Plant Cell Physiol. 43(5): 467–478 (2002)
JSPP © 2002

The ASYMMETRIC LEAVES2 Gene of Arabidopsis thaliana, Required forFormation of a Symmetric Flat Leaf Lamina, Encodes a Member of a Novel
Family of Proteins Characterized by Cysteine Repeats and a Leucine Zipper
Hidekazu Iwakawa 1, Yoshihisa Ueno 1, Endang Semiarti 1, 4, Hitoshi Onouchi 1, 5, Shoko Kojima 2, Hirokazu
Tsukaya 3, Mitsuyasu Hasebe 3, Teppei Soma 1, Masaya Ikezaki 1, Chiyoko Machida 2 and Yasunori Machida 1, 6
1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
College of Bioscience and Biotechnology, Chubu University and CREST, Japan Science and Technology Corporation, 1200 Matsumoto-cho,Kasugai, Aichi, 487-8501 Japan
3
National Institute for Basic Biology, 38 Nishigounaka, Myo-daiji-cho, Okazaki, 444-8585 Japan2;
The nucleotide sequence of the AS2 cDNA has been submitted to
GenBank under accession number AB080802.

The ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana is involved in the establishment of the leaf venation system, which includes the prominent midvein, as wellas in the development of a symmetric lamina. The geneproduct also represses the expression of class 1 knox homeobox genes in leaves. We have characterized the AS2 gene,which appears to encode a novel protein with cysteine
repeats (designated the C-motif) and a leucine-zipper-likesequence in the amino-terminal half of the primary
sequence. The Arabidopsis genome contains 42 putative
genes that potentially encode proteins with conserved
amino acid sequences that include the C-motif and the leucine-zipper-like sequence in the amino-terminal half. Thus,
the AS2 protein belongs to a novel family of proteins that
we have designated the AS2 family. Members of this family
except AS2 also have been designated ASLs (AS2-like proteins). Transcripts of AS2 were detected mainly in adaxial
domains of cotyledonary primordia. Green fluorescent protein-fused AS2 was concentrated in plant cell nuclei. Overexpression of AS2 cDNA in transgenic Arabidopsis plants
resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by lossof-function mutation of AS2. Our results suggest that AS2functions in the transcription of a certain gene(s) in plant
nuclei and thereby controls the formation of a symmetricflat leaf lamina and the establishment of a prominent midvein and other patterns of venation.

Introduction
Leaves of angiosperms, which are relatively flat organs,
exhibit remarkable diversity in terms of their shape and complexity. Nonetheless, the basic structure of each leaf can generally be described in terms of three axes: the proximal-distal,
medial-lateral and adaxial-abaxial axes (Steeves and Sussex
1989, Waites et al. 1998, Hudson 2000, Byrne et al. 2001).
Thus, plants appear to exploit common mechanisms that are
responsible for the establishment of these axes during leaf
development.
Leaves develop as lateral organs from a shoot apicalmeristem (SAM). Various mutants have been isolated withalterations in leaf morphology that are related to the development of shape along each of three axes, to adaxial-abaxial identity, and to the overall shapes of leaves. Some genes responsible for the mutant phenotypes have been cloned andcharacterized (Hake et al. 1989, Conway and Poethig 1997,
Hofer et al. 1997, Kim et al. 1998, Berná et al. 1999, SerranoCartagena et al. 1999, Timmermans et al. 1999, Tsiantis et al.
1999). PHANTASTICA (PHAN) of Antirrhinum majus (Waites
et al. 1998), PHABULOSA (PHB) and PHAVOLUTA (PHV) of
Arabidopsis (McConnell et al. 2001), which appear to encode
myb-like (PHAN) and homeobox-containing transcription
factors (PHB and PHV) are involved in adaxial cell fate.
FILAMENTOUS FLOWER (FIL; Sawa et al. 1999), YABBY3(YAB3; Siegfried et al. 1999), CRABS CLAW (CRC; Bowman
and Smyth 1999) and KANADI (KAN; Kerstetter et al. 2001),
which also appear to encode transcription factors, are involvedin the specification of abaxial cell fate in the leaf lamina. Mutations in these genes convert flat expanded leaves to filamen-

Key words: Arabidopsis thaliana — ASYMMETRIC LEAVES2— C-motif — knox — Leaf development — Leucine zipper.
Abbreviations: AS1; ASYMMETRIC LEAVES1, AS2; ASYMMETRIC LEAVES2, BAC; bacterial artificial chromosome, bp; base pairs,
DAPI; 4¢,6-diamidino-2-phenylindole, GFP; green fluorescent protein, kbp; kilo base-pairs, knox; knotted-like homeobox, ORF; open
reading frame, SAM; shoot apical meristem.
4
5
6

Current address: Faculty of Biology, Gadjah Mada University, Sekip Utara, Yogyakarta 55281, Indonesia.
Current address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
Corresponding author: E-mail, yas@bio.nagoya-u.ac.jp; Fax, +81-52-789-2966.
467

468

ASYMMETRIC LEAVES2 gene for leaf development

tous and rod-shaped structures and they also distort adaxialabaxial identity. Thus, it is likely that the mediolateral development of the leaf lamina might be coupled with the determination by these genes of adaxial-abaxial identity.
With regard to leaf shape along the medial-lateral axis,
the leaves of many plant species commonly exhibit obvious
but approximate left-right symmetry, with the rachis as theaxis (Ogura 1962, Hickey 1973, Hickey 1979, Sinha 1999,
Semiarti et al. 2001a), even though exceptions have beenreported (Whaley and Whaley 1942, Lieu and Sattler 1976,
Dengler 1999). Such symmetry is independent of the complexity of leaf shape (e.g. simple or compound).
To clarify the mechanisms responsible for the development of symmetrical leaves, several groups including our own
have taken advantage of the asymmetric leaves2 (as2) andasymmetric leaves1 (as1) mutants of Arabidopsis. The phenotypes of such mutants are very similar in terms of the asymmetric shape of the lamina and a malformed vein system but theabnormalities are not absolutely identical (Byrne et al. 2000,
Ori et al. 2000, Semiarti et al. 2001a, Sun et al. 2002). The
abnormalities can be summarized as follows. (1) Leaves of as1
and as2 mutants have asymmetric lobes and exhibit downward
curling that is bilaterally asymmetric. (2) They fail to produce athick and distinct midvein and the pattern of the secondaryveins is asymmetric (Semiarti et al. 2001a, Sun et al. 2002). (3)Leaf sections, cultured in vitro on phytohormone-free medium,
regenerate shoots at a higher frequency than sections fromwild-type leaves (Semiarti et al. 2001a). (4) Transcripts of class
1 knox genes, such as KNAT1, KNAT2 and KNAT6, accumulate
in the leaves of the mutants (Semiarti et al. 2001a). (5) Transcripts of AS1, which encodes a myb-like transcription factor
that is related to the products of ROUGH SHEATH2 (RS2) ofmaize and PHAN, accumulate around vascular tissues in cotyledonary and leaf primordia (Byrne et al. 2000).
The observations summarized above suggest that AS1 andAS2 might be involved in the establishment of the entire venation system, which includes the prominent midvein as the
structural axis of left-right symmetry of the leaf, as well as in
the development of lamina symmetry (Semiarti et al. 2001a).
They might also function in maintaining leaf cells in a developmentally determinate state, probably by repressing expressionof class 1 knox genes. Although the roles of these genes in theestablishment of the venation system might be tightly correlated with their roles in maintaining the determinate state ofleaf cells, such correlations remain to be investigated. The similarities among abnormalities in as1, as2, and as1 as2 doublemutant plants have led to the proposal that AS1 and AS2 mightsomehow interact genetically (Semiarti et al. 2001a, Byrne etal. 2002). To obtain a further insight into the mechanisms
whereby AS1 and AS2 control lamina formation, it is obviously important to characterize both the AS2 and the AS1
genes.
In the present study, we isolated and characterized the AS2
gene. This gene encodes a putative novel protein that contains

