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First published online 11 February 2009
doi: 10.1242/dev.028613
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1 National Institute for Basic Biology, Nishigo-naka 38, Myodaiji-cho, Okazaki,
Aichi, 444-8585 Japan.
2 Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku,
Tokyo, 113-0033 Japan.
Author for correspondence (e-mail:
ghori{at}rikkyo.ac.jp)
Accepted 15 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cell proliferation and expansion, Compensation syndrome, Heteroblasty, Leaf development, miR156
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In addition to these external cues, internal
signals that arise at certain developmental stages have a role in the
regulation of leaf size and shape. In most species, some leaf traits change as
a plant passes through developmental phases, such as embryogenic, juvenile
vegetative, adult vegetative and reproductive phases
(Tsukaya and Uchimiya, 1997;
Kerstetter and Poethig, 1998;
Tsukaya et al., 2000). This
phenomenon is called phase change, or heteroblasty. In Arabidopsis
thaliana (Arabidopsis, hereafter), lamina size, leaf
length/width ratio, petiole length, serration number, and production of
abaxial trichomes change in relation to heteroblasty. Cell number and cell
size in leaves might also change with heteroblasty, as an increase in cell
number and a decrease in cell size in higher node leaves is observed in
various species (Ashby, 1948;
Granier and Tardieu, 1998;
Cnops et al., 2004;
Cookson et al., 2007).
However, reduced water availability caused by water deprivation to leaves at
lower nodes, or diffusive inhibitory signals from lower leaves were previously
thought to account for the reduction in cell size in leaves on higher nodes
(Ashby, 1948); whether this
phenomenon is physiological or genetically controlled remains to be
answered.
Recent molecular genetic studies indicate that miRNAs and trans-acting
siRNAs regulate heteroblasty. miR172 in maize promotes the
juvenile-to-adult phase change by repressing its target, Glossy15, an
APETALA2-like gene required for juvenile leaf traits
(Lauter et al., 2005). By
contrast, miR156 inhibits this phase change by repressing its target,
SBP-box-containing genes, when overexpressed in a Corngrass1 mutant
(Chuck et al., 2007). Also, in
Arabidopsis, constitutive overexpression of miR156 severely
inhibits the progression of heteroblasty. Moreover, overexpression of the
miR156-insensitive form of the Arabidopsis SBP-box gene
SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3),
SPL4 or SPL5 accelerates the juvenile-to-adult phase change
(Wu and Poethig, 2006). In
addition, involvement of tasiR-ARF and its target ETTIN
(ETT)/AUXIN RESPONSE FACTOR 3 (ARF3) and
ARF4 in the regulation of heteroblastic traits, such as abaxial
trichome production, is also proposed
(Peragine et al., 2004;
Hunter et al., 2006). However,
how these genes regulate leaf size and/or shape is still unknown.
Leaf size and shape regulation is dependent on the control of cell
proliferation and cell expansion (Tsukaya,
2006), and spatial and temporal regulation of these two processes
is crucial for leaf development. In eudicot species, cell proliferation first
occurs throughout the leaf primordium and is gradually restricted to the
proximal part (Donnelly et al.,
1999). Arrest of cell cycle and subsequent post-mitotic cell
expansion occur from the distal to the proximal part of a young leaf. The
regulatory mechanisms of cell proliferation and cell expansion have been
investigated in many studies. An interesting topic is how these two cellular
processes are coordinated in the context of leaf development. The first
example of such coordination was in barley leaves irradiated with 
rays, which show no cell division during development but develop significantly
larger cells (Haber, 1962).
