Download A leucine-rich repeat containing receptor-like kinase

2049
Development 124, 2049-2062 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV0091
A leucine-rich repeat containing receptor-like kinase marks somatic plant
cells competent to form embryos
Ed D. L. Schmidt, Flavia Guzzo, Marcel A. J. Toonen and Sacco C. de Vries*
Department of Molecular Biology, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands
*Author for correspondence (e-mail: [email protected])
SUMMARY
The first somatic single cells of carrot hypocotyl explants
having the competence to form embryos in the presence of
2,4-dichlorophenoxyacetic acid (2,4-D) were identified
using semi-automatic cell tracking. These competent cells
are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the
provascular elements of the hypocotyl. A search for marker
genes to monitor the transition of somatic into competent
and embryogenic cells in established suspension cell
cultures resulted in the identification of a gene transiently
expressed in a small subpopulation of the same enlarged
single cells that are formed during the initiation of the
embryogenic cultures from hypocotyl explants. The
predicted amino acid sequence and in vitro kinase assays
show that this gene encodes a leucine-rich repeat contain-
ing receptor-like kinase protein, designated Somatic
Embryogenesis Receptor-like Kinase (SERK). Somatic
embryos formed from cells expressing a SERK promoterluciferase reporter gene. During somatic embryogenesis,
SERK expression ceased after the globular stage. In plants,
SERK mRNA could only be detected transiently in the
zygotic embryo up to the early globular stage but not in
unpollinated flowers nor in any other plant tissue. These
results suggest that somatic cells competent to form
embryos and early globular somatic embryos share a highly
specific signal transduction chain with the zygotic embryo
from shortly after fertilization to the early globular
embryo.
INTRODUCTION
explants used, and it appears that there is quite some variability in the tissue that responds first. A further complicating factor
in unraveling the early stages of somatic embryogenesis is the
fact that there is almost no evidence to show that the cellular
changes observed in particular cells are indeed directly responsible or even necessary for embryo formation.
In Daucus carota (carrot), the formation of embryogenic cell
cultures usually commences with the incubation of seed-derived
seedling hypocotyl explants in auxin-containing medium (De
Vries et al., 1988a). As in other species, following an increase
in cytoplasmic content, cell division is resumed in provascular
cells, but not in cortical or in epidermal cells (Guzzo et al., 1994).
Continued cell divisions then result in the formation of masses
of small isodiametric cells. These cells can then enlarge and
finally detach into the culture medium to contribute to the developing embryogenic cell culture (Guzzo et al., 1994). Such an
embryogenic culture contains morphologically and biochemically different cell types, that can be present as single cells or
cell clusters (Van Engelen and De Vries, 1992), of which only
1-2% of the cells are actually embryogenic (De Vries et al.,
1988a). The term ‘embryogenic’ is defined as the ability to form
somatic embryos without further exogeneous application of
growth regulators (De Jong et al., 1993). The origin of embryogenic cells, that are usually present as clusters of small
cytoplasm-rich cells (Komamine et al., 1990) is not clear and is
Somatic or asexual embryogenesis is the process whereby
somatic cells develop into plants via characteristic morphological stages. The later stages, in particular, closely resemble
zygotic embryo development, and in dicots pass through the
globular, heart and torpedo stage. Somatic embryos have been
obtained in many different plant species and from a wide variety
of starting materials such as microspores, protoplasts, immature
embryos, tissue explants and in vitro cultured cells (see for
recent reviews Dudits et al., 1995; Mordhorst et al., 1997). The
events that take place during the period in which plant cells
undergo the transition from somatic to embryogenic cell are
poorly understood (reviewed by De Jong et al., 1993). In tissue
explants, the first response is often noted to be the rapid
replacement of the vacuole with cytoplasm, followed by the first
division. In Dactylis glomerata, leaf mesophyll cells respond
this way (Trigiano et al., 1989), while in Sorghum bicolor also
vascular tissue, close to the wound surface, responds (Wernicke
and Brettel 1980). The same responses are observed in
Cichorium (Dubois et al., 1991), but in contrast to these studies,
in Medicago sativa (Dos Santos et al., 1983) and in Ranunculus sceleratus (Konar et al., 1972), particular types of epidermal
cells were noted to be the responsive ones. In all of these
examples, in vitro regenerated plantlets were the source of the
Key words: Daucus carota L., somatic embryo, embryogenic cell,
leucine-rich repeat receptor-like kinase, zygotic embryo, SERK
2050 E. D. L. Schmidt and others
thought to involve an auxin dependent transition stage occurring
in single cells. Cells in this transition between the somatic and
embryogenic cell state are defined as competent cells
(Komamine et al., 1990; Toonen et al., 1994), which is an operational definition based on the requirement for exogenous auxin.
Recording of the developmental fate of many thousands of individual carrot cells from established embryogenic suspension
cultures by cell tracking revealed that competent cells have a
highly variable appearance that prevents their identification on
the basis of morphology (Toonen et al., 1994). Using this system
with cells from an activated carrot hypocotyl explant revealed
that a small subset of a particular type of elongated single cells
are the ones that first acquire the competence to form embryogenic cells. Apart from the ability to form embryogenic cells,
these cells are indistinguishable from the majority of the
elongated single cells.
It is generally assumed that the formation of plant embryos
requires the activation of specific sets of genes (reviewed by
Goldberg et al., 1994; Thomas et al., 1993) and many studies have
employed differential screening techniques to identify such genes
(eg. Wilde et al., 1988; Aleith and Richter, 1990; Wurtele et al.,
1993; Heck et al., 1995). The corresponding expression pattern
of these genes during zygotic embryogenesis allowed classification into several groups (Goldberg et al., 1989; Sterk and de
Vries, 1992). While many of the genes found to be expressed in
early somatic embryos appeared to encode genes normally
expressed late in zygotic embryogenesis or throughout plant
development (reviewed by Zimmerman, 1993), others such as
LTP (Sterk et al., 1991) and EMB-1 (Wurtele et al., 1993) have
been shown to be expressed at the corresponding, globular, stage
in zygotic embryogenesis. Embryo-expressed MADS box-containing regulatory proteins have been identified in both Brassica
napus (Heck et al., 1995) and in Arabidopsis thaliana (Rounsley
et al., 1995). The Arabidopsis gene, AGL15, is expressed as early
as the 8-celled zygotic embryo, and is also present uniformly in
torpedo stage embryos, in seedlings and to a lower level in leaves
(Rounsley et al., 1995). Several genes have been reported
(reviewed by Zimmerman, 1993) that are putative markers for
embryogenic cell clusters, but none have been described to date
that are reliable markers for the preceding stage of competent
cells. There may be several reasons for this, such as the bias in
many screening procedures towards more abundantly expressed
genes, the low number of competent cells present in embryogenic
cultures and the unavailability of rapid and reliable procedures to
detect gene expression in single cells. To overcome these difficulties screens were carried out employing a series of carrot cell
cultures with widely differing numbers of single competent cells
as the starting material. A small number of genes were found for
which expression is detectable in single competent cells in
embryogenic cell cultures. One of these genes was investigated
in more detail and found to encode a receptor-like kinase that
appears to mark competent and embryogenic cells.
MATERIALS AND METHODS
Plant material, cell cultures, hypocotyl explant activation
and cell tracking
Seedlings of Daucus carota cv. Flakkese were grown for 2-3 weeks in
vermiculite, while adult plants of this cultivar were obtained from S&G
Seeds (Enkhuizen). Controlled pollination was performed by hand, and
complete umbels removed at various days after pollination (DAP).
Flower RNA was obtained from three complete umbels for each time
point and contained all flower organs including residual pollen grains.
Cell cultures were derived from Daucus carota cv. Flakkese and maintained as previously described (De Vries et al., 1988a). Cell suspension
culture was carried out at high cell density in B5 medium (Gamborg et
al., 1968) supplemented with 2 µM 2,4-D (2,4-dichlorophenoxy-acetic
acid; B5-2 medium). Embryo cultures with globular, heart and torpedostage somatic embryos were derived from <30 µm sieved cell cultures
cultured at low cell density (100,000 cells / ml) in B5 medium without
2,4-D (B5-0). For hypocotyl explant induction experiments, plantlets
were obtained from seed of Daucus carota cv. S. Valery as described
previously (Guzzo et al., 1994). The hypocotyls of 1-week old plantlets
were divided in segments of 3-5 mm, incubated for various periods of
time in B5-2 medium and returned to B5-0 medium. Seven days after
explantation and exposure to 2,4-D, the hypocotyl segments were
washed in B5-2 medium and subsequently fragmented on a 170 µm
sieve and the resulting cells collected to form a fine cell suspension.
Immobilization of these cells in B5-0.2 medium was performed in a
thin layer of phytagel (Toonen et al., 1994). After 1 week of further
culture, 2,4-D was removed by washing the plates with B5-0 medium.
This allowed embryos to develop beyond the globular stage. Development of the immobilized cells was recorded using a modified procedure
described by Toonen et al. (1994). The main change involved a new
MicroScan program for automatic 3-axis movement to scan all cells in
the phytagel (Toonen and de Vries, 1997).
Nucleic acid isolation and analysis
RNA was isolated from cultured cells and plant tissues as described
by De Vries et al. (1988b). Poly(A)+ RNA was obtained by purification by oligo(dT) cellulose (Biolabs). For RNA gel blot analysis
samples of 10 µg total RNA were electrophoresed on formamide gel,
and transferred to Nytran-plus membranes (Schleicher & Schuell).
For spot-dot northern analysis, 5 µg of total RNA was denatured and
spotted onto nytran-plus filters using a hybridot manifold (BRL).
Hybridization of RNA blots took place at 42°C in hybridization buffer
containing 50% formamide, 6× SSC, 5× Denhardt, 0.5% SDS and 0.1
mg/ml salmon sperm DNA. Genomic DNA was isolated according to
the method of Sterk et al. (1991). Samples of 10 µg genomic DNA
were digested with different restriction enzymes and separated on
agarose gel, and transferred to Nytran-plus membranes. Hybridization
of DNA blots was performed as previously described (Sterk et al.,
1991). Following hybridization, filters were washed under stringent
conditions (3× 20 minutes in 0.1% SSC, 1% SDS, at 65°C). Filters
were exposed to Kodak XOmat AR film. Nucleotide sequence
analysis was performed on an ABI 373A automated DNA sequencer
(Applied Biosystems). The sequences reported here have been
deposited in the GenBank database, accession number U93048.
