The Plant Journal (2000) 22(3), 257±264
SHORT COMMUNICATION
Disruption of an Arabidopsis cytoplasmic ribosomal protein
S13-homologous gene by transposon-mediated
mutagenesis causes aberrant growth and development
Takuya Ito1, Gyung-Tae Kim1,² and Kazuo Shinozaki1,2,*
1
Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, The Institute of Physical and Chemical Research
(RIKEN), 3±1-1 Koyadai, Tsukuba, Ibaraki 305±0074, Japan, and
2
Plant Mutation Exploration Team, Plant Functional Genomics Research Group, RIKEN Genomic Sciences Center,
3-1-1 Koyadai, Tsukuba, Ibaraki 305±0074, Japan
Received 8 October 1999; revised 15 February 2000; accepted 22 February 2000.
*For correspondence (fax +81 298 36 9060; e-mail [email protected]).
²
Present address: Center for Bio-Environmental Research, National Institute for Basic Biology, 38 Nishigounaka, Myodaiji-cho, Okazaki 444±8585, Japan.
Summary
We identi®ed a Dissociation (Ds) transposon-inserted Arabidopsis mutant of a gene (AtRPS13A)
homologous to cytoplasmic ribosomal protein (RP) S13. We named our mutant pointed ®rst leaf (p¯) 2
because of its similar phenotype to the p¯1 mutant of the RPS18 gene. This mutant caused multiple
phenotypic changes, including aberrant leaf and trichome morphology, retarded root growth, and late
¯owering. Microscopic analysis showed that the ®rst leaf blade of p¯2 contained a signi®cantly reduced
number of palisade cells, which suggests that the mutant phenotype was caused by reduced cell
division. However, no phenotypic changes were observed during reproductive growth. In Arabidopsis,
the RPS13 protein was encoded by a small expressed gene family including AtRPS13A. A p¯1 p¯2 double
mutant showed no additive effect. These results suggest that RPS13 functions in quantitative and
pleiotropic ways during growth and development, and that mutations at different kinds of RP gene loci
are accumulatable without serious growth defects because they belong to small gene families.
Introduction
Ribosomes are involved in protein biosynthesis in all living
cells. The 40S and 60S subunits of eukaryotic ribosomes
contain 3 or 4 RNAs and 60±80 proteins. The ribosomal
proteins (RPs) are major components of the biosynthetic
machinery, representing up to 15% of cellular protein
(Mager, 1988). Some RP genes are regulated both developmentally and environmentally in plants. In maize, RPS14
was transiently accumulated in the endosperm of 12-dayold developing kernels, just before synthesis of seed
storage protein began (Larkin et al., 1989). In Arabidopsis,
RPS18 was active after wounding (Van Lijsebettens et al.,
1994), and two members of RPL16 were active when lateral
roots were initiated (Williams and Sussex, 1995). In
tobacco, RPL25 was inducible by wounding, auxin and
cytokinin (Gao et al., 1994). These expression analyses
enable us to understand the function of the RP genes in
plant growth and development, but it is also necessary to
ã 2000 Blackwell Science Ltd
analyse mutants at these gene loci. However, in plants,
only three mutants at RP gene loci have been reported,
although a number of RP genes have been cloned.
Mutations at one Arabidopsis cytoplasmic RPS18 gene
(RPS18A) and one RPS27 gene (ARS27A) cause `pointed
®rst leaf' (RPS18A; Van Lijsebettens et al., 1994) and
genotoxic stress sensitivity (ARS27A; Revenkova et al.,
1999), and a mutation at a putative Arabidopsis gene
(SSR16) homologous to mitochondrial S16 causes an
embryo-defective phenotype (Tsugeki et al., 1996).
We report an Arabidopsis mutant in which a
Dissociation (Ds) transposon is inserted into a cytoplasmic
40S RPS13 gene homologue named AtRPS13A. The
phenotype of this mutant resembles that of a mutant at
the RPS18A gene locus (Van Lijsebettens et al., 1994), in
that both have a pointed ®rst leaf, but the phenotypic
description of the RPS18A mutant is limited. To study the
257
258 Takuya Ito et al.
function of RP genes in plant growth and development, we
analysed the AtRPS13A mutant more precisely. This
mutant exhibits multiple phenotypic changes, such as
aberrant leaf and trichome morphology, and late ¯owering. The data presented here suggest that RPS13 may
function in quantitative and pleiotropic ways during
growth and development.
