Protoplasma (1996) 191: 70-78 PROTOP $MA @ Springer-Verlag 1996 Printed in Austria

A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata

Bernard L. Epel 1' *, Jan W. M. van Lent 2, Lilla Cohen 1, Guy Kotlizky 1, Aviva Katz 1, and Avital Yahalom 1

Botany Department, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv and 2 Department of Virology, Wageningen

Agricultural University, Wageningen

Received June 14, 1995 Accepted October 9, 1995

Summary. Plasmodesmata, dynamic pore structures that traverse plant cell walls, function in cytoplasmic transport between contigu- ous cells. Cell walls containing embedded plasmodesmata were iso- lated from mesocotyls of etiolated maize seedlings. Proteins asso- ciated with the isolated walls were separated by SDS-PAGE and antibodies were generated against a 41 kDa protein, one of several associated with this wall fraction. Immunoblot analysis showed that the 41 kDa polypeptide was also associated with other subcellular fractions obtained following tissue homogenization and differential centrifugation. The wall associated 41 kDa protein is apparently a peripheral membrane protein since it could be extracted by high salt and high pH. Silver-enhanced immunogold light microscopy showed that the 41 kDa protein was associated with the cell walls of cells both in the stele and cortex. The immunolabeling pattern was trans- wall and punctate. Electron microscopic immuno-gold labeling localized the polypeptide to plasmodesmata and to electron dense cytoplasmic structures that are apparently Golgi membranes. The significance of the presence of this protein in the Golgi is discussed.

Keywords: Cell wall; Golgi apparatus; Immunolocalization; Meso- cotyl; Plasmodesmata; Plasmodesmatal associated proteins; Zea

mays.

Abbreviations. ER endoplasmic reticulum.

Introduction

The regulation of symplastic cell-to-cell transport via plasmodesmata has been suggested to play an impor- tant role in the control of cellular activities and in the regulation of plant development. Plasmodesmata are aqueous gatable pores that traverse cell walls and generally allowing for the direct cytoplasmic move- ment of molecules of less than a 1000 kDa (Baron- Epel et al. 1988; Erwee and Goodwin 1983; Goodwin

* Correspondence and reprints: Botany Department, Tel Aviv Uni- versity, Tel Aviv 69978, Israel.

1983; Meiners et al. 1988; Robards and Lucas 1990; Terry and Robards 1987; Tucker 1982, 1988). During stress or as the result of certain environmental signals, the size exclusion limit may change and plasmodes- mata may either close (Epel and Erlanger 1991, Epel 1994, Oparka and Prior 1992) or dilate, allowing for the passage of larger molecules (Tucker 1993, Cle- land et al. 1994). In addition to their normal physio- logical function, plasmodesmata can be altered and exploited by viruses as conduits for viral spread from cell to cell (Atabekov and Taliansky 1990, Deom et al. 1987, Epel 1994, Hull 1989, Maule 1991). Current models of plasmodesmatal substructure (Botha et al. 1993, Ding et al. 1992) describe the plas- modesma as a complex plasma membrane lined pore containing a central membranous axial component termed the desmotubule which is considered to be derived from and continuous with cortical ER. Parti- cles, presumably proteinaceous, were depicted to be embedded in the inner leaflet of the plasma mem- brane (Ding et al. 1992), within the cytoplasmic sleeve (Botha et al. 1993), and both in the outer and inner leaflets of the desmotubule (Ding et al. 1992, Botha et al. 1993). The central region of the desmotu- bule, the so-called rod, was depicted as a series of particles, probably protein embedded in the lipid of the fused inner leaflet of the ER. The identity of these protein particles is unknown. Information with regard to protein composition of plasmodesmata has only recently been obtained (Hunte et al. 1992; Schulz et al. 1992; Meiners and Schindler 1987, 1989; Meiners et al. 1991; Monzer

