functional compartmentation of the golgi apparatus of plant cells

Plant Physiol. (1992) 99, 1070-10830032-0889/92/99/1070/1 4/$01 .00/0

Received for publication January 4, 1992Accepted March 17, 1992

Functional Compartmentation of the Golgi Apparatus ofPlant Cells'

Immunocytochemical Analysis of High-Pressure Frozen- and Freeze-Substituted SycamoreMaple Suspension Culture Cells

Guo Feng Zhang and L. Andrew Staehelin*

Department of Molecular, Cellular, and Developmental Biology, University of Colorado,Boulder, Colorado 80309-0347

ABSTRACT

The Golgi apparatus of plant cells is engaged in both the proc-essing of glycoproteins and the synthesis of complex polysaccha-rides. To investigate the compartmentalization of these functionswithin individual Golgi stacks, we have analyzed the ultrastructureand the immunolabeling patterns of high-pressure frozen andfreeze-substituted suspension-cultured sycamore maple (Acerpseudoplatanus L.) cells. As a result of the improved structuralpreservation, three morphological types of Golgi cisternae, desig-nated cis, medial, and trans, as well as the trans Golgi network,could be identified. The number of cis cisternae per Golgi stackwas found to be fairly constant at approximately 1, whereas thenumber of medial and trans cisternae per stack was variable andaccounted for the varying number of cisternae (3-10) among themany Golgi stacks examined. By using a battery of seven antibodieswhose specific sugar epitopes on secreted polysaccharides andglycoproteins are known, we have been able to determine in whichtypes of cisternae specific sugars are added to N-linked glycans,and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The,8-1,4-linked D-glucosyl backbone of xyloglucan is synthesized intrans cisternae, and the terminal fucosyl residues on the trisaccha-ride side chains of xyloglucan are partly added in the trans cister-nae, and partly in the trans Golgi network. In contrast, the poly-galacturonic/rhamnogalacturonan-I backbone is assembled in cisand medial cisternae, methylesterification of the carboxyl groupsof the galacturonic acid residues in the polygalacturonic aciddomains occurs mostly in medial cisternae, and arabinose-contain-ing side chains of the polygalacturonic acid domains are added tothe nascent polygalacturonic acid/rhamnogalacturonan-l mole-cules in the trans cisternae. Double labeling experiments demon-strate that xyloglucan and polygalacturonic acid/rhamnogalactu-ronan-I can be synthesized concomitantly within the same Golgistack. Finally, we show that the xylosyl residue-linked ,-1,2 to the,-linked mannose of the core of N-linked glycans is added inmedial cisternae. Taken together, our results indicate that in syca-more maple suspension-cultured cells, different types of Golgicisternae contain different sets of glycosyl transferases, that thefunctional organization of the biosynthetic pathways of complexpolysaccharides is consistent with these molecules being processed

' This work was supported by National Institutes of Health grantGM 18639 to L.A.S., by Department of Energy grant DE-FGO9-85ER13426 to A. Darvill, and by Department of Energy grant DE-FGO9-86ER 13497 to M. Chrispeels.

in a cis to trans direction like the N-linked glycans, and that thecomplex polysaccharide xyloglucan is assembled exclusively intrans Golgi cisternae and the trans Golgi network.

The Golgi apparatus of plant cells serves two major syn-thetic functions: it assembles and processes the oligosaccha-ride side chains of glycoproteins and proteoglycans, and itsynthesizes the complex polysaccharides of the cell wallmatrix (3, 31). Whereas the first function is common to plantsand animals, the second is a unique feature of plant cells. Weare interested in determining how these two types of syn-thetic pathways are spatially and functionally organizedwithin the confines of an individual plant Golgi stack.The initial glycosylation of N-linked glycoproteins occurs

in the ER and involves the transfer of a high mannose 14-sugar oligosaccharide from a dolichol carrier to defined as-paragine residues of the nascent polypeptide. Following theremoval of three terminal glucosyl residues, the glycoproteinis transported to the Golgi apparatus, where it is exposed toa battery of glycosidases and glycosyltransferases that processthe oligosaccharide side chain(s) in a specific manner. Manyof the enzymes involved in these reactions as well as theirintermediary products have been identified, and the sequencein which they operate has been determined (21). Althoughthis pathway was initially elucidated in animal cells, subse-quent studies have shown that in plant cells, N-linked gly-coproteins are processed in a nearly identical fashion, withmost of the differences involving the use of different typesof terminal sugars (9).The two major classes of complex polysaccharides synthe-

sized by the Golgi apparatus in dicotyledonous plants are theneutral hemicelluloses and the acidic pectic polysaccharides(3). XG2 is the most abundant hemicellulose of the cell wallof growing dicotyledonous plants. This large polysaccharide(degree of polymerization up to 2200) is composed of a 3-

2 Abbreviations: XG, xyloglucan; PBST, phosphate buffered salineplus Tween-20; PGA/RG-I, polygalacturonic acid/rhamnogalactu-ronan-I; TGN, trans Golgi network.

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FUNCTIONAL COMPARTMENTATION OF PLANT GOLGI APPARATUS

1,4-linked glucosyl backbone that is decorated at regularintervals with xylosyl and xylosyl-galactosyl-fucosyl sidechains to form characteristic hepta- and nonasaccharide re-peats (14). Its main function is to produce cross-links betweencellulose fibrils and thereby contribute to the overall strengthof the wall and permit cells to regulate the rate and degreeof their expansion (14).PGA/RG-I is the most abundant pectic polysaccharide. It

consists of two covalently-linked domains, blocks of PGAand blocks of RG-I. Most of the PGA blocks are methylester-ified prior to secretion, and are acted upon by a pectinmeth-ylesterase after arrival in the cell wall (18). RG-I is composedof a backbone of alternating galactosyl and rhamnosyl resi-dues, with about half of the rhamnosyl residues carryingoligosaccharide side chains (1). Pectic polysaccharides definethe pore size and degree of hydration of plant cell walls andcontribute to cell adhesion (2, 18). Specific fragments of XGand PGA/RG-I have also been shown to serve as hormone-like regulatory molecules (40).

