47
Development 106, 47-56 (1989)
Printed in Great Britain <Q The Company of Biologists Limited 1989
A set of cell surface glycoproteins forms an early marker of cell position,
but not cell type, in the root apical meristem of Daucus carota L.
J. PAUL KNOX, SUSAN DAY and KEITH ROBERTS
Department of Cell Biology, John Innes Institute, Colney Lane, Norwich, NR4 7UH, UK
Summary
A monoclonal antibody (JIM4) has been derived that
recognizes a series of glycoproteins associated with the
plasma membrane of a suspension-cultured carrot cell
line and also an arabinogalactan proteoglycan secreted
by the cultured cells. Immunocytochemistry indicated
that the plasma membrane antigen(s) recognized by
JIM4 are specific to certain cells of Daucus carota L.
seedlings. In the root apex JIM4 labelled two segments of
the stele. These were centred upon the poles of the
protoxylem. An axis of unlabelled cells connected the
two phloem regions. Two sections of the pericycle with
characteristic oblique longitudinal divisions were particularly reactive with JIM4. This pattern of reactive
cells, reflecting cell position rather than a specific future
cell type, would appear to be a unique observation in
plants. The association of JIM4 antigens with these
vascular tissues is maintained through the transition
from root to the shoot tissue of the cotyledons and the
mature plant. Examination of J1M4 labelling upon
Introduction
All of the cells in a higher plant derive ultimately from
the dividing cell populations in the shoot and root apical
meristems. These meristems, in giving rise to the organs
of the plant, produce cell lineages that develop into the
characteristically patterned vasculature of these structures. Despite our knowledge of the physical structure
of many apical meristems and some of the physical
determinants of their anatomy, the molecular processes
that determine plant morphology and cell differentiation remain unknown.
As dividing cells in the meristematic region of a root
move back from the apex, as files of related cells, two
patterns are established that reflect the mature root
morphology. A concentric pattern underlies the classical division into three tissue systems - epidermis, cortex
and stele. Within the stele there is a species-specific
radial pattern of vascular development into phloem and
xylem. In the case of carrot {Daucus carota L.), the root
shows a bilaterally symmetrical diarch structure. The
establishment of such tissue patterns and subsequent
ultrathin frozen sections of the carrot seedling root
apical meristem indicated that the expression of the
antigen is a very early event in root development. Cells
express the surface epitope, within one or two cells of the
dome of apical initials, before the pattern of future
vascular tissue can be discerned and well before its
actual differentiation.
Abbreviations: AGPs, arabinogalactan proteins; DAPI,
4',6-diamidino-2-phenyl-indole; ELISA, enzyme-linked
immunosorbent assay; McAb, monoclonal antibody; PBS,
phosphate-buffered saline; SDS-PAGE, sodium
dodecylsulphate-polyacrylamide gel electrophoresis; TBS,
Tris-buffered saline.
Key words: arabinogalactan proteins, Daucus carota L.,
monoclonal antibody, pattern formation, plasma
membrane, root meristem.
differentiation requires the acquisition of some form of
identity by cells or cell lineages, although the chemical
nature of any such identity is unknown. To understand
how these patterns of xylem and phloem are established, we need molecular markers of early pattern
formation, or cell identity, at a time well before the cells
finally differentiate into recognizable cell types.
Arabinogalactan proteins (AGPs) form a very large
and diverse group of macromolecules in plants and can
be subdivided most readily into extracellular proteoglycans and membrane-associated glycoproteins (Fincher
etal. 1983; Pennell etal. 1989). No clear function for any
of these molecules has emerged.
AGPs are antigenic and capable of generating monoclonal antibodies (McAb) with reactivities inhibitable
by L-arabinose, D-galactose and/or associated disaccharides (Anderson et al. 1984). Such antibodies have
been generated in response to complex membranous
plant immunogens (Meyer et al. 1987; Bradley et al.
1988; Brewin et al. 1988). In one case an antibody has
been shown to be specific for AGPs and not to crossreact with other cell surface arabinosylated and hy-
48
/. P. Knox, S. Day and K. Roberts
droxyproline-containing glycoproteins such as the
extensins and the lectins of the Solanaceae (Pennell et
al. 1989).