SERK promoter-luciferase expression
A 2200 bp HindIII/DraI genomic DNA fragment, with the DraI site 42
bp upstream of the predicted translational start codon of SERK, was
cloned into the binary vector pMT500 (Toonen et al., 1996b) containing the firefly luciferase reporter gene (Millar et al., 1992). The resulting
construct was transformed into carrot cells as described for the AtLTP1
promoter-luciferase constructs (Toonen et al., 1996b). A subpopulation
of <50 µm diameter cells from a primary transformed suspension
culture was embedded in phytagel with B5-0 medium at a concentration of 100,000 cells/ml. In single suspension cells, luciferase activity
was not detectable with the CCD camera system described (Toonen and
de Vries, 1997), even after a one hour exposure. It was only possible
to measure luciferase activity in single cells by using a CCD camera
(Photometrics) without a microscope, directly over the cells. Since
luciferase is quickly deactivated in the presence of luciferin, measurements were made immediately after the addition of luciferin (Promega)
to a final concentration of 20 µM in order to obtain the signal from the
accumulated luciferase. The spatial resolution in the digitized images
of the CCD camera is restricted to a pixel size of 30 by 30 µm. To dis-
Somatic embryo receptor-like kinase 2051
tinguish luciferase activity from e.g. cosmic radiation, only signals
observed in three consecutive 1-hour exposures were retained and positioned over the microscopic images of the same area of the culture dish.
Development of the cells was recorded for 13 days as described by
Toonen and de Vries (1997). A detailed analysis of the SERK promoter
and of the luciferase-expressing cells will be presented elsewhere.
Screening procedures
Two independent cDNA libraries were constructed with equal amounts
of poly(A)+ RNA from total established cell cultures grown for 6 days
in B5-2 medium, sieved <125 µm cell cultures grown for 6 days in B50 medium, and sieved <30 µm cell cultures grown for 6 days in B5-0
medium. cDNA synthesis and cloning into the Uni-ZAPTM XR vector
was performed according to the manufacturers protocol (Stratagene).
Differential screening of the cDNA libraries was performed essentially as described by Scott et al. (1991). RNA was isolated from either
three embryogenic or three non-embryogenic cell cultures, which
were grown for 7 days in B5-2 after sieving through a 30 µm mesh.
First strand cDNA synthesis was performed on 4 µg total RNA using
AMV reverse transcriptase (RT; Gibco BRL). [32P]dATP labeled
probes were prepared using random prime labeling on first strand
cDNA. Pooled probes from high embryogenic or non-embryogenic
cell populations were hybridized to two pairs of nitrocellulose filters,
each containing 1000 plaques from one cDNA library. After washing
for 3× 20 minutes in 0.1% SSC, 1% SDS at 65°C, hybridization was
visualised by autoradiography for 2 days on Kodak XOmat film.
Plaques that only showed signal with the embryogenic transcript
probe were purified by two further rounds of screening.
In order to identify cDNA clones which are expressed at low levels
in the <30 µm sieved cell population, cold plaque screening was
performed as described by Hodge et al. (1992). Plaques from the differential screening that did not show any signal after 7 days of autoradiography were purified by two further rounds of screening. The
resulting clones were used as probes for characterization of the
expression pattern of the corresponding genes.
Differential display RT-PCR
Differential display (dd) of mRNA was performed essentially as
described by Liang and Pardee (1992). cDNA synthesis took place by
annealing 1 µg of total RNA in 10 µl buffer containing 200 mM KCl,
10 mM Tris-HCl (pH 8.3), and 1 mM EDTA with 100 ng of one of
the following anchor primers: (5′-TTTTTTTTTTTGC-3′), (5′TTTTTTTTTTTCTG-3′), (5′-TTTTTTTTTTTCA-3′). Annealing
took place by heating the mix for 3 minutes at 83°C followed by incubation for 30 minutes at 42°C. Annealing was followed by the addition
of 15 µl prewarmed cDNA buffer containing 16 mM MgCl2, 24 mM
Tris-HCI (pH 8.3), 8 mM DTT, 0.4 mM dNTP, and 4 Units AMV
reverse transcriptase (Gibco BRL). cDNA synthesis took place at 42°C
for 90 minutes. First strand cDNA was phenol/chlorophorm extracted
and precipitated with ethanol using glycogen as a carrier. The PCR
reaction was performed in a reaction volume of 20 µl containing 10%
of the synthesized cDNA, 100 ng of anchor primer, 20 ng of one of
the following 10-mer primers: (5′-GGGATCTAAG-3′), (5′TCAGCACAGG-3′), (5′ GACATCGTCC-3′), (5′-CCCTACTGGT-3′),
(5′-ACACGTGGTC-3′), (5′-GGTGACTGTC-3′), 2 µM dNTP, 0.5
Unit Taq enzyme in PCR buffer (10 mM Tris-HCl (pH 9.0), 1.5 mM
MgCl2, 50 mM KCl, 0.01% gelatin and 0.1% Triton X-100) and 6 nM
[α-32P]dATP (Amersham). PCR parameters were 94°C for 30 seconds,
40°C for 1 minute, and 72°C for 30 seconds for 40 cycles using a Cetus
9600 (Perkin-Elmer). Amplified and labeled cDNAs were separated on
a 6% denaturing DNA sequencing gel. Gels were dried without fixation
and bands were visualized by 16 hours of autoradiography using
Kodak XOmat film. Bands containing differentially expressed cDNA
fragments of 150-450 nucleotides were cut out of the gel and DNA
was extracted from the gel slices by electroelution onto DE-81 paper
(Whatmann). Reamplification of the cDNA fragments was performed
using the same PCR cycling parameters as described above but with
reaction buffer containing 2.5 µM of both the 10-mer and the anchor
oligo and 100 µM dNTP. Reamplified PCR products were cloned into
pBluescript vector II SK (Stratagene).
RT-PCR
2 µg of total RNA from adult plant tissue or cell cultures was annealed
at 42°C with 50 ng oligo (5′-TCTTGGACCAGATAATTC-3′) in 10
µl annealing buffer (250 mM KCl, 10 mM Tris-HCl pH 8.3, 1 mM
EDTA). After 30 minutes annealing, 10 units AMV-reverse transcriptase were added in a volume of 15 µl cDNA buffer (24 mM TrisHCl pH 8.3, 16 mM MgCl2, 8 mM DTT, 0.4 mM dNTP). The reverse
transcription reaction took place for 90 minutes at 42°C. PCR amplification of SERK cDNA was carried out with two specific oligos for
the SERK kinase domain, (5′-CTCTGATGACTTTCCAGTC-3′) and
(5′-AATGGCATTTGCATGG-3′). Amplification was carried out with
30 cycles of 30 seconds at 94°C, annealing at 54°C for 30 seconds
and extension at 72°C for 1 minute, followed by a final extension for
10 minutes at 72°C. SERK PCR products were then separated by
agarose gel electrophoresis, blotted and hybridized with a radiolabeled kinase domain of SERK cDNA. Following hybridization,
filters were washed under stringent conditions (3× 20 minutes in 0.1%
SSC, 1% SDS, at 65°C). The resulting hybridizing band of 680 bp
was in agreement with the expected size of the PCR product. Independent RT-PCR experiments were performed twice with similar
results. Hybridization with an 18S ribosomal probe of a northern blot
loaded with similar amounts of RNA as used for the RT-PCR reactions
confirmed the integrity and amounts of RNA used in the experiment.
Whole-mount in situ hybridization
Whole-mount in situ hybridizations were performed essentially as previously described (Engler et al., 1994). Cell cultures and somatic
embryos were immobilized on poly-L-lysine coated slides during
fixation to improve handling. Whole-mount in situ hybridization on
explants took place by embedding hypocotyls from 7-day old plantlets
in 3% Seaplaque agarose (Duchefa) and processing them in Eppendorf
tubes. Transverse as well as longitudinal sections were made with a
vibrotome (Biorad Microcut). Sections of 50-170 µm thick were
incubated in B5-2 medium for a minimum of 3 days to induce
formation of embryo-forming cells. Optimal induction was achieved
with longitudinal hypocotyl sections with a thickness of at least 90 µm.
To obtain proliferating, non-embryogenic cell cultures, hypocotyl
sections were exposed to 2,4-D for only 1 day, and subsequently transferred to B5-0 medium (Guzzo et al., 1994). Whole-mount in situ
hybridization on developing seeds was performed by removing the
chalazal end of the seeds to allow easier probe penetration. After
hybridization, the enveloping layers of integuments and endosperm
were carefully removed to expose the developing embryos. All samples
were fixed for 60 minutes in PBS containing 70 mM EGTA, 4%
paraformaldehyde, 0.25% glutaraldehyde, 0.1% Tween 20, and 10%
DMSO. Samples were then washed, treated with proteinase K for 10
minutes, again washed and fixed a second time. Hybridization solution
consisted of PBS containing 0.1% Tween 20, 330 mM NaCl, 50 µg/ml
heparin, and 50% deionized formamide. Hybridization took place for
16 hours at 42°C using digoxigenin-labeled sense or antisense riboprobes (Boehringer Mannheim). After washing, the cells were treated
with RNase A, and incubated with anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim) which had been preabsorbed with a plant protein extract. Excess antibody was removed by
washing, followed by rinsing in staining buffer (100 mM Tris-HCl pH
9.5, 100 mM NaCl, 5 mM MgCl2, 1 mM levamisole) and the staining
reaction was performed for 16 hours in a buffer containing 4-nitroblue
tetrazolium chloride and 5-bromo-4-chloro-3-indoyl-phosphate.
Observations were performed using a Nikon Optiphot microscope
equipped with Nomarski optics or brightfield optics.