the cysteine repeats, designated the C-motif, and a leucinezipper-like motif. A database search revealed that AS2 belongs
to a large family of proteins that we designated the AS2 family.
Transcripts of AS2 were mainly detected in adaxial domains ofcotyledonary primordia during embryogenesis. Green fluorescent protein-fused AS2 was localized in plant nuclei eventhough AS2 does not include an obvious nuclear localization
signal. Overexpression of AS2 cDNA resulted in a decrease in
the efficiency of generation of transgenic shoots. Transgenic
Arabidopsis plants that overexpressed the AS2 cDNA produced upwardly curled leaves and, occasionally, rod-shaped
leaves without proper expansion of the lamina. Thus, it appears
that AS2 might control the transcription of certain genes in the
nucleus, the development of a symmetric flat leaf lamina, andthe establishment of leaf venation.

Results
Isolation of the AS2 gene by map-based cloning
The as2 locus was previously mapped between two derivatives of the Ac transposable element known as dAc-I-RS in
two transgenic lines of Arabidopsis, #14-22.4.4W3 (T-1200)
and #14-68.1.4 (T-1700), which were generated in our laboratory (Machida et al. 1997; Fig. 1A). Each transposable element
included a hygromycin-resistance gene. Thus, for map-based
cloning, we used these two dAc-I-RS elements as genetic andmolecular markers, in addition to other PCR-RFLP markers.
As shown in Fig. 1A, the AS2 locus was finally mapped to a
32-kbp region between two PCR-RFLP markers (Marker 1 andMarker 4) in the BAC clone F5I14. We next performed PCRRFLP analysis and sequenced this region of DNA from an as2-1
mutant. We found a 13-bp deletion in the open reading frame(ORF) corresponding to the ORF15 of BAC clone F5I14. In
addition, we found that all other available mutant alleles, suchas as2-2, as2-4 and as2-5, had mutations in this ORF (fordetails, see Fig. 2A and below). To determine whether a mutation in the ORF15 might be responsible for the as2 phenotype,
we introduced an ApaI–HpaI fragment of the wild-type
genome (fragment I; 6.16 kb), which included all of ORF15,
into as2-1 plants (Fig. 1B). We also transformed as2-1 plants
with fragment II, which lacked the region upstream of ORF15,
and with fragment III, which lacked the upstream region of
ORF15 as well as the region from the middle of ORF15 to the3¢ end of fragment I (Fig. 1B). Only fragment I reversed theas2 phenotype. ORF15 is the only reading frame in fragment I
that can encode a protein of more than 3,000 Da. Thus, theresults indicate that ORF15 corresponds to the AS2 gene. The
failure of fragment II to complement the as2 phenotype suggested that the region between ApaI and Bsu36I of fragment I
might be required for the expression of AS2.
Characterization of as2 alleles
We isolated a cDNA clone that corresponded to theASYMMETRIC LEAVES2 gene for leaf development

Fig. 1 Structure of the region of the Arabidopsis genome that
includes the AS2 locus, which is located at the lower part of chromosome 1, and the results of complementation analysis with segments ofthe genome. (A) Map-based cloning of the AS2 gene. The AS2 locus
was mapped between two markers, Marker 1 and Marker 4 on bacterial artificial chromosome (BAC) clone F5I14. Other BAC clones thatoverlapped the region of interest are also shown. Numbers above themarkers indicate numbers of recombinants between respective markers and the AS2 locus (in most cases, we examined more than 10,000
chromosomes). T-1200 and T-1700 show the positions of the dAc-I-RS
transposable elements with the hygromycin-resistance (Hygr) genefrom which we started the chromosome walking. Marker 1, Marker 2,
Marker 3, and Marker 4 are RFLP markers that we generated for the
study. (B) Magnified view of the chromosomal region around AS2 andthe results of complementation analysis. The structure of AS2 cDNAis shown under the genomic structure. Restriction fragments that wereused in complementation analysis are indicated by horizontal lines
labeled I, II, and III. Symbols + and – on the right indicate positive andnegative results, respectively, of complementation tests with the corresponding DNA fragments.

ORF15. Comparison of the nucleotide sequence of the AS2
gene with that of the cDNA suggested that the mRNA contains
two introns in the 5¢-untranslated region but no introns in thecoding region (Fig. 1B) and, furthermore, that AS2 encodes anovel protein with 199 amino acid residues (Fig. 2A). The
as2-1 and as2-2 alleles both had 13-bp deletion at the same
position upstream of the coding sequence for the leucinezipper-like sequence (for details, see below; Fig. 2A, C). This

469

deletion should result in production of a short polypeptide thatlacks of most of the carboxyl-terminal (C-terminal) half of AS2
but contains 13 additional amino acid residues (Fig. 2A). In theas2-4 mutant, there is one bp deletion in the middle of thecoding region that corresponded to the C-terminal half of AS2
and this deletion generated a frame-shift mutation (Fig. 2A),
which resulted in replacement of the C-terminal 48 amino acidresidues of AS2 with the 63 new residues. In the as2-5 mutant,a single guanine nucleotide was replaced by an adenine nucleotide in the coding region for the amino-terminal (N-terminal)
half of the protein, resulting in substitution of the glycine residue at position 46 by a glutamic acid residue (Fig. 2A). This
glycine residue is conserved in all members of the AS2 family,
as described below. Therefore, this glycine residue is essential
for the function of AS2.
Structural characteristics of AS2 and related proteins
The predicted AS2 protein contained a leucine-zipper-likesequence, from residue 81 to residue 109, which included five
repetitions of hydrophobic amino acid residues, such as valine,
isoleucine and leucine, with six-residue intervals (Fig. 2A). A
search of databases revealed that the N-terminal half of AS2
was similar, in terms of amino acid sequence, to the N-terminal halves of a number of putative proteins encoded by hypothetical genes and ESTs of Arabidopsis and other plant species(Oryza sativa, Glycine max, Lycopersicon esculentum, etc.;
Fig. 2B). The public databases of sequences of the Arabidopsis
genome include at least 41 ORFs that are predicted to encode
proteins with N-terminal halves that are related to that of AS2.
Of these proteins, several appear to be only distantly related toAS2 (Fig. 2B).
As shown in Fig. 2B, comparative analysis of the putative
AS2-like proteins revealed that the Cx2Cx6Cx3C sequence(where x is an unconserved residue; designated the C-motif)
was completely conserved in the N-terminal halves of all 41
predicted proteins identified in the database search and AS2(positions from 10 to 24 in AS2). In addition to the C-motif,
the leucine-zipper-like sequence was also strongly conserved inmost of the 41 proteins. More than a half of the proteins,
including AS2, had additional conserved sequences, as follows: PCAACKFLRRKCxxxCVFAPYFP in and around the C-

