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First published online 21 March 2007
doi: 10.1242/dev.003533
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1 College of Bioscience and Biotechnology, Chubu University and CREST, Japan
Science and Technology Agency, 1200 Matsumoto-cho, Kasugai 487-8501,
Japan.
2 Division of Biological Science, Nagoya University, Nagoya 464-8602,
Japan.
3 Center for Integrative Bioscience, National Institute for Basic Biology,
Okazaki 444-8585, Japan.
Author for correspondence (e-mail:
yas{at}bio.nagoya-u.ac.jp)
Accepted 25 February 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Intercellular signaling, Epidermis, Tissue specification, Embryonic patterning, Arabidopsis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Stern, 2005).
In higher plants, specification of outer (protoderm) and inner (provascular
and ground) tissues takes place during early embryogenesis in a way that is
apparently different from the mechanisms involved in animal systems; the
tissue specification in higher plants does not involve cell ingression
(Esau, 1977;
Jürgens and Mayer, 1994;
Ito et al., 2002). Instead, in
both monocotyledonous and dicotyledonous plants, cells at the outermost part
of the early embryo, via currently unknown mechanism(s), acquire distinct
characteristics, including the formation of epidermal cuticle and of a
regulated cell-division plane perpendicular to the surface of the embryo
(anticlinal division), which enables self-maintenance of the protodermal
cell-layer that generates cuticle. During post-embryonic development,
epidermal cells of most aerial organs are derived from the protoderm of the
shoot apical meristem (SAM; L1 layer)
(Satina et al., 1940).
In Arabidopsis thaliana, the first indication of the morphological
differentiation of the protoderm is visible in the embryo at the 16-cell
stage, when outer and inner cells are generated
(Jürgens and Mayer,
1994). Outermost cells of early embryos express a specific set of
genes, which include those for homeodomain transcription factors
(ATML1 and PDF2) (Lu et
al., 1996; Abe et al.,
2003), for fatty-acid metabolism (FDH)
(Yephremov et al., 1999), for
a putative extracellular protein (PDF1)
(Abe et al., 1999) and for a
receptor-like protein kinase (RLK; ACR4)
(Tanaka et al., 2002;
Gifford et al., 2003). The
homeodomain proteins ATML1 and PDF2 are essential for the differentiation of
the epidermis, the expression of PDF1 and ACR4 in seedlings
(Abe et al., 2003), and for
maintaining ATML1 promoter activity
(Takada and Jürgens,
2007). ATML1 and PDF2 proteins bind to a cis-regulatory
element, the L1 box, which is found in protoderm-specific genes such as
PDF1 and FDH, as well as in the genes for ATML1 and PDF2
themselves (Abe et al., 2001;
Abe et al., 2003). Thus, it
seems possible that the expression of protoderm-specific genes involves
positive-feedback regulation by ATML1 and PDF2. Our understanding of the
regulatory mechanisms that control the activity and/or expression of ATML1 and
PDF2 is, however, very limited.
We have shown that the abnormal leaf shape 1 (ale1)
mutation in Arabidopsis results in impaired cuticle formation, in the
adhesion of endosperm and embryo, and in the fusion of cotyledons and leaves
(Tanaka et al., 2001). The
ALE1 gene encodes a member of the subtilisin-like serine protease
family and is preferentially expressed within the endosperm
(Tanaka et al., 2001). These
observations imply that a signal from the endosperm is required for the
formation of cuticle around the embryo. Mutations in the ACR4 gene,
which encodes a RLK, affect the organization of cell layers in ovules and at
sepal margins, as well as affecting cuticle formation in leaves and ovules
(Gifford et al., 2003;
Watanabe et al., 2004). Thus,
signaling systems that include the subtilisin-like serine proteases and RLKs
have been shown to be involved in epidermal differentiation in both animal and
plant embryos, yet our understanding of this signaling pathway in plants is
still very limited.
We isolated a novel Arabidopsis mutant, designated abnormal leaf shape 2 (ale2), which has epidermal defects similar to those of ale1 and/or acr4 mutant plants. We describe here the molecular cloning of the ALE2 gene and show that it encodes a novel RLK. Our genetic analysis revealed that ALE2, ALE1 and ACR4 play collectively essential roles in protoderm specification, and in the formation of the primordia of cotyledons, during embryogenesis. Furthermore, our results suggest that ALE2 and ACR4 act in close harmony whereas ALE1 functions in a different manner to ensure robust facilitation of the differentiation of plant epidermis.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Watanabe et al., 2004). The
ale2-1 mutant was isolated from a T-DNA-tagged population with a
Columbia-6 background
(http://www.arabidopsis.org/abrc/jack.jsp).