Biochemical fractionation, histochemical, and immunocy-tochemical studies have demonstrated that the Golgi appa-ratus of animal cells is composed of four types of cistemae,cis, medial, and trans, and the TGN, based on their positionwithin a stack and the different sets of enzymes they contain(7, 13). In conjunction with pulse chase experiments, theseinvestigations led to the postulate that newly synthesizedglycoproteins are processed sequentially in cis, medial, andtrans cistemae, and that the final products are sorted andpackaged in the TGN for transport to their final destination(33). For example, there is general agreement that mannosi-dase I is a cis cisterna enzyme, that N-acetyl-glucosaminyl-transferase II is resident in medial cisternae, and that galac-tosyltransferase is localized to trans cistemae of the animalGolgi apparatus. This knowledge of the functional organi-zation of the Golgi apparatus has proven immensely valuablefor designing experiments to explore mechanisms of traffick-ing through the Golgi apparatus (24, 39).The fact that all plant Golgi cistemae possess very similar

densities, together with the lack of proven enzymic markers,has hampered efforts to study subcompartmentation of Golgistacks derived from plant cells (4, 42), even though theyexhibit a pronounced morphological polarity in electron mi-croscopic images (36). Furthermore, because the structure ofmany types of complex polysaccharides is still poorly defined,it is difficult to even contemplate in a more than superficialmanner how the glycoprotein and complex polysaccharidesynthesis pathways might be integrated in plants. Compli-cating matters further, a study of high-pressure frozen/freeze-substituted root tips of Arabidopsis and Nicotiana hasdemonstrated that the architecture of plant Golgi stacks variesin a cell type-specific manner (41). This observation suggeststhat the functional organization of plant Golgi stacks mightbe tailored to meet the specific needs of different types ofcells, an hypothesis that has received support from two recentimmunocytochemical investigations. Thus, polyclonal anti-bodies raised against the pectic polysaccharide, PGA/RG-I,have been reported to bind specially to cis and medial Golgicisternae of root tip cortical cells (31) and to trans cistemaeand the TGN of peripheral root cap cells of clover (25).To circumvent potential problems arising from tissue-spe-

cific differences in plant Golgi stacks, we have focused ourcurrent investigation on the functional organization of theGolgi apparatus of suspension-cultured sycamore maple cells.Using a battery of well-characterized antibodies, we haveimmunolabeled the high-pressure frozen/freeze-substitutedcells to determine in which types of morphologically definedGolgi cistemae specific sugars are added to glycoproteins andcomplex polysaccharides. Our data imply that the assemblyof pectic polysaccharides involves cis, medial, and trans typesof Golgi cistemae, whereas the synthesis of the hemicellulose,XG, is confined to trans Golgi cistemae and the TGN. More-over, the addition of xylose to N-linked glycoproteins in themedial cistemae suggests that the enzymes of the N-linkedglycosylation pathway are similarly organized in the plantand animal Golgi apparatus.

MATERIALS AND METHODS

Plant Materials and Culture Conditions

Suspension-cultured sycamore maple (Acer pseudoplatanusL.) cells were kindly provided by Dr. Michael G. Hahn(Complex Carbohydrate Research Center, University of Geor-gia, Athens, GA) and cultured in modified M6 medium (44)on a shaker (125 rotations/min) at 250C in the dark. The cellswere subcultured every 7 to 10 d.

High-Pressure Freezing

Five- to seven-day-old cultures were harvested by centrif-ugation (lOOOg) and mixed in a ratio of 1:10 (v/v) with 25%(w/v) aqueous dextran (Mr 38,800) or 200 mm sucrose infresh culture medium to optimize the percentage of well-frozen cells. The concentrated cell suspension was thenpoured onto a 30 ,tm nylon mesh in a Petri dish, andimmediately prior to their transfer to the specimen cups forfreezing, the cells were further concentrated by lifting acorner of the nylon mesh out of the fluid. Specimens werefrozen in a Balzers HPM 010 high-pressure freezing appara-tus as described by Craig and Staehelin (6), and were storedin liquid nitrogen prior to freeze-substitution.

Freeze-Substitution, Infiltration, and Embedding

The frozen specimen cups were transferred to precooled1.8-mL cryogenic vials filled with 2.0% (w/v) osmium tet-roxide and 8.0% 2,2-dimethoxypropane in acetone as a sub-stitution medium. Substitution was carried out at -78.60C ina dry ice/acetone bath for 2.5 to 3.0 d. After slow warming(2 h at -200C, 2 h at 40C, and 2 h at room temperature), thesubstitution medium was discarded and the samples rinsedin acetone. To ensure proper infiltration of the cell walls withthe resin, the following infiltration schedule was used: 10%Epon 812:acetone for 14 to 16 h, 25%, 50%, 75% Epon812:acetone each for 24 h, and 100% Epon 812 resin for 48h with a change of resin after 24 h. The Epon-embeddedsamples were polymerized under vacuum at 550C for 22 h.Specimens to be embedded in LR White were first subjectedto an ethanol wash after the acetone rinse and then infiltratedwith mixtures of the same percentages of LR White:ethanoland times as for the Epon-embedded samples. Polymerizationwas carried out at 520C for 24 h.

1071

ZHANG AND STAEHELIN

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Thin sections (about 0.1,um) were cut on a Reichert Ultra-cut E (American Optical, Buffalo, NY) and picked up onfomvar-carbon-coated copper or nickel grids (300 mesh,Polysciences, Warrington, PA). The Epon-embedded sectionswere mounted on copper grids and counterstained with 2%(w/v) uranyl acetate in water for 5 min and lead citrate for50 s, and examined at 80 kV in a Philips electron microscope.Nickel grids were used exclusively for the antibody labelingexperiments.

Antibody Probes

The rabbit polyclonal anti-XG and anti-PGA/RG-I anti-bodies were raised against purified sycamore maple XG andRG-I, respectively (29). Their characterization has been de-scribed in great detail by Lynch and Staehelin (25). Thepolyclonal anti-3Xyl antiserum was kindly provided by Dr.Maarten Chrispeels (University of Califomia San Diego, LaJolla, CA) and has been characterized as described in Lauriereet al. (22). The rat monoclonal JIM7 antibody that recognizesmethylesterified PGA (19) was a generous gift of Dr. PaulKnox (John Institute, Norwich, England). Dr. Michael Hahnkindly supplied us with aliquots of the CCRC-M1, CCRC-M2, and CCRC-M7 mouse monoclonal antibodies. TheCCRC-M1 antibody (35) recognizes the terminal fucosyl res-idue of the trisaccharide side chain of XG in the context ofan extended XG chain, but not in the context of an isolatednonasaccharide fragment. CCRC-M2 is an RG-I-specific an-tibody that does not bind PGA, CCRC-M7 recognizes anarabinosyl-containing epitope on RG-I, which suggests thatit recognizes a side chain of RG-I, but the precise structureof the epitope has not yet been determined (M. Hahn, per-sonal communication).

