The Recessive Epigenetic swellmap Mutation Affects the
Expression of Two Step II Splicing Factors Required for the
Transcription of the Cell Proliferation Gene STRUWWELPETER
and for the Timing of Cell Cycle Arrest in the Arabidopsis Leaf
Nicole K. Clay and Timothy Nelson1
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104
Generally, cell division can be uncoupled from multicellular development, but more recent evidence suggests that cell cycle
progression and arrest is coupled to organogenesis and growth. We describe a recessive mutant, swellmap (smp), with
reduced organ size and cell number. This defect is partially compensated for by an increase in final cell size. The mutation
mutationprovedtobeanepigeneticmutation(renamed smpepi)thatdefined asinglelocus,SMP1,butaffected theexpression
of both SMP1 and a second very similar gene, SMP2. Both genes encode CCHC zinc finger proteins with similarities to step II
splicing factors involved in 39 splice site selection. Genetic knockouts demonstrate that the genes are functionally redundant
cycle regulator genes CYCD3;1 and CDC2A but affects expression of the cell proliferation gene STRUWWELPETER (SWP)
whose protein has similarities to Med150/Rgr1-like subunits of the Mediator complex required for transcriptional activation.
also restored in these lines, suggesting a physical interaction among the three proteins and/or genes. We propose that step II
splicing factors and a transcriptional Mediator-like complex are involved in the timing of cell cycle arrest during leaf
Plant cells do not move and are surrounded by a rigid cell wall,
and for this reason, cell division rates and patterns were thought
to be directly responsible for generating new structures during
development. However, despite genetic manipulation of either
cell proliferation or cell expansion, the resulting organs and/or
organisms often attain the normal size (Hemerly et al., 1995;
Smith et al., 1996; Cleary and Smith, 1998; Jones et al., 1998;
Wang et al., 2000; De Veylder et al., 2001; Autran et al., 2002).
They may consist of fewer but larger cells, ormore numerous but
iterative plant development. Similarly, in animals, generally,
changes in cell size can be compensated for by changes in cell
number to maintain the final size of an organism, suggesting
a lack of correlation between cell division and the formation of
complex structures during development (reviewed in Day and
Lawrence, 2000; Weinkove and Leevers, 2000). Furthermore,
forexample, therearenofixed patternsofcelldivision,butrather
a stochastic gradient of cell division arrest from the distal tip to
the base of the leaf (Donnelly et al., 1999).
The accumulating evidence of cell division–independent
mechanisms of development has lead to a long debate on the
actual function or necessity of cell division in plant development
(reviewed in Doonan, 2000). However, more recent evidence has
assigned developmental significance to cell cycle progression
in particular DNA synthesis inhibitors have shown that, unlike
cytokinesis, cell cycle progression is coupled to meristem
growth and patterning (Grandjean et al., 2004). Also, the over-
expression of the D-type cyclin CYCD3;1, unlike most other cell
cycle proteins, inhibited several cell differentiation pathways in
et al., 2003). More specifically, CYCD3;1 overexpression re-
duced the proportion of cells in the G1phase of the cell cycle,
suggesting that cell cycle exit at the G1phase is required for
proper execution of differentiation in the leaf (Dewitte et al.,
2003). Similarly, in mammals, cell cycle exit has been shown
to be required for skeletal myogenesis (Skapek et al., 1995;
Zachsenhaus et al., 1996; Guo and Walsh, 1997) and lens fiber
cell differentiation (Zhang et al., 1998). Most likely, there exists
a subtle and close interplay between growth/organogenesis and
cell cycle progression/exit, and this relationship is defined by
1To whom correspondence should be addressed. E-mail timothy.
firstname.lastname@example.org; fax 203-432-5711.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Timothy Nelson
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 17, 1994–2008, July 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
The relationship among D-type cyclins, G1phase cell cycle
exit, and leaf cell differentiation is intriguing because in all
eukaryotes, the G1phase seems to be a major checkpoint in
the decision to stoportocontinue cell proliferation. Inplants, like
in animals, the initiation of cell division probably involves the
inactivation of the retinoblastoma protein by the appropriate
cyclin-dependent kinases and D-cyclins. This would involve two
transcription factors, E2F and Dpa, which, also in plants, are
involved in the activation of the cell cycle genes and in the
maintenance of meristematic competence (De Veylder et al.,
2002). During cell differentiation, the opposite process is thought
Figure 1. General Plant Phenotype of the smp Mutant.