In this report we describe the specific, restricted and
novel distribution of a set of plasma membrane antigens
in seedlings of Daucus carota L. The McAb JIM4
recognized an epitope common to a set of glycoproteins
of the plasma membrane of a carrot cell line and an
arabinogalactan protein secreted by the cell line. We
demonstrate that in the carrot seedling the most abundant expression of the membrane-associated epitope
occurs in the vascular tissues. Examination of the root
apical meristem of carrot seedlings indicated that the
expression of the epitope is a very early event in the
continuing development of the root meristem and
seems to reflect early stages in the formation of the
vascular pattern.
Materials and methods
Plant materials and cell culture
Carrot {Daucus carota L. cv. Early Nantes) seeds were
germinated on moist tissue paper and grown for 5 days in the
dark at 22°C before use. Mature carrot plants were collected
locally. A suspension culture of carrot cells (Lloyd et al. 1979)
was maintained in Murashige and Skoog medium supplemented with lmgl" 1 2,4-dichlorophenoxy-acetic acid and
25 gl" 1 sucrose. Cells were subcultured every 7 days and used
for experimental purposes 5 or 6 days after subculture.
Immunization and production of hybridoma
The hybridoma secreting the monoclonal antibody J1M4 was
developed from a series of immunizations of rats with intact
protoplasts prepared from the carrot suspension cell culture.
The protoplasts (lxlO 6 cells in 300 jil phosphate-buffered
saline, PBS) were injected intraperitoneally into a male
LOU/c rat (5 weeks old) on days 0, 22, 56 and 103. Spleen
lymphocytes were collected and fused with the IR983F
myeloma cell line (Bazin, 1982) on day 106. Procedures used
for the fusion, HAT selection and maintenance of hybridomas
were essentially as described by Galfre & Milstein (1981).
The J1M4 McAb was selected as a plasma membrane
reactive antibody by its ability to bind to a preparation of
membranes from the carrot cell line and indirect immunofiuorescence on carrot protoplasts prepared from the cultured
cells. The JIM4 hybridoma secretes immunoglobulins of class
IgM.
Preparation of protoplasts
The procedures used for the preparation of protoplasts from
the suspension-cultured cells and their use for obtaining
indirect immunofluorescence with plasma membrane-reactive
antibodies are described elsewhere (Pennell et al. 1989).
Preparation of microsomal membrane fractions
Protoplasts, prepared from the carrot cell line or the intact
carrot seedlings, were homogenized in 4mlg~ 1 fresh weight
of 50 mM-Tris(hydroxymethyl)aminomethane-HCl (TRIS)
buffer pH7-5 containing 0-25M-sucrose, 3mM-disodium ethylenediaminetetra-acetate (EDTA), 2-5rtiM-dithiothreitol
(DTT) and 1 mM-phenylmethylsulphonyl fluoride (PMSF).
Homogenization was performed with a glass hand-held homogenizer. A pellet collected by centrifugation at 5000g for
lOmin was discarded and the subsequent pellet at 100 000 g
(1 h) was washed in the homogenization buffer and resuspended in 5 mM-potassium phosphate buffer pH7-8 (containing the additions of the homogenization buffer other than the
EDTA) and retained as the membrane fraction and stored at
—20°C. All procedures were performed at 4°C. Protein was
determined according to the method of Lowry et al. (1951).
Preparation of a fraction containing the
arabinogalactan protein of the carrot culture medium
The culture medium of the carrot cell line, conditioned by 5
days growth subsequent to subculture was separated from the
cells by centrifugation and filtered through Whatman No. 1
paper and brought to 90 % (v/v) acetone at 4°C. After 30 min
the precipitate was collected by centrifugation, extracted with
water and the soluble components lyophilized.