Antibody Labeling Procedures

The LR White sections were first blocked for 30 min in a5% (w/v) (for polyclonal antibodies) or a 3% (w/v) (formonoclonal antibodies) solution of nonfat dried milk in PBST(10 mm Na-phosphate, 500 mm NaCl, pH 7.2, 0.1% Tween-20; however, the salt concentration in PBST for monoclonalantibodies was 50 mm NaCl). After wicking away the block-ing solution, the grids were incubated on the primary anti-serum diluted either 1:10 (anti-XG, anti-PGA/RG-I, anti-BXyl, CCRC-M1, CCRC-M2, and CCRC-M7) or 1:1 JIM7)in PBST. Incubation was for 2 h at room temperature for thepolyclonal antibodies, and overnight at 40C for the mono-clonal antibodies. The grids were washed in a continuousstream of PBST containing 0.5% Tween-20 for 30 s and thentransferred onto a secondary dilution in PBST containingeither colloidal gold (10, 15, or 20 nm) coupled to protein A(Janssen Pharmaceuticals, B-2430 Olen, Belgium) for the anti-XG, anti-PGA/RG-I, and anti-,BXyl antibodies, and to goatanti-rat antibodies (Biocell Research Laboratories, Cardiff,UK) for the JIM7 antibodies, or to goat anti-mouse immuno-globulin G + immunoglobulin M (Biocell Research Labora-tories) for the CCRC-M1, M2, and M7 antibodies. After 45min on the secondary solution, the grids were first rinsed for

30 s with PBST (0.5% Tween-20) and then with dH20 for 30s. After immunolabeling, the sections were stained with 2%aqueous uranyl acetate for 4 min, and Reynold's lead citratefor 15 to 20 s. For the deesterification experiments, thesections were first incubated with 0.1 M Na2CO3 overnight at40C prior to the blocking step. Unless otherwise noted, allincubations were at room temperature.The double labeling protocol for JIM7 and anti-XG anti-

bodies is a modification of the procedure of Moore (28).Sections were incubated with 3% milk in PBST solution, JIM7antibody, and washed as above. Following this first primaryantiserum, the grids were incubated in the smaller size goldprobe (10 nm) for 45 min, washed with PBS (as above)containing 0.5% Tween-20, and blocked with an excess ofprotein A (0.15 mg/mL) for 20 min followed by 20 minincubation in a 5% milk solution. The grids were then incu-bated with the second primary antiserum for 2 h, washed,and treated with the large gold probe (20 nm) for 45 min.The grids were washed and counterstained as above.

Quantitative AnalysisThe statistical analyses were performed on micrographs of

cross-sectioned Golgi stacks randomly selected from wholesections, and which possessed the clarity with which thecisternae could be discerned. The morphological criteria usedfor identifying cis, medial (early and late), and trans types ofGolgi cisternae, as well as elements of the TGN, are definedin 'Results.' Generally, cis cisternae possess the most irregularcontour and are the most difficult to visualize. Some small-sized Golgi stacks lack identifiable cis cisternae due to theabsence of differences in staining pattem between the cisface region and adjacent regions in the cytoplasm. Medialand trans cisternae were easily distinguished. Identificationof the TGN was based on the intercisternal spacing betweenthe two structures and the presence of rounded, branchedvesicles.To count the number of gold particles over the Golgi stack

and the associated TGN, about 50 micrographs of Golgistacks with clearly defined cisternae and heavy labeling(except in the case of the anti-PGA/RG-I antibody-labeledsamples before Na2CO3 treatment) were chosen for eachanalysis. Gold particles over intercisternal spaces werecounted as 'half' particles for each type of compartment.

RESULTS

Variation in Golgi Stack Size and Morphology

The morphology of the Golgi stacks of suspension-culturedsycamore maple cells is remarkably variable (Figs. 1-5). Thisvariability expresses itself most notably in the number ofcisternae, but also in their diameter, the ratios of cisternaltypes, and the presence/absence and size of the TGN.Whereas the greatest differences are seen between Golgistacks of different cells, it is not uncommon to observe Golgistacks with different numbers of cisternae and with differentdiameters within a single cell.

Figures 1 and 2 depict typical images of Golgi stacks offreeze-substituted and high-pressure frozen suspension-cul-tured sycamore maple cells embedded in Epon 812. Their

1 072 Plant Physiol. Vol. 99, 1992


Figures 1 and 2. Thin section electron micro-graphs of Golgi stacks with (Fig. 1) and without(Fig. 2) a typical TGN in suspension-culturedsycamore maple cells preserved by high-pres-sure freezing/freeze-substitution techniques(embedded in Epon 812). Both of the stacksconsist of five cisternae and display a distinctstructural polarity that can be used to distin-guish one cis, two medial (one early [E] medialand one late [L] medial), and two trans cister-nae. The intercisternal spacing and stainabilityof cisternal lumina increase sequentially fromthe cis to the trans face, whereas the intracis-ternal spacing decreases in the same direction.Note that the late medial cisterna possessesnearly the same intracisternal spacing as theearly medial cisterna, but its lumen is filled withmore densely staining material. Bar, 0.2 Mum.

cisternae display a cis to trans variation in morphology, andwith five cisternae they fall into the category of 'average'Golgi stacks in terms of size. Characteristic of the Golgi stacksof the suspension-culture cells used in this study, the one

shown in Figure 1 displays a distinct TGN close to the trans-most cisterna, whereas the stack illustrated in Figure 2 ex-

hibits no TGN. Indeed, within a random sample of 236 Golgistacks examined, only 137 (52%) displayed any associationwith membrane structures resembling a TGN on the transside of the cistemal stack. To what extent the TGN can

become detached from a Golgi stack and float around as a

separate unit in the cytoplasm, as suggested by Hilmer et al.(16), has not been determined for these cells. As documentedin Figures 3 and 4 and quantitated in Figure 5, the numberof cisternae per Golgi stack and the ratios of cistemal types(see below) can vary dramatically in sycamore maple suspen-sion culture cells. Thus, about 75% of the stacks contain 5 or

6 cisternae, less than 10% 3 or 4 cisternae, and close to 20%as many as 7 to 10 cisternae.