Lines were backcrossed at least three times with a wild-type strain [Columbia
(Col-0), Landsberg erecta (Ler) or Wassilewskija (Ws)], as
indicated in the text and Figures. The ale2-2 mutant was isolated by
reverse-genetic screening of a T-DNA-tagged population, generated at the
University of Wisconsin (Krysan et al.,
1996), with following gene specific primers: UWBC-ALE2#1,
5'-GAGGAAGCTATATGGTTACACCTTTGGTT-3'; and UWBC-ALE2#4,
5'-ACCGGAAATCTTTACCCAGATGAACCGAA-3'. Plants were grown as
described elsewhere (Tanaka et al.,
2004b).
Analysis of cuticle defects
To assess the permeability to water-soluble molecules of the leaf surface,
we immersed plantlets in an aqueous solution of toluidine blue (TB test), as
described by Tanaka et al. (Tanaka et al.,
2004b). For quantification of the amount of TB bound to plant
tissues, the aerial parts of each plant were washed and ground thoroughly in a
microtube that contained 200 µl of buffer [200 mM Tris-HCl (pH 8.0), 250 mM
NaCl, 25 mM EDTA]. Next, 400 µl of ethanol was added, with vortex mixing,
and plant debris was pelleted by centrifugation. The supernatant was examined
spectrophotometrocally and the amount of TB was determined by the absorbance
at 630 nm (A630). The major peak of absorbance due to plant
material (A435) was used for normalization. Relative levels of TB
were calculated as the ratio of A630:A435. When it was
necessary to determine the genotype of a particular plant (e.g. to identify a
segregating ale2 mutation), DNA was extracted from the pellet.
Transmission electron microscopy was performed as described previously
(Watanabe et al., 2004).
Cloning and sequencing of the ALE2 gene
An F2 mutant population was generated by genetic crossing of
ale2-1 (isolated in ecotype Col) and Ler. Rough mapping of
ALE2 was performed with CAPS and SSLP markers, available from TAIR
(http://www.arabidopsis.org).
CAPS markers between PHYB and ER were designed on the basis
of information from the CEREON collection of polymorphisms
(http://www.arabidopsis.org/Cereon/index.jsp).
Meiotic-recombination breakpoints were generated near ALE2 by
screening 142 ale2 mutants in the F2 generation for
recombinants between the ale2-1 mutation and flanking markers. The
interval of recombination breakpoints closest to the ale2 mutation
was narrowed down to a 55 kb region that was located on one BAC (F11A3).
Overlapping DNA fragments covering this region were amplified from
ale2-1 genomic DNA by PCR and DNA sequences were determined with
automated sequencers (ABI3100; Applied Biosystems). Reverse transcriptase
(RT)-PCR was performed as described previously
(Watanabe et al., 2004).
ALE2 cDNA, covering the entire coding region, was amplified with
primers ALE2RT-5' (5'-GAGGCTTGGTGTCTCCGTTATTGACTAAT-3') and
ALE2RT-3' (5'-CCTCCTTTTCCCTTTTCTTC-3') and cloned into
pBluescript SK (-) (Stratagene), and its nucleotide sequence was determined.
For the complementation experiment, a fragment of genomic DNA covering the
entire coding region of ALE2 (nucleotides -2179 to 4389 relative to
the initiation codon) was cloned into the binary vector pGreen0029
(Hellens et al., 2000)
(http://www.pgreen.ac.uk/)
and introduced into ale2-1/+ plants by
Agrobacterium-mediated transformation.