Electrophoresis and immunoblotting
Sodium dodecylsulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed using 10% or 8% (w/v)
acrylamide slab gels according to the method of Laemmli
(1970). Gels were blotted on to nitrocellulose by means of a
semi-dry electroblotting system (Sartorius, Goettingen,
FRG). Nitrocellulose sheets were blocked with 5 % (v/v) calf
serum in PBS for at least 1 h before incubation with a 100-fold
dilution of McAb culture supernatant in the same buffer
overnight at 4°C. Extensive washing in PBS containing 0-05 %
Tween 20 was performed before and after incubation with a
2000-fold dilution of rabbit anti-rat Ig linked to alkaline
phosphatase (Sigma) in the PBS containing calf serum for 2 h.
The enzyme substrate was developed according to the manufacturer's method.
Enzyme-linked immunosorbent assays
ELISAs were performed in microtitre plates coated with the
membrane preparation at SOmgl"1 protein (18 h at 4°C) and
blocked for at least 1 h with 5 % (v/v) calf serum in PBS. JIM4
binding was detected by means of a second antibody (rabbit
anti-rat Ig linked to horseradish peroxidase, ICN Biomedicals) developed by conventional reactions. The dilution of
JIM4 giving 90 % of maximal binding was used for assessment
of hapten and glycoprotein inhibition. In certain cases, the
immobilized antigens were treated prior to antibody incubations with Pronase E (Sigma) at 1 gl" 1 in 50 mM-TRIS-HCl
buffer pH7-5 or 25mM-sodium metaperiodate in 50 mMsodium acetate buffer, pH4-3 for l h in the dark. After such
treatments the plates were washed extensively in water and
re-blocked prior to antibody incubations and assessment of
antigen degradation.
Immunogold electron microscopy
Thefixationand subsequent treatment of suspension-cultured
carrot cells and carrot seedlings for the analysis of JIM4
binding by means of immunogold electron microscopy is
described elsewhere (Pennell et al. 1989).
Tissue fixation and microtomy
Root apices of 5-day-old carrot seedlings were excised and
immersed in freshly prepared 4% (w/v) formaldehyde in
fixation buffer (50mM-piperazine-A',A/'-bis[2-ethanesuUonic
acid] {Pipes}, 5mM-MgSO4 and 5mM-ethylene glycol bis[/3aminoethylether]A',/V,./v",iV'-tetra acetic acid {EGTA},
pH6-9) for 2h, washed in fixation buffer and infused for at
least 72 h with l-5M-sucrose and 0-5% formaldehyde in the
same buffer. Before sectioning, root apices were trimmed to
approx. lmm, transferred to freezing stubs and plunged into
liquid ethane. The stub with frozen tissue was fitted to an
Pattern formation in the root apical meristem
49
Fig. 1. (A) Immunofluorescence
generated by JIM4 binding to the
surface of intact protoplasts
prepared from the carrot cell line.
Note the agglutination of the
protoplasts. Bar, 10/an.
(B) Immunogold localization of
JIM4 antigens to the plasma
membrane (pm) of carrot
suspension cell, w, cell wall;
c, cytoplasm. Bar, 0-1 /an.
(C) Immunogold electron
microscopy confirms J1M4 binding
to the plasma membrane of cells
from a carrot seedling root, w, cell
wall; c, cytoplasm. Bar, 0 1 /an.
Ultracut EFC 4D cryoultramicrotome (Reichert-Jung, UK,
Slough) and the tissue sectioned at -110°C at a thickness of
0-5 /on. Sections were collected on small drops of 2M-sucrose
in water and settled on to multiwell slides. Before use the
slides were extensively washed with Tris-buffered saline
(TBS). Plant tissues other than root apices were similarly
immersed in 4% formaldehyde in fixation buffer for at least
2h before embedding with OCT compound (Miles Scientific,
Illinois, USA) and frozen at -20°C. Sections (5-10/im) were
made at —20°C using a Bright 5030 cryomicrotome (Cambridge, UK) and collected on multiwell slides coated with
poly-L-lysine and allowed to dry. Before use the slides were
extensively washed with water to remove the OCT compound.
