Development 106, 47-56 (1989)Printed in Great Britain <Q The Company of Biologists Limited 1989

47

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 thatrecognizes a series of glycoproteins associated with theplasma membrane of a suspension-cultured carrot cellline and also an arabinogalactan proteoglycan secretedby the cultured cells. Immunocytochemistry indicatedthat the plasma membrane antigen(s) recognized byJIM4 are specific to certain cells of Daucus carota L.seedlings. In the root apex JIM4 labelled two segments ofthe stele. These were centred upon the poles of theprotoxylem. An axis of unlabelled cells connected thetwo phloem regions. Two sections of the pericycle withcharacteristic oblique longitudinal divisions were par-ticularly reactive with JIM4. This pattern of reactivecells, reflecting cell position rather than a specific futurecell type, would appear to be a unique observation inplants. The association of JIM4 antigens with thesevascular tissues is maintained through the transitionfrom root to the shoot tissue of the cotyledons and themature plant. Examination of J1M4 labelling upon

ultrathin frozen sections of the carrot seedling rootapical meristem indicated that the expression of theantigen is a very early event in root development. Cellsexpress the surface epitope, within one or two cells of thedome of apical initials, before the pattern of futurevascular tissue can be discerned and well before itsactual differentiation.

Abbreviations: AGPs, arabinogalactan proteins; DAPI,4',6-diamidino-2-phenyl-indole; ELISA, enzyme-linkedimmunosorbent assay; McAb, monoclonal antibody; PBS,phosphate-buffered saline; SDS-PAGE, sodiumdodecylsulphate-polyacrylamide gel electrophoresis; TBS,Tris-buffered saline.

Key words: arabinogalactan proteins, Daucus carota L.,monoclonal antibody, pattern formation, plasmamembrane, root meristem.

Introduction

All of the cells in a higher plant derive ultimately fromthe dividing cell populations in the shoot and root apicalmeristems. These meristems, in giving rise to the organsof the plant, produce cell lineages that develop into thecharacteristically patterned vasculature of these struc-tures. Despite our knowledge of the physical structureof many apical meristems and some of the physicaldeterminants of their anatomy, the molecular processesthat determine plant morphology and cell differen-tiation remain unknown.

As dividing cells in the meristematic region of a rootmove back from the apex, as files of related cells, twopatterns are established that reflect the mature rootmorphology. A concentric pattern underlies the classi-cal division into three tissue systems - epidermis, cortexand stele. Within the stele there is a species-specificradial pattern of vascular development into phloem andxylem. In the case of carrot {Daucus carota L.), the rootshows a bilaterally symmetrical diarch structure. Theestablishment of such tissue patterns and subsequent

differentiation requires the acquisition of some form ofidentity by cells or cell lineages, although the chemicalnature of any such identity is unknown. To understandhow these patterns of xylem and phloem are estab-lished, we need molecular markers of early patternformation, or cell identity, at a time well before the cellsfinally differentiate into recognizable cell types.

Arabinogalactan proteins (AGPs) form a very largeand diverse group of macromolecules in plants and canbe subdivided most readily into extracellular proteogly-cans and membrane-associated glycoproteins (Fincheretal. 1983; Pennell etal. 1989). No clear function for anyof these molecules has emerged.

AGPs are antigenic and capable of generating mono-clonal antibodies (McAb) with reactivities inhibitableby L-arabinose, D-galactose and/or associated disac-charides (Anderson et al. 1984). Such antibodies havebeen generated in response to complex membranousplant immunogens (Meyer et al. 1987; Bradley et al.1988; Brewin et al. 1988). In one case an antibody hasbeen shown to be specific for AGPs and not to cross-react with other cell surface arabinosylated and hy-

48 /. P. Knox, S. Day and K. Roberts

droxyproline-containing glycoproteins such as theextensins and the lectins of the Solanaceae (Pennell etal. 1989).

In this report we describe the specific, restricted andnovel distribution of a set of plasma membrane antigensin seedlings of Daucus carota L. The McAb JIM4recognized an epitope common to a set of glycoproteinsof the plasma membrane of a carrot cell line and anarabinogalactan protein secreted by the cell line. Wedemonstrate that in the carrot seedling the most abun-dant expression of the membrane-associated epitopeoccurs in the vascular tissues. Examination of the rootapical meristem of carrot seedlings indicated that theexpression of the epitope is a very early event in thecontinuing development of the root meristem andseems to reflect early stages in the formation of thevascular pattern.