Recent studies using various mutant or transgenic plants have provided several
lines of evidence for the presence of organ-level coordination of cell
proliferation and cell expansion (Tsukaya,
2002; Beemster et al.,
2003; Tsukaya,
2003; Horiguchi et al.,
2006a; Ferjani et al.,
2007). When cell number is reduced by a mutation in a
cell-proliferation-promoting gene, the size of individual cells often
increases. This phenomenon, called compensation syndrome, is observed in
several Arabidopsis mutants. However, overexpression of such a gene
[for example, ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING
FACTOR 1 (GIF1) or AINTEGUMENTA (ANT)] results
in an increase in cell number, but cell size does not change
(Mizukami and Fischer, 2000;
Kim and Kende, 2004;
Horiguchi et al., 2005). These
observations indicate that cell number and cell size in a leaf might not be
determined by a simple trade-off between cell proliferation and cell
expansion. The precise mechanism coordinating these two processes is
unknown.
To further investigate the regulation of cell proliferation and cell
expansion, we isolated a number of mutants with altered cell number, size or
both (Horiguchi et al., 2006a;
Horiguchi et al., 2006b;
Ferjani et al., 2007;
Fujikura et al., 2007). Here,
we report a new class of mutants named more and smaller cells
(msc) that have increased cell number and decreased cell size, the
opposite phenotype to compensation syndrome. These mutants also show
accelerated heteroblasty. Analysis of leaves at various nodes demonstrated
that adult leaves had an increased cell number and decreased cell size
compared with those in juvenile leaves. This indicates that heteroblasty plays
an important role in the regulation of cell number and size. Cloning of
MSC genes and subsequent molecular and genetic analyses demonstrated
that miR156 and its target SPL genes are involved in the
regulation of heteroblastic change of cell number and size, whereas another
group of heteroblasty-related genes (RDR6, SGS3, ZIP, ARF3 and
ARF4) might not be involved.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Before analyses, all of the mutants were backcrossed to
the wild-type Col at least three times. We also used an3-4
(Horiguchi et al., 2005),
ant-1 (Mizukami and Fischer,
2000), fugu1, fugu2-1, fugu3-D, fugu4-D, fugu5-1
(Ferjani et al., 2007),
psd-1, psd-6 (Hunter et al.,
2003a; Li and Chen,
2003), rdr6-11, sgs3-11
(Peragine et al., 2004),
zip-1 (Hunter et al.,
2003b) and axr1-3
(Estelle and Somerville, 1987)
mutants and the T-DNA insertion mutants arf3-2 (CS24604)
(Okushima et al., 2005),
arf4-2 (SALK_070506C) (Alonso et
al., 2003) and sqn-5 (SALK_033511)
(Prunet et al., 2008). The
sqn-1 (Berardini et al.,
2001) mutant and the transgenic plants that overexpress
miR156, SPL3m, SPL3
, SPL4 or
SPL5 were gifts from G. Wu and R. S. Poethig
(Wu and Poethig, 2006). All of
these mutants and transgenic plants were in the Col background. Plants were
grown on rock wool at 22°C under a 16 hour light/8 hour dark photoperiod
at a light intensity of approximately 40 µmol m–2
s–1.
Phenotypic analyses
For histological analysis of cells, first, third or fifth leaves of
30-day-old seedlings were collected. Collected leaves were fixed in
formalin/acetic acid/alcohol and cleared using chloral solution, as described
by Tsuge et al. (Tsuge et al.,
1996). Whole leaves and cells were observed as previously
described (Fujikura et al.,
2007). Because expansion of first, third and fifth leaves was
already completed at that stage, cell area was uniform in all parts of the
leaf (see Fig. S1 in the supplementary material). Areas of 20-30 cells were
measured for each leaf and averaged. Mean ± s.d. of average cell areas
from six individual plants are indicated in the figures. To calculate the
total cell number in leaves, we measured cell density of observed images of
cells, and multiplied the cell density by the area of the same leaf. For
analysis of abaxial trichomes, plants were grown until the first few flowers
opened.
Genetic mapping
The msc loci were genetically mapped using various genetic markers
according to the sequence information available in The Arabidopsis
Information Resource (TAIR) database
(http://www.arabidopsis.org/).