Immunocytochemistry
The sections were treated with a 5-fold dilution of hybridoma
culture supernatant into 5 % (v/v) calf serum in TBS for up to
12 h at 4°C. The sections were rinsed in TBS before treatment
with goat anti-rat Ig linked to fluorescein isothiocyanate,
(ICN Biomedicals) diluted 100-fold into the TBS with calf
serum, for at least 2h. The final, extensive washing of the
slides in TBS also included a 30s incubation with 4',6diamidino-2-phenyl-indole (DAPI) at lmgl" 1 in TBS. Final
mounting was with Citifluor anti-fade mountant (Citifluor,
London, UK) and the sections were observed on a Zeiss
Photomicroscope i n equipped with epifluorescence irradiation.
Results
Characterization of JIM4 antigens of the plasma
membrane of suspension-cultured carrot cells
The McAb JIM4 bound to the surface of protoplasts
prepared from the carrot cell line as revealed by indirect
immunofluorescence (Fig. 1A). The specific localization of the JIM4 epitope to the plasma membrane of
these cells was confirmed by means of immunogold
electron microscopy performed on ultrathin sections of
such cells retaining an intact cell wall (Fig. IB).
Immunoblotting of electrophoretically separated
membranes indicated that JIM4 recognized a series of
discrete bands in the range of Mr 20000 to 60000
(Fig. 2). These corresponded with the binding pattern
of an anti-AGP McAb MAC 207 (Pennell et al. 1989). In
-205
-116
-66
-45
-29
Fig. 2. Immunoblotting of JIM4 (lane a) and MAC 207
(lane b) to the membrane preparation of carrot protoplasts
separated by SDS-PAGE and transferred to nitrocellulose.
Loading was at 50/ig protein per lane. JIM4 also reacted
with the arabinogalactan protein (Afr70000 to 100000) in an
acetone precipitate of the conditioned medium of the carrot
cells (lane c, 10/ig: protein loading). Positions of protein
markers (M r xl0 ) and the dye front (arrowhead) are
indicated.
addition, JIM4 also reacted with an arabinogalactan
protein (Pennell et al. 1989), derived from the culture
medium of the carrot cells, which was electrophoretically resolved as a smear of MT 70000 to 100000
(Fig. 2).
Further analysis of the JIM4 antigen and epitope was
by means of an enzyme-linked immunosorbent assay
(ELISA) of its binding to membranes prepared from
protoplasts of the carrot cell line. Treatment of the
immobilized membranes with either periodate or a
protease reduced JIM4 binding by 76 % and 68 %,
respectively, indicating that the antigen contained both
carbohydrate and protein components. The binding of
JIM4 to the membranes was inhibited by 50 % in the
50
/. P. Knox, S. Day and K. Roberts
Pattern formation in the root apical meristem
Future phloem
region
Future xylem
region
Fig. 3. (A) Immunofluorescence generated by JIM4 on a
transverse section of a carrot root, 50-100 ^m from the
apical initials is restricted to two segments of the vascular
cylinder and is most abundant in the cells of the pericycle
(p). At the centre of the cylinder 3 xylem vessel mother
cells are weakly labelled (The centre cell is indicated with
x). The adjacent regions that will develop into the phloem
(ph) are unlabelled. c, cortex. The arrow and arrowhead
refer to the sections shown in Fig. 4. Bar, 50/zm.
(B) Immunofluorescence generated by MAC 207 on a
comparable section to A reveals reactivity with all cells.
Bar, 50/an. (C) Anatomical diagram of transverse section
through the carrot root apex to show position of JIM4reactive cells (fine shading) in relation to future vessel
elements. Inner emphasized line is the boundary between
future pericycle and endodermis. Outer emphasized line is
the boundary between root epidermis and ensheathing root
cap. Adapted from Esau (1940).
presence of the exuded AGP of Acacia Senegal at
(SOmgF1, but not by the Solanum tuberosum lectin at
up to 2gl - 1 . These characteristics are essentially similar
to those of MAC 207 (Pennell et al. 1989) but differ in
our observation that J1M4 displayed no hapten inhibition by any tested mono- or disaccharides, (see
Anderson et al. 1984 and Pennell et al. 1989).
Distribution of JIM4 antigens in root apex
Indirect immunofluorescence, utilizing J1M4, on sections prepared from the root apices of carrot seedlings,
revealed a specific and restricted distribution of its
antigens. This is in stark contrast to MAC 207 which
recognizes the plasma membrane of all cells in the
carrot root (Pennell et al. 1989, and see Fig. 3B).