Materials and methods

Plant materials and cell cultureCarrot {Daucus carota L. cv. Early Nantes) seeds weregerminated on moist tissue paper and grown for 5 days in thedark at 22°C before use. Mature carrot plants were collectedlocally. A suspension culture of carrot cells (Lloyd et al. 1979)was maintained in Murashige and Skoog medium sup-plemented with lmgl"1 2,4-dichlorophenoxy-acetic acid and25 gl"1 sucrose. Cells were subcultured every 7 days and usedfor experimental purposes 5 or 6 days after subculture.

Immunization and production of hybridomaThe hybridoma secreting the monoclonal antibody J1M4 wasdeveloped from a series of immunizations of rats with intactprotoplasts prepared from the carrot suspension cell culture.The protoplasts (lxlO6 cells in 300 jil phosphate-bufferedsaline, PBS) were injected intraperitoneally into a maleLOU/c rat (5 weeks old) on days 0, 22, 56 and 103. Spleenlymphocytes were collected and fused with the IR983Fmyeloma cell line (Bazin, 1982) on day 106. Procedures usedfor the fusion, HAT selection and maintenance of hybridomaswere essentially as described by Galfre & Milstein (1981).

The J1M4 McAb was selected as a plasma membranereactive antibody by its ability to bind to a preparation ofmembranes from the carrot cell line and indirect immunofiu-orescence on carrot protoplasts prepared from the culturedcells. The JIM4 hybridoma secretes immunoglobulins of classIgM.

Preparation of protoplastsThe procedures used for the preparation of protoplasts fromthe suspension-cultured cells and their use for obtainingindirect immunofluorescence with plasma membrane-reactiveantibodies are described elsewhere (Pennell et al. 1989).

Preparation of microsomal membrane fractionsProtoplasts, prepared from the carrot cell line or the intactcarrot seedlings, were homogenized in 4mlg~1 fresh weightof 50 mM-Tris(hydroxymethyl)aminomethane-HCl (TRIS)buffer pH7-5 containing 0-25M-sucrose, 3mM-disodium eth-ylenediaminetetra-acetate (EDTA), 2-5rtiM-dithiothreitol(DTT) and 1 mM-phenylmethylsulphonyl fluoride (PMSF).Homogenization was performed with a glass hand-held hom-ogenizer. A pellet collected by centrifugation at 5000g forlOmin was discarded and the subsequent pellet at 100 000 g

(1 h) was washed in the homogenization buffer and resus-pended in 5 mM-potassium phosphate buffer pH7-8 (contain-ing the additions of the homogenization buffer other than theEDTA) and retained as the membrane fraction and stored at—20°C. All procedures were performed at 4°C. Protein wasdetermined according to the method of Lowry et al. (1951).

Preparation of a fraction containing thearabinogalactan protein of the carrot culture mediumThe culture medium of the carrot cell line, conditioned by 5days growth subsequent to subculture was separated from thecells by centrifugation and filtered through Whatman No. 1paper and brought to 90 % (v/v) acetone at 4°C. After 30 minthe precipitate was collected by centrifugation, extracted withwater and the soluble components lyophilized.

Electrophoresis and immunoblottingSodium 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 asemi-dry electroblotting system (Sartorius, Goettingen,FRG). Nitrocellulose sheets were blocked with 5 % (v/v) calfserum in PBS for at least 1 h before incubation with a 100-folddilution of McAb culture supernatant in the same bufferovernight at 4°C. Extensive washing in PBS containing 0-05 %Tween 20 was performed before and after incubation with a2000-fold dilution of rabbit anti-rat Ig linked to alkalinephosphatase (Sigma) in the PBS containing calf serum for 2 h.The enzyme substrate was developed according to the manu-facturer's method.

Enzyme-linked immunosorbent assaysELISAs were performed in microtitre plates coated with themembrane preparation at SOmgl"1 protein (18 h at 4°C) andblocked for at least 1 h with 5 % (v/v) calf serum in PBS. JIM4binding was detected by means of a second antibody (rabbitanti-rat Ig linked to horseradish peroxidase, ICN Biomedi-cals) developed by conventional reactions. The dilution ofJIM4 giving 90 % of maximal binding was used for assessmentof hapten and glycoprotein inhibition. In certain cases, theimmobilized antigens were treated prior to antibody incu-bations with Pronase E (Sigma) at 1 gl"1 in 50 mM-TRIS-HClbuffer pH7-5 or 25mM-sodium metaperiodate in 50 mM-sodium acetate buffer, pH4-3 for lh in the dark. After suchtreatments the plates were washed extensively in water andre-blocked prior to antibody incubations and assessment ofantigen degradation.