RNA analyses
Total RNA was extracted from leaves using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA) following the manufacturer's instructions. For quantitative
RT-PCR, total RNA was treated with amplification grade DNase I (Invitrogen)
before reverse transcription. Reverse transcription was performed with the
oligo(dT)20 primer using the SuperScript III First-Strand Synthesis
System for RT-PCR (Invitrogen). Quantitative PCR was performed with
Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA,
USA). The UBIQUITIN10 (UBQ10) gene was used as an internal
control. The primers used are listed in
Table 1. At least three
biologically independent samples were analyzed and triplicated reactions were
performed with each sample. Reactions performed without reverse transcription
did not result in any amplifications (data not shown). For 5'-RLM-RACE,
total RNA was extracted from the first and second rosette leaves of 14-day-old
plants and 5'-RLM-RACE was carried out using the GeneRacer Kit
(Invitrogen). For small RNA blots, 5 µg of total RNA was separated using 8
M urea-denaturing polyacrylamide gels and electrically transferred to a
Hybond-N+ membrane (GE Healthcare, Buckinghamshire, UK). Blots were
hybridized with a [-32P]ATP labeled
miR156-complementary oligonucleotide probe
5'-GTGCTCACTCTCTTCTGTCA-3' at 40°C in ULTRAhyb-oligo
hybridization buffer (Ambion, Austin, TX, USA). A U6
snRNA-complementary oligonucleotide probe
5'-TCATCCTTGCGCAGGGGCCA-3' was used as a loading control.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Horiguchi et al., 2006b), we
selected three mutants that had increased cell number and decreased cell size
(Fig. 1C,D). We named these
more and smaller cells (msc) mutants. All msc
mutants had larger first leaves than the wild type
(Fig. 1B). msc1 had no
obvious phenotype other than a larger leaf area and a slightly earlier
flowering phenotype (Fig. 2E,
reduced total rosette leaf number). msc2 showed a delay in the
initiation of the first pair of rosette leaves, as if the shoot apical
meristem were inactive for a few days (Fig.
1E). msc3 had a delay in leaf emergence and expansion,
but the final leaf size was larger than in the wild type
(Fig. 1F). msc2 and
msc3 were inherited as recessive mutations and msc1 was
semi-dominant, and was thus designated as msc1-D.
msc2 and msc3 are allelic to paused (psd) and squint (sqn) mutants, respectively
We cloned MSC1-MSC3 genes using a map-based approach.
msc2 and msc3 were found to be new alleles of previously
reported mutants showing accelerated heteroblasty. These findings suggest that
msc1-D might have a genetic lesion in the heteroblasty-related gene;
therefore, we first described MSC2 and MSC3 genes and then
considered MSC1.
The mutation in MSC2 was mapped to the PSD gene
(At1g72560) (Hunter et al.,
2003a; Li and Chen,
2003). msc2 has an 870 bp deletion spanning the seventh
intron to the eleventh exon of PSD and an insertion of T-DNA in this
region (Fig. 2A). msc2
had a very similar phenotype to the psd mutant [meristem pause
(Fig. 1E), pointed leaves (data
not shown) and accelerated heteroblasty
(Fig. 2E, see below)].
Therefore, we tested for the cell number and size of psd-1 and
psd-6 alleles and found that they had increased cell number and
decreased cell size, as found in msc2 (see Fig. S2 in the
supplementary material). F1 plants from a cross between msc2 and
psd-6 failed to complement their phenotypes (see Fig. S2 in the
supplementary material). We concluded that msc2 was a new allele of
psd (psd-16).
The mutation in MSC3 was mapped to the SQN gene
(At2g15790) (Berardini et al.,
2001). msc3 had a 127 bp deletion from the seventh intron
through the eighth exon of SQN
(Fig. 2A). msc3 and
sqn mutants also shared other phenotypes [leaf size and shape
(Fig. 1F), disturbed flower
phyllotaxis (data not shown) and accelerated heteroblasty
(Fig. 2E, see below)]. We then
examined the number and size of cells in sqn-1 and sqn-5
leaves and confirmed that they had similar phenotypes to that of msc3
(see Fig. S2 in the supplementary material). F1 plants from the cross between
msc3 and sqn-1 did not complement their phenotypes (see Fig.