B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls 71

and Kloth 1991; Turner et al. 1994; Xu et al. 1990; Yahalom et al. 1991; Kotlizky et al. 1992; White et al. 1994). Immunological data suggested that dicotyle- donous plants contained a peptide homologous to the mammalian gap junction protein, connexin32 (Mei- ners and Schindler 1987, 1989; Meiners et al. 1991; Xu et al. 1990; but see Mushegian and Koonin 1993). Using indirect immunogold labeling of ultrathin sec- tions, we were able to show that maize mesocotyl plasmodesmata contain two different proteins with apparent molecular weights of 26 and 27 kDa that cross-react with connexin gap junction antibodies (Yahalom et al. 1991). These two plasmodesmatal associated proteins were termed, respectively, PAP26 and PAP27. White et aI. (1994) showed the presence of actin in plasrnodesmata. A major obstacle in the biochemical characterization of plasmodesmata has been the difficulty in the isola- tion of these structures. Recently, progress toward this goal was achieved with the development of a pro- cedure for the isolation of maize cell walls that con- tain embedded plasmodesmata. Isolated walls were found to be virtually devoid of other membranes (Kotlizky et al. 1992). A number of proteins were found to be associated with this wall fraction. Here we present evidence that one of these wall associated proteins, a 41 kDa polypeptide, is a plasmodesmatal associated protein.

Material and methods Plant material

Maize seeds (Zea mays L. cv. Jublee; Roger Bros. C., Idaho Falls, ID, U.S.A.) were imbibed in running tap water overnight, planted in moist vermiculite and grown in the dark for 5 days at 25 ~

Preparation of cell walls

Clean maize mesocotyl cell walls were prepared by a modifidation of a procedure previously described by Kotlizky et al. (1992). In brief, mesocotyl segments were frozen in liquid nitrogen, pulverized to a fine powder and homogenized in media HM/8.5 (2 ml/g tissue) con- sisting of 20 mM Tris-HCl pH 8.5, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, I mM phenyl methyl sulphonyl fluoride (PMSF). Cell walls were separated by filtration onto nylon cloth (200 mesh) and rehomogenized with HM/7.5 which was identical to HM/8.5 except the pH was 7.5. The homogenate was loaded into a nitrogen disrup- tion bomb (Parr Cell Disruption Bomb, Model 4635; Parr Instrument Co., Mokine, IL, U.S.A.) incubated for 30 rain at l0 MPa, and then slowly passed through the release valve. The walls were recovered by filtration as above. Homogenization in HM/7.5, disruption and fil- tration were repeated 3 more times.

Electrophoresis and immunoblot analysis

Proteins were extracted from indicated fraction by boiling in sample buffer (SB) containing 2.2% (w/v) sodium dodecyl sulfate (SDS)

11% glycerol (v/v), 50 mM Tris-HCt pH 6.8 and 5.5% (v/v) 2 mer- captoethanol for 5 rain and the extract cleared of insoluble material by centrifugation at 5000 g. Extracted proteins were separated by SDS-PAGE (15% polyacrylamide) according to Laemmli (1970). The gels were either silver stained (Blum et al. 1987) or the proteins were electrotransferred to nitrocellulose by a modification of the method of Szewczyk and Kosloff (1985). The nitrocellulose blots were blocked, immunoprobed with specified polyclonal rabbit anti- serum, and visualized with alkaline phosphatase conjugated goat anti-rabbit. Protein content analysis was determined by a modifica- tion of the method of Marder et al. (1986). Electrophoresis, electro- transfer, protein determination, and immunoblot protocols were essentially as previously described (Yahalom et al. 1991).

Antibodies

The antiserum against the 41 kDa polypeptide (S-41) was prepared according to Knudsen (1985). Briefly, nitrocellulose strips (2 mm • 15 cm) containing 20-40 pg of the immobilized 41 kDa protein were dried thoroughly, cut into small fragments, and dis- solved in 250-500 ~.d dimethylsulfoxide (DMSO). The dissolved nitrocellulose fragments were mixed with 500 pl Freund's adjuvant (Difco Laboratories, Detroit, MI, U.S.A.). The mixture was injected subcutaneously into 10-20 sites on the back of i month old female New Zealand rabbits. The first injection mixture was prepared with Freund's complete adjuvant while the 2 subsequent booster injec- tions, spaced 10 to 28 days apart, were prepared with Freund's incomplete adjuvant. The rabbits were bled from the ear 10 days after each boost and the serum was tested for antibody response. The antiserum obtained after the second boost was employed at 1 : 20,000 dilution in Tris buffered saline (TBS) containing 5% milk powder and 0.02% azide. The antibody CL-100 which cross-reacts with PAP26 (Yahalom et al. 1991) is a site-directed antibody raised against a predicted cytoplasmic loop peptide (residue 100-122) of connexin43. The antibody, prepared by Dr. Dale Laird, was generat- ed against a synthetic peptide synthesized according to the cDNA clone made by Beyer et al. (1987) and affinity purified as previously described (Laird and Revel 1990). The CL-100 was used in blot overlays at a concentration of 1-2 ~g/ml in TBS containing 5% milk powder and 0.02% azide.