Autophosphorylation assay
A 1.4 kb SspI cDNA fragment of the SERK cDNA encoding most of
2052 E. D. L. Schmidt and others
the open reading frame was cloned into the pGEX expression vector
(Pharmacia). A fusion protein consisting of SERK and the glutathione
S-transferase (GST) gene product was isolated and purified as
described previously (Horn and Walker, 1994). Purified fusion protein
was coupled to glutathione agarose beads (Sigma) and incubated for
20 minutes at 20°C in a volume of 10 µl buffer: 50 mM Hepes (pH
7.6), 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 1 µCi [γ-32P] (3,000
Ci/mmol). Excess label was removed by washing the fusion
protein/glutatione agarose beads three times for 5 minutes in 50 mM
Tris-HCl (pH 7.3), 10 mM MgCl2 at 4°C. Protein was removed from
the beads by cooking in SDS-PAGE loading buffer. Equal amounts of
protein were separated by SDS-PAGE and protein autophosphorylation was visualized by autoradiography.
RESULTS
Isolation of cDNA clones that are preferentially expressed
in embryogenic cell cultures of carrot
In order to increase the chance of obtaining genes expressed in
carrot suspension cells competent to form embryos, the number
of such cells in a series of established cell cultures was determined. A subpopulation of cells that had passed through a 30
µm nylon sieve was isolated from eight different cultures ranging
in age between 2 months and 4 years. In these <30 µm populations, the number of embryos formed from the single cells and
small cell clusters was determined and expressed as a percentage of the total number of cells present at the start of embryogenesis. Three sieved <30 µm cultures able to form somatic
embryos at a frequency of more than 100 embryos per 10,000
cells were then used as a source for competent cells, and three
other cultures that produced less than 1 embryo per 10,000 cells
were used as non-embryogenic controls. The assumptions made
were that a 100-fold difference in the embryogenic capacity
would indicate a similar difference in the number of competent
cells, and that this would be sufficient to detect the mRNA of a
hypothetical gene only expressed in competent single cells with
the screening methods employed. As main selection strategies,
cold plaque screening (Hodge et al., 1992) and differential
display (dd) RT-PCR (Liang and Pardee, 1992) were used
besides conventional differential screening of cDNA libraries.
Labeled probes for differential screening were obtained from
RNA out of a <30 µm sieved subpopulation of cells from either
embryogenic or non-embryogenic cell cultures. Employing
these probes in a library screen of approximately 2000 plaques
yielded 26 plaques that failed to show any hybridization to
either probe. These so-called cold plaques were purified and
used for further analysis. From the total number of plaques that
did hybridize, about 30 did so only with the probe from
embryogenic cells. Differential display reverse transcriptionpolymerase chain reactions (ddRT-PCR) using a combination
of one anchor primer and one decamer primer were performed
on mRNA isolated from three embryogenic, and three nonembryogenic suspension cultures. About 50 ddRT-PCR
fragments were obtained from each reaction. Using combinations of three different anchor and six different decamer
primers, a total of approximately 1000 different cDNA
fragments was visualized. Six of these PCR fragments were
only found in lanes with mRNA from <30 µm populations of
cells from embryogenic cultures and with oligo combinations
of the anchor primer (5′-TTTTTTTTTTTGC-3′) and the
decamer primers (5′-GGGATCTAAG-3′), (5′-ACACGTGGTC-3′), (5′-TCAGCACAGG-3′). Because differential PCR
fragments often consist of several unresolved cDNA fragments
(Li et al., 1994), cloning proved to be essential prior to undertaking further characterization of the PCR fragments obtained.
All clones obtained were subjected to a second screen that
consisted of spot-dot northern hybridization performed under
conditions of high stringency. This method, which used RNA
from entire unsieved embryogenic and non-embryogenic suspension cultures, proved to be a fast and reliable additional
selection method. Only one clone (22-28) of the 30 clones
obtained after differential screening, proved to be restricted to
embryogenic cell cultures while the majority was constitutively
expressed. The 26 clones obtained from the cold plaque screening
required very long exposure times in the spot-dot northern
analysis. Six of these clones failed to show any hybridization
signal and 19 proved to be expressed in both embryogenic and
non-embryogenic cell cultures (results not shown). One clone
(31-50) showed low expression in all embryogenic cultures and
in one non-embryogenic culture, but not in the others (Fig. 1). Of
the six cloned fragments obtained by ddRT-PCR display, four
showed hybridization more or less restricted to transcripts present
in embryogenic cultures (6-11, 7-13, 10-25, 11-21; Fig. 1). All
clones that passed through the second screening were sequenced.
The clones 6-11 and 7-13 were identical to the carrot Lipid
Transfer Protein (LTP) gene, previously identified as a marker for
embryogenic carrot cell cultures. LTP expression is restricted to
embryogenic cell clusters and the protoderm of somatic and
zygotic embryos from the early globular stage onwards (Sterk et
al., 1991). Therefore, while the LTP gene is not a marker for
competent cells, its appearance in the screening confirms the
validity of our methods with respect to the cloning of genes
expressed early during somatic embryogenesis.
cDNA clone 31-50 encodes a leucine-rich repeat
containing receptor-like kinase
The predicted amino-acid sequence of the cDNA clone 31-50
(Fig. 2A) shows homology with the structural features of plant
and animal receptor protein kinases. Because clone 31-50 is
Fig. 1. Screening for competent cell markers. 5 µg of total RNA,
from non embryogenic cultures with less than 1 embryo per 10,000
cells (−), embryogenic cultures with apprimately 20 embryo per
10,000 cells (+), and high embryogenic cultures with over 100
embryos per 10,000 cells (++), was immobilized and hybridized to
labeled DNA probes. The control panel shows hybridization to a
constitutively expressed ubiquitin transcript. Exposure of the
autoradiographs was for 16 hours, except for clone 31-50 encoding
SERK, of which the autoradiograph was exposed for 14 days.
Somatic embryo receptor-like kinase 2053
A
B
-32
ctcatttaattttactttaaaaaataattctatATGAATCGTAACAGTATAAATATATTA
M N R N S I N I L
27
28
AATTACATGCAGTTCACTGATGCTTACCTTGACAAATATGGGGTTCTTATGACATTGGAG
N Y M Q F T D A Y L D K Y G V L M T L E
87
88
CTTTACAGCAATAACATAAGTGGACCAATTCCTAGTGATCTTGGGAATCTGACAAATTTG
L Y S N N I S G P I P S D L G N L T N L
*
*
GTGAGCTTGGACCTATACATGAATAGCTTCTCTGGACCTATACCGGACACATTAGGAAAG
V S L D L Y M N S F S G P I P D T L G K
147
SERK
P**L**L**L**L*L*NN*LSGPI
5
207
RLK5
P**L**L**L**L*L**N*LSG*I
21
ERECTA
P**LG*L**L**L*L**N*L*G*I
19
CTTACAAGGCTAAGATTCTTGCGTCTCAACAACAACAGCCTCTCTGGTCCAATTCCAATG
L T R L R F L R L N N N S L S G P I P M
*
TCACTGACTAATATTACAACTCTTCAAGTCCTGGATTTATCAAACAATCGGCTATCAGGA
S L T N I T T L Q V L D L S N N R L S G
*
CCAGTACCGGATAATGGCTCATTTTCTTTGTTTACACCTATCAGTTTTGCCAATAATTTG
P V P D N G S F S L F T P I S F A N N L
*
AATTTATGTGGACCCGTAACTGGGAGGCCCTGCCCTGGATCTCCCCCATTTTCGCCACCA
N L C G P V T G R P C P G S P P F S P P
o o
CCTCCGTTCATCCCACCATCAACAGTACAGCCTCCAGGACAAAATGGTCCCACTGGAGCT
P P F I P P S T V Q P P G Q N G P T G A
o o
ATTGCTGGGGGAGTAGCTGCTGGTGCTGCTTTACTGTTTGCTGCACCTGCAATGGCATTT
I A G G V A A G A A L L F A A P A M A F
267
PRK1
P**L**L**L**L*L**NN**G*I
5
CF-9
PS*L**L**L**LDLSSNNL*G*I
26
TOLL
P**LF*H**NL**L*L**N*L**L
15
568
GCATGGTGGCGGAGAAGAAAACCGCGAGAACATTTCTTTGATGTGCCAGCTGAAGAGGAC
A W W R R R K P R E H F F D V P A E E D
627
628
CCAGAAGTGCACCTTGGTCAACTGAAGAGGTTTTCTCTGCGAGAATTGCAAGTCGCAACG
P E V H L G Q L K R F S L R E L Q V A T
687
688
GATACTTTTAGTACCATACTTGGAAGAGGTGGATTTGGTAAGGTGTATAAGGGACGCCTT
D T F S T I L G R G G F G K V Y K G R L
747
748
GCTGATGGCTCACTTGTAGCAGTTAAAAGGCTTAAAGAAGAACGAACACCAGGTGGTGAG
A D G S L V A V K R L K E E R T P G G E
807
808
CTGCAGTTTCAAACAGAGGTGGAAATGATTAGCATGGCTGTGCATCGAAATCTTCTGCGT
L Q F Q T E V E M I S M A V H R N L L R
867
868
CTACGTGGTTTCTGCATGACACCAACAGAGCGGCTTCTTGTATATCCATACATGGCTAAT
L R G F C M T P T E R L L V Y P Y M A N
*
GGAAGTGTTGCGTCGTGTTTAAGAGAGCGTCAGCCATCAGAACCTCCCCTTGATTGGCCA
G S V A S C L R E R Q P S E P P L D W P
927
988
ACTAGGAAGAGGATTGCACTAGGATCTGCTAGGGGGCTTTCTTATTTGCATGACCATTGT
T R K R I A L G S A R G L S Y L H D H C
104
1048
GATCCCAAGATTATCCATCGTGATGTAAAAGCTGCAAATATATTATTGGACGAAGAATTT
D P K I I H R D V K A A N I L L D E E F
110
1108
GAGGCTGTTGTAGGTGATTTTGGGTTAGCTAGGCTCATGGATTACAAGGATACCCATGTT
E A V V G D F G L A R L M D Y K D T H V
116
1168
ACAACTGCTGTAAGGGGTACCTTGGGCTACATAGCTCCCGAGTACCTCTCGACTGGAAAG
T T A V R G T L G Y I A P E Y L S T G K
122
1228
TCATCAGAGAAGACCGATGTCTTTGGTTATGGGATTATGCTCTTAGAGCTCATTACTGGA
S S E K T D V F G Y G I M L L E L I T G
128
1288
CAGAGAGCTTTTGATCTTGCTCGCCTTGCGAACGATGATGATGTTATGTTGTTGGATTGG
Q R A F D L A R L A N D D D V M L L D W
134
1348
GTTAAAAGCCTTTTGAAAGAGAAAAAGTTGGAGATGCTGGTCGATCCTGACCTGCAGAAC
V K S L L K E K K L E M L V D P D L Q N
140
1408
AATTACATTGACACAGAAGTTGAGCAGCTTATTCAAGTAGCATTACTCTGTACCCAGGGT
N Y I D T E V E Q L I Q V A L L C T Q G
146
1468
TCGCCAATGGAGCGGCCTAAGATGTCAGAGGTAGTCCGAATGCTTGAAGGTGATGGCCTT
S P M E R P K M S E V V R M L E G D G L
152
1528
GCAGAAAAGTGGGACGAGTGGCAAAAAGTAGAAGTCATCCATCAAGACGTAGAATTAGCT
A E K W D E W Q K V E V I H Q D V E L A
158
1588
CCACATCGAACTTCTGAATGGATCCTAGACTCGACAGATAACTTGCATGCTTTTGAATTA
P H R T S E W I L D S T D N L H A F E L
164
1648
TCTGGTCCAAGAtaaacagcatataaaatgtgaatgaaattaatattttttatggttaaa
S G P R ***
170
1708
aaaaaaaaaaaaaaa
148
208
268
328
388
448
508
Protein
327
Consensus Sequence
387
447
507
567
C
I
II
1722
III
SERK
OsPK10
PRK1
PELLE
ATDTFSTILGRGGFGKVYKGRLAD
..SN.CNK..Q....S..L.T.P.