Results
Identi®cation of a Ds insertion mutant of an
RPS13-homologous gene
Arabidopsis lines containing transposed Ds elements were
generated by using an Activator (Ac)/Ds tagging system
(Ito et al., 1999; Smith et al., 1996). Ds-transposed lines
were generated from a Ds donor line (transformant
Ds4 391-20). Genomic DNA was extracted from leaves of
individual transposed lines, and Ds-¯anking genomic DNA
was obtained from the 94 lines by using thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR)
(Liu and Whittier, 1995). The partial sequences of the TAIL±
PCR products were then determined. Among these lines,
we identi®ed a Ds insertion mutant (line 155-1) of an RPS13
gene homologue (EST clone 186H13T7). This heterozygous mutant plant with a wild-type phenotype was selfpollinated. Resultant seeds of the next generation were
sown on agar plates containing hygromycin B (Hyg), which
is a transposon marker. Hyg-sensitive (HygS) and Hygresistant (HygR) seedlings were scored, and segregated as
1:2.6 (68 HygS and 178 HygR; c2 (1:3) = 0.92, P > 0.05). This
indicates that Ds was inserted into a single locus in line
155-1.
Phenotypic description of the insertion mutant
We sowed identical seeds on agar plates without Hyg and
identi®ed seedlings with narrow pointed or round ®rst and
second leaves (Figure 1A). Seedlings segregated as 1:3.9
(39 narrow pointed and 151 round), which indicates that
this mutant is a nuclear recessive mutant. We showed that
the Ds insertion caused the mutant phenotype by obtaining revertants from mutant to wild-type phenotype (see
below). Because the phenotype of this mutant (narrow
pointed ®rst and second leaves) resembles that of the
pointed ®rst leaf (p¯) 1 mutant, which is an insertion
mutant of an RPS18 gene (RPS18A) by T-DNA (Van
Lijsebettens et al., 1994), we named our mutant p¯2.
When p¯2 seedlings were grown on, the narrow pointed
shape of later leaves was not so severe (Figure 1A). Leaf
indices (length/width values) for the ®fth leaves were 1.4
(No-0) and 1.7 (p¯2), while those of the ®rst leaves were 1.1
(No-0) and 2.7 (p¯2). p¯2 exhibited a late ¯owering
phenotype under continuous light (Figure 1A,B). Although
p¯2 showed bolting retardation for about 1 week, the
growth rate of the primary shoot after bolting was the same
in wild-type and p¯2 plants (data not shown). The ®nal
primary shoot length was almost the same (data not
shown). We could not distinguish any morphological
difference in ¯oral organs between wild-type and p¯2
plants (data not shown). These results indicate that there
are no phenotypic changes during reproductive growth.
Root growth of the mutant was inhibited (Figure 1C).
We also found differences in trichome morphology
(Table 1 and Figure 1H,I). The ®rst leaf of the mutant had
no trichomes, the ®fth leaf had a reduced number of
trichomes, which had two branches, and the tenth leaf had
both aberrant and normal three-branched trichomes.
These results indicate that PFL2 (RPS13 gene homologue,
AtRPS13A) is essential for normal trichome development.
Anatomical analysis of the p¯2 mutant leaf
The ®rst and second leaves of the p¯2 mutant were
narrower than those of the wild-type (Figure 1A). To
dissect this morphological difference at the cellular level,
we compared the ®rst leaf cells as stained transverse
sections (Figure 1F,G). In the mutant leaf blade, many
enlarged cells and intercellular spaces were present. The
number of sub-epidermal palisade cells of the mutant was
less than that of the wild-type. We also compared adaxial
epidermal cells (Figure 1J,K). Epidermal cells in the mutant
leaf blade were round with fewer protrusions.
The vascular system of the p¯2 ®rst leaf was also
different from that of the wild-type (Figure 1D,E). In the
wild-type, branches of vascular strands were connected to
each other and circled. In the mutant, however, no ®ne
strands developed, and branches of vascular strands were
not connected to each other. This result indicates that
AtRPS13A is necessary for vascular network development
in early leaves. In later leaves, no signi®cant differences in
leaf cell number and vascular network development were
observed (data not shown).