Affinity purification

Antibodies against the 41 kDa protein were affinity purified by a modification of the procedure of Smith and Fisher (1984). In brief, wall proteins were extracted and separated by preparative SDS- PAGE and electrotransferred to nitrocellulose as descibed above. The blot was treated with periodate as described below and the pro- teins on the nitrocellulose were then stained with Ponceau S and the 41 kDa band excised. The strips containing the 41 kDa protein were washed with 10 mM NaOH to remove the Ponceau, washed with water, blocked with 5% nonfat dry milk in TBS for 1 h and then incu- bated for 24 h in a 1 : 2500 dilution of S-41. The strips were washed for 10 min with TNT buffer (10 mM Tris-HC1, t50 mM NaC1 and 0.05% Tween 20), twice again for 10 min each with TNT containing 1% Triton X-100, and finally for 10 rain with TNT. The strips were diced and vortexed for 30 s in 500 pl 0.2 M glycine containing i mM EGTA, pH 2.8 in order to elute the bound antibody. The decanted eluent was immediately neutralized with 62 pl 1 M Tris pH 8.0. The elution was repeated 3 times. The nitrocellulose strips were washed with TNT, re-blocked and used again for further affinity purification. The pooled eluents were concentrated with Centricon-100 microcon-

72

centrator (Amicon, Inc., Beverly, MA, U.S.A.) to a final concentra- tion of about 0.2 to 10 mg protein/ml. The concentrated affinity puri- fied antibody, termed AF-41, was stored at 4 ~ with 0.02% azide. For Western blots the concentration AF-41 employed was 4 ~g/ml, and for LM immuno-labeling about 100 pg/ml.

Concanavalin A detection of glycoproteins on nitrocellulose blots

The detection of Con A binding glycoproteins on nitrocellulose blots was performed by employing biotin-conjugated Con A (Glass et al. 1981, Clegg 1982, Rohringer and Holden 1984). Blots were blocked overnight with 2% Tween-20 in TBS overnight and then incubated for 1 h with shaking with 10 gg/ml biotin labeled Con A (Sigma, St. Louis, MO, U.S.A.; cat. no. C-2272) in TBS containing 50 ~M Mg ++, 50 ~aM Mn ++, and 50 ~aM Ca ++ and were indicated with 0.2 M methyl a-D-mannopyranoside or 0.2 M mannose. The blots after washing 6 times 10 rain each with TBS were incubated for 1 h with monoclonal antibiotin conjugated with alkaline phosphatase (Sigma cat. no. A-6561) diluted in TBS (1 : 400). The blots were washed 3 times 10 min and the alkaline phosphatase was visualized as previ- ously described (Yahalom et al. 1991).

Sodium periodate blot treatment

The oxidation of sugar moieties associated with glycoproteins was peffol~ed in situ on blots with sodium periodate by a modification of the Smith method (Baker et al. 1972). Blots were soaked in a solu- tion of 50 mM sodium acetate pH 4.5 with 20 mM sodium periodate for a period of 15 to 30 rain at room temperature. The blots were then transferred to a solution containing 10 mM sodium borohydride in TBS for 15 min at room temperature and then quickly rinsed in TBS. Controls were subjected to the same processing with the omission of the 20 mM sodium periodate in the first step.

Light microscopy

Fresh segments of roots excised just above the root cap or of meso- cotyl excised directly below the coleoptile node were fixed in FAA (formalin, acetic acid, and alcohol). Routinely, the tissue was dehy- drated in a graded ethanol series, infiltrated and embedded in Para- plast (Lancer, St. Louis, MO, U.S.A.). Sections, 8 pm thick, were mounted onto slides pretreated with Biobond (BioCell, Cardiff, U.K.). The specimens were dewaxed, rehydrated to water and blocked with milk as in immunoblotting. After sections were incu- bated for 36 h with S-41 antiserum diluted in blocking solution (1 : 1000) and washed 5 times in TBS containing 0.1% Tween 20

B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls

(TTBS) for 20 min each, the bound antibody was visualized using alkaline phosphatase conjugated goat anti-rabbit antibody as described in immunoblotting. In labeling experiments in which sec- tions were incubated in affinity purified anti-41 antibody, the sec- tions were washed as above after incubation in the primary antibody and the primary antibody was visualized with a goat anti-rabbit anti- bodY conjugated with 5 nm gold colloid (1 : 100, 2 h) followed by silver enhancement according to the instructions provided with the kit (BioCell). Sections were rinsed with deionized water and mounted in glycerol. Preparations challenged with preimmune anti- serum or secondary antibody alone were used as controls.