LLRASAEV..S.NL.SS..AL.M.
DGWSPDNR..Q....D..R.KWK
SERK
OsPK10
PRK1
PELLE
VHRNLLRLRGFCMT
H.IH.VK.....TE
T.P...P.VAYYYR
R.D.I.A.Y.YSIK
PTERLLVYPYMANGSVASCLRERQ
GPH...A.E......LDKWIFHSK
KE.K....D.AS...L..H.H
GGKPC...QL.KG..LEAR..AHKAQ
VIA
VIB
SERK
R2976
OsPK10
PRK1
PELLE
PSEPPLDWPTRKRIALGSARGLSYLHDHCDPK IIHRDVKAANILLDEEF
...A.............N... ...............D.
EDDHL...D..FN....T.K..A...QD..S. .V.C.I.PE.V...DN.
GNQSR...SS.LK.VK.V.KA.A...NELPSLALP.GHL.SS.V...KYL
NPL.A.T.QQ.FS.S..T...IYF..TARGTPL ..G.I.P......QCL
SERK
RIC2976
OsPK10
PRK1
PELLE
EAVVGDFGLARLMDYKDTHV TTAVRGTLGYIAPEY
...F......KP........ ....H..I.H.....
I.K.S.....K..TREQS.. F.TL...R..L...W
NP.LM.YT.VP.VNLAQ
VQHLLVA.K....
QPKI.....V.EGPKSLDA.VEVNK.F..KI.LP..F
SERK
R2976
OsPK10
PRK1
PELLE
LSTGKSSEKTDVFGYGIMLLELITGQRAFDLA
....IL...................K.V....
.TNYAI...S..YS..MV...I.G.RKSY.PS
AQQ.RITR....WSL..L...TL..KFPTNYL
RNFRQL.TGV..YSF..V...VF..RQVT.RV
SERK
R2976
OsPK10
PRK1
PELLE
KEKKLEMLVDPDLQNNYIDT
EVEQLIQVALLCTQGSPMERPKMSEVVRMLE
GGNAG.I .......K...
..DS....V........LD....VA.A.I.DE
GDLQDIFDAKLKYNDKDG
R..TA.K...W.I.DDFYQ..S..K..Q...
RDNESAFDKEMNTTKDSQ
QIRK.FDIGVA.C.EDLDT.WDLK...QSOQ
RQNRM.L.EKHLAAPMGKELD MCMCA.EAG.H..A.D.QD..S.NA.LKRF.
SERK
R2976
OsPK10
PRK1
PELLE
GDGLAEKWDEWQKVEVIHQDVELA
PHRTSEWILDSTDNLHAFELSGPR*
..A...R.R....I.IVQ.....
GLYKNG.TV...E....V......*
.VCEVLQPPVSSQIGYRLYANAFKSSSEEGTSSGMSDYN.DAL.S.VR.....*
SLNDKDHGHSNSDQMHDAGV*
FTVTD*
IV
928
Extracellular
LRR Repeats
GSLVAVKRLKEERTP
GGELQFQTEVEMISMA
..RI...K. .GIGQ
.KKE.RS..TI.GSI
.QA.V...F. QMNH
VAKED.HEHMRRLGRL
QLD..I.VMNYRSPNIDQK PV...QSYNELKYLNSI
V
987
VII
VIII
IX
X
X
RLANDDDVMLLDWVKSLL
L.RGEG..P...AQTITQ
EISEKAHFPSFAFK.LEE
A.STGYGTE.AT..DTII
PE.ETKKN...Y..QQW
XI
Fig. 2. Comparison between the SERK amino acid sequence and related sequences. (A) The predicted amino acid sequence of SERK is
presented in the single letter code. Asterisks denote potential N-linked glycosylation sites and open circles indicate potential O-glycosylation
sites. The putative membrane-spanning region is underlined. The nucleotides are numbered, starting at 1 from the translational start site.
Capitals mark the open reading frame. (B) The consensus sequences of SERK LRRs compared with other LRR-containing proteins. Nonconserved amino acids are indicated by an asterisk. The other LRR-containing sequences are from the following proteins: Arabidopsis RLK5
(Walker, 1993), Arabidopsis ERECTA (Torii et al., 1996), Petunia PRKI (Mu et al., 1994), Lycopersicon Cf-9 (Jones et al., 1994) and
Drosophila Toll (Hashimoto et al., 1988). (C) Sequence alignment of the predicted kinase domain and the remaining C-terminal part of the
carrot SERK protein with protein kinases from Oryza OsPK10 (Zhao et al., 1994), Oryza EST clone R2976 representing a partial cDNA,
Petunia PRK1 and Drosophila Pelle (Shelton and Wasserman, 1993). The 11 conserved protein kinase subdomains are indicated I to XI (Hanks
et al., 1988). Conserved residues are only indicated in the SERK protein and are represented by a dot in the other sequences. The translation
stop sites are indicated by asterisks.
2054 E. D. L. Schmidt and others
Fig. 3. The kinase domain of SERK. (A) DNA blot hybridization
analysis of carrot genomic DNA hybridized to the HincII-SspI
fragment of the SERK cDNA clone encoding the kinase domain. The
restriction enzymes indicated were used to digest 10 µg of genomic
DNA. (B) Autophosphorylation assays (Mu et al., 1994) were
performed using the SERK kinase domain expressed in E. coli. GSTSERK fusion proteins were tested using the 1.4 kb SspI cDNA
fragment of the SERK cDNA clone in the reverse orientation (first
lane), in the sense orientation in frame with the GST protein
resulting in a 70×103 Mr fusion protein (second lane), the GST
protein alone (third lane), and an E. coli extract from an
untransformed control (fourth lane). Labeled protein was visualized
by autoradiography after SDS-PAGE.
expressed in embryogenic cell cultures it was renamed Somatic
Embryogenesis Receptor Kinase (SERK). The SERK protein
contains an N-terminal domain with five leucine-rich repeats
(LRRs; Fig. 2B) that is proposed to act as a protein-binding
region in LRR receptor kinases (Kobe and Deisenhofer, 1994).
Several potential N-glycosylation sites are present in the LRR
motifs and one in the intracellular kinase domain (Fig. 2A).
Between the extracellular LRR domain of SERK and the
membrane-spanning region there is a 32 amino acid region rich
in prolines (13), that is unique for the SERK protein. Of particular interest is the sequence SPPPP, which is conserved in
extensins, a class of universal plant cell wall proteins (Varner
1
A
B
and Lin, 1989). The significance of this proline-rich box (Fig.
2A) is not clear, it might act as a hinge region by providing flexibility to the extracellular part of the receptor, or act as a region
for interaction with the cell wall. In extensins, usually all
prolines in the SPPPP repeat are hydroxylated and are considered to be used as targets for O-linked glycosylation. Although
a transmembrane domain is present in the protein, the Nterminal amino acids do not clearly display characteristics of a
3
5
7
12
16
18
1
9
Fig. 4. An example of the embryo formation as observed in a cell tracking experiment. The images of the same area of a culture dish are taken
from videotapes used to record development. The number of days of culture after fragmentation of the activated hypocotyl explants is indicated
in each frame. The cells were grown in medium with a low amount of 2,4-D for 7 days after fragmentation. After this period, the 2,4-D was
removed to allow embryo formation to proceed. Bar: 100 µm.
Somatic embryo receptor-like kinase 2055
A
a
❉
eg
Cc
b
B
Ee
d
D
❉
❉
❉
❉
❉
Fig. 5. Examples of cell populations released from activated explants. (A) Based on the size and on the known position of particular cell types
within the hypocotyl before fragmentation, 4 morphologically different types of cells were distinguished, and examples of each are shown:
black circle: small cytoplasm-rich cell; black square: enlarging cell; white circle: enlarged cell; white square: large cell. The enlarged cell
labeled with an asterisk is a competent cell, since it gave rise to an embryo as determined by cell tracking (see Fig. 4). (B-E) Asterisks indicate
some of the 24,722 cells recorded that were observed to form embryos in the cell tracking experiment. Bar: 100 µm.
signal peptide. The proposed intracellular domain of the protein
contains the 11 subdomains characteristic of the catalytic core
of protein kinases (Hanks et al., 1988; Fig. 2C). The core
sequences HRDVKAAN and GTLGYIAPE in respectively the
kinase subdomains VI and VIII suggest a function as a serine /
threonine kinase (Hanks et al., 1988). Another interesting
feature of the intracellular part of the SERK protein is that the
second half of the C-terminal motif resembles an LRR and is
also present in two other plant proteins that resemble protein
kinases (Fig. 2C). This domain may be involved in mediating
the protein-protein interaction necessary for transmission of an
intracellular phosphorylation cascade.