RPS13 genes in Arabidopsis constitute a small expressed
gene family
The nucleotide sequence of the EST clone 186H13T7
(accession no. R90398), into which Ds was inserted, was
determined. We named this gene AtRPS13A and deduced
the amino acid sequence (Figure 2a). This gene was
located on a sequenced BAC clone, F6N15 (accession no.
AF069299), whose position is at 1 cM on chromosome 4 on
the recombinant inbred map initiated by Lister and Dean
(1993). The amino acid sequences of the RPS13 proteins
are highly conserved among plant and animal species, all
being 151 amino acids long (Figure 2b). Ninety-two per
cent of the amino acids (139 of 151) were identical between
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 257±264
Ribosomal protein S13 in growth and development
259
Figure 1. Phenotype of the p¯2 mutant.
(A) Leaves of 1-month-old plants. No-0 (upper) and p¯2 (bottom); left to right, 1st to 14th leaves. Leaf numbers at ¯owering were 9.3 6 0.9 (wild-type; n = 9)
and 16.5 6 1.7 (p¯2; n = 6). (B) Forty-day-old plants of No-0 (left) and p¯2 (right). (C) Seventeen-day-old seedlings of No-0 (left) and p¯2 (right) grown
vertically on an agar plate. (D,E) Vascular systems of No-0 (D) and p¯2 (E) ®rst leaves. Whole-mount preparations, dark-®eld optics. (F,G) Transverse
sections of the ®rst leaf blades of No-0 (F) and p¯2 (G): e, adaxial epidermal cell layer; p, sub-epidermal palisade cell layer. Samples were embedded in
Technovit 7100 resin. Sections (4 mm thick) were stained with toluidine blue. (H,I) Trichome cells on the adaxial side of the ®fth leaf blades of No-0 (H) and
p¯2 (I). (J,K) Adaxial epidermal cells in the ®rst leaf blades of No-0 (J) and p¯2 (K). Whole-mount preparations as described by Tsuge et al. (1996) used
Nomarski differential interference±contrast optics. The leaves (D±K) were photographed at the fully expanded stage (1-month-old plants). Scale bars: 3 cm
(A,C), 10 cm (B), 3 mm (D,E) and 100 mm (F±K).
AtRPS13A and pea RPS13, 87% (131) between AtRPS13A
and maize RPS13 (Joanin et al., 1993), 81% (122) between
AtRPS13A and humus earthworm RPS13, and 79% (119)
between AtRPS13A and rat RPS13 (Suzuki et al., 1990) or
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 257±264
human RPS13 (ChadeÂneau et al., 1993). Ds was inserted
into an exon region of this gene, and an 8 bp duplication
sequence speci®c to the Ac/Ds insertion was detected
(Figures 2A and 3G).
260 Takuya Ito et al.
Table 1. Trichome number of No-0 (wild-type) and p¯2 leaves
Number of trichomesb
(adaxial side)
Organa
Branch number
No-0
p¯2
First leaf
Three branches
Two branches
Total trichomes
Three branches
Two branches
Total trichomes
Three branches
Two branches
No branch
Total trichomes
14 6 4 (n = 5)
0 6 0 (n = 5)
14 6 4 (n = 5)
50 6 18 (n = 5)
9 6 3 (n = 5)
58 6 19 (n = 5)
±c
±c
±c
±c
0 6 0 (n = 8)
0 6 0 (n = 8)
0 6 0 (n = 8)
0 6 0 (n = 7)
4 6 2 (n = 7)
4 6 2 (n = 7)
30 6 10 (n = 5)
61 6 35 (n = 5)
5 6 7 (n = 5)
96 6 48 (n = 5)
Fifth leaf
Tenth leaf
a
Leaves were measured at the fully expanded stage (1-monthold plants).
b
Data are means 6 standard deviation for n plants examined.
c
No-0 bolts before the 10th leaf appears under continuous light
condition.
Because Arabidopsis RP genes (L3, S18 and L16)
constitute a small gene family (Kim et al., 1990; Van
Lijsebettens et al., 1994; Williams and Sussex, 1995), we
examined redundancy in AtRPS13A by Southern analysis.