(A) to (E) Cleared leaves viewed under dark-field illumination. The wild type is in (A), and smp is in (B) to (E). (D) is a close-up of the veins in smp.
Arrowheads point to axialization defects in veins. Arrows point to discontinuities in the venation pattern.
(F) and (G) Leaf shape and size of 2-week-old plants grown on soil. The wild type (left in [F]) and smp ([G] and right in [F]).
(H) Leaf rosettes of 4-week-old wild-type (left) and several smp (right) plants.
(I) Fully-grown wild-type (left) and smp (right) plants.
(J) and (K) Inflorescences of the wild type (J) and smp (K).
(L) and (M) Mature green wild-type (left) and smp (right) siliques were viewed under bright-field illumination (M) or cleared and viewed under dark-field
illumination (L) to look at seed size and number.
(N) and (O) Week-old smp seedlings containing a transgenic copy of SMP gene and exhibiting a partially restored phenotype (C1) or a wild-type
phenotype (C2) were grown on agar media, and the outgrowths of the first two leaves were compared with those of wild-type (top row) and untransgenic
smp (bottom row) seedlings.
(P) Leaf shape and size of 2- to 3-week-old wild-type (left) and C1 (right) plants.
Splicing Factors in Cell Cycle Arrest 1995
in G1, inducing cell differentiation) (Ach et al., 1997). Much more
work needs be done to uncover the players and mechanisms
regulating cell proliferation in organs and/or organisms.
number. This defect is partially compensated for by an increase
in final cell size. The recessive mutation proved to be epigenetic
but affected the expression of both SMP1 and a second very
similar gene, SMP2, both of which encode putative step II splic-
ing factors, which are involved in 39 splice site selection. Genetic
knockouts demonstrate that the genes are functionally redun-
dant and developmentally essential. SMP1 expression is asso-
ciated with regions of cell proliferation in lateral organs, and
overexpression of SMP1 similarly reduced leaf organ size but in-
of the cell proliferation gene STRUWWELPETER (SWP), whose
protein has similarities to Med150/Rgr1-like subunits of the
suggesting that SWP is the direct target of SMP1 and/or SMP2.
smp Mutation Affects Cell Number and Size
A single allele of the smp mutant was identified from a mutant
screen by a reduced amount of leaf venation and a narrow,
pointed venation pattern, both of which were reflected in the leaf
shape and size (Figures 1A to 1H). This phenotype was com-
pletely penetrant and affected all leaf-like organs except for
cotyledons. Besides the reduction in leaf organ size (less than
half that of the wild type for all rosette leaves; Figure 1F), there
were pronounced serrations at the marginal teeth, and the veins
themselves were not properly axialized; that is, the vein cell files
were not aligned from end to end in a smooth, tight bundle and
instead had spaces between them (Figure 1D). Frequently, there
were discontinuities in the midvein at the leaf tip, and the polar
ends of vein cells did not join near the leaf margins (Figures 1B,
1C, and 1E). The mutant phenotype was first evident at the
seedling stage by the retarded outgrowth of the first pair of
leaves and slightly reduced root growth (Figure 1N). Fully grown
smp plants were reduced in stature, had narrower and/or shorter
siliques (seed-bearing pods) that contain fewer but larger seeds
(Figures 1I, 1L, and 1M). Moreover, a few smp floral meristems
produced clustered floral buds (Figure 1K), which were spaced
normally later in development.