Immunogold electron microscopyThe fixation and subsequent treatment of suspension-culturedcarrot cells and carrot seedlings for the analysis of JIM4binding by means of immunogold electron microscopy isdescribed elsewhere (Pennell et al. 1989).

Tissue fixation and microtomyRoot apices of 5-day-old carrot seedlings were excised andimmersed in freshly prepared 4% (w/v) formaldehyde infixation buffer (50mM-piperazine-A',A/'-bis[2-ethanesuUonicacid] {Pipes}, 5mM-MgSO4 and 5mM-ethylene glycol bis[/3-aminoethylether]A',/V,./v",iV'-tetra acetic acid {EGTA},pH6-9) for 2h, washed in fixation buffer and infused for atleast 72 h with l-5M-sucrose and 0-5% formaldehyde in thesame buffer. Before sectioning, root apices were trimmed toapprox. lmm, transferred to freezing stubs and plunged intoliquid ethane. The stub with frozen tissue was fitted to an

Pattern formation in the root apical meristem 49

Fig. 1. (A) Immunofluorescencegenerated by JIM4 binding to thesurface of intact protoplastsprepared from the carrot cell line.Note the agglutination of theprotoplasts. Bar, 10/an.(B) Immunogold localization ofJIM4 antigens to the plasmamembrane (pm) of carrotsuspension cell, w, cell wall;c, cytoplasm. Bar, 0-1 /an.(C) Immunogold electronmicroscopy confirms J1M4 bindingto the plasma membrane of cellsfrom a carrot seedling root, w, cellwall; c, cytoplasm. Bar, 01 /an.

Ultracut EFC 4D cryoultramicrotome (Reichert-Jung, UK,Slough) and the tissue sectioned at -110°C at a thickness of0-5 /on. Sections were collected on small drops of 2M-sucrosein water and settled on to multiwell slides. Before use theslides were extensively washed with Tris-buffered saline(TBS). Plant tissues other than root apices were similarlyimmersed in 4% formaldehyde in fixation buffer for at least2h before embedding with OCT compound (Miles Scientific,Illinois, USA) and frozen at -20°C. Sections (5-10/im) weremade at —20°C using a Bright 5030 cryomicrotome (Cam-bridge, UK) and collected on multiwell slides coated withpoly-L-lysine and allowed to dry. Before use the slides wereextensively washed with water to remove the OCT com-pound.

ImmunocytochemistryThe sections were treated with a 5-fold dilution of hybridomaculture supernatant into 5 % (v/v) calf serum in TBS for up to12 h at 4°C. The sections were rinsed in TBS before treatmentwith goat anti-rat Ig linked to fluorescein isothiocyanate,(ICN Biomedicals) diluted 100-fold into the TBS with calfserum, for at least 2h. The final, extensive washing of theslides in TBS also included a 30s incubation with 4',6-diamidino-2-phenyl-indole (DAPI) at lmgl"1 in TBS. Finalmounting was with Citifluor anti-fade mountant (Citifluor,London, UK) and the sections were observed on a ZeissPhotomicroscope in equipped with epifluorescence ir-radiation.

Results

Characterization of JIM4 antigens of the plasmamembrane of suspension-cultured carrot cellsThe McAb JIM4 bound to the surface of protoplastsprepared from the carrot cell line as revealed by indirectimmunofluorescence (Fig. 1A). The specific localiz-ation of the JIM4 epitope to the plasma membrane ofthese cells was confirmed by means of immunogoldelectron microscopy performed on ultrathin sections ofsuch cells retaining an intact cell wall (Fig. IB).

Immunoblotting of electrophoretically separatedmembranes indicated that JIM4 recognized a series ofdiscrete bands in the range of Mr 20000 to 60000(Fig. 2). These corresponded with the binding patternof an anti-AGP McAb MAC 207 (Pennell et al. 1989). In

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Fig. 2. Immunoblotting of JIM4 (lane a) and MAC 207(lane b) to the membrane preparation of carrot protoplastsseparated by SDS-PAGE and transferred to nitrocellulose.Loading was at 50/ig protein per lane. JIM4 also reactedwith the arabinogalactan protein (Afr70000 to 100000) in anacetone precipitate of the conditioned medium of the carrotcells (lane c, 10/ig: protein loading). Positions of proteinmarkers (Mrxl0 ) and the dye front (arrowhead) areindicated.

addition, JIM4 also reacted with an arabinogalactanprotein (Pennell et al. 1989), derived from the culturemedium of the carrot cells, which was electrophoreti-cally resolved as a smear of MT 70000 to 100000(Fig. 2).

