Institute of Cell and Molecular Biology, Kings Buildings, University of Edinburgh, Edinburgh EH9 3JR, UK
* Author for correspondence (e-mail: Gwyneth.Ingram{at}ed.ac.uk)
Accepted 28 May 2003
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SUMMARY |
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Key words: L1, Cell layer, Integuments, Signalling, Receptor kinase, Ovule, Arabidopsis thaliana
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INTRODUCTION |
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Experiments and observations in many plant species have shown that the
developmental behaviour of cells in meristems and developing organs is largely
dictated by their position rather than by lineage. Thus if the progeny of
cells from one layer invade another layer during development, the displaced
cells differentiate according to their new position
(Stewart and Derman, 1975;
van den Berg et al., 1995
;
Kidner et al., 2000
). For this
developmental plasticity to be achieved, cells must constantly receive and
interpret information from their neighbours. Our understanding of how plant
cell layers communicate is currently limited to a few specific examples. In
Arabidopsis roots, an inside to outside movement of transcription
factors (notably the SCARECROW (SCR) protein) is required for normal
differentiation of ground cell layers
(Nakajima and Benfey, 2002
).
In contrast, inter layer communication in shoot meristems appears to require
the interaction of a diffusible ligand with a cell-autonomous receptor kinase
complex (Fletcher et al.,
1999
). A similar interaction is invoked in the development of
maize leaves and endosperm, where the receptor kinase-encoding
CRINKLY4 (CR4) and the calpain-encoding DEFECTIVE KERNEL
1 (DEK1) genes are required for specification and maintenance of
`outer' cell layer identity during endosperm and leaf development
(Becraft et al., 1996
;
Becraft et al., 2002
;
Lid et al., 2002
). The maize
EXTRA CELL LAYERS 1 (XCL1)
gene seems to be involved in pathways regulating division behaviour in L1
cells during organ formation. The Xcl1 mutant provides intriguing
evidence that cell identity can be uncoupled from positional cues at least
late in development. (Kessler et al.,
2002
).
In a search to identify genes involved in inter-cell layer communication in Arabidopsis, a study of ACR4, an Arabidopsis CR4 homologue, was carried out. ACR4 was found to be required for normal cell organisation during ovule integument development and the formation of sepal margins. Both these tissues are formed exclusively from plates of L1 cells arranged back to back. By isolating the functional ACR4 promoter, ACR4 was shown to be expressed in L1 cells in all apical meristems and young organ primordia, including those of the developing ovule integuments. In addition, ACR4 is expressed in an intriguing pattern in root meristems. The kinase activity of ACR4 was demonstrated and, using fusion proteins expressed under the ACR4 promoter, ACR4 protein localisation was visualised in vivo in the plasma membranes of L1-derived cells. The wide expression pattern of ACR4 compared to its associated mutant phenotype may be a result of functional redundancy with other related proteins or functionally related pathways. Taken together, the data presented indicate a role for ACR4 in the cellular signalling pathways required for correct cell organisation in ovule integuments and sepal boundaries, and may provide important clues as to the types of signalling involved in cell layer maintenance and specification in the wider context of plant development.
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MATERIALS AND METHODS |
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Expression of recombinant proteins in bacteria and kinase assays
To express recombinant GST fusion proteins in bacteria, the ACR4
kinase domain was amplified using 5'-AGGATCCGTCCGGATCTTGATGAG and
5'-GAGCTCGAGTTTCCCATTAGCTGTGC, and cloned as an in-frame fusion with GST
coding sequences in pGEX-3x (Amersham Pharmacia Biotech). Protein expression
and purification using GST-sepharose (Amersham Pharmacia Biotech) was carried
out according to the manufacturer's guidelines. Site directed mutagenesis was
carried out using the QuikChange site-directed mutagenesis kit (Stratagene)
with primer 5'-GGAACCACTGTTGCAGTGATGAGAGCGATAATGTC and its reverse
complement. GST fusion proteins were assayed for kinase activity by incubation
in 30 µl (final volume) with 20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM
MgCl2 with 10 µCi of [-32P]ATP for 1 hour at
room temperature. Samples were boiled in loading buffer and analysed by
SDS-PAGE. Coomassie Blue-stained gels were dried and exposed to film.