S2 in the supplementary material). Thus, we conclude that msc3 is a
new allele of sqn (sqn-6).
msc1-D has a mutation in the miR156 target site of the SPL15 gene
We mapped the msc1-D mutation at low resolution to the lower arm
of chromosome 3, near the SNP marker SGCSNP7. Around the marker, the
SPL15 (At3g57920) gene was found, which has an miR156 target
site (Rhoades et al., 2002).
Recently, SPL15 and its closest homolog SPL9 were reported
to be involved in the regulation of heteroblasty
(Schwarz et al., 2008). Based
on this information, we sequenced the SPL15 gene in the
msc1-D background. We found a C-to-T nucleotide substitution in the
miR156 target site in the msc1-D mutant
(Fig. 2A,C). Although this
mutation does not cause amino acid substitution, it could lead to reduced
efficiency of miR156-targeted mRNA cleavage. To test this, mRNA
cleavage sites of SPL15 transcripts were identified by
5'-RLM-RACE. A distinct band of amplified products of an expected size
was observed in the wild type (Fig.
2B). We found that most cleavage events took place at the ninth
and tenth residues from the miRNA 5' end
(Fig. 2C) as previously
reported (Wu and Poethig,
2006). In msc1-D, however, the amount of amplified
products was significantly reduced (Fig.
2B), and most of the cleavage events occurred outside of the
miR156 complementary site (Fig.
2C). We also tested expression levels of SPL15 by
quantitative RT-PCR. The expression levels in msc1-D were much higher
than in the wild type (Fig.
2D). These results strongly suggest that the mutation in the
miR156 target site of the SPL15 gene leads to reduced
cleavage and increased accumulation of SPL15 mRNA in the mutant. In
addition, msc1-D showed accelerated heteroblasty
(Fig. 2E, see below). As
SPL15 promotes phase change
(Schwarz et al., 2008), the
mutated SPL15 is highly likely to be responsible for the phenotypes
of the msc1-D mutant. We hereafter designate the mutant
spl15-1D/msc1-D.
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; Berardini et al.,
2001). In Arabidopsis, juvenile leaves have trichomes
only on the adaxial side and adult leaves have trichomes on both adaxial and
abaxial sides; thus, trichome presence is a typical marker of heteroblasty. To
investigate whether spl15-1D/msc1-D exhibits accelerated
heteroblasty, we counted rosette leaves without abaxial trichomes (juvenile
leaves) or with abaxial trichomes (adult leaves) and cauline leaves
(reproductive leaves). The wild type produced 6.3 juvenile leaves on average,
whereas the spl15-1D/msc1-D mutant had 4.3, indicating that it shows
accelerated heteroblasty (Fig.
2E). psd-16/msc2 and sqn-6/msc3 produced 4.2 and
4.6 juvenile leaves, respectively, confirming the accelerated heteroblasty
(Fig. 2E).
Leaves at higher nodes have increased cell number and decreased cell size compared with those at lower nodes
Because msc1-msc3 mutants show accelerated heteroblasty, their
leaves might have characteristics of those at higher nodes in the wild type.
If this interpretation is correct, wild-type leaves should exhibit progressive
changes in the number and size of cells during phase change; such changes have
been observed mainly in epidermal cells in previous reports
(Cnops et al., 2004;
Cookson et al., 2007). To
address this possibility, we determined the number and size of palisade
mesophyll cells at various nodes in the wild type. Cell number increased and
cell size decreased in leaves at higher nodes compared with those at lower
nodes (Fig. 3A,B). In
spl15-1D/msc1-D and sqn-6/msc3 mutants, cell number and cell
size resembled those in leaves at higher nodes than the corresponding nodes of
the wild type (Fig. 3A,B).
However, cell number in the third and fifth leaves of psd-16/msc2 did
not exceed that of the wild type (Fig.
3A,B). This might be because of a genetic lesion in the
PSD gene, which encodes a tRNA export mediator exportin-t
(Hunter et al., 2003a;
Li and Chen, 2003) and could
lead to reduced efficiency of protein synthesis, and thus to a decline in
plant growth.