Further analysis of the JIM4 antigen and epitope wasby means of an enzyme-linked immunosorbent assay(ELISA) of its binding to membranes prepared fromprotoplasts of the carrot cell line. Treatment of theimmobilized membranes with either periodate or aprotease reduced JIM4 binding by 76 % and 68 %,respectively, indicating that the antigen contained bothcarbohydrate and protein components. The binding ofJIM4 to the membranes was inhibited by 50 % in the

50 /. P. Knox, S. Day and K. Roberts

Pattern formation in the root apical meristem 51

Future phloemregion Future xylem

region

Fig. 3. (A) Immunofluorescence generated by JIM4 on atransverse section of a carrot root, 50-100 ̂ m from theapical initials is restricted to two segments of the vascularcylinder and is most abundant in the cells of the pericycle(p). At the centre of the cylinder 3 xylem vessel mothercells are weakly labelled (The centre cell is indicated withx). The adjacent regions that will develop into the phloem(ph) are unlabelled. c, cortex. The arrow and arrowheadrefer to the sections shown in Fig. 4. Bar, 50/zm.(B) Immunofluorescence generated by MAC 207 on acomparable section to A reveals reactivity with all cells.Bar, 50/an. (C) Anatomical diagram of transverse sectionthrough the carrot root apex to show position of JIM4-reactive cells (fine shading) in relation to future vesselelements. Inner emphasized line is the boundary betweenfuture pericycle and endodermis. Outer emphasized line isthe boundary between root epidermis and ensheathing rootcap. Adapted from Esau (1940).

presence of the exuded AGP of Acacia Senegal at(SOmgF1, but not by the Solanum tuberosum lectin atup to 2gl - 1 . These characteristics are essentially similarto those of MAC 207 (Pennell et al. 1989) but differ inour observation that J1M4 displayed no hapten inhi-bition by any tested mono- or disaccharides, (seeAnderson et al. 1984 and Pennell et al. 1989).

Distribution of JIM4 antigens in root apexIndirect immunofluorescence, utilizing J1M4, on sec-tions prepared from the root apices of carrot seedlings,revealed a specific and restricted distribution of itsantigens. This is in stark contrast to MAC 207 whichrecognizes the plasma membrane of all cells in thecarrot root (Pennell et al. 1989, and see Fig. 3B).Immunogold electron microscopy confirmed the plasma

membrane location of the JIM4 antigens in the carrotseedling (Fig. 1C).

The place and plane of sectioning that provided themost informative and clearly resolved specificity of therestricted distribution of the JIM4 antigens was atransverse section through the root apex as shown inFig. 3A. At this region, 50-100 fxm from the most apicalmeristematic cells, the predominant feature was thelabelling of the plasma membranes of cells in twosegments of the stele. The segments are centred uponthe protoxylem poles of the diarch xylem. The largercells of the future xylem axis, weakly labelled in thecentre of the stele (Fig. 3A), are the vessel mother cellsof the future xylem plate (Esau, 1940). The sites of theprotophloem lie perpendicular to this region and areessentially unlabelled with JIM4. The most intenselabelling with JIM4 occurred in two arcs of pericyclecells with oblique longitudinal divisions whereas theadjacent cells of the endodermis are labelled onlyweakly (Fig. 3A). Esau (1940) has described the occur-rence of such divisions of the pericycle cells, but doesnot appear to have noted their restriction to the tworegions that can be clearly seen in Fig. 3A. It is ofinterest that the MAC 207 epitope appeared moreabundant in these regions although also occurring onthe plasma membrane of every cell of the root apex(Fig. 3B). The distribution of JIM4-reactive cells inrelation to the tissues of the carrot root apex is showndiagrammatically in Fig. 3C which is adapted from Esau(1940).