Electron microscopy

In situ detection of the 41 kDa protein was performed on ultrathin sections of maize mesocotyl and root tissue. For this, 0.5 mm thick slices of the tissue from young maize plants were fixed for 3 h in 3% (w/v) paraformaldehyde/0.02% glutaraldehyde in a phosphate/citrate buffer (0.1 M Na2HPO4 �9 2H20, 9.7 mM citric acid pH 7.2), contain- ing CaC12, dehydrated in series of ethanol and infiltrated with LR Gold resin (London Resin Company) containing 0.5% (w/v) benzil at -20 ~ The resin was polymerized with ultraviolet light (360 nm) for 24 h at -25 ~ and 48 h at room temperature. Fixation and immu- nogold localization protocols were essentially as described by Van Lent et al. (1990). Antiserum against the 41 kDa protein and the pre- immune serum (as a control) were used in a dilution of 1 : 500 and 1 : 1000 in PBS-BSA.

Results

Isolation and characterization of wall associated 41 kDa protein

A silver stain of proteins extracted from a clean cell wall fraction preparation from etiolated maize meso- cotyl tissue and separated by SDS-PAGE is shown in Fig. 1 A. About 12 to 20 proteins were apparently associated with such a clean cell wall fraction. One of the major proteins associated with this fraction was a protein with an apparent molecular weight of 41 kDa (Fig. 1 A). Antibodies against this polypeptide were generated and employed to examine the distribution of this polypeptide among different cell fractions. The

Fig. 1 A-C. SDS-PAGE and Western blot analysis of poly- peptides associated with isolated maize mesocotyl walls. A SDS-PAGE analysis of proteins extracted from etiolated maize mesocotyl cell walls. Proteins were visualized by sil- ver staining. 41 kDa polypeptide marked with arrow. B Western blot analysis of 41 kDa protein in different cell fractions. Blots were probed with S-41 antiserum. 1 Wall fraction; 2 heavy membrane fraction; 3 light membrane frac- tion; 4 soluble fraction. C Western blot analysis of wall asso- ciated proteins probed with affinity purified antibodies against 41 kDa protein

B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls

Fig. 2. Western blot analysis of polypeptides associated with the wall fraction of roots (1), mesocotyl (2), and of leaves (3) probes with A CL-100, B S-4I

antiserum S-41 detected the presence o f the 41 kDa

polypept ide not only in the wall fraction but also in the heavy membrane, light membrane and soluble

fractions (Fig. 1 B). In should be noted that the S-41 ant ibody in addit ion

to labeling the 41 kDa polypept ide extracted f rom

mesocoty l cell walls also cross-reacted with the plas-

modesmata l protein PAP26 (Fig. 1 B, lane 1). Even fol lowing affinity purif ication o f the antiserum on the

73

41 kDa protein, the affinity purified ant ibody still

cross-reacted with PAP26 (Fig. 1 C).

Western blot analysis o f root, mesocoty l and leaf tis-

sues with C L - t 0 0 , an ant ibody against a cytoplasmic

loop of connexin43 which cross-reacts with PAP26

(Yahalom et al. 1991) and with S-41, showed that

PAP26 is highly expressed in leaf and mesocoty l tis-

sue but is undetectable in roots (Fig. 2 A, B).

To determine whether this cross-reactivi ty between

the 41 kDa polypept ide and the PAP26 was due to

c o m m o n sugar side groups, a blot o f corn mesocoty l

wall polypept ides was probed with Con A-biot in con-

jugate which binds to mannose and glucose contain- ing glycoproteins. The binding was visualized with an

antibiotin conjugated to alkaline phosphatase. Figure

3 shows the 41 kDa protein was not Con A labeled. In

contrast, a posit ive reaction was observed for proteins

with molecular weights o f 78, 56, 51, 35, and 32 kDa.

Neither the p lasmodesmata l associated protein

PAP26, nor the 41 kDa protein were labeled by Con A. Furthermore, when blots were pretreated with

sodium periodate to oxidize prosthetic sugar groups,

cross-reactivi ty was still apparent (data not shown).