Kinase assays in vitro
An ALE2 cDNA fragment encoding the protein kinase domain (residues
324-619) was PCR amplified and cloned into the GST fusion vector pGEX4T-3
(Amersham Pharmacia Biotech). To generate kinase-inactive ALE2, a cDNA
fragment corresponding to residues 289-619 was PCR amplified, a point mutation
at the 377th codon (AAA to TGG) was introduced with mutagenic primers, and
this fragment was then cloned into the pGEX4T-3 vector. An ACR4 cDNA
fragment encoding the protein kinase domain
(Watanabe et al., 2004) was
cloned into the NusA fusion vector pET50 (Novagen). Recombinant proteins (1
µg each) and myelin basic protein (MBP; 10 µg) was incubated in 20 µl
of kinase buffer (Sasabe et al.,
2006) containing 50 µM ATP and 10 µCi of
[
-32P] ATP. The reaction products were then separated by
SDS-PAGE on polyacrylamide gels.
Histological analysis
For observations of cell morphology, we prepared semi-thin sections (500 nm
thickness) as described previously
(Watanabe et al., 2004).
Morphology of ovules and embryos were examined as described by Tanaka et al.
(Tanaka et al., 2004a).
Analysis of patterns of gene expression
In situ hybridization was performed essentially as described previously
(Tanaka et al., 2001).
Template DNA for transcription in vitro was amplified by RT-PCR and cloned
into the EcoRV site of pBluescript SK (-). For ATML1, we
used primers ATML1-5'D (5'-AGCCTAAGACCAAGTCCGAT-3') and
ATML1-3'U (5'-CCAGTAGTAGTAACCACTTCAAGA-3') for RT-PCR, and
we cloned the product of PCR into the vector to generate pNU562. For
FDH, we used primers FDH-5'D
(5'-CGGTGAACTTGAAGTACGTGA-3') and FDH-3'U
(5'-ACACGTGTCTTCTCGAAGAGTT-3'), generating plasmid pNU823. For
PDF1, we used primers PDF1-5'D
(5'-CGTAAGGTTTGAGGATGCCA-3') and PDF1-3'U
(5'-TCCAAGCAAGCCCCATATCA-3') to generate pNU563. We generated an
antisense probe for ATML1 by linearizing pNU562 with
HindIII, with subsequent synthesis of RNA by T3 RNA polymerase. We
generated an antisense probe for FDH by linearizing pNU823
(FDH) with ClaI, with subsequent transcription by T3 RNA
polymerase. For the PDF1 probe, we digested pNU563 with SmaI
and transcribed the probe with T7 RNA polymerase. For fusion of the
PDF1 promoter (pPDF1) with a gene for green fluorescent
protein (GFP), a 1595 bp fragment (nucleotides -1602 to -8 relative
to the initiation codon) containing the cis-regulatory sequence of
the PDF1 gene (Abe et al.,
1999) was amplified by PCR and cloned upstream of
sGFP(S65T) (Chiu et al.,
1996) in pGreen. Two independent transgenic lines (ecotype
Ler) were crossed with ale1-1; ale2-1/+ plants and the
fluorescence due to GFP was examined under a fluorescence
stereomicroscope.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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At the reproductive stage, ale2 plants generated malformed and
fused floral organs (Fig. 1E).
In addition, ale2 plants were sterile. Reciprocal crosses between
ale2 and wild-type plants indicated that sterility was mainly due to
defects in female reproductive tissue(s), although male fertility was also
reduced. Therefore, we maintained ale2 mutants as the self-progeny of
heterozygous plants or via genetic crosses with pollen from homozygous
parents. In order to identify the primary defect responsible for the female
sterility of ale2 plants, we examined the morphology of female
gametophytes (ovules). Ovules from wild-type parents were oval with a smooth
surface (Fig. 1F), reflecting
the organized development of flattened layers of epidermal cells. The inner
cells - including the adaxial outer integumental cells, inner integumental
cells and endothelial cells - are derived from the protoderm (L1 layer)
(Jenik and Irish, 2000) and
form organized layers (Fig. 1H)
that cover the female gametes (embryo sacs;
Fig. 1H, asterisk). Scanning
electron microscopy revealed that adjacent ovules generated from ale2
parents had fused to one another. Moreover, their surfaces were rough and
disorganized as a consequence of the swollen outer cells
(Fig. 1G). Histological
analysis confirmed that organization in the integument and endothelium had
been disturbed by the ale2 mutation, which had induced irregularly
orientated cross walls and the deformation of cells
(Fig. 1I). In addition, mature
ovules from ale2 plants contained degenerated embryo sacs
(Fig. 1I), suggesting that the
sterility of ale2 plants was due, at least in part, to the failure of
ovules to develop. These observations suggested that the ALE2 gene is
involved in the regulation of cell morphology, of the plane of cell division
and of cuticle formation in epidermis-related tissues.