Isolation and phenotypic characterisation of mutant alleles
To isolate acr4-1 the Wisconsin collection was screened with
oligos 5'-TGCCATCTCAGTACTTCATGACTCTCTCT and
5'-CTCTCTGCCTCTTTGTTACTTTCCTGCCT as described previously
(Krysan et al., 1999). The
mutants acr4-2, acr4-3, acr4-4 were identified on the Syngenta
website (Sessions et al.,
2002
). To estimate insertion number, probes against the GUS marker
gene or BAR selection gene were made by amplifying the GUS ORF with primers
5'-GTGGGAAAGCGCGTTACAAGAAAGC and 5'-CACCATTGGCCACCACCTGCCAGTC or
the BAR ORF with 5'-CGTACCGAGCCGCAGGAAC and
5'-ATCTCGGTGACGGGCAGGAC. For histological analysis, tissue was submerged
overnight in 84 mM Pipes (pH 6.8) solution containing 4% acrolein, 1.5%
glutaraldehyde 1% paraformaldehyde and 0.5% Tween 20. Tissue was rinsed
several times in 100 mM Pipes and dehydrated using an ethanol series. JB4
resin was infiltrated into the tissue over a period of 2 weeks before
embedding. 4.5 µm sections were stained in Toluidine Blue and visualised
using a Leica standard light microscope. For creation of the ATML1
marker line, the ATML1 ORF was amplified by reverse transcription PCR
and cloned into pGEMT-easy using oligos ATML1A and ATML1B
(Abe et al., 2001
). GFP was
amplified using 5'-AGCTAGCATGAGTAAAGGAGAAGAAC and
5'-AGCTAGCGTGTTTGTATAGTTCATC, and cloned pGEM-9z (Promega). The
ATML1 ORF was fused downstream of GFP and the fused construction was
cloned downstream of the pAS99 HindIII insert [containing the full
ATML1 promoter (Sessions et al.,
1999
)] in pBIBHyg (pL178).
Brefeldin A experiments
Roots were incubated for 2 hours in 100 µM brefeldin A (BFA) (B7651,
Sigma-Aldrich). The working BFA solution was made by diluting a 10 mM DMSO
stock 1:100 in water. Control roots were incubated for the same period of time
in a 1:100 dilution of DMSO in water.
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RESULTS |
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In the roots of plants transactivating H2B::YFP, marker expression was observed in the quiescent centre (QC) central cells, columella initials and cells below the QC, the lateral root cap (LRC) and the initial cells destined to give rise to the root epidermal cell file and the LRC (Fig. 1J). However, expression was not observed in epidermal cells until the point where they emerged from under the LRC (Fig. 1K). This transition was sharp, with cells initiating expression as soon as they started to lose contact with the LRC. Expression in the root epidermis was maintained into the elongation zone, where it diminished. In more distal positions on the root, initiating lateral root primordia could be identified on the basis of their expression of H2B::YFP. Expression initiated in lateral root primordia at the four- to eight-cell stage, usually in a double file of cells (not shown). Expression in lateral roots resembled that observed in apical root meristems. The expression pattern of ACR4 in roots differed from that in apical regions, firstly, in that a population of meristematic L1 cells (epidermal cell file under LRC) did not express ACR4, and secondly in that populations of `internal' cells (QC, and lateral root primordium initials) expressed ACR4.
In contrast to in situ hybridisation results, H2B::YFP remained visible in developing organs until relatively late in development. To investigate this phenomenon, a sequence encoding a cytoplasmically localised version of mGFP6 was placed under the control of the 1.9 kb ACR4 promoter. Lines expressing this construction showed expression in the same meristematic zones observed for lines expressing H2B::YFP, although fluorescent protein `leaked' from outer cell layers into internal cell layers, especially in young embryos and floral/inflorescence meristems. GFP expression was not maintained in mature organs indicating that in some tissues H2B::YFP may persist in nuclei after gene expression has terminated.
The ACR4 promoter is restricted to an 857 bp region upstream
of the ATG
To determine the extent of the functional ACR4 promoter, the 1.9
kb full-length promoter was reduced distally from -1849 (where -1 is the base
before the ATG) to give a -1026, a -857 and a -405 deletion. These fragments
were placed directly upstream of the H2B::YFP reporter gene
previously described, and transformed into plants. Their ability to drive
L1-specific expression was assessed in young roots, developing seeds and
inflorescence meristems, and compared to that of the full-length promoter.
-1026 and
-857 both gave expression patterns identical to that
shown by the full-length promoter in roots, embryonic and meristematic tissues
(verified in 20 independent transformants).
-405 gave no detectable
H2B::YFP expression (40 independent transformants screened). Thus all
sequences required for normal ACR4 expression were located in the
first 857 bases of the promoter.