When an AtRPS13A-speci®c probe was used, a single band
was detected (Figure 2c, lanes 1 and 2). On the other hand,
when a full-length cDNA probe was used, extra bands
appeared (lanes 4 and 5). The genomic DNA region
covered by the full-length cDNA probe contains neither
BamHI nor HindIII sites, indicating that the extra bands
were not of AtRPS13A origin. From this result, we
conclude that AtRPS13A is a member of a small gene
family (three copies at most). We also examined the Ds
copy number of the mutant at the p¯2 locus. By using the
speci®c probe, we detected a band shift corresponding to a
Ds insertion (approximately 6 kb long) in the mutant
(Figure 2c, lanes 2 and 3). Ds does not contain a HindIII
site. This result indicates that a single copy of Ds was
inserted at the p¯2 locus.
To examine whether multiple members of the RPS13
gene family are expressed, we carried out Northern
analysis using AtRPS13A-speci®c and full-length cDNA
probes. When the speci®c probe was used, no transcript of
AtRPS13A was detected in the p¯2 mutant (Figure 2d,
lane 1), but a small amount of the transcript was detected
in the wild-type (lane 2). When the full-length cDNA probe
was used, equal amounts of RPS13 transcripts were
detected in both (lanes 3 and 4). This result indicates that
multiple members of the RPS13 gene family are expressed. Indeed, at least one EST group homologous to
cytoplasmic RPS13 other than AtRPS13A exists in the
database (EST clone 111E16T7, accession no. T42230).
Tissue-speci®c GUS staining using the enhancer trap
system
The transposon-tagging system used in this study
(transformant Ds4 391-20) contains enhancer trap apparatus (Fedoroff and Smith, 1993). Within the transposon
is a coding sequence for GUS fused to a core sequence
for cauli¯ower mosaic virus 35S promoter. The p¯2
mutant showed GUS staining in the shoot apical region,
young leaves, vascular tissue in young hypocotyls,
axillary buds and young ¯ower buds (Figure 3A,D±F).
Because RPL16 gene expression was induced when
lateral roots were initiated (Williams and Sussex,
1995), we examined the inducibility of AtRPS13A by
using auxin to induce lateral roots. Indol-3-acetic acid
(IAA) induced GUS staining in lateral root primordia
(Figure 3C). These results indicate that GUS staining is
observed in meristematic tissues. This meristematic
staining pattern is similar to the patterns in other RP
genes, RPS18A, RPL16A and RPL16B (Van Lijsebettens
et al., 1994; Williams and Sussex, 1995).
However, we cannot immediately draw the conclusion
that AtRPS13A is expressed in meristematic tissues,
because the enhancer trap may be responding to additional enhancers or silencers at some distance from the
insertion site. Indeed, some enhancer trap patterns are not
speci®c to meristematic tissues. For example, there was
GUS staining at the tip of the leaf which is the ®rst part of
the leaf that stops dividing (Figure 3D). Additionally, some
enhancer trap patterns do not correlate with the mutant
phenotypes observed in p¯2. For example, we could not
detect any staining in root tissues (Figure 3B,E), although
p¯2 exhibited retarded root growth (Figure 1C).
Isolation of revertants from mutant to wild-type
phenotype
To con®rm that Ds insertion was responsible for the p¯2
mutant phenotype, we isolated revertants from the mutant
phenotype to the wild-type phenotype. We isolated four
seedlings with narrow pointed leaves, which contained the
Ac transposase gene. These mutant plants were selfpollinated and the resultant seeds were sown on agar
plates. Among the seedlings, we found three revertants
with round leaves, indicating that the mutant phenotype of
p¯2 was due to Ds insertion into AtRPS13A. The footprint
sequences of the revertants were determined (Figure 3G).
The sequences from the revertants restored a putative
wild-type reading frame. This result is consistent with
sequence analysis showing that Ds had been inserted into
a translated region of AtRPS13A (Figure 2a).
AtRPS13A proteins of the three revertants contained one
or two additional amino acids (Figure 3H). Because the
total amino acid number (151) of the RPS13 proteins is
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Ribosomal protein S13 in growth and development
261
Figure 2. Molecular analyses of AtRPS13A.