Immunogold electron microscopy confirmed the plasma
51
membrane location of the JIM4 antigens in the carrot
seedling (Fig. 1C).
The place and plane of sectioning that provided the
most informative and clearly resolved specificity of the
restricted distribution of the JIM4 antigens was a
transverse section through the root apex as shown in
Fig. 3A. At this region, 50-100 fxm from the most apical
meristematic cells, the predominant feature was the
labelling of the plasma membranes of cells in two
segments of the stele. The segments are centred upon
the protoxylem poles of the diarch xylem. The larger
cells of the future xylem axis, weakly labelled in the
centre of the stele (Fig. 3A), are the vessel mother cells
of the future xylem plate (Esau, 1940). The sites of the
protophloem lie perpendicular to this region and are
essentially unlabelled with JIM4. The most intense
labelling with JIM4 occurred in two arcs of pericycle
cells with oblique longitudinal divisions whereas the
adjacent cells of the endodermis are labelled only
weakly (Fig. 3A). Esau (1940) has described the occurrence of such divisions of the pericycle cells, but does
not appear to have noted their restriction to the two
regions that can be clearly seen in Fig. 3A. It is of
interest that the MAC 207 epitope appeared more
abundant in these regions although also occurring on
the plasma membrane of every cell of the root apex
(Fig. 3B). The distribution of JIM4-reactive cells in
relation to the tissues of the carrot root apex is shown
diagrammatically in Fig. 3C which is adapted from Esau
(1940).
Serial longitudinal sectioning through the root meristem of a 5-day-old carrot seedling was performed to
determine the expression of the JIM4 antigens in
relation to the developing meristem. Immunofluorescent micrographs of two non-median and a median
section through the most apical region of the root apex
are shown in Fig. 4. The photographs are reproduced to
allow observation of labelled cells in relation to nonlabelled cells. The plane of sectioning in relation to the
JIM4 pattern of labelling of the transverse section is
indicated in Fig. 3A. The specific labelling of certain
cell lineages was observed to be a very early event of
development, being observed to within one or two cells,
20^m, of the most apical cells. The cells labelled in
Fig. 4C are of the developing pericycle. In no cases
were cells of the root cap observed to be labelled.
Distribution of JIM4 antigens in carrot plants
The pattern of labelling observed in the root apex was
reflected in other regions of the seedling and mature
carrot plant. Ultramicrotomy was not suitable for other
tissues, and immunofluorescent labelling could not be
so readily resolved on thicker sections. However, the
two segments of labelling, each centred upon an end of
the xylem axis, can be clearly seen in the stele in the
hypocotyl (Fig. 5A). An equivalent section, with JIM4
omitted from the labelling procedure, is shown in
Fig. 5B. The association of JIM4 antigens with vascular
tissue was observed through the region of the transition
of the vascular pattern from the root to that of the shoot
(occurring in the upper hypocotyl), and its association
52
/. P. Knox, S. Day and K. Roberts
Fig. 4. JIM4-generated immunofiuorescence restricted to cells of the stele in serial longitudinal sections of a carrot apex.
The plane and direction of sectioning in relation to Fig. 3A is shown in that figure with the arrowhead. (A) A non-median
section in which all the cells of the vascular cylinder appear to be labelled as well as isolated groups of distal cells
(arrowheads) and certain epidermal cells (e). (B) A further non-median section closer to the centre of the root, r, root cap.
(C) A median section (position indicated by the arrow in Fig. 3A) indicating JIM4 reactivity with two lineages of pericycle
cells to within 20/zm of the apical initials (a). (D) DAPI staining of DNA in the section shown in C. Bar, 100/an.
Pattern formation in the root apical meristem
53
Fig. 5. (A) Immunofluorescence generated by JIM4 on a transverse section of the hypocotyl of a 5-day-old carrot seedling is
restricted to two segments of the vascular tissue and also the epidermis (e). c, cortex; vc, vascular cylinder. Bar, 100/an.