Definition of cis, Medial, and trans Golgi Cisternae

The assignment of cis, medial, and trans labels to subtypesof Golgi cisternae in the micrographs of high-pressure frozen,

freeze-substituted, and Epon-embedded sycamore maplecells is based largely on the morphological criteria defined byStaehelin et al. (41) for the Golgi stacks of similarly processedroot tips of Arabidopsis and Nicotiana, and not on markerenzymes as has been done for animal cells (21). These criteriaare as follows.

cis cisternae occupy the end of the stack opposite the mostdensely staining trans cisternae. Both their membranes andtheir contents pick up little stain, which often makes themdifficult to discern (compare Figs. 1-4), and difficult to distin-guish from ER membranes, which stain equally poorly infreeze-substituted cells processed according to standard pro-

tocols. cis cisternae also possess the widest lumen, but thefenestrations even in their central region often make themappear like rows of distorted vesicles. As illustrated in Figure5, nearly all Golgi stacks contain only one cis-type cisterna.As their name suggests, the medial cisternae occupy the

central region of the stack. They consist of a well-definedcentral domain where the spacing between the two mem-

branes of each cisterna, as well as the spacing betweenadjacent cistemae, is remarkably uniform. The staining of thelumenal contents tends to be mottled and in most instancesthe medial cisterna adjacent to the first trans cisterna is much

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Figures 3 and 4. Two sizes of Golgi stacks observed in sycamoreculture cells. The smaller stack (Fig. 3) consists of four cisternae,one cis, one early medial, one late medial, and one trans cisterna;and the large-sized one (Fig. 4) consists of eight cisternae, one cis,two early medial, one late medial, and four trans cisternae. The ciscisternae possess a relatively wide, unstained lumen and irregularcontours. Early medial cisternae have lumina that are slightly nar-

rower than cis cisternae and partially filled with stainable products.Late medial cisternae exhibit a more consistently dense stainingpattern than early medial cisternae but the same intracisternalspacing. Trans cisternae can be recognized by their tightly ap-pressed membranes that form a single densely stained line. CW,Cell wall. Bar, 0.2 gm.

darker than those closer to the cis cisterna (Figs. 1-4). Becauseof the consistency with which these differences are displayed,we propose that the more cis located, and more lightly stainedmedial cisternae be called 'early medial' cisternae, and themore trans located, and more darkly stained medial cisternae,'late medial' cisternae. Occasionally, the late medial cisternaeexhibit a central domain with a staining pattern of an earlymedial cisterna (Figs. 1-3). The contents of the bulbousmargins of the medial cisternae tend to appear darker thanthe contents of the more appressed central domains. Some

of the swollen margins carry nonclathrin-type fuzzy coats,but in these sycamore cells, the coats are never as clearlyresolved as those seen in the root tips of Nicotiana (41), even

though the same staining procedures were employed in bothstudies. Unfortunately, we have not yet been able to incor-porate the distinction of 'early' and "late' medial cisternae tothe immunocytochemical studies (see below) due to the lossof the characteristic staining patterns in LR White-embeddedsamples.

Typical of trans cisternae is a collapsed lumen, where thecisternal membranes are tightly appressed and the lumen isfilled with uniformly dense products, giving rise to 4 to 6 nmdark lines in cross-sectional views. The spacing between thecisternae is quite uniform and slightly greater than betweenthe medial cisternae (Figs. 2-4). Blebbing vesicles withdensely staining contents are often associated with the cister-nal margins.

In some instances, such as in Figures 1 and 3, it is fairlyeasy to distinguish the TGN from the trans cisternae basedon the spacing between the two structures, the more roundedand often branched configuration of the vesicles budding offthe TGN, and the clathrin coats on some of them. Where no

distinct TGN is seen, the trans-most trans cisterna oftenassumes an intermediate morphology by displaying some

rounded and/or branching vesicular configurations (Figs. 2and 4), suggesting that in these Golgi stacks the trans-mostcisterna(e) may assume some TGN function. However, more

detailed studies are needed to clarify this point.The histograms shown in Figure 5 illustrate not only the

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Figure 5. Histograms illustrating the variability in Golgi structure ofsuspension-cultured sycamore maple cells in terms of total numbersof cisternae per stack and numbers of specific types of cisternae.Stacks of all sizes usually have only one cis cisterna. Larger stackshave increased numbers of medial (especially early) and transcisternae. For medical cisternae, dark shading = early; light shading= late.

l1074 Plant Physiol. Vol. 99, 1992


percentages of Golgi stacks in sycamore maple suspension-culture cells containing specific numbers of cisternae, but alsoa breakdown of the average number of cis, medial, and transcistemae in Golgi stacks of different size. Most Golgi stacks,irrespective of size, possess only one and never more thantwo cis-type cisterna. Thus, the increased number of cisternaeof larger stacks is due to a proliferation of both medial andtrans types of cisternae in roughly equal proportions. Theaverage number of less than one cis cisternae for small Golgistacks is due to the fact that very small cis cisternae aredifficult to discern in our electron micrographs.

Nature of the Epitopes Recognized by theAnti-Glycan Antibodies

The greatly improved structural preservation of the Golgistacks in freeze-substituted samples (Figs. 1-4), and the re-sulting ability to identify subtypes of cisternae based on theirposition, dimensions, and staining patterns offers researchersunique opportunities to determine the functional organiza-tion of Golgi stacks by immunocytochemical means. To thisend, we have employed a battery of both monoclonal andpolyclonal antibodies raised against defined complex poly-saccharides and glycoproteins. The specific epitopes recog-nized by the antibodies are illustrated in Figures 6 to 8. Thepolyclonal anti-XG antibodies have been shown to recognizenearly exclusively the 13-1,4-linked glucose backbone of XG(25), most likely in its twisted solution form (23); they alsobind to solubilized cellulose molecules but not to cellulosefibrils. The CCRC-M1 monoclonal antibody appears to rec-ognize specifically the terminal fucosyl residue of the trimericside chain of XG in the context of an extended XG chain (35).The polyclonal anti-PGA/RG-I antibodies appear to recog-nize the transition region between deesterified PGA and thefirst a-1,2-linked rhamnosyl residue of RG-I (25, 29). TheJIM7 monoclonal antibodies recognize methylesterified PGAdomains (19). The monoclonal antibody CCRC-M7 recog-nizes an arabinosyl-containing epitope on RG-I, consistentwith this epitope being part of a side chain, but the exactnature of the epitope remains to be determined (M. Hahn,personal communication). Similarly incomplete is the infor-mation on CCRC-M2, which binds to RG-I. The major

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1 075

ZHANG AND STAEHELIN

(>95%) epitope recognized by the anti-,3Xyl antibodies is thexylose residue linked p3-1,2 to the p3-linked mannose of theglycan core of N-linked oligosaccharides (22); the remaining<5% of the polyclonal antiserum binds to another but un-determined epitope of complex glycans (M. Chrispeels, per-sonal communication).