(a) cDNA sequence of AtRPS13A (Columbia ecotype) and predicted amino acid sequence. The in-frame stop codon upstream of the putative start codon is
underlined. An 8 bp sequence duplicated by Ds insertion is double-underlined. The speci®c probe used in (c) and (d) is indicated with a broken line. The
triangles mark the positions of introns as deduced by comparison between the cDNA and genomic DNA sequences. (b) Comparison between amino acid
sequences of RPS13 proteins deduced from Arabidopsis (AtRPS13A), pea (Z25509), maize (X62455), human (L01124), rat (X53378) and humus earthworm
(AJ011706) cDNAs. Identical amino acids to Arabidopsis protein are indicated by dots. The N-terminal methionine of the rat RPS13 protein is removed
after translation (Suzuki et al., 1990). (c) Southern analysis of AtRPS13A. Southern blot containing No-0 (wild-type) and p¯2 (mutant) DNA digested with
either BamHI (B) or HindIII (H) and hybridized to AtRPS13A cDNA probe. The cDNA region used for the speci®c probe is shown in (a). (d) Northern
analysis of AtRPS13A. Total RNA prepared from 3-week-old No-0 (wild-type) and p¯2 (mutant) plants was hybridized with AtRPS13A cDNA probes.
strictly conserved among plant and animal species
(Figure 2b), we examined the effect of the amino acid
additions to the AtRPS13A proteins of the revertants. At
the seedling stage, we could not ®nd any morphological
differences between the wild-type and the revertants
(Figure 3I). These revertants also restored wild-type bolting
time, root growth, vascular network development and
trichome morphology (data not shown).
p¯1 p¯2 double mutant analysis
p¯1 is a T-DNA insertion mutant of RPS18A, which is one
of three expressed RPS18 genes (Van Lijsebettens et al.,
1994). RPS13 and RPS18 proteins are components of the
same cytoplasmic 40S small subunit of ribosomes. To
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 257±264
examine the effect of double mutations at AtRPS13A and
RPS18A on growth and development, we produced a p¯1
p¯2 double mutant. We compared the morphology of the
single and double mutants at the vegetative stage. We
could not ®nd any differences between the double mutant
and any single mutant grown at 22°C (data not shown). As
the phenotypic differences between the p¯1 mutant and
the wild-type grown at 13°C were more dramatic (Van
Lijsebettens et al., 1994), we also compared phenotypes of
each mutant plant grown at 13°C. When grown at 13°C, the
phenotype of p¯2 was more severe than that of p¯1, which
suggests that p¯1 is a leaky allele. The phenotype of the
p¯1 p¯2 double mutant was similar to that of the p¯2
single mutant (data not shown). These results indicate that
there is no additive effect of the mutations.
262 Takuya Ito et al.
Figure 3. Histochemical analysis of GUS staining in the p¯2 mutant using the enhancer trap system (A±F) and revertant analysis (G±I).
(A) Apical region of a 9-day-old seedling. (B) Root tip of a 9-day-old seedling. (C) Lateral root of a 16-day-old seedling 72 h after treatment with 30 mM IAA.
(D) Apical region of a 3-week-old seedling. (E) Junction region of hypocotyl (h) and root (r) of a 9-day-old seedling. (F) In¯orescence of a 1.5-month-old
plant. (G) The nucleotide sequence of AtRPS13A (from 5¢ to 3¢) around the Ds insertion site is shown for wild-type, p¯2 and revertants. The 8 bp
duplication sequence when Ds was inserted is boxed. D represents the change in nucleotide length compared with the wild-type. (H) Deduced amino acid
sequences of AtRPS13A proteins for wild-type and revertants around the Ds insertion site. The numbers indicate the positions of the deduced amino acid
sequences of the wild-type protein. Asterisks indicate amino acids identical to those of wild-type protein. Gaps (±) were introduced to maximize homology
between wild-type and revertant proteins. (I) Fourteen-day-old seedlings of wild-type (No-0), p¯2 and revertants. Arrows indicate ®rst and second leaves.
Scale bars: 500 mm (A±C,E), 5 mm (D,F) and 1 cm (I).