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The predicted sequence of the ALE2 protein contains a hydrophobic sequence at the N-terminus and a hydrophobic sequence in the middle region (Fig. 2C). ALE2 also includes an amino acid sequence in the C-terminal region that is strongly conserved in protein serine/threonine kinases (Fig. 2C). These structural features are typical of those of RLKs, and it is likely that the N-terminal region between the two hydrophobic regions serves as the extracellular region. When we used the predicted amino acid sequence of the putative extracellular domain to search available databases, several protein sequences predicted either from cDNA and/or genomic DNA sequences were found to contain sequences significantly homologous to that of ALE2. These sequences were rice XP473126 and XP468388, and Arabidopsis At5g56885. All these sequences had a cluster of basic amino acid residues (5-12 residues in each member), followed by sequences containing four cysteine residues (Fig. 2D), which are somewhat similar to that of the processing sites in animal insulin-related peptides. Sequences of these four proteins exhibited significant similarity between the basic region and putative transmembrane domain (Fig. 2D).
To examine whether ALE2 is a functional protein kinase, a recombinant protein in which glutathione S-transferase (GST) was fused to the putative cytoplasmic region that covered the kinase domain (KD) of the wild-type ALE2 protein (GST:ALE2KD-WT) was synthesized in E. coli cells. As a negative control, a mutant fusion protein in which the lysine residue at position 377 in the potential ATP-binding domain was replaced with a tryptophan residue (GST:ALE2KD-KW) was also synthesized. In vitro phosphorylation experiments revealed that the wild-type fusion protein, GST:ALE2KD-WT, had activity to phosphorylate itself and myelin basic protein (MBP), whereas GST:ALE2KD-KW did not (Fig. 3A).
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; Watanabe et al.,
2004), could phosphorylate one another. A recombinant protein that
contained the putative cytoplasmic region of ACR4 with the protein-kinase
domain was synthesized in E. coli cells as a histidine- and
NusA-tagged fusion (His-Nus:ACR4KD-WT), and was purified. A mutant fusion
protein in which the lysine residue in the putative ATP-binding site of ACR4
was replaced with a tryptophan residue (His-Nus:ACR4KD-KW) was also
synthesized (Fig. 3B). As shown
in Fig. 3C, His-Nus:ACR4KD-WT
and GST:ALE2KD-WT proteins phosphorylated the kinase-inactive recombinant
proteins GST:ALE2KD-KW and His-Nus:ACR4KD-KW, respectively. When ALE2KD-WT and
ACR4KD-WT were used for the reaction, levels of phosphorylation of both
proteins significantly increased compared with reactions with either type of
kinase-negative proteins (Fig.
3C,D). Thus, ALE2 and ACR4 appear to have the potential to
phosphorylate one another.
The ale2 mutation is semi-dominant in the presence of the acr4 mutation
The potential biochemical interaction, as well as the similarity between
the phenotypes of ale2 and acr4 mutants, prompted us to
perform phenotypic analysis of an ale2 acr4 double mutant. We found
that ale2 acr4 plants were viable and able to produce sterile
flowers, as was the ale2 single mutant
(Fig. 4A,B). With respect to
the number of seeds per silique and the morphology of ovules, the effects of
the acr4 mutation were weaker than those of ale2, whereas
plants that were double homozygous for the ale2 and acr4
mutations had a phenotype similar to that of the ale2 single mutant
(Fig. 4A-C). We next examined
the phenotypes of plants that were homozygous for one mutation and
heterozygous for the other (i.e. ale2/+; acr4 and
ale2; acr4/+). On the homozygous ale2 mutant
background, the effect of a heterozygous acr4 mutation was unclear
(Fig. 4A,B). By contrast, on
the acr4 homozygous mutant background, the effects of the
ale2 mutation were semi-dominant in terms of reduced fertility
(Fig. 4B, lanes 7-9).