Because of their conspicuous and developmentally familiar
smp mutation. Transverse sections through smp leaves revealed
that the leaves were wider and that aside from vascular cells,
which were normally sized, all other leaf cells were fewer in
number and larger in size (Figure 2D). Cell numbers were
from the second pair of leaves to arise from each genotype
Figure 2. Leaf Cell Number and Size Are Affected in the smp Mutant.
(A) and (B) Scanning electron micrographs of the adaxial surface of fully expanded rosette leaves. (A) Wild-type; (B) smp. White bar on the bottom
right ¼ 10 mm.
(C) and (D) Transverse sections through fully expanded rosette leaves. (C) Wild-type; (D) smp. Bar ¼ 50 mm.
1996The Plant Cell
blotting membrane (Bio-Rad, Hercules, CA), UV cross-linked, and baked
for 2 h at 808C. Blots were hybridized to32P-labeled DNA probes
overnight at 428C, washed twice at 558C for 10 min in 13 SSC/1%
SDS, and exposed to Eastman Kodak X-OMAT AR film (Rochester, NY)
Total RNA Isolation and RNA Gel Blot Analysis
Total RNA was isolated from the aerial part of 38-d-old plants with
TRIzol (Gibco BRL, Cleveland, OH) according to the manufacturer’s
instructions. Ten micrograms of total RNA was electrophoresed in 1.2%
formaldehyde-agarose gel, transferred to ZetaProbe GT blotting mem-
brane (Bio-Rad), UV cross-linked, and baked for 2 h at 808C. Blots were
stained with 0.02% methylene blue/0.3 M sodium acetate, pH 5.2, for
3 min, and destained with 20% ethanol for 10 to 15 min, and the resulting
rRNA bands were visualized with a Gel Doc 2000 (Bio-Rad). Blots were
then hybridized to32P-labeled DNA probes overnight at 428C, washed at
exposed to Eastman Kodak X-OMAT AR film at ?708C.
Two micrograms of total RNA was reverse-transcribed with 200 units of
were treated with 10 units of DNase I (Roche, Indianapolis, IN) for 30 min
at 378C, purified on Qiaquick PCR column (Qiagen, Valencia, CA), and
used as template to PCR amplify SMP1 (40 cycles), SMP2 (40 cycles),
SWP (40 cycles), and eIF4A (39 cycles). PCR conditions are as follows:
948C for 15 s, 528C for 15 s, and 728C for 20 s. PCR products (350 to
440 bp) were electrophoresed in 1.5% agarose gel and visualized with a
Gel Doc 2000. Primer sequences for SMP1 are 59-GACCATAGGA-
AGCAAATTGA-39 and 59-AAGATCTATCACACGATGGT-39; for SMP2
are 59-GATCACAGGAAGAAATTAGAA-39 and 59-ACGATCTACCACAT-
GACGGT-39; and for SWP are 59-TCTGCTCTTGTTGGTCGAG-39 and
RNA in Situ Hybridization
SMP1 cDNA (nucleotides 439 to 640 relative to ATG) was PCR amplified
with primerscontaining engineered T7 and T3 RNA polymerase promoter
sites and used as template to generate dioxigenin-labeled sense and
antisense RNA probes. In situ hybridizations and labeling reactions were
performed as described by Jackson (1991) with some modifications,
which were described by Clay and Nelson (2002).
For each genotype, transverse sections through a total of three adult
leaves were used for cell counts per defined area. The defined area
encompassed a region just right or left of the midvein, and the average of
two serial sections was used to calculate the mean cell number per area
for each leaf. This average was then used to calculate the mean cell num-
ber per area for all three leaves.
Sequencing analysis was performed by the HHMI Biopolymer/Keck
Foundation Biotechnology Resource Lab (Yale University, New Haven,
CT). We thank James A. Sullivan for his generous gift of the PJIM19
vectors. This work was supported by National Science Foundation
Grants IBN-0114648 and IBN-0416731 to T.N.
Received March 18, 2005; revised May 9, 2005; accepted May 12, 2005;
published June 3, 2005.
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2008The Plant Cell