Hybridization of the SERK cDNA clone to the carrot
genome revealed the presence of only a single main hybridizing band, indicating a single SERK gene in the carrot genome
(Fig. 3A). No signal was observed after northern blotting of
mRNA from embryogenic cell cultures and hybridization
with labeled SERK probes (results not shown), reflecting the
Table 1. Correspondence between number and cell type of embryo-forming cells and cells expressing the SERK gene
Cell tracking
In situ hybridization
Total no. of cells
No. of cells forming
embryos (% of total)
Total no. of cells
No. of cells expressing
SERK (% of total)
4976
0
325
0
Enlarging
(±16×40 µm)
10764
0
763
0
Enlarged
(±35×90 µm)
3511
20 (0.56%)
1593
7 (0.4%)
Large
(>60×140 µm)
5471
0
850
0
Cell type
Small cytoplasm rich
(±16×16 µm)
2056 E. D. L. Schmidt and others
Fig. 6. SERK gene expression during hypocotyl
explant activation. Gene expression is visible as a
purple precipitate in individual cells. (A-E) Cell
population obtained by mechanical fragmentation of
the 2,4 D-treated hypocotyls. Only a few of the
enlarged cells show SERK expression (asterisks).
(F) Longitudinal vibrotome section of a hypocotyl
before 2,4-D treatment shows complete absence of
SERK expression in any type of cell.
(G-I) Proliferating cell masses that originate from
the provascular tissue (longitudinal section). In G a
single enlarged cell shows SERK expression while
the adjacent rows of morphologically identical cells
do not express the gene. In H a single enlarged
SERK-expressing cell is in the process of
detachment from the surface of the proliferating
mass. In I a small cluster of enlarged cells showing
SERK expression is present at the surface of
proliferating tissue. (J) Proliferating mass of cells
developed from the provascular tissue of the
hypocotyl 10 days after the beginning of a short (24
hours) 2,4-D treatment followed by culture in basal
medium. The lateral root primordia and the enlarged
cells detaching from the surface are devoid of SERK
mRNA. Bar: 50 µm.
low levels of transcript present in these cultures. Detection of
the SERK transcript on the original spot-dot northerns was
only possible after long exposure times compared with other
probes (Fig. 1).
Recently a number of LRR receptor-like kinases have been
isolated from other plant species (Walker, 1993, 1994).
Homology between the SERK protein and receptor-like
kinases from Petunia (Mu et al., 1994) and Oryza (Zhao et al.,
1994) could be observed (Fig. 2C), as well as with some of the
recently identified pathogen resistence gene products (Fig. 2B)
like tomato Cf-9 (Jones et al., 1994). BLAST database searches
resulted in the identification of the dbEST clone R2976. This
partial rice clone shows a 74% identity on the amino acid level
with SERK, suggesting that this gene is highly conserved
between monocots and dicots. The ability of the SERK protein
to autophosphorylate was investigated in vitro, using a previously described autophosphorylation assay (Mu et al., 1994),
with a bacterial fusion protein that contained the complete
intracellular region of the SERK protein. The bacterially
expressed SERK fusion protein indeed proved to be able to
autophosphorylate (Fig. 3B), indicating that the SERK protein
is able to fulfill a role as a protein kinase.
Expression of the SERK gene corresponds to the
first appearance of competent cells during
hypocotyl activation
The formation of competent cells was determined after
Fig. 7. SERK gene expression in cell cultures. RT-PCR products
obtained from RNA of entire hypocotyls after 10 days of culture are
present in lane one (after 2 days of 2,4-D treatment, nonembryogenic culture) and lane two (after 3 days of 2,4-D treatment,
embryogenic culture). Lane three shows RT-PCR products obtained
from an established non-embryogenic cell culture and lane four an
established embryogenic culture. The electrophoresis pattern of the
resulting 680 bp PCR fragment was blotted and hybridized with a
probe containing the kinase domain of SERK. As a control, a
northern blot with similar amounts of RNA as used for the RT-PCR
reaction, was hybridized with an 18S ribosomal probe.
exposing seed-derived carrot hypocotyl explants to 2,4-D
(Guzzo et al., 1994). When carrot hypocotyls are induced with
Somatic embryo receptor-like kinase 2057
2,4-D, only the cells of the provascular tissue proliferate. This
up the newly initiated embryogenic suspension culture are
suggests that the cells derived from this tissue form all the
actually competent to form embryogenic cells. The expression
different cells, including the embryogenic ones, that are
of the SERK gene was determined by whole-mount in situ
present in a newly initiated suspension culture. Explant cells
hybridization in a population of cells similar to the one used
of cortical and epidermal origin only expand, and are quickly
for the cell tracking experiment. Examples of the results
lost upon subculture. The duration of 2,4-D treatment is
obtained are shown in Fig. 6A-E, and the results of 3,531 cells
important in the formation of embryogenic cells in activated
hybridized with the SERK antisense riboprobe are included in
hypocotyls (Guzzo et al., 1994), and has to be at least 3 days,
Table 1. Expression of the SERK gene was found to be
with an optimal period of around 7 days. In the presence of
restricted to only 0.44% of the enlarged cell type. Therefore,
2,4-D, the formation of competent cells and the transition
the expression of the SERK gene appears to be closely corretowards embryogenic cells is initiated. After
removal of 2,4-D, the formation of somatic
embryos from embryogenic cells occurs after 2-3
weeks. The first appearance of single competent
cells in the explant was determined experimentally
by semi-automatic cell tracking (Toonen et al.,
1994) and was performed on large populations of
immobilized cells. Hypocotyl explants activated
with 2,4-D for 7 days were first washed to remove
previously released cells and to ensure that only
cells still in the explant were included in the
analysis. After mechanical fragmentation of the
explants, samples of the resulting population of
mainly single cells were immobilized to allow
recording of their development by cell tracking. A
typical example of the recordings made is shown in
Fig. 4. In the immobilized cell populations obtained
in this way all the morphologically discernible cell
types were present that were also seen in the unfragmented activated hypocotyls. Fig. 5A-E shows the
different types of cells, which can be divided into
four groups according to mean size and cytoplasmic content (Table 1). Because the same cell types
were observed in sections of activated hypocotyl
explants (Guzzo et al., 1995), it was possible to
predict the position of each type of cell in the
explant. Small cytoplasm-rich cells (16×16 µm) are
found as the proliferating cells that surround the
vascular elements. Enlarging vacuolated cells
(16×40 µm) are encountered on the surface of the
mass of proliferating cells and these can detach
from the surface when fully enlarged (35×90 µm).
Large vacuolated cells (more than 60×140 µm) are
the non-proliferating remnants of the hypocotyl
epidermis and cortical parenchyma. The shape of
the enlarging and fully enlarged cells ranged from
oval to elongate or triangular. Cell tracking on a
total of 24,722 cells released from 7-day activated
hypocotyls showed that only 20 single cells formed
a somatic embryo. Because of their dependance on
Fig. 8. SERK gene expression in established embryogenic cell cultures and in
zygotic embryos. Gene expression is visible as a purple precipitate. (A-D) Very
continued 2,4-D treatment, the embryo-forming
few cells of all morphologically recognizable types of single cells in
single cells are still in the competent cell stage. All
embryogenic suspension cultures show SERK expression. (E-I) Embryo cell
of the embryo-forming single cells belonged to the
culture. SERK transcripts are not detectable in large clusters. (E) SERK
category of 3,511 enlarged cells (Fig. 5B-E) that
transcripts are present in small clusters. (F,G) SERK expression is detected in
therefore contained competent cells in a frequency
small globular embryos. No signal could be detected using a sense SERK
of 0.56%. The cell tracking experiment clearly
riboprobe (H) or during later stages of somatic embryogenesis (I). (J) SERK
reveals that none of the highly cytoplasmic and
transcript is detectable in an approximately 8-celled embryo from a seed
rapidly proliferating cells has reached the
collected at 14 DAP. Note the absence of any hybridization in the other tissues
competent cell state, but that only after elongation
of the seed. (K) An early globular embryo at 17 DAP of approximately 100
the first competent cells form. It is also evident that
cells. (L) An embryo at the globular-heart transition stage at 17 DAP. Bar: A-I,
50 µm; J-L, 100 µm.
only a very limited number of the cells that make
2058 E. D. L. Schmidt and others
and the first appearance of competent cells in explants treated
for at least 3 days with 2,4-D before being returned to basal
medium. Hybridization of northern blots, derived from RNA
electrophoresis patterns on formamide gels, never gave any
signal after hybridization with SERK cDNA probes, not even
after prolonged exposure in a PhosphorImager (results not
shown), in line with the very restricted expression pattern of
the SERK gene.
Fig. 9. SERK gene expression in plant organs. RT-PCR products
obtained from flower/seed RNA at 0 to 20 days after pollination
(lanes one to five), from leaves (lane six), seedling stems (lane seven)
and seedling roots (lane eight). The electrophoresis pattern of the
resulting 680 bp PCR fragment was blotted and hybridized with a
probe containing the kinase domain of SERK. As a control, a
northern blot with similar amounts of RNA as used for the RT-PCR
reaction, was hybridized with an 18S ribosomal probe.
lated, both qualitatively and quantitatively, with the presence
of competent single cells.
To obtain insight into the temporal regulation of SERK
expression during explant activation, whole mount in situ
hybridization was performed on entire intact or handsectioned explants treated for different periods with 2,4-D
(Fig. 6F-J). Representative samples were collected from
explants that were untreated (Fig. 6F) and treated for 7 days
(Fig. 6G, H) or 10 days (Fig. 6I) with 2,4-D. While enlarged
cells became present after the first 5 days of culture, the first
few SERK-expressing enlarged cells were found after 7 days
of culture in the presence of 2,4-D (Fig. 6H). These cells were
present at the surface of the mass of proliferating cells originating from the provascular tissue. In the hypocotyls treated
for 10 days with 2,4-D, the number of SERK-positive cells had
increased to about 3% and included at this stage cells also
present in small clusters (Fig. 6I). No SERK transcript was
ever detected in small cytoplasm-rich cells or large vacuolated
cells. Hypocotyls treated for only 1 day with 2,4-D and subsequently cultured in hormone free medium for 10 days
showed proliferating explant cells that gave rise to roots and
non embryogenic cell cultures. SERK expression could never
be detected in such explants (Fig. 6J). The in situ hybridization results described above were obtained from a relatively
small number of explants, so RT-PCR followed by Southern
hybridization was performed to obtain more quantitative
results. These are shown in Fig. 7 and confirm the close
temporal correlation between the expression of the SERK gene
The SERK gene is expressed in established
embryogenic cell cultures and transiently during
somatic and zygotic embryogenesis
While the results described so far indicate that competent and
embryogenic cell formation is restricted to a particular class
of enlarged cells during explant activation, the situation in an
established embryogenic cell culture is more complex.