ACR4 is necessary for normal seed development
To gain material for functional analysis of ACR4, collections of
T-DNA insertion lines were screened. One insertional mutant in ACR4
was identified in the Wisconsin population
(Krysan et al., 1999) and
shown to be heterozygous for a double (back to back) T-DNA between bases 1066
and 1100 of the ORF. This allele was designated acr4-1. Three mutant
lines were uncovered in the Syngenta collection
(Sessions et al., 2002
): the
acr4-2 allele contained a T-DNA insertion at base 249 of the
ACR4 ORF, acr4-3 contained an insertion 570 bp downstream of
the ACR4 ORF and acr4-4 housed two insertions in the
ACR4 promoter, one 1.6 kb and one 810 bp upstream of the start of
transcription. PCR and subsequent Southern blot analysis confirmed that the
progeny of heterozygous acr4-1, -2, -3 and -4 plants
segregated wild-type, heterozygous and homozygous individuals in a 1:2:1
ratio. Southern blot analysis also showed that the acr4-1 and
acr4-2 and backgrounds contained no other T-DNA insertions than those
at the ACR4 locus, but that both the acr4-3 and
acr4-4 backgrounds contained multiple independently segregating
TDNAs. The positions of the insertions in acr4-1 and acr4-2
would be predicted to give strong mutant alleles and were therefore of
particular interest for functional studies.
Segregating populations carrying acr4-1, acr4-2, acr4-3 and acr4-4 were analysed to identify potential mutant phenotypes associated with disruption of the ACR4 gene. No differences in gross plant morphology between homozygous mutants and wild-type plants were noted in any of the four populations. However, all acr4-1 and acr4-2 homozygotes showed abnormalities in both the shape and texture of developing seeds. Instead of being elliptical and smooth, the developing seeds were rounded and rough in appearance. In addition, seeds were heterogeneous in their development compared to wild type, and siliques contained unfertilised ovules and aborted seeds at a rate of 40-85% (Fig. 2A,B). The developmental stage of seed abortion varied from just after pollination to just prior to maturity. When selfed heterozygous plants were analysed, no seed abnormalities were found, indicating that the phenotypes described were due to the maternal genotype. No seed defects were observed in the siliques of homozygous acr4-3 and acr4-4 plants.
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To understand the developmental basis of the observed seed phenotype, ovule morphology in mutant plants was analysed. Mutant ovules displayed phenotypes of varying severity (Fig. 3B-D). All ovules showed epidermal irregularities, including abnormal cell size and shape, callus-like outgrowths, and occasional inappropriate cell types such as stomata. Ovules sometimes fused together (Fig. 3D). In most (>90%) of mutant ovules the abaxial zone of the integuments failed to elongate sufficiently to give the curvature seen in wild-type ovules. In some cases the embryo sac/nucellus protruded from the shortened integuments (Fig. 4H,J). In addition to disruption in ovule epidermal organisation, lack of organisation of integument cell layers was observed, with some ovules showing loss of cell layers, and others showing sporadic over-proliferation of integument cells. A varying proportion (20-50%) of ovules lacked a recognisable embryo sac (Fig. 3C,D). In extreme cases the endothelium was absent or reduced to a few disorganised cells. In other cases the endothelium cells enclosed differentiated/divided cells, or an empty space. In 30-50% of mutant ovules the egg apparatus (synergids, egg cell and polar nucleus) could be distinguished (Fig. 3B).
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Defects observed in ovules were maintained in developing seeds when fertilisation had been possible. In particular, the texture of the seed coat was abnormal, with outgrowths observed, particularly in retarded seeds. A lack of proximodistal elongation of the mutant embryo sac after fertilisation caused the mutant endosperm to develop in a reduced volume giving seeds a round rather than elliptical shape (Fig. 2D). Although defects in embryo organisation were not observed, seeds with more severe defects in integument organisation were also retarded in embryo and endosperm development. Histological analysis supported the hypothesis that these seeds were those observed to abort.