Serial longitudinal sectioning through the root meris-tem of a 5-day-old carrot seedling was performed todetermine the expression of the JIM4 antigens inrelation to the developing meristem. Immunofluor-escent micrographs of two non-median and a mediansection through the most apical region of the root apexare shown in Fig. 4. The photographs are reproduced toallow observation of labelled cells in relation to non-labelled cells. The plane of sectioning in relation to theJIM4 pattern of labelling of the transverse section isindicated in Fig. 3A. The specific labelling of certaincell lineages was observed to be a very early event ofdevelopment, being observed to within one or two cells,20^m, of the most apical cells. The cells labelled inFig. 4C are of the developing pericycle. In no caseswere cells of the root cap observed to be labelled.

Distribution of JIM4 antigens in carrot plantsThe pattern of labelling observed in the root apex wasreflected in other regions of the seedling and maturecarrot plant. Ultramicrotomy was not suitable for othertissues, and immunofluorescent labelling could not beso readily resolved on thicker sections. However, thetwo segments of labelling, each centred upon an end ofthe xylem axis, can be clearly seen in the stele in thehypocotyl (Fig. 5A). An equivalent section, with JIM4omitted from the labelling procedure, is shown inFig. 5B. The association of JIM4 antigens with vasculartissue was observed through the region of the transitionof 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-mediansection 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 pericyclecells 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 isrestricted 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 distributionof the mature xylem vessels. Bar, 100/an. (C) JIM4 immunofluorescence on a longitudinal and median section through thecotyledons 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 apetiole from a mature carrot plant is predominantly associated with the xylem tissues (x) of the vascular bundles, theepidermis and the outer parenchyma cells but not the collenchyma (cl). ad, adaxial epidermis. Bar, 500/an. (F) Acomparable section displaying MAC 207 immunofluorescence. All tissues are reactive including the phloem and thecollenchyma bundles. Bar, 500 ym.

54 /. P. Knox, S. Day and K. Roberts

a b

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Fig. 6. Immunoblotting of MAC207 (lane a) and JIM4 (lane b)against a membrane preparationfrom 5-day-old carrot seedlingsseparated by SDS-PAGE. Loading(50/ig protein per lane) andantibody incubations wereequivalent. Reactivity occurredthroughout the indicated MT rangeand the difference in the antibodieswas predominantly quantitative.Positions of protein markers(Mrxl0~3) and dye front(arrowhead) are indicated.

JIM4 appeared to be relatively species specific in thatit did not react with sections of Pisum or Allium roots orNicotiana petioles. It did, however, cross-react withPetroselinum crispum. (Not shown).

Electrophoretic analysis of JIM4 reactive membraneantigens in intact seedlingsImmunoblotting of membranes prepared from 5-day-old carrot seedlings indicated a wide range of bands andsmears reactive with MAC 207 (Fig. 6). A smear of Mr50000 to 150000 was most intensely labelled, butreactivity extended from the dye front to Mr of 200 000.JIM4 reactivity, with equivalent loadings and incu-bations, appeared to differ only quantitatively from thatof MAC 207 with a much reduced signal (Fig. 6). Noobvious qualitative differences in the binding of thesetwo McAb to the electrophoretically separated mem-branes were detectable.

with the several separate cotyledonary traces are shownin Fig. 5C. In Daucus the primary vascular tissue of thehypocotyl diverges entirely into the cotyledons and isthus not continuous with that of the epicotyl (Havis,1939; Esau, 1940). This can be seen in a medianlongitudinal section through the cotyledons at thecotyledonary node and shoot apex (Fig. 5C). No label-ling of cells in the shoot apex at this stage can be seen.The position of the apical meristem can be seen moreclearly in the DAPI-stained image of the same section(Fig. 5D). Subsequently, of course, the vascular tissuesof the root and shoot are continuous. JIM4 was found tobe expressed in the shoot tissues of a mature plant.Contrasting immunofluorescent micrographs of sec-tions of a petiole from a mature carrot labelled withJIM4 and MAC 207 are shown in Fig. 5E and 5F. JIM4can be seen to be reactive with the xylem and associatedtissues of the vascular bundles and not the surroundingparenchyma cells. In contrast MAC 207 reacted with allthe tissues of the petiole and the phloem regions of thevascular bundles and the collenchyma bundles wereparticularly strongly labelled.