(B) A comparable section to A in which the JIM4 incubation was omitted. Autofluorescence indicates the diarch distribution
of the mature xylem vessels. Bar, 100/an. (C) JIM4 immunofluorescence on a longitudinal and median section through the
cotyledons at the cotyledonary node indicates reactivity remains with the vascular tissue as it diverges into the cotyledons
(cd). A region of the adaxial cotyledon epidermis adjacent to the shoot apex (a) was also reactive (arrowheads). Bar,
100 fun. (D) DAPI staining of the section in 4C. (E) JIM4 immunofluorescence on a transverse section through a region of a
petiole from a mature carrot plant is predominantly associated with the xylem tissues (x) of the vascular bundles, the
epidermis and the outer parenchyma cells but not the collenchyma (cl). ad, adaxial epidermis. Bar, 500/an. (F) A
comparable section displaying MAC 207 immunofluorescence. All tissues are reactive including the phloem and the
collenchyma bundles. Bar, 500 ym.
54
a
/. P. Knox, S. Day and K. Roberts
JIM4 appeared to be relatively species specific in that
it did not react with sections of Pisum or Allium roots or
Nicotiana petioles. It did, however, cross-react with
b
-116
-97
-66
-45
-29
Petroselinum crispum. (Not shown).
Fig. 6. Immunoblotting of MAC
207 (lane a) and JIM4 (lane b)
against a membrane preparation
from 5-day-old carrot seedlings
separated by SDS-PAGE. Loading
(50/ig protein per lane) and
antibody incubations were
equivalent. Reactivity occurred
throughout the indicated MT range
and the difference in the antibodies
was predominantly quantitative.
Positions of protein markers
(M r xl0~ 3 ) and dye front
(arrowhead) are indicated.
with the several separate cotyledonary traces are shown
in Fig. 5C. In Daucus the primary vascular tissue of the
hypocotyl diverges entirely into the cotyledons and is
thus not continuous with that of the epicotyl (Havis,
1939; Esau, 1940). This can be seen in a median
longitudinal section through the cotyledons at the
cotyledonary node and shoot apex (Fig. 5C). No labelling of cells in the shoot apex at this stage can be seen.
The position of the apical meristem can be seen more
clearly in the DAPI-stained image of the same section
(Fig. 5D). Subsequently, of course, the vascular tissues
of the root and shoot are continuous. JIM4 was found to
be expressed in the shoot tissues of a mature plant.
Contrasting immunofluorescent micrographs of sections of a petiole from a mature carrot labelled with
JIM4 and MAC 207 are shown in Fig. 5E and 5F. JIM4
can be seen to be reactive with the xylem and associated
tissues of the vascular bundles and not the surrounding
parenchyma cells. In contrast MAC 207 reacted with all
the tissues of the petiole and the phloem regions of the
vascular bundles and the collenchyma bundles were
particularly strongly labelled.
However, in these tissues, certain cells of the epidermal layer were also labelled with JIM4. The pattern of
labelling could not always be clearly defined and in
certain instances isolated cells or discrete groups of cells
were observed to express the antigen (Fig. 4A, 5A,C).
At the cotyledonary node, no continuous labelling of
the epidermal tissues of the hypocotyl occurred. A very
reactive region occurred on the adaxial epidermal
tissues for the 100 /an adjacent to the shoot apex
(Fig. 5C). Serial sectioning revealed that the reactive
tissue was continuous through the node, around the
apex, connecting the two regions labelled in Fig. 5C
(data not shown). In the petiole the outer region of
parenchyma cells was also labelled in addition to the
epidermal and vascular tissue (Fig. 5E). Labelling of
isolated groups of cells was observed in the root apex
and the abrupt labelling of the epidermal cells was
observed in certain instances but in all cases at least
100 (xm from the apical initials (Fig. 4A).
Electrophoretic analysis of JIM4 reactive membrane
antigens in intact seedlings
Immunoblotting of membranes prepared from 5-dayold carrot seedlings indicated a wide range of bands and
smears reactive with MAC 207 (Fig. 6). A smear of Mr
50000 to 150000 was most intensely labelled, but
reactivity extended from the dye front to Mr of 200 000.
