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 Arrest1995
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
(vascular cells were excluded), and the mean cell number in the
mutant (95 cells, SD of 8.3; 0.11 mm2) was found to be 45.2% of
that in the wild type (210.2 cells, SD of 5; 0.11 mm2). Similarly,
scanning electron micrographs of smp leaves confirmed that the
that this increase in cell size was not enough to fully restore final
this reduction was partially compensated for by an increase
in final cell size. Interestingly, although the longitudinal vascular
pattern was severely reduced, the transectional vascular pattern
(the arrangement of xylem and phloem) was normal except that
the veins were slightly thicker.
to a contraction of the cell proliferation phase or to a reduction in
the rate of division during organ development, transverse sec-
tions of leaf primordia were taken at various time points during
the cell proliferation phase of leaf development. Meristematic
cells appear as small, densely stained cells in contrast with
primordia had significantly more cells that were vacuolated and
larger sized, suggesting that those cells had prematurely exited
the cell proliferation phase (Figures 3D to 3F) Also, the sections
revealed large intercellular spaces developing within the organ
(Figures 3G to 3I), suggesting that meristematic cells were not
proliferating fast enough to keep up with the expanding epider-
defect led to such a schizogenous-like separation of tissues.
Whatever the cause of these large intercellular spaces, their
development accounts for the disorganization of the inner cell
layers (particularly the palisade layer) seen later on in mature
leaves (Figure 2D).
To see if smp’s effect on cell proliferation extended beyond
leaves, we examined the structure of the inflorescence shoot
apical meristem (SAM), the formation of floral organs at the
just below the youngest silique. Longitudinal sections through
smp SAMs indicated that the organization into layers (at least for
the first two layers) was not disrupted, although the layers
consisted of fewer meristematic cells (Figure 9C). Floral organ
formation appeared normal, albeit with reduced cell numbers
(Figure 9C). The same is true for the stem (data not shown).
SMP1 and SMP2 Encode Putative Step II Splicing Factors
A map-based cloning strategy was used to isolate the gene.
Genetic analysis of F2 progeny indicated that the smp mutant
phenotype segregated as a single recessive locus. The smp
mutation was mapped initially between simple sequence length
polymorphic markers nga111 and AthGENEA on the bottom of
chromosome 1 and then finely mapped to a region on BAC
F1E22 that spanned five predicted genes. Sequencing of all five
genes in the mutant background did not reveal any detectable
encodes a CCHC zinc finger protein that is 22% identical to the
yeast SLU7 protein, a step II splicing factor involved in 39 splice
site selection (Figure 4B; Frank and Guthrie, 1992). Constitutive
expression of a single locus of the wild-type transgene fully
complemented the smp mutation in at least two independent
lines (Figure 1O). Furthermore, expression of this gene by its
Figure 3. Precocious Arrest of Cell Proliferation in Leaf Primordia of the smp Mutant.
Transverse sections through leaf primordia of 7- to 9-d-old wild-type ([A] to [C]) and smp ([D] to [I]) seedling were arranged in developmental order from
(A) to (C) and from (D) to (F). (G) and (H) are serial sections of the same plant. Arrowheads point to large intercellular spaces. Bar ¼ 50 mm.
Splicing Factors in Cell Cycle Arrest 1997
native promoter (810 bp of upstream sequence) gave partial
rescue during the first week of growth and then a full rescue
hereafter (Figures 1N and 1P). The constitutive expression of
a C-terminal translational fusion of the putative SMP gene to
smGFP (SMP:GFP) also fully rescued the mutant in at least two
independent lines. Thus, At1g65660 was identified as the SMP
gene and is very similar to another unlinked gene, At4g37120
(Figure 4B; 90% identity on an amino acid level). Hereafter, we
will refer to At1g65660 and At4g37120 as SMP1 and SMP2,
There was a discrepancy between the sequence of the full-
length SMP1 cDNA in GenBank (accession number BT002797)
and the genomic sequence of the predicted SMP1 coding region
in that the cDNA sequence in BT002797 contained an additional
exon further upstream of the predicted start codon and a single
base pair change in the fourth exon. To confirm that the
sequence in BT002797 is correct, SMP1 cDNA was indepen-
dently isolated from wild-type RNA, and its sequence matched
that in BT002797. Thus, SMP1 coding sequence consists of
10 exons that span a 2.6-kb genomic region (Figure 4A).
The CCHC zinc finger motif (CX2CX4HX4C), also known as
retroviral-type zinc finger, is found in the nucleocapsid pro-
teins of RNA retroviruses (e.g., Moloney murine leukemia virus
[Shinnick et al., 1981], Rous sarcoma virus [Schwartz et al.,
1983], and human immunodeficiency virus [Wain-Hobson
et al., 1985]); in transposable elements (e.g., Drosophila Copia
1988]); and in developmental proteins (e.g., Drosophila Nanos
[Curtis et al., 1997], human CNBP [Rajavashisth et al., 1989],
and Caenorhabditis elegans glH-1[Roussell and Bennett, 1993]).
All of the above have been demonstrated to bind to single-
stranded nucleic acids. Based on the sequence similarity to
the yeast step II splicing factor SLU7 outside the zinc finger
motif, SMP1 and SMP2 most likely bind RNA (Figure 4B). SMP1
Figure 4. SMP1 Encodes a Putative Step II Splicing Factor.
(A) Genomic structure of the SMP1 and SMP2 genes. Black boxes indicate exons. Arrows indicate direction of transcription. SALK T-DNA insertions are
indicated for the smp1 and smp2 insertion alleles.
(B) Alignment of the deduced amino acid sequences of SMP1, AT4G37120 (SMP2), yeast SLU7 (Frank and Guthrie, 1992), and human hSLU7 (Chua and
Reed, 1999). The CCHC domain is underlined. Shared amino acids are shaded.
1998 The Plant Cell