Fig. 2 The predicted amino acid sequence of AS2 and comparison of the amino acid sequences of the AS2 domains of AS2 and AS2-like proteins. (A) Predicted amino acid sequence of AS2. The positions of cysteine residues in the C-motif and hydrophobic residues in the leucine-zipper-like sequence are shown by asterisks and dots, respectively. Mutated amino acid residues in various as2 alleles are indicated in italics above
the wild-type sequence. (B) Comparison of the amino acid sequences of the AS2 domains in members of the AS2 family. The sequence from residue 8 to residue 109 of AS2 is aligned with sequences from corresponding regions of members (designated ASL; see text) of the AS2 family.
Amino acid residues conserved in more than 20 members are indicated by white characters on a black background. The consensus sequences of
the C-motif and hydrophobic residues in the leucine-zipper-like sequence are indicated by asterisks and dots, respectively. The glycine residue
that is conserved in all members of the family and mutated in the as2-5 allele is marked by two asterisks. The members of the Arabidopsis AS2
family can be divided into two classes (class I and class II). Amino acid residues conserved in more than three members of class II are shaded ingray. O. s., Oryza sativa; G. m., Glycine max; and L. e., Lycopersicon esculentum. (C) Domain organization and characteristic features of AS2.
The region indicated by a bracket below the box is the AS2 domain. The shaded box and the striped box represent the C-motif and the leucinezipper-like sequence, respectively. Sites of as2-1, as2-2, as2-4, and as2-5 mutations are indicated. Numbers below the box indicate positions ofamino acid residues.

470

ASYMMETRIC LEAVES2 gene for leaf development

Fig. 2

motif; FAxVHKVFGASNVxKLL between the C-motif and theleucine-zipper-like sequence; and RxxAVxSLxYEAxARxRDPVYGCVGxISxLQxQL(V or I)xxLQxxLxxxxxxL(V or I) in
and around the leucine-zipper-like sequence (Fig. 2B). Despite

the strong conservation of amino acid sequence at the Nterminus of AS2, the amino acid sequence of the C-terminal
region of AS2 was unlike those of the other AS2-like proteins
and other proteins in the database (Fig. 2C).

ASYMMETRIC LEAVES2 gene for leaf development

Fig. 3 An unrooted maximum-likelihood tree for 42 members of theAS2 family of proteins from Arabidopsis, as generated by a local rearrangement search. Numbers on branches represent local bootstrap values, which were calculated with the ProtML program. The length ofeach horizontal branch is proportional to the estimated evolutionary
distance. The brackets on the right indicate the classification of members of the AS2 family.

In AS2, the N-terminal sequence is characterized by the
C-motif and the leucine-zipper-like sequence. We propose that
this characteristic region be designated the AS2 domain andthat proteins that include this domain be designated membersof the AS2 family. According to this designation, the genomeof Arabidopsis contains 42 ORFs that potentially encode proteins that belong to the AS2 family. As shown in Fig. 2B, wechose the designation AS2-like genes (ASLs) for the genes or
putative genes for the 41 proteins that resembled AS2 and numbered these genes 1 to 41, respectively. Nucleotide sequences ofsome cDNAs of these genes have been submitted to the GenBank database and named LOB and LBD by Shuai, B. andSpringer, P.S. (2001; Table 2).
The AS2 family consists of at least two major classes of proteins
Members of the AS2 family can be divided into at least
two classes, class I and class II (Fig. 2B). Class I consists of
AS2 and 35 proteins (from ASL1 through ASL35) that include471

Fig. 4 Sites of accumulation of AS2 transcripts. (A) Northern blotting analysis of poly(A)+ RNA from various organs of Arabidopsis.
Aliquots of 1.0 mg of poly(A)+ RNA were prepared from roots (lane1), cotyledons (lane 2), leaves (lane 3), and shoot apices (lane 4) of 12day-old plants; from flower buds (lane 5) of 28-day-old plants; andfrom rosette leaves (lane 6), cauline leaves (lane 7), inflorescence (I)stems (lane 8), and siliques (lane 9) of 35-day-old plants. The same
blot was reprobed with a gene for Éø-tubulin (TUBA) as a control.
The numbers on the right indicate the sizes of marker RNA molecules. (B) Detection of AS2 transcripts by in situ hybridization. (a–d)
Patterns of distribution of AS2 transcripts obtained with an antisense
probe. (a) Globular stage; (b) triangular stage; (c) heart-shaped stage;(d) torpedo stage; (e) sense control for panels (a) through (d). Reddish
brown coloration in the cell layer beneath the seed coat (indicated bywhite asterisks) was not specific to AS2 RNA because it was generated by both probes. Scale bars: 20 mm.

Fig. 5 Subcellular localization of AS2::GFP in onion epidermal
cells. Plasmids that carried 35S::AS2::GFP (A, B), 35S::NLS::GFP (C,D) and 35S::GFP (E, F) were introduced by bombardment into onion
epidermal cells and introduced tissues were cultured for 16 h at 28°C
(see text for details). Fluorescence was monitored under a fluorescence microscope (left panels). Nuclei stained with 1 mg ml–1 DAPI
are shown in the right panels. Scale bars: 100 mm.

472

ASYMMETRIC LEAVES2 gene for leaf development

Fig. 6 Phenotypes of transgenic Arabidopsis calli and plants that overexpressed AS2 cDNA and accumulation of transcripts of AS2 gene(s). (A)Results of transformation of root segments from Col-0 with the empty vector pSK1 (a) and with pSK35S::AS2 (b). Transformed root segments
were incubated as described in the text. Photographs were taken 21 days after the start of incubation. Scale bars: 10 mm. (B) Phenotypes of transgenic Arabidopsis plants. (a) A 35-day-old plant transformed with the empty vector pSK1; (b) a 35-day-old plant transformed with pSK35S::AS2;(c) a 25-day-old plant transformed with pSK35S::AS2; (d) the plant in panel c was grown for a further 15 d. Scale bars: 10 mm. (C) Analysis by
RT-PCR of transcripts of the AS2 gene(s) in transgenic and wild-type (Col-0) plants. See Materials and Methods for details of RT-PCR. The
number of cycles is indicated at the right of each panel. Amplified DNA fragments were separated by electrophoresis on an agarose gel and visualized by staining with ethidium bromide. Lane 1, the transgenic plant shown in panel Bd; Lane 2, wild-type plant (Col-0). Products that were
amplified with primers specific for the region of open reading frame of AS2 transcripts (a; ORF), 5¢- and 3¢-untranslated regions of the AS2 transcripts (b; UTR), and transcripts of the gene for a-tubulin (c; a-tubulin) are shown. (D) Leaf phenotypes. The fifth leaves from a typical 35-dayold wild-type Col-0 plant (a) and as2-1 plant (b) are shown. Typical leaves of transgenic plants that exhibited mild phenotypic changes (c) andsevere phenotypic changes (d) are shown. The positions of these latter leaves could not be determined. Scale bars: 5 mm.

a C-motif, a leucine-zipper-like sequence and most of the
major conserved residues noted above. Class II consists of six
proteins (from ASL36 through ASL41). These proteins include
a C-motif and an incomplete leucine-zipper-like sequence, inwhich the fourth hydrophobic residue is missing, and there is
weaker sequence conservation in the AS2 domain. Furthermore, most amino acid residues are conserved among all members of class II, with only a few respective residues being different in each protein in this class.
As shown in Fig. 3, a phylogenetic tree confirmed thatclass II is only distantly related to class I and suggested thatmembers of class I can be divided further into sub-classes,
namely class Ia and class Ib, with weaker sequence conservation but a complete leucine-zipper-like sequence.