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;
Wu and Poethig, 2006). We
investigated these two pathways in relation to the heteroblastic regulation of
cell number and size. Initially, we observed accelerated heteroblasty mutants
rdr6, sgs3 and zip, in which tasiR-ARF expression
was not detected (Hunter et al.,
2006). As shown in Fig.
4A,B, rdr6 and sgs3 had a slightly increased
cell number, although cell size was not significantly affected, except for the
fifth leaf of sgs3. The zip mutant, by contrast, showed no
alteration in cell number and size (Fig.
4A,B). Next, we investigated the arf3-2 and
arf4-2 mutants, in which heteroblasty is retarded
(Hunter et al., 2006), and
found that they showed no change in cell number or size in any leaves compared
to the wild type (Fig. 4C,D).
These results suggest that tasiR-ARF-mediated regulation of
ETT/ARF3 and ARF4 might not be required for the
heteroblastic change in cell number and size. In the arf3-2 arf4-2
double mutant, although cell number was reduced in all leaves tested, an
increase in cell number in the leaves at higher nodes occurred in a similar
manner to the wild type (Fig.
4C,D). Moreover, cell size did not change compared to the wild
type.
We then examined miR156 constitutively overexpressing plants. In
these plants, cell number and size in the first leaf were the same as in the
wild type (Fig. 4E,F). However,
the cell number increase and cell size reduction in leaves at higher nodes
were considerably inhibited (Fig.
4E,F), indicating that heteroblastic change in cell number and
size was severely inhibited. By contrast, transgenic plants constitutively
overexpressing SPL3 with a mutation in the miR156 target
site (35S:SPL3m) or SPL3 or 4 with a deletion of
the miR156 target site (35S:SPL3 and
35S:SPL4) (Wu and Poethig,
2006) had a significantly increased cell number and decreased cell
size, most remarkably in the first leaf
(Fig. 4E,F). Those
overexpressing SPL5 with a deletion of the miR156 target
site (35S:SPL5) showed no significant alteration in cell
number but a considerable reduction in cell size
(Fig. 4E,F). These results
indicate that miR156 and its target SPL genes (SPL3
subclass) might be involved in the regulation of heteroblastic change in cell
number and size.
We further investigated the expression levels of miR156 or various SPL genes in msc1-msc3 mutants. miR156 levels were partially reduced in psd-16/msc2 and sqn-6/msc3 (Fig. 5A). By contrast, we found preferential upregulation of specific SPL genes among 10 SPL genes with the miR156 target sequence: SPL13 and SPL15 were upregulated 2.6- and 3.6-fold, respectively, in psd-16/msc2, and SPL3 and SPL13 were upregulated 3.9- and 5.6-fold, respectively, in sqn-6/msc3 (Fig. 5B). In spl15-1D/msc1-D, only SPL15 was markedly upregulated, as expected (Fig. 5B). These results suggest that altered cell number and size in msc mutants might have arisen from upregulation of a few SPL genes, although the relative importance of individual SPL genes might differ in the respective msc mutants. To test whether the upregulation of these SPL genes accounts for the phenotypes of psd-16/msc2 or sqn-6/msc3, we crossed them with 35S:miR156. The phenotypes of psd-16/msc2 and sqn-6/msc3 were effectively suppressed by miR156 overexpression (Fig. 6A,B), suggesting that their phenotypes are dependent on upregulated SPL genes.
Heteroblasty in other known mutants
Our finding that cell number and size change in relation to heteroblasty
allowed us reconsider the phenotypes of two groups of other known mutants. The
first group includes mutants exhibiting compensation syndrome and the second
is an auxin resistant 1 (axr1) mutant.