Competent single cells in such cultures do not appear to
belong to one cell type in particular, but have been shown to
originate from all morphologically different cell types.
Embryogenic cells, that do not require exogenous auxin
treatment, are thought to be present only in the form of
clusters of at least 3-4 cells and not as single cells (Toonen
et al., 1994). SERK expression was found in all morphologically discernible single cell types that were present in an
embryogenic cell culture (Fig. 8A-D) at a frequency between
0.1 and 0.5% (results not shown) depending on the cell type.
In non-embryogenic cultures, SERK-expressing cells were
never encountered. Unfortunately, the non-embryogenic
culture in which the original spot-dot northern showed SERK
expression was lost and could not be included in this analysis.
We expect that in this particular cell culture the competent
cell stage is initiated but not completed. As was observed in
the activated explants, SERK expression was not restricted to
single cells, but also occurred in small clusters of 2 to 8 cells
(Fig. 8E). Since clusters of this size are known to consist of
embryogenic cells, these data show that SERK expression is
not restricted to competent single cells, but may persist in
small clusters of embryogenic cells. SERK expression was
also followed during the course of somatic embryo development. While, in small globular somatic embryos of up to
about 100 cells, there is a high level of expression (Fig. 8F,G),
no SERK expression was encountered during the mid to late
globular, heart (Fig. 8I) and torpedo-stages of somatic
embryogenesis.
Whole-mount in situ hybridization on partially dissected
carrot seeds showed that the SERK gene was only expressed in
early embryos up to the globular stage (Fig. 8J,K). No expression
luciferase
day 1
day 1
day 2
day 3
day 6
day 9
day 13
Fig. 10. Luciferase expression under control of the SERK promoter. Luciferace activity of immobilised cells was recorded at day 1 with a CCD
camera (left image). The single pixel in the left image measures 30 by 30 µm. Video cell-tracking of the cells was performed for a period of 13
days (light microscopic images). The light microscope images were sized to match the CCD image.
Somatic embryo receptor-like kinase 2059
was seen in seedlings, roots, stems, leaves, developing and
mature flower organs, pollen grains and stigmas before and after
fertilization (results not shown). Tissues in the developing seed
such as seed coat, integuments, all embryo sac constituents
before fertilization, as well as the endosperm at all stages of
development investigated, did not show any SERK expression.
Later stages of carrot zygotic embryos (Fig. 8L) were also completely devoid of SERK mRNA. These results were confirmed
using RT-PCR (Fig. 9) and indicate that no SERK mRNA accumulates in any of the adult plant organs nor in flowers prior to
pollination. The first occasion when SERK expression can be
detected is in flowers at 3 days after pollination (DAP), at which
stage fertilization has taken place and endosperm development
has commenced. SERK mRNA remains present in flowers up to
20 DAP, corresponding to the early globular stage of the zygotic
embryo (Lackie and Yeung, 1996). Therefore, the SERK mRNA
as detected by RT-PCR in flowers at 3 and 7 DAP is likely to
come from SERK gene expression in the zygotes, because in
carrot the zygote remains undivided up to 1 week after pollination (Lackie and Yeung, 1996). Attempts to perform wholemount in situ hybridisation on seeds containing only zygotes
have so far been unsuccessful.
SERK promoter-luciferase expression during
somatic embryogenesis
To determine directly whether SERK-expressing cells indeed
develop into somatic embryos, transformed carrot suspension
cultures containing a SERK promoter-luciferase construct
were analysed for luciferase expression in cell cultures sieved
through a 50 µm mesh to enrich for single cells and small cell
clusters. Development of the immobilized cells after recording
the luciferase images was determined using automated cell
tracking (Toonen et al., 1994, 1997). The origin of nine
torpedo stage somatic embryos was determined this way. Of
these, three developed from a single cell that showed
luciferase activity at day 1, four developed from cell clusters
consisting of 2-6 luciferase-expressing cells while two
embryos developed from single cells that failed to show a
detectable level of luciferase activity at day 1. The somatic
embryo shown in Fig. 10 originated from a luciferase-expressing two-celled cluster. These results demonstrate that most
somatic embryos develop from single cells and small cell
clusters expressing SERK at day 1. Somatic embryos beyond
the globular stage did not show luciferase expression, confirming the transient SERK gene expression pattern (results
not shown).
DISCUSSION
The SERK gene as a marker for the competent cell
stage in somatic embryogenesis
In plants, embryo formation in the absence of the fusion of
two gametes is a widespread phenomenon. It occurs naturally
in certain species in the developing ovule, as exemplified by
apomictic embryogenesis (Koltunow, 1993), or on the surface
of leaves as in Malaxis (Taylor, 1967). More commonly,
embryogenesis can be induced experimentally with a wide
variety of tissue explants as the starting material after
treatment of explants with synthetic growth regulators. It is
generally accepted that the genetic and physiological consti-
tution of the donor plant as well as the age and type of the
explant are important parameters in succesfully inducing
somatic embryogenesis. However, knowledge about the first
events that take place during the transition of somatic cells
into embryogenic cells is largely lacking. There appear to be
two main reasons for this. The first is that cellular changes
observed in somatic explant cells that have responded to the
inducing treatment in general have not been proved to be
essential for the formation of embryogenic cells. The second
is that no specific markers have been described so far that
reliably predict which explant cells will become embryogenic.
The process of cellular reactivation and the subsequent
formation of embryogenic cells in carrot explants has been
described by Guzzo et al. (1994, 1995). That work showed
that a particular elongated cell type appeared in culture,
derived from small rapidly proliferating cytoplasmic cells
that themselves derived from reactivated provascular cells. It
was further shown cytologically, that some of the elongated
cells underwent an asymmetrical division. After continued
culture, small clusters of dividing cytoplasmic cells appeared
that resembled the proembryogenic masses seen in established embryogenic suspension cultures (Guzzo et al., 1994,
De Vries et al., 1988a). The fact that only a limited number
of cells actually undergoes the transition of somatic into
embryogenic cell is postulated to be the result of the presence
of different sets of auxin receptors (Filippini et al., 1992).
One of the goals of the work presented here was to determine
which cells of a carrot hypocotyl explant have completed the
transition of a somatic cell into an embryogenic cell. The
results revealed that the first cells to become competent
belong to a type of enlarged cells that are detaching from the
surface of the mass of proliferating cells, confirming the
previous predictions (Guzzo et al., 1994). All other cells,
including the majority of the enlarged cells present, were
completely unresponsive. This result is in contrast to the
generally accepted idea that a population of small rapidly
dividing meristematic cells are the ones that are competent to
become embryogenic. Instead, the results presented here,
together with the cytological observations (Guzzo et al.,
1994), demonstrate that competent cells arise first as elongating cells, when still attached to the explant. This is a
situation that is strikingly similar to the rapid increase in cell
volume observed in plant egg cells after fertilization
(Mansfield and Briarty, 1991). Unlike the zygote, it was so
far not possible to predict which elongated cell on the surface
of the explant will become competent. This raises the
question whether the observed correspondence between
elongation and competent cell formation is causal or merely
reflects a particular state common to most cells present on the
surface of the explant. To answer this question, it was
essential to achieve another goal of this work: to obtain
markers that are able to distinguish precisely between
competent and non-competent cells. The expression of the
SERK gene described here was found to be very tightly correlated with the ability of cells of the correct morphology to
attain the competent cell state. In line with the much less
defined morphology of competent single cells in established
suspension cultures (Toonen et al., 1994), no clear cell type
specificity was apparent in cells expressing the SERK gene in
those cultures. The difference between these findings and the
2060 E. D. L. Schmidt and others
results obtained employing activated explants is at present not
explained. In comparison with other markers that have been
suggested to distinguish individual embryo-forming cells,
such as callose (Dubois et al., 1991) and the monoclonal
antibody JIM8 (Pennell et al., 1992), the SERK gene appears
to be quite specific under culture conditions. While the
presence of the JIM8 epitope was restricted to embryogenic
cell cultures, cell tracking of cells labeled with this antibody
failed to show a correlation with the ability of these cells to
develop into somatic embryos (Toonen et al., 1996a). As
shown in this work, such a correlation was established for the
SERK gene by cell tracking of cells expressing luciferase
under the control of the SERK promoter. The availability of
a vital marker for competent cells offers the possibility of
following, with great precision, the events that take place
during formation of such cells. It could for instance be a
useful tool to help determine the frequency of pseudomeiotic
segregation events proposed to accompany embryogenic cell
formation (Giorgetti et al., 1995).
The identification of the SERK gene and other markers for
competent cells was facilitated by the availability of a range
of suspension cultures differing strongly in the number of
competent single cells present. This strategy avoided the
potential problem that a non embryogenic cell line would be
employed in which competent cell formation had occurred,
but in which a later stage in embryogenic cell formation
would have been inhibited, thus reducing the chance of
finding genes expressed during the early competent cell
states. In addition, the availability of in situ hybridization
methods that allowed the visualization of gene expression in
single suspension cells while preserving cell morphology,
together with cloning methods aimed at avoiding selection
towards abundantly expressed genes, explains why similar
experiments carried out previously only yielded genes
expressed in later stages, such as proembryogenic masses
(Choi and Sung, 1984; Wilde et al., 1988; Aleith and Richter,
1990).