Because all of the antibody probes employed in this studywere directed against sugar residues, we have been able touse the same freeze-substitution protocol, including the 2%OS04 as for the morphological studies, without destroyingthe antigenicity of the glycoprotein and complex polysaccha-ride epitopes. This has ensured an overall excellent structuralpreservation of the Golgi stacks despite the use of LR Whiteas an embedding medium for the immunolabeling experi-ments. Thus, as shown in Figures 9, 10, 12, 13, 15, 17, 19,and 20, even though the structural preservation of the LRWhite-embedded specimens does not quite match the qualityof the Epon-embedded samples (compare Figs. 1-4 with Figs.9 and 10), it is still possible to structurally identify thesubtypes of Golgi cisternae in the LR White sections afterimmunolabeling.

Immunolocalization of XG in Golgi Stacks

As already demonstrated by Moore and Staehelin (30) andmentioned above, the polyclonal anti-XG antibodies and themonoclonal antibody CCRC-M1 (Fig. 6) can be used toimmunolocalize the sites for synthesizing and assembling theneutral hemicellulose, XG. Figure 9 illustrates the immuno-

Figures 9 and 10. Anti-XG (Fig. 9) and CCRC-Ml (Fig. 10) labeled Golgi stacks of suspension-cultured sycamore maple cells embedded inLR White. The majority of anti-XG and CCRC-Ml antibody label is seen over trans cisternaeand the TGN (arrows). As confined in Figure11, the CCRC-M1 antibodies bind more heavilyto the TGN than the trans cisternae. Bar,0.2 Am.

labeling pattern of the anti-XG antibodies over a Golgi stackof a suspension-cultured sycamore maple cell. Note that allof the gold particles appear over trans cisternae and the TGN.A similar distribution of label over trans cisternae and theTGN is also observed with the CCRC-M1 (anti-terminalfucose of XG) antibodies (Fig. 10). The results of a quantitativeanalysis of these binding patterns can be seen in the histo-gram Figure 11, which confirms that >90% of the labeling ofboth types of anti-XG antibodies is confined to the transcisternae and the TGN. Interestingly, the anti-XG backboneantibodies label the trans cisternae more heavily than theelements of the TGN (approximately 60% versus approxi-mately 40%), whereas CCRC-M1 antibodies exhibit the op-posite distribution, i.e. a proportionally greater amount oflabeling over the TGN.

Distribution of Deesterified, Methylesterified, and MaturePectic Polysaccharides in Golgi Stacks

Pectic polysaccharides are a very complex constituent ofplant cell walls. A number of antibodies that recognize dif-ferent domains of pectic polysaccharides (Fig. 7) are availablefor immunolabeling experiments. The binding patterns of thepolyclonal anti-PGA/RG-I antibodies before and afterNa2CO3 treatment are shown in Figures 12 and 13. Thebinding of anti-PGA/RG-I antibodies to the Golgi cisternaeand the TGN is altered by treatment with Na2CO3, whichdeesterifies methylesterified PGA. Thus, in untreated sam-ples, most of the meager (only 30% of the stacks carried two

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1076 Plant Physiol. Vol. 99, 1992

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wuJ0-JLum-J

60

50

40

30

20

10

cis medial trans TGN

CISTERNAL POSITION IN GOLGI STACK

Figure 11. Histogram illustrating the immunolabeling patterns ofGolgi cisternae and the TGN of suspension-cultured sycamoremaple cells treated with anti-XG and CCRC-M1 antibodies. Both ofthe antibodies label the same sets of Golgi cisternae but differ inratio of the labeling over trans cisternae and the TGN.

or more gold particles) labeling is seen over cis and medialcisternae of the Golgi stacks, whereas after the Na2CO3treatment, the much heavier labeling (about 100% of Golgistacks labeled) shifts more toward the medial and transcisternae as well as the TGN (Fig. 13). This suggests that thedeesterification treatment uncovers cryptic anti-PGA/RG-Iepitopes in the later compartments (Fig. 13). These qualitativeresults are confirmed in the quantitative analysis shown inthe histogram Figure 14.The immunolabeling patterns with anti-pectic polysaccha-

ride antibodies JIM7, CCRC-M2, and -M7 are depicted inFigures 15 to 17. As seen in Figure 15, the JIM7 antibody,which recognizes methylesterified PGA residues (Fig. 7), bindmost heavily to the medial cisternae and in decreasingamounts to the trans cisternae and the TGN. The validity ofthis pattern is confirmed by the quantitative analysis shownin the histogram Figure 18. Thus, approximately 45% of the



Figure 14. Histogram showing the binding patterns of anti-PGA/RG-I antibodies to Golgi cisternae and the TGN in sycamore mapleculture cells before and after Na2CO3 treatment. In untreatedsections, most of the label appears over cis and medial cisternae,whereas after deesterification of PGA by Na2CO3 treatment, addi-tional labeling is seen over the medial and trans cisternae as well as

the TGN.

JIM7 labeling occurs over medial cisternae, about 40% over

trans cisternae, and about 15% over the TGN. Virtually no

label was present over the cis cistemae. In contrast, theCCRC-M2 and the CCRC-M7 antibodies that both appear torecognize side chains of RG-I (Fig. 7) only detect their epi-topes in trans cisternae and the TGN. The labeling patternsfor both types of antibodies appear very similar on theelectron micrographs (Figs. 16 and 17), and this is confirmedin the histogram Figure 18.To determine whether hemicellulose and pectic polysac-

charides are synthesized in the same Golgi stack, the same

section was subjected to two rounds of labeling with JIM7and anti-XG antibodies. Both types of label were observedover individual Golgi stacks (data not shown), indicating thata given Golgi stack can produce both hemicellulose and pecticpolysaccharides simultaneously.

.0,

I

cis

medtr fan-

44Ri-G4p;- (-N*C 0 )

12

A .. c-.

+lti-PtNRG4'. +Na203).

0 .

;..

O w~~~~A~Ca.Rw

13 ,GN~~~'A

Figures 12 and 13. Golgi stacks of sycamore maple culture cells immunolabeled with anti-PGA/RG-I antibodies without (Fig. 12) and with(Fig. 13) Na2CO3 treatment that deesterifies the PGA domains of pectic polysaccharides. The gold particles appear over cis and medialcisternae (arrow) in the absence of Na2CO3. After treatment, they are found across the whole stack as well as over the TGN (arrows).Bar, 0.2 ,um.