Site of expression of the AS2 gene and nuclear localization of
the gene productWe examined the sites of accumulation of AS2 transcripts
in wild-type plants. We purified poly(A)+ RNA from roots, cotyledons, leaves, and shoot apices of 12-day-old plants, fromflower buds of 28-day-old plants, and from rosette leaves,cauline leaves, influorescence stems and siliques of 35-day-old
plants and then subjected the samples of RNA to Northern blot
analysis with AS2 cDNA as the probe. We detected AS2 transcripts in all samples analyzed, with the exception of thosefrom inflorescence stems (Fig. 4A). The level of AS2 transcripts was highest in the sample from shoot apices that
included small developing leaves. These data are consistent
with the previous observation that as2 mutations are evident as

ASYMMETRIC LEAVES2 gene for leaf development

Table 1Frequency of generation of transformed shoots

Plasmid

Masses of roots examined

pSK1
pSK1::35S::AS2
a

18
21

Numbers ofGreen calli
Shoots76 (4.2)
31 (1.5)a45 (2.5)
14 (0.67)

473

Shoots with stems
22 (1.2)
4 (0.19)

Numbers in parentheses are average numbers of transformants per mass of roots.

mutant phenotypes in all leaf-like organs, such as cotyledons,rosette leaves, cauline leaves, sepals, and petals, but not instems (Semiarti et al. 2001a). However, although AS2 transcripts were detected in roots, the roots of as2 plants resembled wild-type roots.
We also examined sites of expression of AS2 during
embryogenesis by in situ hybridization with AS2 cDNA as theprobe. In embryos of the globular stage, AS2 transcripts weredetected in all cells (Fig. 4Ba). As bulges formed at the upperparts of triangular embryos, AS2 transcripts accumulated in theuppermost regions of the embryos (Fig. 4Bb). From the heartshaped stage to the torpedo stage, we detected signals in protoderm cells on the adaxial side of cotyledonary primordia (Fig.
4Bc, Bd). After the walking-stick stage, the accumulation of
AS2 transcripts was detected throughout cotyledonary primordia without any obvious concentration in a specific region (datanot shown). After germination, no specific hybridization signals were detected in situ around shoot apical meristems, in leaf
primordia and in mature leaves. However, the accumulation of
AS2 transcripts was confirmed by Northern blotting (Fig. 4A).
Nuclear localization of AS2
The predicted AS2 protein does not include an obvious
nuclear localization signal (NLS, Fig. 2A ). However, to determine whether AS2 is localized in plant cell nuclei, we generated a fusion construct in which AS2 cDNA was fused to the
DNA for green fluorescent protein (GFP), with transcription
under the control of the 35S promoter of cauliflower mosaic
virus (CaMV) (35S::AS2::GFP). The fusion construct was
introduced into onion epidermal cells by particle bombardment
and fluorescence due to GFP in the cells was monitored. As
shown in Fig. 5, fluorescence signals due to AS2-GFP, as wellas similar signals due to NLS-GFP, prepared with the NLS of
SV40, were detected mainly in nuclei (Fig. 5A, C). When the
35S::GFP construct was introduced into onion cells, signals
due to GFP were detected both in the nucleus and the cytoplasm(Fig. 5E). These results suggest that AS2 is a nuclear protein.
Phenotypes of Arabidopsis plants that overproduce AS2
To investigate the function of the AS2 gene in further
detail, we fused AS2 cDNA to the 35S promoter (35S::AS2)
and attempted to generate transgenic Arabidopsis plants thatoverexpressed AS2. Our preliminary data demonstrated that it
was difficult to generate transformed shoots (Fig. 6A). Therefore, we first determined the approximate efficiency of genera-tion of transformed shoots by a conventional root-transformation procedure. We counted the numbers of transformed green
calli, regenerated shoots, and shoots with inflorescence stems
per mass of roots used for transformation. Table 1 shows thatwhen 35S::AS2 was introduced into root tissue, the efficiency
of formation of green calli was lower than that when empty
vector plasmid pSK1 was used for transformation. The efficiencies of regeneration of transgenic shoots and shoots withinflorescent stems were 5- to 10-fold lower (Table 1). These
data suggested that overexpression of AS2 cDNA had an inhibitory effect on cell proliferation in green tissues and/or theregeneration of shoots.
We obtained two transgenic lines that overexpressed AS2
cDNA. We confirmed that AS2 transcripts that were derived
from the cDNA were accumulated in one of the transgeniclines that exhibited the severer phenotype (Fig. 6C). One lineof transgenic plants had a mildly dwarf phenotype and generated leaves that curled upward (Fig. 6Bb). The other line oftransgenic plants, with a severely abnormal phenotype, generated leaves with a very narrow lamina at the early stages ofplant growth (Fig. 6Bc, Dc). The leaf lamina failed to expand
and generated rod-like leaves (Fig. 6Bd, Dd).

Discussion
Functional domains in the AS2
The AS2 gene appears to encode a novel protein of 199
amino acid residues that includes a C-motif (defined as
Cx2Cx6Cx3C) and a leucine-zipper-like sequence in its Nterminal half and an apparently unique sequence in its Cterminal half (Fig. 2A, C). It is generally accepted that aleucine-zipper sequence is involved in protein–protein interactions (Ellenberger et al. 1992) and it seems likely that theleucine-zipper-like sequence of AS2 might also play a role in
the association of AS2 with some other protein(s). Such interaction(s) might be essential for actions of AS2.
The C-motif was identified for the first time in this study
but it is rather similar to a zinc finger, which generally has theconsensus sequence Cx2-3CxnCx2-3C (n >12) and which functions in interactions with other macromolecules (Pavletich andPabo 1991, Wang et al. 1998). It is likely that the C-motif isalso involved in association(s) with other macromolecules,
such as DNA and proteins, and with AS1, in particular (Semiarti et al. 2001a, Byrne et al. 2002, our unpublished data). The
identification and characterization of the molecules with which

474

Table 2ASYMMETRIC LEAVES2 gene for leaf development

Members of the AS2 family of proteins in A. thaliana

Member BAC code a Gene code b EST ID c

LOB d

AS2
ASL1
ASL2
ASL3
ASL4
ASL5
ASL6
ASL7
ASL8
ASL9
ASL10
ASL11
ASL12
ASL13

F5I14.15
MUD21.13
F26B6.31
MGF10.6
MDC12.5
T27E13.13
T19E23.11
T17D12.6
F24B9.1
F3O9.33
T9D9.15
T2P4.18
F9F8.10
MLJ15.5

LBD6

ASL14
ASL15
ASL16
ASL17
ASL18
ASL19
ASL20
ASL21
ASL22
ASL23
ASL24
ASL25
ASL26
ASL27
ASL28
ASL29
ASL30
ASL31
ASL32
ASL33
ASL34
ASL35
ASL36
ASL37
ASL38
ASL39

