordinated during plant organ growth. However, the rela-
tionship between cell division and cell growth in the
context of plant growth is far from clear. Recent research
®ndings have provided different and often contradictory
views regarding the role of cell division in plant growth, a
long-standing and fundamental question (Hemerly et al.,
1999; Jacobs, 1997; Kaplan and Hagemann, 1991;
Meyerowitz, 1996).
The relationship between cell division and plant growth
can be examined by altering the number of cell divisions.
Prior to identi®cation of cell-cycle genes, external means
were sought to arrest the cell cycle. When cell division was
inhibited by radiation in wheat seedlings, the outgrowths
for leaf primordia and lateral roots were still initiated
without mitosis (Foard, 1971; Foard et al., 1965), indicating
that cell division was not the determining factor for cell
growth and organ formation.
The identi®cation of cell-cycle regulators provides a
convenient and more precise means to address this
question. Hemerly et al. (1995) expressed a dominant
negative mutant of Arabidopsis cdc2a in transgenic
tobacco plants, resulting in decreased cell number.
However, the smaller number of cells was compensated
by larger cell size with no signi®cant change in plant
growth, indicating that inhibition of cell division did not
affect plant growth. Cell division in tobacco plants could,
on the other hand, be stimulated by yeast Cdc25, a mitotic
inducer that functions to phosphorylate Cdc2 (Bell et al.,
1993). Plants or tissues from either constitutive or
inducible expression had smaller cells (Bell et al., 1993;
McKibbin et al., 1998). When a mitotic cyclin gene,
Figure 5. Cell size in control and transgenic35S-ICK1 plants.Fully expanded leaves 5, 6, 7 and 8 of 30-day-old plants were used. Pavement cellson the adaxial side within a given area atthe middle third portion of each leaf weremeasured individually. Average cell sizewith standard error in mm2 is presented foreach leaf number of the same type ofplants. The average sample size for eachdatum presented is n = 136 cells. Theoverall average cell size for all four leaves(number in brackets) is listed under theplant type. Plant type: Wt, wild type; GUS,35S-GUS; the other four are different 35S-ICK1 lines.
CDK inhibitor ICK1, cell division and plant development 619
ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 613±623
Arabidopsis CycB1 (Cyc1At) was ectopically expressed
using the Arabidopsis cdc2a promoter, root growth was
enhanced (especially when exogenous auxin was applied)
as a result of increased cell division in transgenic plants,
but there was no change in cell size (Doerner et al., 1996).
During submission of this paper, Cockcroft et al. (2000)
reported that expression of Arabidopsis CycD2 resulted in
a faster cell division cycle and increased plant growth.
Results from the present study show for the ®rst time
that expression of a plant cell-cycle regulator results in
modi®cations of all three processes ± cell division, cell
growth, and plant growth. Concurrent with the reduction
in cell number, there is a general increase in cell size in
35S-ICK1 plants. These results indicate a certain degree of
uncoupling between cell size (at least by the measurement
of ®nal cell size) and cell division. Alternatively, if we
consider that there exists a normal balance (co-ordination)
between cell size and cell division in wild-type plants, this
balance must have been altered and a new balance is
reached in transgenic plants due to ICK1 overexpression.
On the other hand, overexpression of cyclins had no
signi®cant effect on cell size (Cockcroft et al., 2000; Doerner
et al., 1996).
The difference in the effect on plant growth between this
study and the study of transgenic tobacco plants express-
ing the dominant negative cdc2a mutant (Hemerly et al.,
1995) is interesting, considering that CDK activities and cell
division were inhibited in both cases. Different plant
systems may be one reason for this discrepancy.
Alternatively, ICK1 might have stronger (and broader)
inhibitory activities and, as a result, cell division was more
signi®cantly affected. The changes in cell number
observed in 35S-ICK1 plants were probably greater than
that observed in plants obtained by Hemerly et al. (1995). It
is conceivable that there is a limit on the capacity of a plant
to use cell size increase to compensate for growth inhib-
ition caused by reduced cell number. Beyond that limit, a
reduced cell number inevitably leads to growth inhibition.
Cell division can also be altered by mutations or
transgenic expression of other genes involved in develop-
ment of plant meristems. Mutations in CLV1 and CLV3
resulted in increased cell division and enlarged ¯oral
meristems (Clark et al., 1993; Clark et al., 1995), and muta-
tion of CAF converted ¯oral meristems from the determi-
nate state to the indeterminate state (Jacobsen et al.,
1999). The functions of CLV and CAF appear to suppress
(directly or indirectly) cell division in ¯oral meristems to
maintain the normal size and state in wild-type plants. On
the other hand, the loss-of-function of ANT, a transcription
factor belonging to the AP2 family, resulted in decreased
cell number and reduced size of organs, while over-
expression of ANT increased organ growth by increasing
cell number (Mizukami and Fischer, 2000). It is clear from
these results that changes in cell number resulted in
growth modi®cations. However, the question as to the role
of cell division is not addressed speci®cally, as these
regulators probably affect both cell division and organ
growth (Fletcher et al., 1999; Mizukami and Fischer, 2000).