However, in these tissues, certain cells of the epider-mal layer were also labelled with JIM4. The pattern oflabelling could not always be clearly defined and incertain instances isolated cells or discrete groups of cellswere observed to express the antigen (Fig. 4A, 5A,C).At the cotyledonary node, no continuous labelling ofthe epidermal tissues of the hypocotyl occurred. A veryreactive region occurred on the adaxial epidermaltissues for the 100 /an adjacent to the shoot apex(Fig. 5C). Serial sectioning revealed that the reactivetissue was continuous through the node, around theapex, connecting the two regions labelled in Fig. 5C(data not shown). In the petiole the outer region ofparenchyma cells was also labelled in addition to theepidermal and vascular tissue (Fig. 5E). Labelling ofisolated groups of cells was observed in the root apexand the abrupt labelling of the epidermal cells wasobserved in certain instances but in all cases at least100 (xm from the apical initials (Fig. 4A).

Discussion

JIM4 labels a novel distribution of plasma membraneantigensThe plasma membrane location of the JIM4 epitope isclearly seen in Figs 1 and 3A. This epitope was restric-ted to specific regions of the carrot seedling. The moststriking and easily discernible distribution of this anti-gen is as a cell surface determinant on a series of cellsassociated with, but not unique to, the xylem andpericycle tissue in the root.

Certain cells of the pericycle, distinctive due to aseries of oblique longitudinal divisions, first discernibleat 40 pern from the most apical cells (Esau, 1940), areparticularly reactive with JIM4. Serial sectioning ofsuch a primary root meristem revealed that labelling ofthe pericycle cells occurred to within one or two cells ofthe apical initials. It is at this distance from the apexthat the first cells of the stele, the pericycle cells,become individualized and provide the first indicationof pattern arising from the meristem (Esau, 1940). It isat 30 /xm (3 to 4 cells) from the apex that vessel mothercells of the future xylem plate become enlarged. Thesecells can be seen, though only weakly labelled, inFig. 3A. However, the first vascular elements to reachmaturity are the two protophloem sieve tubes at ap-proximately 300 /an from the apex (Esau, 1940). It isthus clear that the specific expression of JIM4 antigensby certain cells of the stele occurred in a region of theroot apical meristem well before differentiation of thevessel elements and appeared to occur in conjunctionwith the beginnings of pattern formation. The bilateralsymmetry of JIM4 labelling within the stele reflects theposition and orientation of the future vascular patternbut is not correlated directly with a certain future celltype or tissue such as the xylem, phloem or pericyclecells. These distinct groups of reactive cells appear to berelated to the inheritance of JIM4 antigen expressionwithin certain cell lineages.

An esterase activity has been shown to be an early

Pattern formation in the root apical meristem 55

marker of meristematic cells that will form the tissues ofthe stele in roots of Pisum sativum, but the precisepattern of activity in relation to future cell types isunclear (Gahan, 1981; Rana & Gahan, 1982). Thereactivity of McAb JIM4 with the cell surface wouldseem to be unique as a marker of cell position in thepattern-forming systems of plants. There are, however,parallels with animal systems. Antigens have beendetected that are markers for cell position and not celltype in retina (Trisler et al. 1981) and in the earlydevelopment of chick limb buds (Ohsugi & Ide, 1986),although in these cases position is reflected in terms of alinear gradient of antigen rather than a symmetricaldistribution.

JIM4 reactivity with epidermal tissues was not con-sistent and is not so readily interpreted. In certaininstances, labelling of epidermal cells was observed tobegin abruptly behind the root apex. A very activeregion of expression was located on the adaxial epider-mal tissues of the cotyledonary node around the shootapex but not of other epidermal tissues in this region. Inthe petiole of a mature plant outer cortical cells carriedthe JIM4 epitope in addition to the epidermal cells.

JIM4 recognizes an epitope ofAGPsThe McAb JIM4 recognized a series of glycoproteins ofthe plasma membrane of a suspension-cultured carrotcell line and also an extracellular AGP derived from theculture medium of the cell line. These are exactly thesame series of antigens recognized by the L-arabinoseinhibitable anti-AGP McAb MAC 207 (Pennell et al.1989).