MLJ15.1
At3g26620
MHK10.16 At2g42440
F9D24.100 At3g58190
F16D14.15 At2g31310
MHK10.15 At2g42430
F6N15.4
At4g00220
F4L23.41 At2g45420
F20H23.21 At3g03760
F6N15.25 At4g00210
F4L23.45 At2g45410
K16F4.4
At5g06080
T20E23.110 At3g50510
T12H17.90 At4g22700
MIK22.21 At5g35900
K24A2.3
At3g27940
T23J7.200 At3g47870
MCP4.8
At3g13850
F3N23.18 At1g72980
F9P14.14
At1g06280
T22A15.8 At1g36000
F3P11.11
At2g19510
F6F22.15
At2g19820
T26J14.8
At1g68510
F5A8.2
At1g67100
F16B3.18 At3g02550
K8K14.16 At5g67420

ASL40
ASL41

F3A4.20
F19F18.30

At1g65620
At5g66870
At2g23660
At3g27650
At5g63090
At2g30130
At1g31320
At2g28500
At1g07900
At1g16530
At2g30340
At2g40470
At3g11090
At3g26660

Z33806, Z25656

LBD25
LOBa

AV554524, AV551296, AV538901
AI993799, N97300, BE529105

LBD13
LBD15

AV519677, AV565474, AV555285, AV560157, AV556223, AV557695, AV535896,
AV564540, AV565846, AV439949, AV561987, AV567074, AV519558

AV553261, AV544763

LBD29

AI994962, AV563330
BE520513, BE521898, BE521897

LBD16
LBD30
LBD18

AV559860
LBD42
BE520541, BE520808, BE520344, BE521846, BE524729, BE520809, BE520810
AV537704, AI996685, AV549715, H76116, AV563797, T42227, AV563144
LBD41
AV525400, AV526154, T41721, AV551431, H36818, F14269, AV550089, T14105, LBD37
N38449, F14441, AI996949, N65652
At3g49940 T76164, Z29130, F13856
At4g37540 AI994989, R65200

a, bd

ORF number with BAC clone name and corresponding gene code obtained from TAIR database. c EST ID obtained from EMBL database.
Some of ASL proteins have been submitted to the GenBank database as LOB domain (LBD) proteins (see text).

AS2 interacts will be critical to the elucidation of the functionsof AS2.
The sequence of the C-terminal half of AS2 is unlike thatof any protein in the databases, including known motifs (Fig.
2C). Since the as2-4 allele had a frame-shift mutation in thisregion and the phenotype due to this allele is similar to thoseASYMMETRIC LEAVES2 gene for leaf development

due to other as2 alleles (Semiarti et al. 2001a), it seems that the
C-terminal half is essential for the functions of AS2, in addition to the N-terminal half, which contains the newly characterized AS2 domain.
AS2 is a nuclear protein that negatively regulates expression ofclass 1 knox genes
Both AS1 and AS2 repress the expression of class 1 knox
homeobox genes (Byrne et al. 2000, Ori et al. 2000, Semiarti etal. 2001a), but it is unclear whether such repression is directly
or indirectly attributable to AS1 and AS2. In the present study,
we showed that the AS2::GFP fusion protein was localized in
the nuclei of plant cells (Fig. 5), as would be expected for a
protein that is involved in repression of class 1 knox genes innuclei via interactions with other, as yet unidentified nuclear
proteins. The AS1::GFP fusion protein was also localized innuclei (our unpublished data). This observation is consistent
with the hypothesis that AS1 might be one of the moleculeswith which AS2 interacts in nuclei.
AS2 is involved in the development of a flat leaf lamina
Overexpression of AS2 cDNA induced the upward curling of leaves and cotyledons (Fig. 6). By contrast, loss-offunction mutations in AS2 result in the downward curling ofleaves and cotyledons (Semiarti et al. 2001a). Therefore, it
seems plausible that AS2 might be involved in suppression of
the growth of the adaxial domain of the leaf lamina or in stimulation of the growth of the abaxial domain. Considering the
growth-inhibitory effects that we observed upon transformation (Fig. 6B) and the expression of AS2 in the adaxial domain
in the cotyledonary primordia in embryos (Fig. 4B), we canspeculate that AS2 might function in suppression of the adaxial domain. It will be of interest to determine whether such suppression is achieved via the inhibition of the proliferation or the
expansion of cells.
Whatever the details of the activity of AS2 at the molecular level, the present study of overproducers and previous analysis of as2 mutant leaves indicate that AS2 is responsible forthe expansion of a flat leaf lamina, as well as for leaf symmetry. As described in the Introduction, mutations in genes thatare responsible for the adaxial-abaxial fates of leaf domains
interfere with the fate determination of leaves and often induce
formation of rod-shaped leaves (Waites et al. 1998, Sawa et al.
1999, McConnell et al. 2001). There might be a relationshipbetween AS2 and these genes. In this context, it is worth noting that AS1 is an Arabidopsis homolog of PHAN, eventhough it remains to be determined whether the functions of
AS1 are analogous to those of PHAN.
Possible functions of ASL proteins
The conservation of amino acid sequence in the N-terminal halves (the AS2 domains) of AS2-like proteins led us topropose that these proteins might form a new family of plant
protein, designated as the AS2 family. We named Arabidopsis

475

proteins that are members of this family, with the exception ofAS2, ASL (AS2-like) proteins (Fig. 2B). The similarities
among amino acid sequences and a phylogenetic tree of AS2
domains of ASLs revealed that ASL proteins could be divided
into class I, with sub-classes class Ia and class Ib, and class II(Fig. 2B, 3).
The functions of members of the AS2 family, with the
exception of AS2, remain to be identified. AS2 is required forthe formation of a symmetric, flat, round leaf lamina and forthe establishment of venation patterns that include prominent
midveins (this study and Semiarti et al. 2001a). In addition,
AS2, acting in conjunction with AS1, has the ability to repress
the expression of class 1 knox homeobox genes (Ori et al. 2000,
Byrne et al. 2000, Semiarti et al. 2001a, Byrne et al. 2002).
Since substitution of the conserved glycine residue with theglutamic acid residue in the AS2 domain resulted in generationof mutant phenotypes (Fig. 2), it is clear that this residue aswell as the AS2 domain play an important role in the functionof AS2. Although some members of class Ia exhibit strong
conservation of amino acid sequence of AS2 domains (forexample, ASL1 and ASL2), they might have distinct functions
from AS2, because the C-terminal region of AS2, which is
unique, is required, in addition to the N-terminal AS2 domain
for the functions of AS2 (Fig. 2; Semiarti et al. 2001a, ourunpublished data). Clearer insight into the roles of these closely
related ASLs requires further genetic and molecular experiments.
It was proposed recently that the LOB gene (accession
number AF447897), which corresponds to the gene for ASL4,
is normally expressed at the boundary between meristems andorgan primordia but is absent in as1 and greatly reduced in as2(Byrne et al. 2002). The developmental role of the LOB gene,
however, remains to be determined.
The six ASLs that constitute class II have characteristic
structural features that are distinct from class I (Fig. 2B) andtheir amino acid sequences are very similar to each other.
Expression of these genes has been reported (Table 2), suggesting that they are functional genes. Therefore, it seems likelythat members of class II might play distinct roles during development in Arabidopsis.