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ALE1 and ALE2 genes are essential for organized development of the protoderm
Because ale1 ale2 double mutants had impaired epidermal function
and impaired organ formation, we postulated that the double mutants might be
defective in the differentiation of epidermal precursor cells, which may be
important for the formation of leafy organs. To test this hypothesis, we
examined the morphology of cells in sections of shoot apices. In sections of
wild-type SAM, the L1 layer consists of cells that are relatively regular and
rectangular in profile, resulting in the generation of the smooth surface of
the SAM (Fig. 5E). The L1 layer
in ale1 or ale2 single-mutant plants was mainly a single
layer of cells (Fig. 5E),
whereas, in the shoot apex of ale1 ale2 double mutants, the outermost
cells did not form an organized layer and each cell was swollen, with a
rounded appearance (Fig.
5E).
We followed the course of embryogenesis in the wild type and the mutants,
focusing on the morphology of protodermal cells and the development of
cotyledons. As we reported previously, the ale1 single mutant was
indistinguishable from wild type in terms of the morphology of the embryos
before the heart stage (Tanaka et al.,
2001) (Fig. 6A,B).
In the case of the ale2 single mutant, we examined embryos obtained
from heterozygous parents, because homozygous parents were sterile. We
observed a change in the morphology of protodermal cells in approximately 2%
of embryos at the globular stage; in these embryos, the protodermal cells were
slightly swollen (Fig. 6C,D,E).
At the triangular and the heart stages, we observed embryos with a rough
surface at frequencies of approximately 13 and 26%, respectively
(Fig. 6C,E). In spite of the
malformed protodermal cells, ale2 embryos formed normal cotyledons
(Fig. 6C). Morphological
defects in embryos were obvious from the globular stage in the case of embryos
obtained from ale1; ale2/+ parents
(Fig. 6D). At the triangular
stage and the heart stage, significant fractions of embryos (approximately 13
and 16%, respectively) had very deformed protodermal cells, whose heights were
obviously greater than that of anticlinal cell walls of the protoderm
(Fig. 6D,E). As the
normal-looking siblings developed to the heart and torpedo stages, the
deformation of cotyledons became more obvious, with the occasional loss of
bulging of either one or both primordia
(Fig. 6D). The frequencies of
severe defects in the morphology of protodermal cells were below 25% at all
developmental stages (Fig. 6E),
suggesting that these phenotypes were present in double-homozygous embryos.
However, when mildly affected embryos were taken into account, the percentage
of embryos with swollen protodermal cells was more than 25%
(Fig. 6E; especially obvious at
the triangular stage), perhaps because of a dosage effect of the ALE2
gene in the absence of activity of the ALE1 gene.
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In each single mutant, we detected PDF1 transcripts in all protodermal cells of heart-stage embryos (Fig. 7B,C). However, in cells in the outermost layer of ale1 ale2 embryos, the signal was often below the limit of detection, although strong signals were detected in some outermost cells (Fig. 7D). We obtained similar results after hybridization with FDH and ATML1 probes (Fig. 7E-H). Moreover, when ale1 ale2 double mutants failed to form one or both cotyledonary primordia, no expression of FDH and ATML1 genes was detectable in the outermost cells in the apical region from which cotyledonary primordia would otherwise have developed (Fig. 7F,H). These results suggest that ALE2 is required for the specification of protodermal cell fate, for the transcription of protoderm-specific genes and/or for the accumulation of the transcripts of protoderm-specific genes in the absence of ALE1 activity.