There is a large range of variation in organ and body
sizes among different and often closely related plant
species. The difference in cell number often accounts for
most of the difference in size. However, the underlying
mechanisms for one plant to have intrinsically more cell
divisions than another have yet to be understood. The
present experimental results point to one possibility for
size variation among different plants. Increased expression
of a CDK inhibitor such as ICK1 could produce a plant of
much smaller size, thus creating a signi®cant size differ-
ence between closely related plants.
In a developmental context, it is not known how the cell
number is reduced in 35S-ICK1 plants. There are two
possible mechanisms. First, as a result of ICK1 over-
expression, fewer cells are able to enter mitosis in the
meristems. Second, the cells in meristems of transgenic
plants may take longer to divide (due to their slower cycle).
These two mechanisms need not be mutually exclusive.
Thus it is possible that there are fewer cells making new
cells in the meristems, and they take longer to divide.
These aspects await further investigation.
Cell division and plant morphogenesis
Several recent studies suggest that cell division may not
be important for plant development. The tobacco plants
expressing a dominant negative mutant of Arabidopsis
cdc2a had reduced cell numbers, but showed no change in
plant development (Hemerly et al., 1995). Plant develop-
ment was also normal in Arabidopsis plants overexpres-
sing a mitotic cyclin, despite increased root growth
(Doerner et al., 1996). Several other studies describing
mutants with perturbed cell division patterns but no
obvious developmental changes further support the view
that cell division, either number or position, is unimportant
for plant organ development (see Introduction).
One of the ®rst indications that cell-cycle regulators can
directly impact plant development was obtained recently
in transgenic Arabidopsis plants overexpressing a G1
cyclin, Arabidopsis CycD3 (Riou-Khamlichi et al., 1999).
Effects on plant development, such as leaf shape and
¯owering time, were reported, but the detail and extent of
these effects were not described.
Results of the present study clearly show that expres-
sion of a plant CDK inhibitor has strong effects on plant
morphogenesis and development in transgenic plants. It
is a surprise that one cell-cycle gene is capable of
causing such diverse and dramatic morphological
changes. To a large extent, morphology of most organs
in 35S-ICK1 plants was affected. In general, signi®cant
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changes in morphology coincided with a clear reduction
in growth (for example, organ size). The morphological
changes were not merely miniaturization of original
shapes. The 35S-ICK1 plants appeared dramatically
different from control plants (Figure 2). The observed
changes in organ shape, such as serration in leaves and
petals, and length/width ratio, implies a role for cell
division in morphogenesis.
The formation of organ shape can be considered as
selected cell division and subsequent cell expansion in
a de®ned location. This view implies that the underlying
force for organ formation and shape involves factors
promoting cell division. Such a view is at least partially
validated by the enhanced growth from transgenic
expression of a cyclin in Arabidopsis (Doerner et al.,
1996). The present results, however, identify another
possible force at work. The reduction of cell number
due to expression of ICK1 resulted not only in the
alteration of organ growth (changes in size), but also in
alteration of organ shape. If the reduction of growth
had been strictly uniform, leaf shape would have
remained the same, although smaller in size. In the
absence of cell migration and cell removal we cannot
offer an alternative explanation, but conclude that
inhibition of cell division is the cause for the genesis
of strong serration in these leaves and nascent serration
in petals. The signi®cant serration in leaves may have
been due to differences in the extent of cell division
inhibition along the leaf margin. The difference in
inhibiting cell division may also explain the change in
leaf length/width ratio. It is tempting to postulate that
inhibition of cell division by a CDK inhibitor can be
used as one means for a plant to de®ne its organ
shape.
The initiation and development of lateral organs such
as leaves involve the regulation of the knotted-1-like
(knox) genes (Smith et al., 1992). Overexpression of the
knotted-1 (kn1) from maize and a kn1-like gene from
Arabidopsis resulted in strong alterations of leaf morph-
ology in Arabidopsis (Chuck et al., 1996; Lincoln et al.,
1994). Analyses show that knox genes are expressed in
shoot apical meristems and in axillary meristems, but
not in leaves. It is suggested that kn1 may function to
maintain meristematic cells in an indeterminate state
and its downregulation is important for the determin-
ation of a lateral organ (Smith et al., 1992). Recent
results show that knox genes are downregulated by a
myb-domain protein Rough Sheath2 (RS2)
(Timmermans et al., 1999; Tsiantis et al., 1999). These
and other results indicate a tight link between the
development of lateral organs, such as leaves, and the
maintenance of shoot meristems (Christensen and
Weigel, 1998). However, it is not clear how cell division
might be regulated by knox gene products.
Biotechnology implications
The ®nding that plant growth and morphology can be
changed by modulating cell division has many practical
implications for agriculture, horticulture and forestry. The
expression of CDK inhibitor genes such as ICK1 may be
precisely targeted in order to modify a particular organ. As
cell-division mechanisms are well conserved among
higher plants, including crop species, the ability to control
cell division may have wide applications for modifying
different plants.