As three msc mutants show accelerated phase change, one might
assume that mutants exhibiting compensation syndrome that have a decreased
cell number and increased cell size opposite to the msc mutants
should show delayed phase change. To test this possibility, we investigated
phase change in various compensation-exhibiting mutants by counting the number
of rosette leaves with or without abaxial trichomes and cauline leaves. Some
mutants (an3 and fugu1) clearly showed delayed
juvenile-to-adult phase change, while others (ant, fugu2, fugu3-D,
fugu4-D and fugu5) did not
(Fig. 7A). However, note that
the an3 mutant showed rapid leaf production (shorter plastochron) and
similar flowering time as the wild type
(Horiguchi et al., 2005);
therefore, the seemingly delayed phase change in an3 might be an
indirect consequence of the shorter plastochron. By contrast, fugu1
showed markedly delayed flowering (Fig.
7A, increased rosette leaf number), suggesting that both the
juvenile-to-adult vegetative phase change and vegetative-to-reproductive phase
change were delayed. The modes of delay in phase change in these mutants
differed from each other.
The other mutant, axr1, was originally reported as one whose
leaves have fewer but normal-sized cells
(Lincoln et al., 1990).
Subsequently, we reported that the first leaves of axr1 mutants have
smaller cells compared with the wild type
(Horiguchi et al., 2006b). To
resolve this discrepancy, we determined the number and size of cells in the
first, third and fifth leaves. In axr1-3 mutants, cell number is
significantly decreased in the third and fifth leaves
(Fig. 7B). Moreover, cell size
in the first, third and fifth leaves was almost the same in this mutant; as a
consequence, cell size in the first or fifth leaves of axr1-3 was
smaller or larger, respectively, than the corresponding leaves of the wild
type, whereas cell size in the third leaves of axr1-3 and the wild
type was similar (Fig. 7C).
These results reasonably explain the paradoxical results reported previously
and suggest that AXR1 is required for the control of cell size during
phase change. To investigate the relationship between SPL-regulated
heteroblastic change in cell size and AXR1, we examined axr1-3
msc1-D double mutants. The double mutants showed increased cell number
and slightly decreased cell size compared with the parental axr1-3 in
all leaves tested (Fig. 7B,C),
indicating that msc1-D and axr1-3 mutations have additive
effects on cell number and size.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Granier and Tardieu, 1998;
Cnops et al., 2004;
Cookson et al., 2007), the
cause of this phenomenon was unclear until the present study. In this report,
we propose that miR156 and SPL genes are involved in
heteroblastic change of cell number and size (see below). Our findings
highlight the novel roles of SPL transcription factors in the regulation of
heteroblasty.
Regulation of cell number and size in relation to heteroblasty
A simple explanation for the increased cell number and decreased cell size
in leaves at higher nodes is that a prolonged cell proliferation period in
leaves at higher nodes may cause a shortening of the cell expansion period,
resulting in the decreased final cell size. However, we cannot rule out
another possibility: namely that a change in cell size is genetically
separable from that in cell number. A notable example of this possibility is
found in the rotunda2 (ron2) mutant, which is defective in
the transcriptional co-repressor LEUNIG. In ron2, the cell size in
the third leaf is as large as that in the first leaf of the wild type or
ron2, whereas the cell number is almost the same as that of the third
leaf of the wild type (Cnops et al.,
2004). Another intriguing example is the axr1-3 mutant,
in which cell size is almost the same in any leaf tested
(Fig. 7C), suggesting that
auxin signal transduction is required for the heteroblastic regulation of cell
size. Analysis on these types of mutants in relation to the SPL genes
will help us to distinguish these possibilities.
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). By contrast, many other heteroblastic traits are expressed
heterogeneously within leaves produced during the transition from juvenile to
adult phases. In these intermediate leaves, the distal part, which first
arises from the shoot apical meristem, expresses more juvenile traits, whereas
the proximal part, which arises later, expresses more adult traits
(Kerstetter and Poethig,
1998). The highly homogeneous cell size within an intermediate
leaf, such as the fifth leaf (see Fig. S1 in the supplementary material),
suggests that the cell size is controlled at the organ level, whereas other
traits are controlled in a cell-autonomous manner.