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). To determine whether ACR4, like ALE2, is
responsible for the establishment and/or maintenance of the identity of
protoderm in the absence of ALE1 activity, we examined the expression
of protoderm-specific genes in ale1 acr4 embryos. We failed to detect
any hybridization signals due to the ATML1 and FDH probes in
the outermost cell layer of the apical region, which included possible sites
for the formation of cotyledonary primordia in ale1 acr4 embryos
(Fig. 7I-L). Such abnormal
patterns of transcription of the ATML1 and FDH genes in
ale1 acr4 double mutants were similar to those, described above, in
ale1 ale2 embryos. To gain some insight into the possible mode of regulation of protoderm-specific gene expression by ALE1 and ALE2, we used a GFP reporter gene, driven by the PDF1 promoter (pPDF1::GFP), and examined its expression in wild-type and ale1 ale2 double-mutant seedlings. In wild-type seedlings, strong and uniform signals were observed on the surfaces of young leaves (Fig. 7M,N). In ale1 ale2 seedlings, which had deformed leaves, fluorescence was observed in a patchy pattern on the surfaces of the leaves (Fig. 7O,P).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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ALE2 is a member of a previously uncharacterized RLK/RLCK subfamily
The At2g20300 gene, which turned out to be equivalent to the ALE2
gene in this study, was originally defined as the gene for a receptor-like
cytoplasmic kinase (RLCK), which lacks predicted extracellular domain. Our
experiments by RT-PCR and a cDNA sequence from another source (GenBank
AY091071) indicated that the ALE2 gene encodes a protein with a
structure typical of RLKs, resembling the majority of members of the
Arabidopsis RLK gene family (Shiu
and Bleecker, 2003). Thus, ALE2 might function as cell-surface
receptor. The putative extracellular-domain sequence of the predicted ALE2
protein is similar to that of members of the extensin-like RLK subfamily
(Shiu and Bleecker, 2003) (36%
and 18% identity to that of the At5g56885 and At4g02010 gene products,
respectively). The biological functions of other members in this family are
unknown and, thus, ALE2 is unique, at present, insofar as it has a known
biological function. Our results suggest that ALE2 might function in the
perception and transmission of a signal at the cell surface that is required
for the appropriate differentiation and morphogenesis of epidermal cells.
A possible mode of ALE2 action in the differentiation of the protoderm
We reported previously that the ale1 and acr4 mutations
have a synergistic effect on epidermal-surface function, as well as in the
formation of cotyledons (Watanabe et al.,
2004). These genes encode a putative subtilisin-like serine
protease and a RLK, respectively, which both might potentially be involved in
intercellular signaling (Tanaka et al.,
2001; Tanaka et al.,
2002). Because individual mutations seemed to abolish the
functions of the encoded proteins, it seems likely that ALE1 and ACR4 can
promote the formation of epidermis independently. Our present experiments
revealed an effect of the ale2 mutation that was qualitatively
reminiscent of that of acr4, although the phenotype of acr4
was weaker than that of ale2
(Gifford et al., 2003;
Watanabe et al., 2004) (Figs
1,
4). There seemed to be a strict
requirement for ALE2 activity in the absence of ACR4, because ale2
was associated with a semi-dominant phenotype on the acr4 mutant
background (Fig. 4B). In
addition, epidermis-related defects in the ale2 acr4 double mutants
were reminiscent of those in the ale2 single mutant (Figs
4,
5). Taken together, our results
suggest the presence of a single or closely overlapping pathway mediated by
ACR4 and ALE2 (Fig. 8). Because
ALE2 and ACR4 were able to phosphorylate each other in vitro, a possible model
for the mode of their action is that they function as an enzyme-substrate pair
in plants, thereby functioning in a single signaling pathway that may promote
epidermal specification. In this scenario, their expression patterns should
overlap with each other. ACR4 is expressed in epidermis-related
tissues of various organs (Tanaka et al.,
2002; Gifford et al.,
2003). ALE2 transcripts appear to be evenly distributed
throughout the embryo proper during early embryogenesis, and are
preferentially detected both in the outermost cells and in the inner cells of
cotyledonary primordia at the early heart stage (see Fig. S1 in the
supplementary material). Thus, the pattern of ALE2 expression is
overlapped to some extent with that of ACR4. The molecular
interaction of these RLKs must, however, be examined by further experiments.
Meanwhile, our results support a putative scenario wherein ALE1 functions in a
manner that is somehow related to, but is distinct from, the mode of action of
ALE2 and ACR4 (Fig. 8). A
reasonable hypothesis would be that protoderm specification involves an
intertissue communication (i.e. a signal from endosperm that might be
generated by ALE1) and an extracellular signaling molecule regulating ALE2
and/or ACR4 activity. Molecular identification and characterization of such
molecules would provide further insight into intercellular communication
regulating protoderm specification in plants.
Possible mechanism for the regulation of protoderm-specific gene expression and its importance in development
A striking feature of the accumulation of transcripts of protoderm-specific
genes in ale1 ale2 and ale1 acr4 double mutants was the
patchy pattern of such accumulation, with signals as strong as those in
wild-type cells in some cells, and below the level of detection in other
cells, at the outermost margins of embryos
(Fig. 7). A similar patchiness
of expression of the pPDF1::GFP reporter gene was also observed in
leaves (Fig. 7P). These
observations suggest that ALE1 and ALE2 might not be absolutely required for
the transcription of protoderm-specific genes. Rather, they might be involved
in the organized expression of these genes during development. Abe et al.