The SERK gene is transiently expressed in
embryogenesis
The analysis of the expression pattern of the SERK gene during
embryogenic cell formation and during somatic embryogenesis revealed that SERK expression continues during proembryogenic mass formation and also during somatic embryogenesis up to about the 100-celled globular stage. Because after
this stage expression in somatic embryos is completely
abolished, the SERK gene shows a transient expression pattern,
one that was also found in zygotic embryogenesis, perhaps as
early as the zygote. This transient expression pattern, following
the classification system for embryo-expressed genes would
place the SERK gene in class 2, comprising very few genes
exclusively expressed during early embryogenesis (Sterk and
De Vries, 1992). So far, none of the genes identified using
embryo mutational analysis, such as Bio-1 (Schneider et al.,
1989), Prolifera (Springer et al., 1995), EMB30/Gnom (Shevell
et al., 1994), Fusca-1 (Castle and Meinke, 1994), Knolle
(Lukowitz et al., 1996) and STM (Long et al., 1996) exhibit an
expression pattern that is restricted to the embryo. Thus, the
expression pattern of the SERK gene points to a function in a
signal transduction cascade only required for the first seven or
so cell divisions of the plant embryo. While the nature of the
signal, its transduction and its importance are not clear yet, it
is clear that this cascade is reproduced with great fidelity in
somatic embryogenesis.
The possible function of the SERK gene in early
plant embryogenesis
The predicted SERK protein sequence resembles a leucinerich repeat (LRR) receptor kinase protein, a class of plant
proteins that was originally described by Chang et al. (1992).
Some members of this class of plant receptor-like kinases are
known regulators of developmental processes, like the Arabidopsis Clavata protein (Meyerowitz, 1995) and the Arabidopsis Erecta protein (Torii et al., 1996). Others, like the
Petunia PRK1 protein seem to be involved in signal transduction during pollen development or pollination (Mu et al.,
1994). Expression of this particular Petunia gene is restricted
to pollen and pollen tubes prior to fertilization in contrast to
the carrot SERK gene of which no expression can be detected
in pollen. PRK1 and SERK therefore regulate non-overlapping
processes, separated from each other by the process of fertilization. Other plant receptor-like proteins with LRRs are
involved in pathogen resistence, presumably by the specific
binding to elicitors (reviewed by Dangl, 1995). The specificity
of protein-protein interactions mediated by LRR-containing
proteins are most probably due to the composition of the nonconsensus residues within the LRRs (Kobe and Deisenhofer,
1994).
The presence of the perfect consensus sequence SPPPP
found in extensins and some types of arabinoalactan proteins
or AGPs suggests an interaction of the extracellular part of
the SERK protein with components of the cell wall. The
SPPPP domain is considered to be a target sequence for arabinosylation onto hydroxylated prolines (reviewed by
Carpita and Gibeaut, 1993). All prolines in the SPPPP
consensus are normally hydroxylated. Whether a glycosylated SPPPP sequence is mediating a possible anchoring to
specific regions of the cell wall remains to be determined. If
so, it would be an elegant mechanism to prevent free
movement of receptor molecules and yet prevent inflexibility preventing dimerization, which would be difficult to
reconcile with covalent attachment to a cell wall polymer.
While the average primary cell wall has a thickness of
approximately 50 nm (Pruitt et al., 1993) and the maximum
size of the entire extracellular domain can only be about 15
nm when present as an α helix, the extracellular ligand
binding domain is likely to be completely embedded within
the cell wall. The most likely type of ligand for SERK will
therefore consist of a cell wall-diffusible peptide. Peptides
effective in inducing plant responses, such as systemin
(McGurl et al., 1992) and ENOD40 (Van de Sande et al.,
1996) have been described.
Thus, it appears that while LRR containing receptor-like
protein kinases play several roles in plant development, intercellular peptides are now being uncovered that are likely signal
molecules that can activate developmental processes mediated
through such receptors.
Clues about the function of SERK might be found in the
homology between SERK and two proteins in Drosophila that
are required for the establishment of the dorsoventral polarity
in the embryo. The kinase domain of SERK shows homology
with the Drosophila Pelle protein, a serine/threonine kinase
Somatic embryo receptor-like kinase 2061
involved in formation of the dorsoventral axis during embryogenesis (Shelton and Wasserman, 1993). The Drosophila Pelle
protein itself is activated by the Toll transmembrane receptor
(Hashimoto et al., 1988; Govind and Steward, 1991), of which
the ligand-binding domain, as in SERK, consists of LRRs. In
the plant embryo sac and in the activated explant a situation
may exist whereby an unknown inducer is present uniformly,
but embryo formation awaits the presence of the SERK protein.
Such a model may fit with the restricted expression pattern
found for the SERK gene both in vivo and in vitro. It is also in
line with the hypothesis that in plants inductive interactions
mediated by diffusable signal molecules are an important regulatory mechanism (reviewed by Schmidt et al., 1994). Direct
evidence for the existence of cell inductive processes in plants
was recently presented by Van Den Berg et al. (1995) for the
Arabidopsis root. While most of the elements concerning the
origin and targets of processes of cell to cell communication
in early plant embryogenesis are lacking, the SERK gene
described here may represent a significant part of a mechanism
that is essential for the formation of plant cells destined to
become embryos.
We would like to thank Wim Reidt and Conny Eijkelboom for
practical assistance. Marijke Hartog and Tony van Kampen are
acknowledged for nucleotide sequence analysis, Dorus Gadella for
help with luciferase measurement and Olaf Sonneveld (S&G Seeds,
Enkhuizen, the Netherlands) for the carrot plant material. E. D. L. S.
and M. A. J. T. are supported by the Technology Foundation (STW).
F. G. is supported by EMBO, the Italian government and the EC
program PTP-Biotech. The rice clone R2976 was made available by
the Rice Genome Research Program (RGP).
REFERENCES
Aleith, F. and Richter, G. (1990) Gene expression during induction of somatic
embryogenesis in carrot cell suspensions. Planta 183, 17-24.
Chang, C., Schaller, G. E., Patterson, S. E., Kwok, S. F., Meyerowitz, E. M.
and Bleecker, A. B. (1992). The TMK1 gene from Arabidopsis codes for a
protein with structural and biochemical characteristics of a receptor protein
kinase. Cell 4, 1263-1271.
Choi, J. H. and Chung, Z. R. (1984). Two-dimensional gele analysis of carrot
somatic embryonic proteins. Plant Mol. Biol. Rep. 2, 19-25.
Castle, L. A. and Meine, D. W. (1994). A FUSCA gene of Arabidopsis encodes
a novel protein essential for plant development. Plant Cell 6, 25-41.
Carpita, N. C. and Gibeaut, D. M. (1993). Structural models of primary cell
walls in flowering plants-consistency of molecular structure with the physical
properties of the walls during growth. Plant J. 3, 1-30.
Dangl, J. L. (1995). Pièce de Resistance: novel classes of plant disease
resistance genes. Cell 80, 363-366.
De Jong, A. J., Schmidt, E. D. L. and De Vries, S. C. (1993). Early events in
higher plant embryogenesis. Plant. Mol. Biol. 5, 367-377.
De Vries, S. C., Booij, H., Meyerink, P., Huisman, G., Wilde, H. D.,
Thomas, T. L. and Van Kammen, A. (1988a). Acquisition of embryogenic
potential in carrot cell-suspension cultures. Planta 176, 196-204.
De Vries, S. C., Hoge, H., and Bisseling, T. (1988b). Isolation of total and
polysomal RNA from plant tissues. In Plant Molecular Biology Manual (ed.
S. B. Gelvin, R. A. Schilperoort and D. P. S. Verma), pp. 1-13. Kluwer
Academic Publishers, Dordrecht.
Dos Santos, A. V. P., Cutter, E. G., Davey, M. R. (1983). Origin and
development of somatic embryos in Medicago sativa L. (Alfalfa).
Protoplasma 117, 107-115.
Dubois, T., Guedira, M., Dubois, D. and Vasseur, J. (1991). Direct somatic
embryogenesis in leaves of Cichorum – A histological and SEM study of
early stages. Protoplasma 162, 120-127.
Dudits, D., Gyorgyey, J., Bogre, L. and Bako, L. (1995). Molecular Biology
of somatic embryogenesis. In In vitro Embryogenesis in Plants. (ed. T. A.
Thorpe), pp. 267-308. Kluwer Academic Publishers, Dordrecht.
Engler, J. A., Van Montagu M. and Engler G. (1994). Hybridization in situ of
whole mount messenger RNA in plants. Plant Mol. Biol. Rep. 12, 321-331.
Filippini, F., Terzi, M., Cozzani, F., Vallone, D. and Lo Schiavo, F. (1992).
Modulation of auxin-binding proteins in cell-suspension: II. Isolation and
initial characterization of carrot cell variants impaired in somatic
embryogenesis. Theor. Applied Genetics 84, 430-434.
Gamborg, O. L., Miller, R. A. and Ojima, K. (1968). Plant cell cultures: I.
Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell.
Res. 50, 151-158.
Giorgetti, L., Vergara, M. R., Evangelista, M., LoSchiavo, F., Terzi, M. and
Ronchi, V. N. (1995). On the occurrence of somatic meiosis in embryogenic
carrot cell cultures. Mol. Gen. Genet. 246, 657-662.
Goldberg, R. B., Barker, S. J., Perez-Grau, L. (1989). Regulation of gene
expression during plant embryogenesis. Cell 56, 149-160.
Goldberg, R. B., de Paiva, G. and Yadegari, R. (1994). Plant embryogenesis:
zygote to seed. Science 266, 605-614.
Govind, S. and Steward, R. (1991). Dorsoventral pattern formation in
Drosophila. Trends Genet. 7, 119-125.
Guzzo, F., Baldan, B., Mariani, P., LoSchiavo, F. and Terzi, M. (1994).
Studies on the origin of totipotent cells in explants of Daucus carota L. J.
Exp. Bot 45, 1427-1432.
Guzzo, F., Baldan, B., Levi, M., Sparvoli, E., LoSchiavo, F., Terzi, M., and
Mariani, P. (1995). Early cellular events during induction of carrot explants
with 2,4-D. Protoplasma 185, 28-36.
Hanks, S. K., Quinn, A. M. and Hunter, T. (1988). The protein kinase family:
conserved features and deduced phylogeny of the catalytic domains. Science
241, 42-52.
Hashimoto, C., Hudson, K. L. and Anderson, K. V. (1988). The Toll gene of
Drosophila, required for dorsal-ventral embryogenic polarity, appears to
encode a transmembrane protein. Cell 52, 269-279.
Heck, G. R., Perry, S. E., Nichols, K. W. and Fernandez, D. E. (1995).
AGL15, a MADS domain protein expressed in developing embryos. Plant
Cell 7, 1271-1282.