Materials and Methods
Plant strains and growth conditions
A. thaliana ecotype Col-0 (CS1092) and mutants as2-1 (CS3117)
and as2-2 (CS3118) were obtained from the Arabidopsis Biological
Resource Center (Columbus, OH, U.S.A.; ABRC). Our RFLP analysis showed that the background of as2-1 coincided with the Col-0 ecotype (Semiarti et al. 2001a). Ler-0 (NW20) and as2-4 (N463) wereobtained from the Nottingham Arabidopsis Stock Center (Nottingham, U.K.; NASC). The as2-5 allele was isolated from an M2 population of ethylmethanesulfonate-mutagenized Ler-0 seeds, purchasedfrom Lehle Seeds (Round Rock, TX, U.S.A.; Semiarti et al. 2001a).
The transgenic lines #14-22.4.4W3 (T-1200) and #14-68.1.4 (T-1700)
were isolated from the (I-RS/dAc-I-RS)#14 line; they carried a trans-

476

ASYMMETRIC LEAVES2 gene for leaf development

posed dAc-I-RS element (accession no. AB055064) that included agene for hygromycin phosphotransferase (Machida et al. 1997, Semiarti et al. 2001b). The background of #14-22.4.4W3 (T-1200) and #1468.1.4 (T-1700) was the Ler ecotype in both cases. For analysis ofplants, seeds were sown on soil. After 2 d at 4°C in darkness, plants
were transferred to a regimen of white light at 3,000 lux for 16 h daily
at 22°C as described previously (Semiarti et al. 2001a). Ages of plants
are given in terms of numbers of days after sowing.
Cloning of the AS2 gene
Meiotic recombination breakpoints were generated and identified
near the AS2 gene by screening for recombination between the as2-1
mutation and the flanking transposon markers #14-68.1.4 (T-1700)
and #14-22.4.4W3 (T-1200). These recombinants, which were shown
to be resistant to hygromycin and to have the as2-1 phenotype, wereused to map PCR-RFLP markers relative to the AS2 gene. The information related to sequences used as PCR-RFLP markers was obtained
from the web site of the Arabidopsis thaliana Genome Center at the
University of Pennsylvania, TAIR web site (http://www.arabidopsis.org/)
and the nucleotide sequence of the BAC clone F5I14 (GenBank, accession no. AC001229). PCR-RFLP markers were as follows: for Marker
1, 5¢TGTAACTCTTCCGTCCGGTTTG3¢ and 5¢GCAAAGTCCATAGAGGAGCAAG3¢; for Marker 2, 5¢TTGGGTTTGCACCGAAACTCAG3¢ and 5¢AGTGACAGACAGTGACCACAAG3¢; for Marker 3,
5¢TGGTGGGATGAAACTTTGTGAG3¢ and 5¢CTCTCTCTTTCTCACTCTTCTC3¢; and for Marker 4, 5¢AGAAAACTGCTGTCTTCGGGAC3¢ and 5¢TCCAAAGCACTCTCTAGCTTGG3¢ (Fig. 1).
Complementation analysis
To construct the binary plasmids for complementation experiments, the 6.16-kbp ApaI–HpaI fragment (fragment I in Fig. 1), the4.75-kbp Bsu36I–HpaI fragment (fragment II in Fig. 1) and the 2.2kbp Bsu36I–BglII fragment (fragment III in Fig. 1) were inserted separately into the pBI-BAR plasmid, which was a derivative of pBI101(Jefferson et al. 1987) and contained 1¢promoter::bar (Yoshioka et al.
2001). The newly constructed plasmids were introduced into Agrobacterium tumefaciens strain GV3101. Whole plants were then transformed by vacuum infiltration, as described elsewhere (Clough andBent 1998, Galbiati et al. 2000). Transgenic plants were selected on
soil that contained 0.01% Basta (AgroEvo, Frankfurt, Germany),
which is what genotypes of transgenic plants were analyzed by Southern blotting.
Cloning of cDNA
Poly(A)+ RNA was prepared from 16-day-old plants. For reverse
transcription-PCR, first-strand cDNA was generated as described elsewhere (Hamada et al. 2000). Then AS2 cDNA was amplified by PCR
with primers ORF15SF (5¢GGGTCGACATGGCATCTTCTTCAACAAACTCAC3¢) and ORF15NR (5¢GGGCGGCCGCTCAAGACGGATCAACAGTACGGC3¢). The amplified fragment was then ligatedinto the SalI/NotI site of pBluescript SK(–) (Stratagene, La Jolla, CA,
U.S.A.) and its identity was confirmed by nucleotide sequencing. The
5¢-end sequence of the cDNA was determined by 5¢-RACE withprimer ORF15MR (5¢TATCTGAAGCTGACGAAGCTGATG3¢) andan adapter primer. The 3¢-end of the cDNA was determined by 3¢RACE with primer ORF15MF (5¢GATCTCAGCTGTGCTAAATCTGAGC3¢) and an adapter primer. Nucleotide sequences of mutant alleles(as2-1, as2-2, as2-4 and as2-5) were determined by amplifying theregion of the as2 gene from the genomic DNA of each mutant. The
nucleotide sequence of the as2 cDNA obtained from the poly(A)+RNA that has been isolated from each mutant plant was also determined.

Phylogenetic analysis
Arabidopsis genes that resemble AS2 were obtained from the
AGI data set (Proteins from AGI, Total Genome) at TAIR using theTAIR BLAST version 2.0 program (http://arabidopsis.org/Blast/index.html). The AS2-like genes of other plant species were obtained
from the nr data sets at GenomeNet using the GenomeNet BLAST2
program (http://www.blast.genome.ad.jp/). The sequences of AS2
domains of 42 members of the AS2 family from Arabidopsis werealigned using CLUSTAL W, version 1.8 (Thompson et al. 1994). For
construction of the maximum-likelihood (ML) tree, we used a neighborjoining (NJ) tree as the start tree for a local rearrangement search. We
used the NJdist and ProtML programs in the MOLPHY, version 2.3b3,
package (http://www.ism.ac.jp/software/ismlib/softother.html#molphy;
Adachi and Hasegawa 1996). The NJ tree was obtained with NJdist
and the ML tree was obtained with ProtML. The local bootstrap probability of each branch was estimated using the ProtML program (Himiet al. 2001, Sakakibara et al. 2001).
RNA gel blot analysis
For analysis of Col-0 wild-type plants, we used roots, cotyledons, leaves, and shoot apices collected from 12-day-old plants; flower
buds collected from 28-day-old plants; and rosette leaves, cauline
leaves, influorescence stems, and siliques collected from 35-day-old
plants. Aliquots of 1.0 mg of poly(A)+ RNA were isolated and Northern blotting was performed as described previously. Partial cDNA for
AS2 was used as the probe. It was amplified by PCR with primer 1(5¢GATCTCAGCTGTGCTAAATC3¢) and primer 2 (5¢TCAAGACGGATCAACAGTAC3¢) and cloned into the EcoRV sites of pBluescript
SK(–). Its identity was confirmed by nucleotide sequencing. Then a
300-bp fragment, generated by cleavage of the plasmid pAS2c300
with EcoRI and ClaI extended from codon 151 to codon 200 of the
ORF, which has the termination codon, was labeled with [a-32P]dCTP
using a High Prime DNA Labeling Kit (Boehringer Mannheim
Biochemica, Mannheim, Germany) according to the manufacturer’s
instructions. The labeled fragment was used as probe.
In situ hybridization
Details of methods used for fixation of plants, embedding inparaffin and in situ hybridization can be found at http://www.genetics.
wisc.edu/CATG/barton/index.html and were described by Nakashima
et al. (1998). Sections (thickness, 8 mm) were cut with a microtome(ERMA Inc. Tokyo, Japan). The antisense RNA probe was generated
by T3 RNA polymerase after plasmid pAS2c300 has been linearizedwith ClaI. The sense RNA probe was generated by T7 RNA polymerase after linearization of pAS2c300 with EcoRI.
Construction of plasmids
The plasmid pSK35S::AS2 was constructed by inserting a fragment of AS2 cDNA (600-bp), which extended from the initiation
codon to the termination codon, at XbaI and NotI sites of binary vector pSK1 (Kojima et al. 1999) downstream of the CaMV 35S promoter. The plasmid p35S::AS2::GFP was constructed by inserting a
fragment of AS2 cDNA (597 bp) fragment, which extended from theinitiation codon to the 199th amino acid, and three-alanine linker
sequence (5¢GCAGCTGCC3¢) at the SalI and NcoI sites of pTH-2(Chiu et al. 1996). Plasmids that included 35S::NLS::GFP and
35S::GFP were described previously (Nishihama et al. 2001).
Transformation of Arabidopsis
Agrobacterium-mediated transformation of root explants of Arabidopsis was performed as described by Onouchi et al. (1995). Root
explants were cocultured with Agrobacterium LBA4404/pAL4404(Hoekema et al. 1984) cells that harbored pSK35S::AS2 or pSK1