(Abe et al., 2003) proposed
that the expression of ATML1 and PDF2 might involve
self-activating feedback regulation via the L1 box in their promoter regions.
Recently, it has been shown that epidermis-specific expression of
ATML1 is controlled by several regulatory sequences in its promoter
(Takada and Jürgens,
2007). It is tempting to speculate that ALE1, ALE2 and ACR4
impinge on the activity of such regulatory elements, allowing the uniform
expression of protoderm-specific genes. It is increasingly clear that many
genes required for the formation of cuticle are expressed in an
epidermis-specific manner (reviewed by
Tanaka and Machida, 2006;
Kurdyukov et al., 2006) and
that cuticle is essential for the prevention of the fusion of aerial organs
(Tanaka and Machida, 2006).
Remarkably, it has been shown that the function of FDH, expression of
which was seriously affected in ale1 ale2 and ale1 acr4
double mutants, is essential for proper leaf surface function [see Lolle et
al. (Lolle et al., 1998) and
references therein] (Tanaka et al.,
2004b). Therefore, it is likely that the severe cuticular defects
and organ fusions in ale1 ale2 and ale1 acr4 double mutants
are due, at least in part, to the impaired expression of protoderm-specific
genes, including FDH.
In addition, ale1 ale2 and ale1 acr4 double mutants were
defective in the formation of cotyledons, as is the atml1 pdf2 double
mutant (Abe et al., 2003).
Because ATML1 and PDF2 are specifically expressed in
protodermal cells and are required for the expression of protoderm-specific
genes, it is possible that appropriate gene expression in the protoderm is a
prerequisite for the formation of cotyledons. Our results suggest that the
failure of cotyledon formation in ale1 ale2 and in ale1 acr4
double-mutant embryos was associated with a loss of detectable levels of
expression of protoderm-specific genes. Another gene that plays a role in the
regulation of protoderm-specific gene expression is AtDEK1 (also
known as DEK1), which encodes a protein with a calpain-protease
domain. The product of this gene is essential for the expression of reporter
genes that are driven by the promoters of protoderm-specific genes, such as
ATML1 and ACR4 (Johnson
et al., 2005). In the atdek1 mutant, development is
arrested at the globular stage, without the formation of cotyledons
(Johnson et al., 2005). Thus,
currently available circumstantial evidence supports the hypothesis that
properly differentiated protoderm is somehow essential for the initiation
and/or continuous growth of the cotyledons. A further genetic link between the
functional differentiation of the epidermis and cotyledon formation has been
provided by the analysis of the gurke (gk; also known as
acc1) mutant: strong gk mutant alleles are associated with
the failed formation of cotyledons
(Torres-Ruiz et al., 1996;
Baud et al., 2004;
Kajiwara et al., 2004); and
leaky gk mutations allow the formation of plantlets but result in the
adhesion of aerial organs (Torres-Ruiz et
al., 1996) (H. Tanaka and Y.M., unpublished).
In addition to the defect in cotyledon formation, the organization of cell
layers in the SAM was severely perturbed in ale1 ale2 double mutants
(Fig. 4E). Such mutants often
ceased development after germination, although some eventually generated
several small leaf-like organs (Fig.
5A, Fig. 7O). Such
morphological and growth defects were associated with the appearance of
swollen spherical cells in the outermost layer of the SAM and with a
disorganized pattern of expression of the pPDF1::GFP. These
observations support the hypothesis that proper differentiation of the
epidermal cell layer is required for the functions of the SAM and for the
subsequent development of leaves. The plant hormone auxin plays a crucial role
in the generation of aerial organs (Okada
et al., 1991; Reinhardt et
al., 2000). The patterns of expression and the subcellular
localization of proteins involved in auxin transport indicate that auxin is
transported towards the incipient primordia of leafy organs through the
protoderm and epidermis (Benková et
al., 2003; Reinhardt et al.,
2003) (reviewed by Tanaka et
al., 2006). Thus, the relationships between the differentiation of
the protoderm, the regulation of polar auxin transport, and organ formation
might be an interesting topic for future research.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1643/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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