Discussion
Effects of mutation at AtRPS13A on plant growth and
development
We showed that disruption of AtRPS13A caused aberrant
leaf and trichome morphology in the early vegetative
phase, and late ¯owering phenotype (Figure 1 and Table 1),
but we could detect no remarkable phenotypic changes in
the reproductive phase. These results indicate that the
contribution of AtRPS13A to growth and development is
greater in the vegetative phase. AtRPS13A is a member of
a small gene family consisting of at most three expressed
copies (Figure 2c,d). This suggests that the RPS13 gene
product functions quantitatively in these pleiotropic
phenotypes.
Many gene families of mammalian RP constitute a single
expressed gene and multiple processed pseudo-genes
(Dudov and Perry, 1984; Wagner and Perry, 1985;
Wiedemann and Perry, 1984). In plants, however, multiple
expressed copies exist, such as RPS14 in maize (Larkin
et al., 1989), RPS18 in Arabidopsis (Van Lijsebettens et al.,
1994;), RPL16 in Arabidopsis (Williams and Sussex, 1995),
and RPS13 in Arabidopsis (this study). Two models have
been proposed to explain why there are multiple expressed
copies of RP genes (Larkin et al., 1989). One is a gene
dosage model. At some stage in the plant life cycle,
maximum growth rate requires the supply of RP expressed
from multiple gene copies. We observed that p¯2 caused
phenotypic changes by disrupting only one copy
(AtRPS13A) in the early vegetative phase (aberrant leaf
and trichome morphology) and in the transition from the
vegetative to the reproductive phase (late ¯owering). Based
on the gene dosage model, it is possible that maximum
RPS13 gene expression from all copies is necessary during
these speci®c phases. The other model hypothesizes that
individual copies are differently regulated in different cell
types and at different developmental stages to control the
RP level precisely. According to this model, AtRPS13A may
play more important roles than other RPS13 genes in the
early vegetative phase and in the transition from the
vegetative to reproductive phase. Knock-out mutants of
other RPS13 genes would elucidate which model is correct.
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 257±264
Ribosomal protein S13 in growth and development
Several rat RPs, including S13, and 5.8S ribosomal RNA
form a ribonucleoprotein complex. This complex binds to
ternary complexes of eIF-2:Met-tRNA:GTP and EF-1a:PhetRNA:GTP, thus S13 may form a part of the amino acyltRNA binding domain in the ribosome (Chan et al., 1982).
This biochemical study indicates that RPS13 protein plays
an important role in translation. In Arabidopsis, disruption
of the putative mitochondrial unique gene SSR16 was
lethal to embryos (Tsugeki et al., 1996), but disruption of
the cytoplasmic AtRPS13A, which is one of multiple
copies, had limited effect such as aberrant leaf and
trichome morphology and late ¯owering. From these
observations, it is possible that disruption of all copies of
RPS13 may cause more severe phenotypes such as
`embryo lethal'.
As for mutations of Drosophila Minute genes which
encode RPs (Lambertsson, 1998), the effects of p¯1 and
p¯2 were not additive at the seedling stage (data not
shown). This suggests that AtRPS13A and RPS18A
proteins are components of the same assembly.
Role of AtRPS13A in trichome and leaf development
The endoreplication step affects trichome development
(Folkers et al., 1997; HuÈlskamp et al., 1998). GLABRA1 (GL1)
is required to initiate trichome cell morphogenesis and
concomitant induction of endoreplication rounds.
GLABRA3 (GL3) is required to trigger a fourth round of
endoreplication. Thus, it is feasible that the ®rst, second
and third rounds of endoreplication affect trichome initiation, and the fourth round affects the number of branches.
We observed that trichomes of the p¯2 mutant developed
differently from those of the wild-type (Table 1 and
Figure 1H,I). In the mutant, the ®rst leaf had no trichomes
and the ®fth leaf had a reduced number of trichomes, which
had only two branches. The palisade cell number of the ®rst
leaf blade of the mutant was signi®cantly reduced
(Figure 1F,G). This suggests that cell division activity in
the ®rst leaf was reduced. Taken together, these results
suggest that reduced cell division during the early vegetative phase in the p¯2 mutant might reduce endoreplication
activity and result in a gl1-like phenotype in the ®rst leaf and
a gl3-like phenotype in the ®fth.
Leaves of the angustifolia (an) mutant are the same
length as but narrower and thicker than wild-type leaves,
but the total number of cells is the same. Thus, AN is
proposed to control cell broadening (Tsuge et al., 1996).