ASYMMETRIC LEAVES2 gene for leaf development(Kojima et al. 1999). Transformants were selected on shoot-induction
medium in the presence of 15 mg liter–1 hygromycin B (Onouchi et al.
1995). Plasmids pSK35S::AS2 and pSK1 were used to transform A.
tumefaciens strain GV3101. Whole plants were then transformed by
vacuum infiltration. Transgenic plants were selected on MS medium
contained 15 mg liter–1 hygromycin B and 300 mg liter–1 carbenicillin.
Subcellular localization of the AS2::GFP protein
Cells in epidermal layers of onion bulbs were transformed with
p35S::AS2::GFP, p35S::NLS::GFP or p35S::GFP, as described previously (von Arnim and Deng 1994). Fluorescent signals were recordedwith a fluorescence microscope (Axioplan2; Carl Zeiss, Oberkochen,
Germany) equipped with a cooled CCD camera system (Photometrics,
Tucson, AZ). Pseudocoloring of the images and measurements ofextents of signals from GFP and cell widths were performed with the
IPLab software program (Scanalytics, Fairfax, VA). The same cells
were stained with 1 mg ml–1 4¢,6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence was recorded similarly.
Reverse transcription-polymerase chain reaction (RT-PCR)
Flower buds of a transgenic plant and wild type were harvested,frozen immediately in liquid nitrogen and stored at –80°C. Poly(A)+RNA was purified and the first strand of cDNA was synthesized. Sample volumes were normalized for equal amplification of DNA fragments with primers specific for a-tubulin cDNA. Then, PCR was performed as described by Semiarti et al. (2001a). To amplify DNA
segments specifically derived from transcripts of the endogenous AS2
gene, we selected sites for the design of primer sets in 5¢- and 3¢untranslated regions of AS2 transcripts, both of which were absent in
the cDNA used for generation of transgenic plants. The primer pairs
were as follows: for a-tubulin, pU51 (5¢-GGACAAGCTGGGATCCAGG-3¢) and pU52 (5¢-CGTCTCCACCTTCAGCACC-3¢); foruntranslated regions of cDNA of AS2 gene, pU73 (5¢-CCCCTCTGAGCAACAGAAGC-3¢) and pU74 (5¢-CCAAAACCCTAAAATCTCAAGACGG-3¢); for the region of the open reading frame of AS2
cDNA, pU328 (5¢-GTGTTTGGAGCAAGTAACGT-3¢) and pU315 (5¢AAACCTAGGAGACGGATCAACAGTACGGCG-3¢). Details of theprocedure that we used here will be sent on request.
GenBank accession numbers
The GenBank accession numbers for nucleotide and amino acidsequences reported herein are as follows: AS2 (AB080802); and BACs
F5I14 (AC001229), F1E22 (AC007234), and F12P19 (AC009513).
Information about members of the AS2 family of proteins from Arabidopsis is summarised in Table 2.

Acknowledgments
The authors thank Dr. Yasuo Niwa for providing pTH-2, Mr.
Takaaki Ishikawa for providing pBI-BAR, and Dr. Hirokazu Tanaka
for screening mutants showed as phenotype. The authors also thankProfessor Fumio Osawa (Aichi Institute of Technology and the
Research Development Corporation of Japan) for his encouragement.
E. S. was supported by a fellowship from the Ministry of Education,
Science, and Culture and Sports of Japan. This work was supported inpart by grants from the Research for the Future Program of the Japan
Society for the Promotion of Science (JSPS-RFTF97L00601 to Y. M.
and JSPS-RFTF00L01603 to C. M.), and by Grants-in-Aid for Scientific Research on Priority Areas (no. 10182101 to Y. M. and no.
13044003 to C. M.) and for General Scientific Research (no.
12640598 to C. M.) from the Ministry of Education, Science, and Culture and Sports of Japan, and by Core Research For Evolutional Sci-

477

ence and Technology (CREST) of the Japan Science and Technology
Corporation.

References
Adachi, J. and Hasegawa, M. (1996) MOLPHY version 2.3: programs formolecular phylogenetics based on maximum likelihood. Comput. Sci.
Monogr. 28: 1–150.
Berná, G., Robles, P. and Micol, J.L. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics 152: 729–742.
Bowman, J.L. and Smyth, D.R. (1999) CRABS CLAW, a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein withzinc finger and helix-loop-helix domains. Development 126: 2387–2396.
Byrne, M., Timmermans, M., Kinder, C. and Martienssen, R. (2001) Development of leaf shape. Curr. Opin. Plant Biol. 4: 38–43.
Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A. andMartienssen, R.A. (2000) Asymmetric leaves1 mediates leaf patterning andstem cell function in Arabidopsis. Nature 408: 967–971.
Byrne, M.E., Simorowski, J. and Martienssen, R.A. (2002) ASYMMETRICLEAVES1 reveals knox gene redundancy in Arabidopsis. Development (inpress).
Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. (1996)
Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325–330.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–
743.
Conway, L.J. and Poethig, R.S. (1997) Mutations of Arabidopsis thaliana thattransform leaves into cotyledons. Proc. Natl. Acad. Sci. USA 94: 10209–
10214.
Dengler, N.G. (1999) Anisophylly and dorsoventral shoot symmetry. Int. J.
Plant Sci. 160: S67–S80.
Ellenberger, T.E., Brandl, C.J., Struhl, K. and Harrison, S.C. (1992) The GCN4
basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell 71: 1223–1237.
Galbiati, M., Moreno, M.A., Nadzan, G., Zourelidou, M. and Dellporta, S.L.
(2000) Large-scale T-DNA mutagenesis in Arabidopsis for functional
genomic analysis. Funct. Integr. Genomics 1: 25–34.
Hake, S., Vollbrecht, E. and Freeling, M. (1989) Cloning Knotted, the dominantmorphological mutant in maize using Ds2 as a transposon tag. EMBO J. 8:
15–22.
Hamada, S., Onouchi, H., Tanaka, H., Kudo, M., Liu, Y.G., Shibata, D., MacHida, C. and Machida, Y. (2000) Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves.
Plant J. 24: 91–101.
Hickey, L.J. (1973) Classification of the architecture of dicotyledonous leaves.
Amer. J. Bot. 60: 17–33.
Hickey, L.J. (1979) A revised classification of the architecture of dicotyledonous leaves. In Anatomy of the Dicotyledons. Edited by Metcalfe, C.R. andChalk, L. pp. 25–39. Oxford University Press, New York.
Himi, S., Sano, R., Nishiyama, T., Tanahashi, T., Kato, M., Ueda, K. andHasebe, M. (2001) Evolution of MADS-box gene induction by FLO/LFY
genes. J. Mol. Evol. 53: 387–393.
Hoekema, A., Hooykaas, P.J. and Schilperoort, R.A. (1984) Transfer of theoctopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumor- or root-inducing plasmids: generality of virulence systems.
J. Bacteriol. 158: 383–385.
Hofer, J., Turner, L., Hellens, R., Ambrose, M., Matthews, P., Michael, A. andEllis, N. (1997) UNIFOLIATA regulates leaf and flower morphogenesis inpea. Curr. Biol. 7: 581–587.
Hudson, A. (2000) Development of symmetry in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 349–370.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO J. 6: 3901–3907.
Kerstetter, R.A., Bollman, K., Taylor, R.A., Bomblies, K. and Poethig, R.S.
(2001) KANADI regulates organ polarity in Arabidopsis. Nature 411: 706–
709.
Kim, G.T., Tsukaya, H. and Uchimiya, H. (1998) The ROTUNDIFOLIA3 geneof Arabidopsis thaliana encodes a new member of the cytochrome P-450