In summary, no information has been available to date
on the relationship of plant CDK inhibitors and cell division
in vivo. The present results show that the size of a whole
plant can be greatly reduced solely by ICK1 expression. In
35S-ICK1 plants the size of most organs was reduced; this
reduction was not due to a reduction in cell size, as cells in
all 35S-ICK1 plants were larger than cells of control plants
(Figures 4 and 5). The present results demonstrate that
expression of ICK1 is able to inhibit cell division and
reduce the number of cells in transgenic plants. Therefore,
for the ®rst time, the following cause±effect relationship
has been demonstrated in plants: expression of a CDK
inhibitor ® reduction of CDK activity ® reduction of cell
division ® inhibition of plant growth.
Experimental procedures
Plant materials
Arabidopsis thaliana `Columbia' was used. Unless otherwisestated, plants were grown in pots in growth chambers (20°Cconstant, 16/8 h day/night photoperiod).
Construct preparation and plant transformation
ICK1 cDNA (Wang et al., 1997) was cloned and linked transcrip-tionally behind the 35S promoter in pBI121 (Clontech, Palo Alto,CA, USA). The resulting plasmid and control pBI121 wereintroduced in Agrobacterium tumefaciens strain GV3101.Transformation of Arabidopsis was performed based on thein®ltration method (Bechtold et al., 1993), except the surfactantSilwet-40 (0.01±0.05%) (Clough and Bent, 1998) was added to the®nal suspension for in®ltration. Seeds (T1) were harvested andselected on 0.5 MS basal medium (Sigma-Aldrich Canada,Oakville, Ontario, Canada) containing 50 mg l±1 kanamycin and300 mg l±1 Timentin. Kanamycin-resistant plants were transferredto soil in 4 inch pots and grown in growth chambers. A largenumber of transformants were obtained with both 35S-ICK1 andcontrol 35S-GUS constructs. Transformants with single inserts(one or two copies determined by Southern blotting) were usedfor further detailed characterization.
Plant growth and development
Seeds of T2 transgenic 35S-ICK1 lines were planted in soil. Thesegregating wild-type plants from the heterozygous lines couldbe easily distinguished from 35S-ICK1 plants as the latter had
CDK inhibitor ICK1, cell division and plant development 621
ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 613±623
altered morphology. They were further veri®ed by PCR ampli®-cation of inserted marker gene. At the 3 week stage, the above-ground portion (including cotyledons, leaves and shoot) wasremoved and the fresh weight determined. Wild-type plants andtransgenic plants carrying the 35S-GUS construct were used ascontrols. Seedlings were also grown in petri plates using non-kanamycin plates for the wild-type plants and kanamycin platesfor transgenic 35S-GUS and 35S-ICK1 lines. The fresh weight ofwhole seedlings was determined after 13 days' growth. For plantdevelopment, the number of days to ¯ower and leaf number(rosette plus in¯orescence leaves on the primary axis) wereobtained.
Northern and kinase analyses
Southern hybridization for gene integration and copy number,Northern hybridization for mRNA expression, and assay forp13Suc1-associated Cdc2-like kinase activity were performed asdescribed (Wang et al., 1998).
Structural characterization by SEM and light microscopy
Plant tissue samples were taken from 35S-ICK1 and control plantsgrown under the same conditions. For SEM of tissues such asleaves and hypocotyls, the epoxy replica method (Fowke et al.,1994; Green and Linstead, 1990) was used. The impressionmoulds were prepared using dental impression material (GCExa¯ex vinyl silicone, GC America, Alsip, IL, USA). The mouldswere then used to make replicas of original samples with epoxycement. Some tissues (e.g. ¯owers) were ®xed and critical pointdried as described previously (Fowke et al., 1994). The epoxyreplicas and critical point-dried specimens were mounted on SEMstubs, coated with gold in an Edwards sputter coater (ModelS150B) and then examined in a Philips 505 scanning electronmicroscope. For light microscopy of methacrylate sections,samples (leaf tissues) were ®xed, dehydrated and embedded inmethacrylate according to the method of Baskin et al. (1992).Sections 1.5±2.5 mm were stained with toluidine blue beforeexamination.
Determination of leaf size and cell size
Leaves (numbers 5±8) of 30-day-old plants grown in a growthchamber were used, as leaves of younger plants (e.g. 15-day-old)may continue to expand. At this stage both the 35S-ICK1 andcontrol plants were ¯owering. Leaves were excised and scanned®rst on an Epson ¯at-bed scanner (model 1200S) to determine theleaf size. Epoxy replicas of the adaxial surface were prepared andSEM photographs taken. Two non-marginal sectors from thewidest part of each leaf were photographed and »20 pavementcells of each photographed sector were chosen at random. Guardcells were excluded from measurements. The surface area of eachcell was determined by IMAGEJ (http:/rsb.info.nih.gov/ij/docs/intro.html). Results from the same transformed lines were pooledfor each leaf number. To determine the leaf size, the margins ofscanned individual leaf images were traced and areas weredetermined using IMAGEJ.
Acknowledgements
L.C.F. gratefully acknowledges ®nancial support from the NaturalSciences and Engineering Research Council of Canada and the
University of Saskatchewan. We thank Ralph Underwood forphotographic assistance with Figures 2 and 3. This is SRCpublication number 1385.
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