60

0Lu

m0-J

-J

50

40

30

20

10

1 077

--

I T,1*.0;

1. .,ilK.t,:.-.-J..4" .' -

,-c * .,

ZHANG AND STAEHELIN

Characterization of Antibody Labeling with Anti-,8Xyl inGolgi Stacks

In plants, most complex N-linked oligosaccharides containa terminal 3-1,2-xylosyl residue (Fig. 8; see ref. 9). Thisxylosyl residue, which is not found in animal glycoproteins,appears to be a major antigenic site of N-linked glycoproteinsof plants (M. Chrispeels, personal communication), and isthe dominant epitope recognized by the anti-i3Xyl antibodies.Figure 19 demonstrates binding of these antibodies through-out the stack, but with a slightly greater amount of labelingover medial cisternae. This impression is validated by thequantitative analysis of the anti-#Xyl antibody binding pat-terns shown in Figure 20, which demonstrate little labelingover cis cisterna, the highest percentage of labeling (approx-imately 45%) over medial cisternae, and decreasing percent-ages of gold label over the trans cisternae and the TGN.

J1M7.A

mea,

jtrans

DISCUSSION

Preservation of Ultrastructure and Antigenic Sites of GolgiStacks in High-Pressure Frozen Cells

Like all ultrarapid freezing techniques, high-pressure freez-ing is superior to chemical fixation in its ability to preservethe ultrastructure of plant cells and tissues (6, 41). Thisimproved structural preservation is due largely to the factthat ultrarapid freezing techniques can stabilize cells' ordersof magnitude faster than chemical fixatives, and that duringultrarapid freezing all molecules become immobilized simul-taneously, in contrast with the selective immobilization ofmolecules with chemical fixatives (11). These features are ofparticular importance for the preservation of highly dynamicorganelles such as the Golgi apparatus, where membranefission and fusion events occur on a time scale of milliseconds,and where co- and postfixational osmotic events can producemajor structural rearrangements (34). The advantage of high-pressure freezing for preserving the macromolecular architec-ture of plant Golgi stacks in intact tissues was recentlydemonstrated by Staehelin et al. (41). Here we illustrate howthe much improved structural preservation obtained usinghigh-pressure freeze/freeze-substitution techniques can beexploited in conjunction with immunocytochemical tech-niques to obtain remarkably detailed information on thecisternal distribution of putative functional activities in Golgistacks. We show that both polyclonal and monoclonal anti-bodies that recognize carbohydrate epitopes of either complexpolysaccharides or glycoproteins all bind with high specificityto the Golgi stacks of cryofixed and freeze-substituted cells.

CCRC-M2

7i meo,

". I..tan

IIQ. 'TGN

'nCRC1-M7

liz CIS.::i mned L.

* 'an5. .

?

Variability in Size and Morphology of Golgi Stacks

We chose the suspension-cultured sycamore maple cellsfor this study because they are the source of complex poly-saccharide antigens used for making several of the antibodyprobes employed in this study. In addition, the cell lineappealed to us because of the uniform appearance of thecells, which have been in culture for over 30 years and havelost their ability to differentiate. These latter features seemedimportant considering the findings of Staehelin et al. (41)that in high-pressure frozen and freeze-substituted root tips

Figures 15-17. Immunolabeling of Golgi stacks and the TGN ofsycamore maple cells with monoclonal JIM7 (Fig. 15), CCRC-M2(Fig. 16), and CCRC-M7 (Fig. 17) antibodies. With JIM7 (Fig. 15),labeling is observed mostly over medial and trans Golgi cisternaeand lightly over the TGN (arrows). With the CCRC-M2 and -M7antibodies (Figs. 16 and 17), the heavier labeling is seen over thetrans cisternae and the TGN (arrows). Bar, 0.2 Mm.


w",.

'AW j. 6-4.-

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GN


JIM-7 (methylestentied polygalacturonic acid residues)

2 CCRC-M2 (side chains of rhamnogalacturonan-1 of dicots but not monocots)60 - a CCRC-M7 (side chains of rharnnogalacturonan-1 common to monocots & dicots)

iu 50

en40m0w,, 30m< 20

10



Figure 18. Histogram showing the labeling patterns of the Golgicisternae and the TGN in suspension-cultured sycamore maplecells with monoclonal JIM7, CCRC-M2, and CCRC-M7 antibodies.JIM7 antibodies heavily label medial and trans cisternae and slightlylabel the TGN, whereas CCRC-M2 and CCRC-M7 antibodies donot label the medial cisternal compartment but heavily label thetrans cisternae and the TGN.

of Arabidopsis and Nicotiana, the morphology of Golgi stacksdiffered substantially in a cell type-specific manner. Theobserved variability of size and morphology of the Golgistacks of our cultured sycamore maple cells was somewhatunexpected, but not totally unpredictable. Hirose and Ko-mamine (17) have demonstrated in conventionally fixed sam-ples that the number and diameter of Golgi cisternae ofcultured Catharanthus roseus cells changes systematically in acell cycle-dependent manner. The morphological variabilityof the Golgi stacks in our unsynchronized sycamore mapleculture cells falls within the parameters defined for the Ca-tharanthus cells. To what extent the absence of a clear-cutTGN in a high proportion of the Golgi stacks of sycamore

50 -

m 40-cn0XJ 30-ULU

m

LL 20-

0

I-

z

o 10-LU

0-

:Hcis medial trans TGN


Figure 20. Histogram showing the labeling pattern over the Golgicisternae and the TGN of sycamore maple culture cells with anti-,BXyl antibodies. The antibodies bind most heavily to medial cister-nae and less to trans cisternae and the TGN.

maple cells is typical of cultured cells is unclear at the presenttime because this aspect of the TGN has not been addressedin comparable studies of cultured cells (16, 17). However,even though the morphological variability of the sycamore

maple Golgi stacks was greater than anticipated, our abilityto distinguish subtypes of cisternae by morphological criteriaother than their position within a stack has enabled us to use

these cells to define functional differences between thesecompartments by means of immunocytochemical techniques.

Assembly of XG Is Confined to trans Cisternaeand the TGN

anti-'fXyI, ;:

cis

med

trans

..s \ /TGN

19

Figure 19. Immunolabeling of Golgi stacks of sycamore mapleculture cells with anti-,3Xyl antibodies. Gold particles are foundmostly over medial cisternae and lightly over trans cisternae andthe TGN (arrows). cis cisternae are rarely labeled. Bar, 0.2 Am.