By contrast, in the carrot seedling, the JIM4 epitopewas restricted to a discrete series of cells as discussedabove. MAC 207 recognized a determinant on all cells.Immunoblotting of membranes prepared from suchseedlings revealed reaction of both antibodies with amore diverse array of membrane glycoproteins com-pared with the carrot cell line, although reactive com-ponents common to both preparations were observed.A predominantly quantitative and not qualitative dif-ference in the binding pattern of these two antibodieswith carrot seedling membranes was observed. Inidentical conditions, the JIM4-induced signal was con-siderably weaker than that of MAC 207. This obser-vation suggests that though JIM4 and MAC 207 recog-nize distinct epitopes, (biotinylated JIM4 could not beinhibited from binding to membranes or the extracellu-lar AGP by MAC 207; data not shown), these epitopesoccur on an identical series of glycoproteins and also theextracellular AGP. Although both epitopes occur on adiverse array of glycoproteins, the evidence reportedhere indicates that the MAC 207 epitope can occur inthe absence of the JIM4 epitope. The expression of theJIM4 reactivity could involve the addition or modifi-cation of existing sugar residues, or its absence couldoccur by masking of the epitope. The absence of theJIM4 epitope results in no change in the electrophoreticmobility of the antigens and presumably results from asubtle change in the glycan component of these glyco-protein antigens. It would thus appear that the plasma

membranes of plant cells contain overlapping series ofantigenic glycoproteins sharing distinct epitopes witheach other as well as with extracellular arabinogalactanproteins.

A recent report indicates the presence on the surfaceof Drosophila neurones of a specific carbohydrateepitope (also occurring on certain mannose-containingplant glycoproteins) that is carried on different proteinsthroughout the development of the neurone and is thuscharacteristic of neural cell surfaces rather than theepitope-bearing proteins, (Katz et al. 1988).

AGPs are widely distributed and organ-specific formsdo occur. They have been demonstrated by electro-phoresis in floral tissues of Gladiolus and Lilium(Gleeson & Clarke, 1980), by Yariv reagent crossedelectrophoresis in Glycine root nodules and floral andvegetative tissues of Lycopersicon peruvianum (Cassab,1986; van Hoist & Clarke, 1986) and by serologicalactivity and chemical composition in tissues of Rapha-nus sativus (Tsumuraya et al. 1988). AGPs of the stigmaand style of Nicotiana alata have been separated bymeans of crossed electrophoresis, and those of thestigma were demonstrated to be developmentally regu-lated (Gell et al. 1986). In all the above cases, it wouldappear to be the soluble extracellular AGPs that areinvestigated, although the contribution of related mem-brane-associated glycoproteins is far from clear. In nocase have the differing forms been localized.

Cell- and tissue-specific carbohydrate antigens, whichmay possibly be AGPs, have been reported in the floraltissues of Nicotiana tabacum (Evans et al. 1988). Aglycoprotein, but not with AGP characteristics, hasbeen isolated from the culture medium of carrot cellsand has been immunologically observed to be restrictedto dermal tissues of carrots, although the subcellularlocation is far from clear (Satoh & Fujii, 1988).

As discussed above, the possible function of cellsurface determinants in such restricted lineages of cellsmay be concerned with cell and tissue identity inrelation to subsequent pattern formation. Such a role incell identity has been suggested for AGPs in whichterminal substituents of the /3-galactan and proteinbackbone may be involved in the expression of identityof tissues or cell type (Clarke et al. 1979; Fincher et al.1983). In animal cells carbohydrate structures are com-mon as developmentally regulated antigens and canexert important roles in cell identity and function(Feizi, 1985; Lefrancois et al. 1985; Katz et al. 1988).

Some form of cell-cell interaction and recognitionmay be important during the development of organizedtissue and it is important not to forget that AGPs wereoriginally characterized as /Mectins as demonstrated bytheir ability to bind artificial carbohydrate antigens(Yariv reagents). However, the extent and nature ofany cell interactions involving cell surface lectin-likemolecules during plant developmental processes areunknown.

It is of interest that clonal analysis indicates that cellfate, in terms of differentiation, may be independent ofcell lineage in the early stages of shoot meristemdevelopment (Poethig, 1987). In the carrot root meris-

56 /. P. Knox, S. Day and K. Roberts

tem, the dependence of cell fate upon cell lineage isunknown. The expression of the JIM4 epitope, reflect-ing the future pattern of vascular element differen-tiation, in cells close to the apical dome, may indicate arole for these cell surface glycoproteins in the determi-nation of the fate of cells.

This work was supported with a grant from the AFRC CellSignalling & Recognition programme. We acknowledge theassistance of J. Cooke of the Food Research Institute,Norwich for the handling of rats and J. King and C. Cooperfor the preparation and maintenance of the hybridoma. Wethank Roger Pennell for the electron micrographs and usefulcriticisms and discussions.

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(Accepted 30 January 1989)