On the other hand, the ®rst and second leaves of the p¯2
mutant are narrower than wild-type leaves, but normal leaf
shape is restored in later leaves. Microscopic analysis
suggested that cell division activity in the ®rst leaf blade
was reduced in the p¯2 mutant. These results suggest that
AN controls leaf width through cell elongation and that
PFL2 (AtRPS13A) exerts its control through cell division.
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 257±264
263
However. it is not clear why PFL2 (AtRPS13A) is speci®c to
width.
We are now examining cell division activity of the p¯2
tissues based on the hypothesis that p¯2 phenotypes are
caused by reduced cell division activity.
Experimental procedures
Plant materials and growth conditions
Ds-transposed lines (background ecotype No-0) and TAIL±PCR
analysis have been described previously (Ito et al., 1999). C24
(stock no. CS906) and p¯1 (stock no. CS5208) were supplied by
Ohio State University's Arabidopsis Biological Resource Center.
No-0 was supplied by Dr T. Araki. Plants were germinated and
grown aseptically on germination medium (GM) containing
Murashige and Skoog salts (Murashige and Skoog, 1962), 3%
(w/v) sucrose and 0.05% (w/v) Mes-KOH (pH 5.7), solidi®ed with
0.8% (w/v) Bacto-agar (Difco, Detroit, MI, USA). The plants were
grown at 22°C or 13°C under continuous illumination of about
50 mmol s±1 m±2. For growth in the greenhouse, 2-week-old aseptic
plants were transferred to vermiculite/perlite (1:1) that had been
moistened with Hyponex diluted 1:1000 (Hyponex Japan, Osaka,
Japan) and were grown at about 23°C under continuous
illumination of about 50 mmol s±1 m±2.
DNA sequencing
The cDNA clone (EST 186H13T7) was supplied by Ohio State
University's Arabidopsis Biological Resource Center. It was
sequenced with a model 373A DNA sequencer (Applied
Biosystems, Tokyo, Japan) and an ABI PRISM Dye Terminator
Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Chiba,
Japan).
Southern analysis
Genomic DNA was isolated using the CTAB method according to
McKinney et al. (1995). RNA was digested by incubation at 37°C
for 5 h with 20 mg ml±1 RNAase A. Aliquots (6 mg) of DNA were
digested, separated on a 0.8% agarose gel, and blotted onto
Hybond N+ ®lter (Amersham). The probe was labelled with [a-32P]
dCTP using a BcaBEST Labelling Kit (Takara, Kyoto, Japan).
Stringent washing conditions were used (Sambrook et al., 1989).
Northern analysis
Total RNA was prepared by the method of Nagy et al. (1988) from
whole plants that had been grown on GM plates for 3 weeks.
Aliquots (40 mg) of total RNA were separated on a 1% agarose gel
containing formaldehyde and blotted onto a Biodyne A nylon
membrane (Pall BioSupport, East Hills, NY, USA). The probe was
labelled with [a-32P] dCTP using a BcaBEST Labelling Kit (Takara).
Hybridization and washing conditions were as described in Ito
et al. (1995).
Histochemical GUS assay
Histochemical GUS analysis of p¯2 plants was done with
X-glucuronide (Chemica Alta, Alberta, Canada) as a substrate,
according to the method of Jefferson et al. (1987).
264 Takuya Ito et al.
Acknowledgements
We thank the Arabidopsis Biological Resource Center (Columbus,
OH, USA) for supplying us with p¯1 and C24 seeds and the EST
clone; Dr T. Araki for providing us with No-0 seeds; Dr K.-I. Taoka
and Dr R. Motohashi for their helpful comments; Dr K.
Yamaguchi-Shinozaki for microscopy; and Ms N. Shiba, Ms S.
Kawamura, Ms H. Kanahara and Ms I. Furukawa for their excellent
technical assistance. This work was supported by a grant for
genome research from RIKEN and in part by the Program for
Promotion of Basic Research Activities for Innovative Biosciences,
the Special Coordination Fund of the Science and Technology
Agency, and a grant-in-aid from the Ministry of Education,
Science, Sports and Culture to K.S. T.I. and G.-T.K. were
supported by a postdoctoral fellowship from the Special Postdoctoral Researchers' Program of RIKEN.
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