The miR156-SPLs pathway is involved in heteroblastic change of cell number and size
We propose that miR156-mediated regulation of SPL genes,
including SPL15 and SPL3, -4 and -5, is
involved in heteroblastic regulation of cell number and size for the following
reasons. The spl15-1D/msc1-D mutant has a mutation in the
miR156 target site of the SPL15 gene, and expression levels
of SPL15 are elevated in this mutant
(Fig. 2A-D). In addition,
miR156-overexpressing plants showed severe defects in heteroblastic
change in cell number and size (Fig.
4E,F). Transgenic plants overexpressing the
miR156-insensitive form of SPL3 or SPL4 have
increased cell number and decreased cell size, particularly in the first leaf
(Fig. 4E,F). Moreover, in the
psd-16/msc2 and sqn-6/msc3 mutants, expression levels of a
few SPL genes are significantly higher than in the wild type
(Fig. 5B).
In the rdr6, sgs3 and zip mutants, in which heteroblasty
is accelerated but miR156 levels are not changed
(Peragine et al., 2004), some
leaves have an increased cell number but do not show reduced cell size, except
for the fifth leaf of sgs3 (Fig.
4A,B). The results indicate that these genes have only minor
effects on the heteroblastic regulation of cell number and size. Because
SPL3 expression levels were slightly (1.2- to 1.5-fold) elevated in
these mutants (Peragine et al.,
2004), such increases could affect cell number and size.
Because tasiR-ARF expression is not detected in rdr6,
sgs3 and zip mutants
(Peragine et al., 2004),
tasiR-ARF-mediated regulation of ETT/ARF3 and ARF4
is unlikely to be involved in heteroblastic change of cell number and size.
This differs from the control of trichome production on the abaxial side of
leaves, which is regulated by both SPL genes and by ETT/ARF3
and ARF4 (Hunter et al.,
2006; Wu and Poethig,
2006). Under our experimental conditions, rdr6 and
sgs3 clearly showed acceleration of abaxial trichome emergence, as
previously reported (Peragine et al.,
2004), although zip did not show this effect for unknown
reasons (see Fig. S3 in the supplementary material). These facts suggest that
heteroblastic regulation of abaxial trichome production and cell number and
size are under the control of different pathways. This is further supported by
the result that neither the arf3 nor the arf4 single mutant,
which exhibit a delay in abaxial trichome emergence
(Hunter et al., 2006), showed
altered cell number or size compared to the wild type
(Fig. 4C,D). The arf3-2
arf4-2 double mutant also showed no change in cell size
(Fig. 4D). Although cell number
in this latter mutant was reduced, heteroblastic change in cell number
occurred in a similar manner to that in the wild type
(Fig. 4C,D). The reduced cell
number could have been caused by disturbed leaf polarity, because the
arf3-2 arf4-2 double mutant shows defects in leaf adaxial/abaxial
polarity and altered leaf morphology
(Pekker et al., 2005).
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). As only SPL15 mRNA level was upregulated in
spl15-1D/msc1-D, the mutated SPL15 could have been the
primary cause of the mutant phenotypes. In addition, because miRNAs, including
miR156, also affect the translation of target genes
(Gandikota et al., 2007;
Brodersen et al., 2008), SPL15
protein level might be upregulated in msc1-D.
In psd-16/msc2 and sqn-6/msc3 mutants, the expression
levels of miR156 slightly decreased and those of a few SPL
genes increased. However, the significance of such a slight reduction in
miR156 levels is not clear because only a subset of SPL
genes was affected in their mRNA accumulation in these mutants
(Fig. 5B). The increased
expression of SPL genes should cause an alteration in cell number and
size in leaves. This is further supported by the fact that increased cell
number and decreased cell size in these two mutants were effectively
suppressed by the overexpression of miR156
(Fig. 6). The PSD gene
encodes exportin-t, which regulates tRNA processing and nuclear export, but
does not affect accumulation or export of miRNA
(Park et al., 2005).
SQN encodes cyclophilin 40, a protein that associates with the Hsp90
chaperone complex (Berardini et al.,
2001), but its precise molecular function in plants is unknown.