JIM4 reactivity, with equivalent loadings and incubations, appeared to differ only quantitatively from that
of MAC 207 with a much reduced signal (Fig. 6). No
obvious qualitative differences in the binding of these
two McAb to the electrophoretically separated membranes were detectable.
Discussion
JIM4 labels a novel distribution of plasma membrane
antigens
The plasma membrane location of the JIM4 epitope is
clearly seen in Figs 1 and 3A. This epitope was restricted to specific regions of the carrot seedling. The most
striking and easily discernible distribution of this antigen is as a cell surface determinant on a series of cells
associated with, but not unique to, the xylem and
pericycle tissue in the root.
Certain cells of the pericycle, distinctive due to a
series of oblique longitudinal divisions, first discernible
at 40 pern from the most apical cells (Esau, 1940), are
particularly reactive with JIM4. Serial sectioning of
such a primary root meristem revealed that labelling of
the pericycle cells occurred to within one or two cells of
the apical initials. It is at this distance from the apex
that the first cells of the stele, the pericycle cells,
become individualized and provide the first indication
of pattern arising from the meristem (Esau, 1940). It is
at 30 /xm (3 to 4 cells) from the apex that vessel mother
cells of the future xylem plate become enlarged. These
cells can be seen, though only weakly labelled, in
Fig. 3A. However, the first vascular elements to reach
maturity are the two protophloem sieve tubes at approximately 300 /an from the apex (Esau, 1940). It is
thus clear that the specific expression of JIM4 antigens
by certain cells of the stele occurred in a region of the
root apical meristem well before differentiation of the
vessel elements and appeared to occur in conjunction
with the beginnings of pattern formation. The bilateral
symmetry of JIM4 labelling within the stele reflects the
position and orientation of the future vascular pattern
but is not correlated directly with a certain future cell
type or tissue such as the xylem, phloem or pericycle
cells. These distinct groups of reactive cells appear to be
related to the inheritance of JIM4 antigen expression
within certain cell lineages.
An esterase activity has been shown to be an early
Pattern formation in the root apical meristem
marker of meristematic cells that will form the tissues of
the stele in roots of Pisum sativum, but the precise
pattern of activity in relation to future cell types is
unclear (Gahan, 1981; Rana & Gahan, 1982). The
reactivity of McAb JIM4 with the cell surface would
seem to be unique as a marker of cell position in the
pattern-forming systems of plants. There are, however,
parallels with animal systems. Antigens have been
detected that are markers for cell position and not cell
type in retina (Trisler et al. 1981) and in the early
development of chick limb buds (Ohsugi & Ide, 1986),
although in these cases position is reflected in terms of a
linear gradient of antigen rather than a symmetrical
distribution.
JIM4 reactivity with epidermal tissues was not consistent and is not so readily interpreted. In certain
instances, labelling of epidermal cells was observed to
begin abruptly behind the root apex. A very active
region of expression was located on the adaxial epidermal tissues of the cotyledonary node around the shoot
apex but not of other epidermal tissues in this region. In
the petiole of a mature plant outer cortical cells carried
the JIM4 epitope in addition to the epidermal cells.
JIM4 recognizes an epitope ofAGPs
The McAb JIM4 recognized a series of glycoproteins of
the plasma membrane of a suspension-cultured carrot
cell line and also an extracellular AGP derived from the
culture medium of the cell line. These are exactly the
same series of antigens recognized by the L-arabinose
inhibitable anti-AGP McAb MAC 207 (Pennell et al.
1989).
By contrast, in the carrot seedling, the JIM4 epitope
was restricted to a discrete series of cells as discussed
above. MAC 207 recognized a determinant on all cells.
Immunoblotting of membranes prepared from such
seedlings revealed reaction of both antibodies with a
more diverse array of membrane glycoproteins compared with the carrot cell line, although reactive components common to both preparations were observed.