478

ASYMMETRIC LEAVES2 gene for leaf development

family that is required for the regulated polar elongation of leaf cells. Genes
Dev. 12: 2381–2391.
Kojima, S., Banno, H., Yoshioka, Y., Oka, A., Machida, C. and Machida, Y.
(1999) A binary vector plasmid for gene expression in plant cells that is stably maintained in Agrobacterium cells. DNA Res. 6: 407–410.
Lieu, S.M. and Sattler, R. (1976) Leaf development in Begonia hispida var. cucullifera with special reference to vascular organization. Can. J. Bot. 54:
2108–2121.
Machida, C., Onouchi, H., Koizumi, J., Hamada, S., Semiarti, E., Torikai, S. andMachida, Y. (1997) Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI. Proc. Natl. Acad.
Sci. USA 94: 8675–8680.
McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J. and Barton, M.K.
(2001) Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411: 709–713.
Nakashima, M., Hirano, K., Nakashima, S., Banno, H., Nishihama, R. andMachida, Y. (1998) The expression pattern of the gene for NPK1 protein
kinase related to mitogen-activated protein kinase kinase kinase (MAPKKK)
in a tobacco plant: correlation with cell proliferation. Plant Cell Physiol. 39:
690–700.
Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T. and Machida, Y.
(2001) The NPK1 mitogen-activated protein kinase kinase kinase is a regulator of cell-plate formation in plant cytokinesis. Genes Dev. 15: 352–363.
Ogura, Y. (1962) Plant Anatomy and Morphology. pp. 102–134 Youkendo, Inc.,Tokyo.
Onouchi, H., Nishihama, R., Kudo, M., Machida, Y. and Machida, C. (1995)
Visualization of site-specific recombination catalyzed by a recombinase from
Zygosaccharomyces rouxii in Arabidopsis thaliana. Mol. Gen. Genet. 247:
653–660.
Ori, N., Eshed, Y., Chuck, G., Bowman, J.L. and Hake, S. (2000) Mechanismsthat control knox gene expression in the Arabidopsis shoot. Development 127:
5523–5532.
Pavletich, N.P. and Pabo, C.O. (1991) Zinc finger-DNA recognition: crystal
structure of a Zif268-DNA complex at 2.1 Å. Science 252: 809–817.
Sakakibara, K., Nishiyama, T., Kato, M. and Hasebe, M. (2001) Isolation of
homeodomain-leucine zipper genes from the moss Physcomitrella patens andthe evolution of homeodomain-leucine zipper genes in land plants. Mol. Biol.
Evol. 18: 491–502.
Sawa, S., Watanabe, K., Goto, K., Liu, Y.G., Shibata, D., Kanaya, E., Morita,
E.H. and Okada, K. (1999) FILAMENTOUS FLOWER, a meristem and organ
identity gene of Arabidopsis, encodes a protein with a zinc finger and HMGrelated domains. Genes Dev. 13: 1079–1088.
Semiarti, E., Onouchi, H., Torikai, S., Ishikawa, T., Machida, Y. and Machida,C. (2001b) The transposition pattern of the Ac element in tobacco cultured

cells. Genes Genet. Syst. 76: 131–139.
Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C. and Machida, Y.
(2001a) The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates
formation of a symmetric lamina, establishment of venation and repression ofmeristem-related homeobox genes in leaves. Development 128: 1771–1783.
Serrano-Cartagena, J., Robles, P., Ponce, M.R. and Micol, J.L. (1999) Genetic
analysis of leaf form mutants from the Arabidopsis Information Service collection. Mol. Gen. Genet. 261: 725–739.
Siegfried, K.R., Eshed, Y., Baum, S.F., Otsuga, D., Drews, G.N. and Bowman,
J.L. (1999) Members of the YABBY gene family specify abaxial cell fate inArabidopsis. Development 126: 4117–4128.
Sinha, N.R. (1999) Leaf development in angiosperms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 419–446.
Steeves, T.A. and Sussex, I.M. (1989) Patterns in Plant Development. Cambridge University Press, Cambridge.
Sun, Y., Zhou, Q., Zhang, W., Fu, Y. and Huang, H. (2002) ASYMMETRICLEAVES1, an Arabidopsis gene that is involved in the control of cell differentiation in leaves. Planta 214: 694–702.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, positions-specific gap penalties and weight matrix
choice. Nucl. Acid. Res. 22: 4673–4680.
Timmermans, M.C., Hudson, A., Becraft, P.W. and Nelson, T. (1999) ROUGH
SHEATH2: a Myb protein that represses knox homeobox genes in maize lateral organ primordia. Science 284: 151–153.
Tsiantis, M., Schneeberger, R., Golz, J.F., Freeling, M. and Langdale, J.A.
(1999) The maize rough sheath2 gene and leaf development programs inmonocot and dicot plants. Science 284: 154–156.
von Arnim, A.G. and Deng, X.W. (1994) Light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its
nucleocytoplasmic partitioning. Cell 79: 1035–1045.
Waites, R., Selvadurai, H.R., Oliver, I.R. and Hudson, A. (1998) The PHANTASTICA gene encodes a MYB transcription factor involved in growth anddorsoventrality of lateral organs in Antirrhinum. Cell 93: 779–789.
Wang, J.H., Avitahl, N., Cariappa, A., Friedrich, C., Ikeda, T., Renold, A.,
Andrikopoulos, K., Liang, L., Pillai, S., Morgan, B.A. and Georgopoulos, K.
(1998) Aiolos regulates B cell activation and maturation to effector state.
Immunity 9: 543–553.
Whaley, W.G. and Whaley, C.Y. (1942) A developmental analysis of inheritedleaf patterns in Tropaeolum. Amer. J. Bot. 29: 105–194.
Yoshioka, Y., Kurei, S. and Machida, Y. (2001) Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression. Genes Genet. Syst. 76: 189–198.

(Received March 6, 2002; Accepted April 2, 2002)