Although how these genes affect the expression levels of miR156 or
SPL genes is unknown, one possibility is that they regulate
expression of a subset of SPL genes by unknown mechanisms. If this is
correct, psd and sqn are useful mutants to clarify the
mechanisms of heteroblasty operating upstream of the SPL genes.
Functions of SPL genes
We propose that the heteroblasty-promoting SPL genes, including
SPL3, -4, -5 and -15, increase cell number
and reduce cell size in leaves. However, how these genes regulate cell number
and size is still unknown. An intriguing possibility is that SPLs
function through auxin signaling. Nevertheless, the additive phenotypes of the
axr1-3 msc1-D double mutant indicate that SPL15 and
AXR1 function in at least partially non-overlapping pathways.
SPLs comprise a family of transcription factors that share the SBP domain,
a DNA-binding domain first identified in a protein that binds to a promoter of
the SQUAMOSA gene in Antirrhinum majus
(Klein et al., 1996;
Cardon et al., 1997;
Cardon et al., 1999). The amino
acid sequence of the SBP domain is highly conserved among SPL proteins, but
the sequence outside the SBP domain is diverse
(Cardon et al., 1999). Although
they are thought to bind to similar DNA sequences
(Cardon et al., 1999), the
molecular functions or target genes of SPLs remain unclear. Among the 10
SPL genes that have the miR156 target site, SPL3,
-4, -5, -9 and -15 are involved in the
heteroblastic regulation of abaxial trichome production
(Wu and Poethig, 2006;
Schwarz et al., 2008). In this
study, we found that heteroblastic change in cell number and size is also
regulated by SPL3, -4, -5 and -15,
indicating that these genes might have overlapping functions. Among six
SPL genes without the miR156 target site, SPL14 is
particularly interesting because the loss-of-function mutant of this gene
seems to have a truncated juvenile vegetative phase
(Stone et al., 2005). SPL14
could have an antagonistic function to other SPL proteins that promote
vegetative phase change. Identifying the targets of SPL transcription factors
in further investigations will be necessary.
Regulation of cell number and cell size in leaves through distinct genetic pathways
Precise regulation of cell number and cell size is essential for leaf
development. However, the genetic network controlling cell number and size in
the context of leaf development is too complex to establish a unified view on
its regulation. Instead, finding a particular pathway that confers unique
effects on leaf development and comparing the relationship among individual
pathways are needed. In the present study, we show that increased cell number
and reduced cell size in msc mutants are caused by accelerated
heteroblasty. In contrast to msc mutants, the compensation syndrome,
in which cell number is reduced and cell size is increased, is not necessarily
caused by a genetic pathway associated with heteroblasty, because most
compensation-exhibiting mutants do not show delayed phase change
(Fig. 7A). Two
compensation-exhibiting mutants (an3 and fugu1) indeed show
a delay in phase change. However, they had an increase in cell number and a
decrease in cell size in leaves at higher nodes than the wild type (see Fig.
S4 in the supplementary material), indicating that they are not deficient in
the heteroblastic change in cell number and size. In addition, the
miR156-overexpressing plant did not show a reduction in cell number
and an increase in cell size in the first leaf
(Fig. 4E,F). In other words,
the first leaves might be in the ground state of the vegetative phase and
cannot revert beyond it to a further immature phase. However, compensation
syndrome can be seen in the first leaves (see Fig. S4 in the supplementary
material). This indicates that cell number and cell size are controlled by at
least two distinct genetic pathways, i.e. one associated with heteroblasty and
one related to the compensation syndrome. Increasing numbers of genes
identified from mutants with altered cell number/size will help us to
understand the details of the genetic network and the mechanisms controlling
cell number and size.
In summary, we conclude that heteroblasty has an important role in the regulation of cell number and cell size. Investigating the factor(s) that regulate cell number and size under the control of heteroblasty, particularly downstream of the SPL genes, is crucial for understanding the mechanisms that regulate cell number, cell size and organ size during leaf development.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/955/DC1
* Present address: Department of Life Science, College of Science, Rikkyo
University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo, 171-8501 Japan 
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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