XG is the major type of hemicellulose of dicot cell walls. Itis composed of p3-1,4-linked D-glucosyl residues of which 60to 75% of residues are substituted at C6 with side chains ofeither a single xylosyl residue or with a xylose, galactosyl,and fucosyl trisaccharide (14). Treatment of XG from soy-bean, pea, and sycamore cell walls with endoglucanase yieldsprimarily the heptasaccharide and nonasaccharide repeatingsubunits shown in Figure 6 in equal amounts, and theserepeats appear to form alternating sequences (14). Anotherlarger-scale repeat of XG has been recently reported byMcCann and colleagues (26), who have shown that XGmolecules isolated from onion cell walls appear to be assem-bled from about 30-nm building blocks.The mechanism of XG biosynthesis is still poorly under-

stood. The in vitro incorporation of glucose and xylose intothe XG backbone occurs efficiently only if both UDP-glucoseand UDP-xylose are present together in the reaction mixture,suggesting that the two sugars are added in a cooperativemanner (15). Whether the glucan synthase I activity (43),which is often used as a marker enzyme for Golgi fractions,corresponds to the XG glucosyl transferase is still a matter ofdebate. Neither galactose nor fucose seem to be required for

v z z .-l lz .1 z I r , z - w z .,.A

1079

ZHANG AND STAEHELIN

the elongation of the XG backbone (12), and although gal-actosylation precedes fucosylation, there is no cooperativelinkage between the two enzymic reactions (5, 8).Much current interest is focused on how these functional

activities are organized in the Golgi stacks. The most recentmodel of Brummell et al. (4) suggests that the glucose back-bone is initiated in the cis cisternae, continued in medialcisternae, and completed in the trans cisternae, where fuco-sylation is also initiated. Completion of the fucosylationreactions is postulated to occur in the secretory vesicles duringtransport to the cell wall.

In this study, we confirm some elements of the Brummellet al. (4) model, and we contradict others. The most notabledifference pertains to the observation that our anti-XG back-bone antibodies only label trans cisternae and the TGN, butnot cis and medial cisternae (Fig. 11). This result suggeststhat the assembly of the ,B-1,4-linked glucosyl backbone ofXG occurs exclusively in the trans cisternae, and that noprecursor forms of XG are made in cis and medial cisternae(Fig. 21). Figure 11 also demonstrates that the terminal fu-cosyl residues on the trisaccharide side chains of XG arepartly added in the trans cisternae and partly in the TGN(Fig. 21). These data are consistent with the biochemicalobservation of Brummell et al. (4) that the XG fucosyl trans-ferase activity of pea microsomal membranes is partitionedbetween the dictyosome (Golgi stack) fraction and the secre-tory vesicle fraction. Obviously, the 'dictyosome fraction'would include the trans Golgi cisternae, where the first fu-cosylation reactions occur, and the 'secretory vesicle fraction'most likely encompasses vesicles derived from the TGN,where the final fucosyl transferase reactions appear to takeplace. Because our immunocytochemical data are based onthe localization of Golgi products carrying specific epitopesand not on the localization of specific enzymes, our resultsdo not provide conclusive evidence for the confinement ofthe XG synthesis pathway to trans cisternae and the TGN.However, because of the strength and the reproducibility ofthe product epitope signal in our samples, we feel confidentthat the specific enzymes will be shown to reside in the samecompartments where their product epitopes first appear inthe Golgi stack. Whether the synthesis of other hemicellulosessuch as xylan (10) occurs exclusively in trans cisternae andthe TGN remains to be determined. Another open questionis where the 30-nm XG building blocks (26) are assembledinto the larger molecules found in the cell walls.The demonstration that the biosynthetic pathway of XG is

confined to the trans Golgi cisternae and the TGN (Fig. 21)is rather unique in that it represents the first example of asecretory product of the Golgi apparatus of either plant oranimal cells whose synthesis is initiated in trans cisternae andapparently does not involve the processing of precursorsproduced in earlier compartments. Why the assembly of XGdoes not involve all Golgi cisternae, whereas the synthesis ofthe pectic polysaccharide, PGA/RG-I, does (see below), re-mains to be elucidated.

Spatial Organization of the Assembly Pathways of PecticPolysaccharides

Pectic polysaccharides are a major component of the matrixof the primary cell wall of dicotyledonous plants. They have

cis

medial 0

elIJ

I

svQ( * glucose

A xylose

* galactose

* fucose

PM;

Figure 21. Model of the assembly pathway of XG in the Golgiapparatus of sycamore maple suspension-cultured cells. Note thatsynthesis of the glucose/xylose backbone is postulated to be limitedto trans cisternae, whereas the fucose-containing side chains areadded both in trans cisternae and the TGN.

been postulated to play an important role in cell wall hydra-tion, filtering, adhesion between cells, and wall plasticityduring growth (2, 18). In addition, pectic fragments can serveas elicitors for plant defense responses (40). Paralleling thisdiversity of function is a variability in structure that makespectins the most complex class of cell wall polysaccharides(27). This complexity is further enhanced by differencesbetween cell types within a plant tissue and changes associ-ated with cell wall development (32).

Pectic polysaccharides are composed of distinct structuraldomains that are covalently linked together in characteristicpatterns. The backbone consists of two types of domains,blocks of 15 to 70 a-1,4-linked D-galacturonosyl residues (thePGA or homogalacturonan domains) that are interspersedperiodically with an a-1,2-linked L-rhamnosyl residue (20),and domains containing up to 300 repeats of alternating a-1,4 galacturonosyl-a-1,2 rhamnosyl residues (the RG-I andRG-II domains). About half of the latter rhamnosyl residuesare substituted mostly at C4, with side chains containing upto 15 glycosyl residues (27). Few of the 30 plus arabinosyl-



and galactosyl-rich side chains have been characterized todate (1). The mol wt of isolated pectic polysaccharides rangesfrom 30,000 to 300,000.The physical properties of the pectic polysaccharides are

dominated by the charged PGA blocks that can bind calciumand form Ca2+-PGA junctions, which induce gelling andadds to the overall strength of the cell wall (18). Methyles-terification and acetylation of the carboxyl groups at C6prevents the formation of these calcium bridges, and it isgenerally believed that PGA is synthesized and secreted intothe wall in a highly methylesterified form, thereby aiding itsdiffusion throughout the tightly knit mesh of cell wall mol-ecules. Upon arrival in the cell wall, individual blocks ofPGA are deesterified by a pectinmethylesterase, which ap-parently cannot cross the a-1,2 rhamnose bridges betweenthe PGA blocks (18).Due to the above-mentioned complexity of pectic polysac-