To study the epidermal abnormalities observed in developing seeds, SEM
analysis of mature mutant seeds was carried out. Although seed coat
abnormalities were observed, particularly at the funiculus abscission scar and
at the micropylar region, the majority of seed coat cells had a similar
structure to those observed in wild-type seeds
(Fig. 2C,D). Because homozygous
seeds still differentiated appropriate epidermal cell types, and even in
ovules, mis-specification of cell types (for example the presence of stomata)
involved epidermal-specific identities, the expression of an L1 marker in
mutant ovules was investigated. Homozygous acr4-2 and acr4-1
plants were crossed to marker lines expressing an N-terminal GFP::ATML1 fusion
protein (unpublished results) under the ATML1 promoter
(Sessions et al., 1999). These
lines expressed nuclear localised fusion protein in the L1-specific pattern
previously reported for ATML1 expression in embryos and meristems
(Lu et al., 1996
;
Sessions et al., 1999
). ATML1
fusion protein expression was observed in the outer cell layer and endothelium
of mature ovules in wild-type plants, with weak expression occasionally
observed in the inner cell layer of the inner integument. In acr4
mutant ovules ATML1 expression was similar to or more widespread than
in wild type. In excrescences on the ovule surface, both protruding
callus-like cells and underlying cells showed expression. Strong expression
was sporadically seen in cells situated between the ovule epidermis and the
endothelium. In several cases, the egg sac space was filled with expressing
cells. This analysis suggests that although mutant ovule integument cells
showed abnormalities in organisation, they did not loose their L1
identity.
Because acr4 mutants showed abnormalities in ovule integuments, sepal margins, which have a similar structure (appressed layers of L1 cells) were examined in more detail. Although no major defects in sepal morphology were observed in acr4 mutants, it was noted that the cells at sepal boundaries appeared less well organised than in wild-type plants, giving a somewhat ragged appearance (Fig. 4K,L). In general the border region was thicker (contained more cells) in the abaxial/adaxial dimension than in wild type, suggesting that outgrowth of sepal margins could be affected. Mutant margin cells were irregularly shaped and showed abnormal `lumpy' areas and regions devoid of the cuticular decoration seen in wild-type cells. No defects at the margins of leaves or petals could be discerned.
Although two independent mutant alleles in two different backgrounds both gave identical phenotypes, a further confirmation that the observed phenotype was due to loss of ACR4 function was obtained by genetic complementation of acr4-2. Homozygous mutants were crossed to hygromycin-resistant transformants carrying a full-length ACR4 promoter driving the ACR4 ORF. Four F2 families corresponding to four independent transformants were selected on hygromycin and PCR-genotyped for homo- or heterozygosity of acr4-2. The phenotypes of homozygous plants were compared with those of heterozygous and wild-type plants in each case. For two families homozygosity of acr4-2 plants was verified by Southern blot. For all four families full phenotypic complementation was apparent in immature and mature seeds of homozygous mutant plants, confirming that the observed mutant phenotypes were due to loss of ACR4 function.
ACR4 encodes an active kinase domain
To establish whether ACR4 protein encodes a functional kinase, as predicted
from its sequence, a GST fusion protein construct was engineered to express
the ACR4 kinase domain in bacteria. A 61 kDa protein encoding the
GST-kinase was expressed and purified (Fig.
5). To act as a control in kinase assays, Lys 540 (a crucial amino
acid in the kinase activation loop) was mutated to methionine. GST-kinase and
GST-kinase-null proteins were subjected to in vitro kinase assays. The kinase
domain showed phosphorylation that was absent in the kinase-null variant
(Fig. 5). Incubation of the
kinase domain with GST protein alone did not result in phosphorylation of GST,
indicating that the kinase domain could autophosphorylate inter or
intramolecularly in vitro (results not shown).
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DISCUSSION |
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Many acr4 mutant ovules are never fertilised because of severe
morphological abnormalities, but of the ones that are, those with more severe
organisational defects abort as developing seeds. Abortion is independent of
zygotic genotype and is, moreover not due to developmental defects in embryo
and endosperm development, although both tissues are retarded at the time of
seed death. Retardation and abortion probably occur because defective seeds
provide insufficient maternal support, in terms of nutrients, for embryo sac
development. Similar retardation and death of embryo/endosperm was observed
when reduced expression of the genes FBP7 and FBP11 led to
developmental abnormalities and degeneration in the endothelium and seed coat
of Petunia (Colombo et al.,
1997). The total lack of zygotically derived embryo development
defects and the observation of seed coat abnormalities in our study
contradicts results obtained using antisense experiments to reduce
ACR4 expression (Tanaka et al.,
2002
).
ACR4, as a membrane-localised receptor-like kinase, probably acts by
perceiving extracellular ligands. Several genes encoding possible ligands, or
ligand processing molecules for CR4 and related proteins have been proposed.