Hodge, R., Paul, Wyatt, Draper, J. and Scott, R. (1992). Cold-plaque
screening: a simple technique for the isolation of low abundance,
differentially expressed transcripts of low abundance, differentially
expressed transcripts from conventional cDNA libraries. Plant J. 2, 257-260.
Horn, M. A. and Walker, J. C. (1994). Biochemical properties of the
autophosphorylaton of RLK5, a receptor-like protein kinase from
Arabidopsis thaliana. Biochim. Biophys. Acta 1208, 65-74.
Jones, D. A., Thomas, C. M., Hammond-Kosack, K. E., Balint-Kurti, P. J.
and Jones, J. D. G. (1994). Isolation of the tomato Cf-9 gene for resistance to
Cladosporium fulvum by transposon tagging. Science 266, 789-792.
Kobe, B. and Deisenhofer, J. (1994). The leucine-rich repeat: a versatile
binding motif. Trends Biochem. 19, 415-421.
Koltunow, A. M. (1993). Apomixis: embryo sacs and embryos formed without
meiosis or fertilization in ovules. Plant Cell 5, 1425-1437.
Komamine, A., Matsumoto, M., Tsukahara, M., Fujiwara, A., Kawahara,
R., Ito, M., Nomura, K. and Fujimura, T. (1990). Mechanisms of somatic
embryogenesis in cell cultures – physiology, biochemistry and molecular
biology. In Progress in Plant Cellular and Molecular Biology (ed. H. J. J.
Nijkamp, L. H. W. Van Der Plas, A. Van Aartrijk), pp. 307- 313. Kluwer
Academic Publishers, Dordrecht.
Konar, R. N., Thomas, E. and Street, H. E. (1972). Origin and structure of
embryoids arising from epidermal cells of the stem of Ranunculus sceleratus
L. J. Cell Sci. 11, 77-93.
Lackie, S. and Yeung, E. C. (1996). Zygotic embryo development in Daucus
carota. Can. J. Bot. 74, 990-998.
Li, F., Barnathan, E. S. and Kariko, K. (1994). Rapid method for screening
and cloning cDNAs generated in differential mRNA display: application of
Northern blot for affinity capturing of cDNAs. Nucl. Acid Res. 22, 17641765.
Liang, P. and Pardee, A. B. (1992). Differential display of eucaryotic
messenger RNA by means of the polymerase chain reaction. Science 257,
967-971.
Long, J. A., Moan, E. I., Medford, J. I. and Barton, M. K. (1996). A member
of the KNOTTED class of homeodomain proteins encoded by the STM gene
of Arabidopsis. Nature 379, 66-69.
Lukowitz, W., Mayer, U. and Jürgens, G. (1996). Cytokinesis in the
Arabidopsis embryo involves the syntaxin-related KNOLLE gene product.
Cell 84, 61-71.
Mansfield, S. G. and Briarty, L. G. (1991). Early embryogenesis in
Arabidopsis thaliana. II. The developing embryo. Can. J. Bot. 69, 461-476.
McGurl, B., Pearce, G., Orozco-Cardenas, M. and Ryan, C. A. (1992).
2062 E. D. L. Schmidt and others
Structure, expression, and antisense inhibition of the systemin precursor
gene. Science 257, 1570-1573.
Meyerowitz, E. M. (1995). Pattern formation in Arabidopsis flower
development. EDBC 95 congress of the European developmental biology
organization. Abstract SI4.
Millar, A. J., Short, S. R., Hiratsuka, K., Chua, N. -H., and Kay, S. A.
(1992). Firefly luciferase as a reporter of regulated gene expression in higher
plants. Plant Mol. Biol. Reporter 10, 324-337.
Mordhorst, A. P., Toonen, M. A. J., de Vries, S. C. (1997). Plant
Embryogenesis. Critical Reviews in Plant Sciences. Ed. Conger, B. V. (in
press)
Mu, J-H., Lee H-S. and Kao, T. -H. (1994). Characterization of a pollenexpressed receptor-like kinase gene of Petunia inflata and the activity of its
encoded kinase. Plant Cell 6, 709-721.
Pennell, R. I., Janniche, L., Scofield, G. N., Booy, H., De Vries, S. C. and
Roberts, K. (1992). Identification of a transitional cell state in the
developmental pathway to carrot somatic embryogenesis. J. Cell Biol. 119,
1371-1380.
Pruitt, R. E., Hulskamp, M., Kopczak, S. D., Ploense, S. E. and Schneitz, K.
(1993). Molecular genetics of cell interactions in Arabidopsis. Development
1993 Supplement, 77-84.
Rounsley, S. D., Ditta, G. S. and Yanofsky, M. F. (1995). Diverse roles for
MADS box genes in Arabidopsis development. Plant Cell 7, 1259-1269.
Schmidt, E. D. L., De Jong, A. J. and De Vries, S. C. (1994). Signal molecules
involved in plant embryogenesis. Plant Mol. Biol. 26, 1305-1313.
Schneider, T., Dinkins, R., Robinson, K., Shellhammer, J. and Meinke, D.
W. (1989). An embryo-lethal mutant of Arabidopsis thaliana is a biotin
auxotroph. Dev. Biol. 131, 161-167.
Scott, R., Dagless, E., Hodge, R., Soufleri, I. and Draper, J. (1991). Patterns
of gene exression in developing anthers of Brassica napus. Plant Mol. Biol.
17, 195-207.
Shelton, C. A. and Wasserman, S. A. (1993). Pelle encodes a protein kinase
required to establish dorsoventral polarity in the Drosophila embryo. Cell 72,
515-525.
Shevell, D. E., Leu, W. -M., Gilimor, C. S., Xia, G., Feldmann, K. A. and
Chua, N. -H. (1994). EMB30 is essential for normal cell division, cell
expansion, and cell adhesion in Arabidopsis and encodes a protein that has
similarity to Sec7. Cell 77, 1051-1062.
Springer, P. S., Richard, W., McCombie W. M., Sundaresan, V. and
Martienssen, R. A. (1995). Gene trap tagging of Prolifera, an essential
MCM-2-3-5-like gene in Arabidopsis. Science 268, 877-880.
Sterk, P., Booij, H., Schellekens, G. A., Van Kammen, A. and De Vries, S. C.
(1991). Cell-specific expression of the carrot EP2 lipid transfer protein gene.
Plant Cell 3, 907-921.
Sterk, P., and De Vries, S. C. (1992). Molecular markers for plant embryos. In
Synseeds: Applications of synthetic seeds to crop improvement (ed. K.
Redenbaugh), CRC Press, London.
Taylor, R. L. (1967). The foliar embryos of Malaxis paludosa. Can. J. Bot. 45,
1553-1556.
Thomas, T. L. (1993). Gene expression during plant embryogenesis and
germination: an overview. Plant Cell 5, 1401-1410.
Toonen, M. A. J., Hendriks, T., Schmidt, E. D. L., Verhoeven, H. A., Van
Kammen, A and De Vries, S. C. (1994). Description of somatic-embryoforming single cells in carrot suspension cultures employing video cell
tracking. Planta 194, 565-572.
Toonen, M. A. J., Schmidt, E. D. L., Hendriks, T., Verhoeven, H. A., Van
Kammen, A. and De Vries, S. C. (1996a). Expression of the JIM8 cell wall
epitope in carrot somatic embryogenesis. Planta 200, 167-173.
Toonen, M. A. J., Verhees, J. A., Schmidt, E. D. L., Van Kammen, A. and De
Vries, S. C. (1996b). AtLTP luciferase expression during carrot somatic
embryogenesis. Submitted.
Toonen, M. A. J., de Vries, S. C. (1997). Use of video tracking to identify
embryogenic cultured cells. Plant Tissue Culture Manual H, (ed. K.
Lindsey), pp. 1-45. In press.
Torii, K. U., Mitsukawa, N., Oosumi, T., Matsuura, Y., Yokoyama, R.,
Whittier, R. F. and Komeda, Y. (1996). The Arabidopsis ERECTA gene
encodes a putative receptor protein kinase with extracellular leucine-rich
repeats. Plant Cell 8, 735-746.
Trigiano, R. N., Gray, D. J., Conger, B. V. and McDaniel, K. J. (1989). Origin
of direct somatic embryos from cultured leaf segments of Actylis glomerata.
Bot. Gaz. 150, 72-77.
Van Den Berg, C., Willemsen, V., Hage, W., Weisbeek, P. and Scheres, B.
(1995). Cell fate in the Arabidopsis root meristem determined by directional
signalling. Nature 378, 62-65.
Van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, K., Schell, J.,
Schmidt, J., Walden, R., Matvienko, M., Wellink, J., Van Kammen, A.,
Franssen, H. and Bisseling, T. (1996). Modification of phytohormone
response by a peptide encoded by ENOD40 of legumes and a nonlegume.
Science 273,370-373.
Van Engelen, F. A. and De Vries, S. C. (1992). Extracellular proteins in plant
embryogenesis. Trends Genet. 8, 66-70.
Varner, J. E. and Lin, L. -S. (1989). Plant cell wall architecture. Cell 56, 231239.
Walker, J. C. (1993). Receptor-like protein kinase genes of Arabidopsis
thaliana. Plant J. 3, 451-456.
Walker, J. C. (1994). Structure and function of the receptor-like protein kinases
of higher plants. Plant Mol. Biol. 26, 1599-1609.
Wernicke, W. and Brettel, R. (1980). Somatic embryogenesis from Sorgum
bicolor leaves. Science 287, 138-139.
Wilde, H. T., Nelson, W. S., Booij H., De Vries, S. C. and Thomas, T. L.
(1988). Gene expression programs in embryogenic and non-embryogenic
carrot cultures. Planta 176, 205-213.
Wurtele, E. S., Wang, H., Durgerian, S., Nikolau, B. J. and Ulrich, T. H.
(1993). Characterization of a gene that is expressed early in somatic
embryogenesis of Daucus carota. Plant Physiol. 102, 303-312.
Zhao, Y., Feng, X. H., Watson, J. C., Bottino, P. J. and Kung, S. D. (1994).
Molecular cloning and biochemical characterization of a receptor-like serine
/ threonine kinase from rice. Plant Mol. Biol. 26, 791-803.
Zimmerman, J. L. (1993). Somatic embryogenesis: a model for early
development in higher plants. Plant Cell 5, 1411-1423.
(Accepted 7 March 1997)