charides and their tissue-specific expression, very little isknown about their biosynthesis (32). Moore et al. (31) haverecently reported in an immunocytochemical study thatPGA/RG-I epitopes are localized to the cis and medial cister-nae of Golgi stacks as well as to a subset of secretory vesiclesin cortical parenchyma root tip cells of clover, but due to alack of knowledge of the exact nature of the epitopes, thespecific type of product being localized in these compart-ments could not be determined. With the further character-ization of these antibodies by Lynch and Staehelin (25), weknow now that they bind mostly to the transition region ofthe deesterified PGA/RG-I domains, and probably to thetransition region of the PGA blocks and the interspersed a-1,2-linked rhamnosyl residues. These latter authors alsoshowed that in mucilage-secreting root cap cells of clover,the PGA/RG-I epitopes are shifted to the trans cistemae andthe TGN, consistent with the idea that plant Golgi stacks canbe functionally reorganized in a cell type-specific manner(41).

In suspension-cultured sycamore maple cells, the anti-PGA/RG-I antibodies label nearly exclusively cis and medialcisternae (Fig. 12) as in the root-tip cortical cells of clover(31), but essentially no secretory vesicles. However, the den-sity of labeling was always very low and only about 30% ofthe Golgi stacks carried two or more gold particles. Thiscontrasts with the labeling pattern of the JIM7 antibodies(Fig. 15) and with the anti-PGA/RG-I labeling of the Na2CO3-deesterified samples (Fig. 13), where close to 100% of thestacks bound antibodies and the general density of goldparticles was higher. These results suggest that the PGA/RG-I backbone is assembled in cis and medial cistemae and thatthe methylesterification of the carboxyl groups of the galac-turonic acid residues occurs nearly simultaneously and veryefficiently in the same Golgi compartments (Figs. 14 and 18).The deesterification experiment demonstrates, furthermore,that the lack of binding of the PGA/RG-I antibodies to transcistemae and the TGN in the control cells is due to theesterification, and that PGA/RG-I epitope-containing mole-cules traverse the whole Golgi stack.The precise epitopes recognized by the CCRC-M2 and

-M7 antibodies have not yet been determined, but the char-acterization to date suggests that they bind to side chains ofthe RG-I domain (Fig. 7; M. Hahn, unpublished results). Ourimmunolabeling results with these antibodies (Fig. 18) pro-

vide strong support for the hypothesis that these chains areadded to the nascent PGA/RG-I molecules in the trans Golgicistemae.Taken together, our immunolabeling experiments of syca-

more maple suspension-cultured cells demonstrate (Fig. 22)(a) that the backbone of PGA/RG-I is assembled in a dees-terified form in cis and medial Golgi cistemae, (b) thatmethylesterification of carboxyl groups occurs nearly simul-taneously in the same compartments, (c) that the side chainsof RG-I are added in the trans cistemae, and (d) that thefinished products pass through the TGN on their way to thecell wall.

Sites of Processing of N-Linked Oligosaccharides

Of all the synthetic activities of the Golgi apparatus ofanimal cells, the processing pathway for N-linked glycopro-

cis

*,

medial 0

o trans

R sugars

IsvQ

* galacturonic acid

t wthylastarifledt galacturonic acid

* rhamnose

R oligosaccharldosidechains

PMP

R R

Figure 22. Model of the biosynthesis pathway of PGA/RG-I in theGolgi apparatus of sycamore maple suspension-cultured cells. Themodel postulates that the backbone is initiated in cis cisternae andextended in medial cisternae, that methylesterification of the gal-acturonic acid residues of the PGA domains occurs in medialcisternae, and that addition of the side chains to the RG-1 domainsoccurs in trans cisternae.

l08l

ZHANG AND STAEHELIN

teins is probably the best understood (reviewed in refs. 21and 33). These studies have led to the division of the Golgistacks into three functionally distinct compartments, the cis,medial, and trans types of cistemae, based on the specificoligosaccharide processing enzymes they contain. Thus,marker enzymes for cis cistemae include Golgi a-mannosi-dase I and N-acetylglucosaminyl-phosphotransferase, formedial cisternae, N-acetylglucosaminyl-transferase I and II,

and for trans cistemae, galactosyltransferase and sialyltrans-ferase (21). The localization of many of these enzymes hasbeen determined both by biochemical and immunocytochem-ical techniques, and in most instances the two approacheshave led to fairly consistent findings (37). However, as shownby Roth et al. (38), there are some tissues such as the absorp-tive cells of the rat colonic epithelium where the distinctsubcompartmentation of certain glycosyltransferases (e.g. thea-2,6 sialyltransferase) does not conform to the generallyaccepted model.

In general terms, the processing of N-linked oligosaccha-rides in plants follows the pathways established for animalcells with several important modifications (9). For example,they do not employ the mannose-6-phosphate system fortargeting proteins to the vacuole, xylose is often linked to the,B-linked core mannose, and sialic acid is not used as a

terminal sugar. Because all attempts to subfractionate plantGolgi stacks into cis, medial, and/or trans cisternal fractionshave failed to date (42), it has not been possible to define inbiochemical terms the enzymic composition of these subcom-partments. By using antibodies that recognize specifically thexylose residue attached to the 3-linked mannose of the N-linked oligosaccharide core (Fig. 8; see ref. 22) in conjunctionwith immunocytochemical techniques, we have been able tocircumvent the fractionation problems and localize the siteof addition of this xylose to the medial cistemae (Fig. 20).Because the xylosyl transferase that carries out this reactionoperates just after the N-acetylglucosamine transferase II (9),which in animal is one of the later-acting medial cistemaenzymes (21), our localization would be consistent with thislatter enzyme also being present in medial cisternae of plantGolgi stacks. This would confine the possible sites of additionof the terminal fucosyl and galactosyl residues to more trans-located cistemae of Golgi stacks. Thus, it seems plausible tosuggest that the terminal fucosyl and galactosyl residues ofN-linked oligosaccharides might also be added in trans Golgicisternae of plants. Further studies with appropriate antibod-ies both against specific glycosyl residues and specific proc-

essing enzymes of N-linked oligosaccharides will, hopefully,enable us to verify and expand on these hypotheses.

ACKNOWLEDGMENTS

Thanks are due to Drs. Thomas Giddings and Samuel Levy as

well as to Margaret Lynch for their comments on the manuscript and

for their help with this project.

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functional compartmentation of the golgi apparatus of plant cells

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