These include the subtilase encoded by the ABNORMAL LEAF SHAPE 1
(ALE1) gene (Tanaka et al.,
2001). During embryo and endosperm development, signals from
surrounding tissues (as could be provided by the action of genes such as
ALE1) might be important in signalling required for `outside' cell
layer specification. However, it seems more likely that in organ primordia, as
has been shown in root cell layer differentiation, an `inside to outside'
signalling process is involved in regulating cell layer behaviour, combined
with a role for signals from neighbouring cells in the same cell layer
(Nakajima and Benfey, 2002
).
Our observation that ACR4 protein is localised on `internal' plasma membranes
of `outside' cells supports the hypothesis that ACR4 may perceive
signals from underlying cells and/or same-layer neighbours. If this is the
case, the restriction of the acr4 phenotype to ovule integuments and
sepal margins could be attributable to the fact that these tissues are unique
in the Arabidopsis plant, in being composed of two appressed layers
of L1 cells. If normal L1 behaviour (i.e. anticlinal divisions giving rise to
a monolayer of L1 cells) were dependent on perception of positional
information both from underlying cells, and from same-layer neighbours, then a
loss in signalling between same-layer neighbours could be compensated for by
signals from underlying cells in most tissues. However, in the case of ovule
integuments and sepal margins, positional information would be effectively
limited to that exchanged between same-layer neighbours. The cells in these
organs would thus be particularly sensitive to disruption of this signalling
pathway, which would be expected to lead to a loss of cellular organisation
and thus abnormalities in organ outgrowth, similar to the phenotype observed
in acr4 mutants.
Other pieces of the puzzle
The restricted mutant phenotype of ACR4 compared to maize
CR4 mutants is surprising since ACR4 appears to be unique in
Arabidopsis in its degree of similarity to maize CR4. Unlike
studies of cr4 in maize, we find no evidence for a loss of epidermal
identity in acr4 mutants, but rather solely a loss of cell
organisation. The cell disorganisation observed in acr4 ovule
integuments and sepal margins is, however, reminiscent of aspects of the
epidermal defects observed in the leaves of maize cr4 mutants.
Notably, both phenotypes involve deregulation of the planes of division, and
organisation of populations of L1 cells. Striking differences in expression
also exist between ACR4 and CR4. In maize, CR4 is
expressed in the aleurone cell layer and one of the major phenotypes
associated with cr4 mutants is a defect in aleurone differentiation
(Becraft et al., 1996).
ACR4 shows no endosperm expression, although it is arguable whether
Arabidopsis can be considered to differentiate a structure analogous
to the cereal aleurone layer (Berger,
1999
). In addition, unlike ACR4, CR4 appears to be
expressed throughout apical meristems, without restriction to the L1 layer
until late in leaf development (Becraft et
al., 1996
), and no CR4 expression has been reported in
maize root tissue.
Functional redundancy between ACR4 and four other
Arabidopsis genes showing weaker similarity to CR4 cannot be
ruled out as an explanation for some of the differences in phenotypic severity
between cr4 and acr4 mutants. The two most closely related
genes encode proteins lacking a conserved kinase catalytic domain required for
kinase activity (domain 8) (Hanks et al.,
1998). ACR4 encodes a functional kinase, and kinase
activity is probably required for at least some of its functions. However,
kinase-inactive receptors can retain partial function, possibly by interaction
with other unrelated kinases. The kinase-null clv1-6 allele, which
causes part of the kinase domain of the CLAVATA1 protein to be deleted, causes
only a weak mutant phenotype. The mutant protein thus retains functions that
are independent of its ability to auto/transphosphorylate itself and other
proteins (Torii and Clark,
2000
). Of the two less similar genes, one encodes a protein
closely related to tobacco CRK1, which has recently be implicated in cytokinin
responses (Schafer et al., 2002). The other shares many more residues with
CRK1 than with ACR4 and CR4, especially in the extracellular domain adjacent
to the trans-plasma membrane domain, where ACR4 and CR4
encode putative TNFR-like repeats.
An alternative explanation for the weak acr4 phenotype could be that although several independent mechanisms regulate L1 behaviour in both Arabidopsis and maize, mechanistic differences in organ primordium development in monocotyledonous and dicotyledonous species have led to less functional overlap in maize than in Arabidopsis. Considering the relatively large numbers of genes expressed in L1 cell layers from early in development in both species, this possibility seems realistic, and will be investigated using ongoing mutagenesis and double mutant analysis approaches in the near future.
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ACKNOWLEDGMENTS |
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