A predominantly quantitative and not qualitative difference in the binding pattern of these two antibodies
with carrot seedling membranes was observed. In
identical conditions, the JIM4-induced signal was considerably weaker than that of MAC 207. This observation suggests that though JIM4 and MAC 207 recognize distinct epitopes, (biotinylated JIM4 could not be
inhibited from binding to membranes or the extracellular AGP by MAC 207; data not shown), these epitopes
occur on an identical series of glycoproteins and also the
extracellular AGP. Although both epitopes occur on a
diverse array of glycoproteins, the evidence reported
here indicates that the MAC 207 epitope can occur in
the absence of the JIM4 epitope. The expression of the
JIM4 reactivity could involve the addition or modification of existing sugar residues, or its absence could
occur by masking of the epitope. The absence of the
JIM4 epitope results in no change in the electrophoretic
mobility of the antigens and presumably results from a
subtle change in the glycan component of these glycoprotein antigens. It would thus appear that the plasma
55
membranes of plant cells contain overlapping series of
antigenic glycoproteins sharing distinct epitopes with
each other as well as with extracellular arabinogalactan
proteins.
A recent report indicates the presence on the surface
of Drosophila neurones of a specific carbohydrate
epitope (also occurring on certain mannose-containing
plant glycoproteins) that is carried on different proteins
throughout the development of the neurone and is thus
characteristic of neural cell surfaces rather than the
epitope-bearing proteins, (Katz et al. 1988).
AGPs are widely distributed and organ-specific forms
do occur. They have been demonstrated by electrophoresis in floral tissues of Gladiolus and Lilium
(Gleeson & Clarke, 1980), by Yariv reagent crossed
electrophoresis in Glycine root nodules and floral and
vegetative tissues of Lycopersicon peruvianum (Cassab,
1986; van Hoist & Clarke, 1986) and by serological
activity and chemical composition in tissues of Raphanus sativus (Tsumuraya et al. 1988). AGPs of the stigma
and style of Nicotiana alata have been separated by
means of crossed electrophoresis, and those of the
stigma were demonstrated to be developmentally regulated (Gell et al. 1986). In all the above cases, it would
appear to be the soluble extracellular AGPs that are
investigated, although the contribution of related membrane-associated glycoproteins is far from clear. In no
case have the differing forms been localized.
Cell- and tissue-specific carbohydrate antigens, which
may possibly be AGPs, have been reported in the floral
tissues of Nicotiana tabacum (Evans et al. 1988). A
glycoprotein, but not with AGP characteristics, has
been isolated from the culture medium of carrot cells
and has been immunologically observed to be restricted
to dermal tissues of carrots, although the subcellular
location is far from clear (Satoh & Fujii, 1988).
As discussed above, the possible function of cell
surface determinants in such restricted lineages of cells
may be concerned with cell and tissue identity in
relation to subsequent pattern formation. Such a role in
cell identity has been suggested for AGPs in which
terminal substituents of the /3-galactan and protein
backbone may be involved in the expression of identity
of tissues or cell type (Clarke et al. 1979; Fincher et al.
1983). In animal cells carbohydrate structures are common as developmentally regulated antigens and can
exert important roles in cell identity and function
(Feizi, 1985; Lefrancois et al. 1985; Katz et al. 1988).
Some form of cell-cell interaction and recognition
may be important during the development of organized
tissue and it is important not to forget that AGPs were
originally characterized as /Mectins as demonstrated by
their ability to bind artificial carbohydrate antigens
(Yariv reagents). However, the extent and nature of
any cell interactions involving cell surface lectin-like
molecules during plant developmental processes are
unknown.
It is of interest that clonal analysis indicates that cell
fate, in terms of differentiation, may be independent of
cell lineage in the early stages of shoot meristem
development (Poethig, 1987). In the carrot root meris-
56
/. P. Knox, S. Day and K. Roberts
tem, the dependence of cell fate upon cell lineage is
unknown. The expression of the JIM4 epitope, reflecting the future pattern of vascular element differentiation, in cells close to the apical dome, may indicate a
role for these cell surface glycoproteins in the determination of the fate of cells.
This work was supported with a grant from the AFRC Cell
Signalling & Recognition programme. We acknowledge the
assistance of J. Cooke of the Food Research Institute,
Norwich for the handling of rats and J. King and C. Cooper
for the preparation and maintenance of the hybridoma. We
thank Roger Pennell for the electron micrographs and useful
criticisms and discussions.
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(Accepted 30 January 1989)