To achieve release of peripheral membrane proteins,

wall fractions were incubated overnight either in the

presence o f 3 M NaC1, 2% Triton X-100, or 100 m M

Fig. 3. Fig. 4

Fig. 3. Western blot of wall associated proteins probed with Con A. Blot of proteins extracted from clean cell wall, separated by SDS-PAGE, overlaid with Con A-biotin conjugate (1) or Con A-biotin conjugate plus 0.2 M methyl a-D-mannopyranoside (2), or 0.2 M mannose (3) and visualized by alkaline phosphatase reaction with antibiotin conjugated to alkaline pilosphatase

Fig. 4 A, B. The 41 kDa wall associated polypeptide is a peripheral membrane protein that is solubilized by 3 M NaC1 and 100 mM Na2CO? but not by 2% Triton X-100. SDS-PAGE analysis (A) and Western blot analysis (B) of proteins remaining associated with cell walls following over- night incubation of wall fraction either in the presence of HM/7.5 (1), 2.2% SDS (2), 3 M NaCt (3), 2% Triton X-t00 (4), and 100 mM Na2CO3 pH 11 (5). The walls were then washed and extracted with SB and the proteins separated by SDS-PAGE and either silver stained (A) or trans- ferred to nitrocellulose and probed with AF-4I (B). Arrow points to 41 kDa protein

74 B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls

phosphatase using the substrates 5-bromo-4-chloro-3- indoyl phosphate and nitro blue tetrazolium showed that neither the fixative or embedding temperature (56 ~ impaired the antigenicity of the polypeptide. The cell walls of essentially all cells were labeled (Fig. 5 b). In sections of mesocotyl tissue immuno- reacted with pre-immune serum, chromophoric prod- ucts of the phosphatase reaction were not observed (Fig. 5 a). Using gold conjugated antibodies directed against the rabbit antiserum enabled more precise localization of the protein (Fig. 5 c). Label associated with sections probed with affinity purified antibody against the 41 kDa protein, was generally associated with the wall and appeared punctate and was trans- wall although some general staining was also evident.

Fig. 5 a-c. Immunolocalization of the 41 kDa in mesocotyl sections at light microscope level. Mesocotyl tissue fixed in FAA was indi- rectly immuno-stained with a preimmune serum, b S-41 antiserum and with goat anti-rabbit IgG conjugated to alkaline phosphatase as the secondary antibody, or c with affinity purified antibody AF-41 and goat anti-rabbit IgG conjugated with 5 nm colloidal gold as the secondary antibody. Gold labeling was followed by silver enhance- ment. a and b, • 80; c, • 7900

Na2CO3 pH 11 (Findley 1987). The walls were then washed and extracted with SB and the proteins separ- ated by SDS-PAGE and silver stained (Fig. 4 A) or transferred to nitrocellulose and probed with affinity purified anti-41 (Fig. 4 B). Most efficient extraction was obtained using 3 M NaC1 and 100 mM NazCO3 (pH 11). Triton X-100 extraction relative to the con- trol in which walls were incubated overnight in tissue homogenization media HM/7.5 showed little effect on extracting the 41 kDa protein.

Silver-enhanced immunogold light-microscopy Immunocytochemical localization of the 41 kDa pro- tein at the light microscope level was performed on paraffin sections of etiolated mesocotyl tissue fixed in FAA. Results of preliminary localization via alkaline

lmmunogold-EM In ultrathin sections of mesocotyl and root tissue treated with anti-41 kDa serum and protein A-gold, gold particles were specifically localized on those cell wall segments that contained plasmodesmata (Figs. 6 and 7 a). The number of gold particles on PD-contain- ing cell wall segments (pit fields) was higher than on cell wall segments without PD-structures. In these pit fields, 75% of the gold particles was located specifi- cally on plasmodesmata (Figs. 6 and 7 a), while the remaining 24% was located on the cell wall but not related to a visible plasmodesmatal structure. From controls labeled with pre-immune serum, it was established that approximately 5% of these wall asso- ciated gold particles represented background label- ing. No labeling of plasmodesmata was found in these control specimens. Another prominent location of the 41 kDa protein was the Golgi apparatus (Figs. 6 b-d and 7 a), while some labeling was also evident in intercellular spaces, primarily associated with trapped fibrous structures (Fig. 7 b). No specific labeling of these structures was observed with pre-immune serum.

Discussion

In a previous study using antibodies to animal gap junction proteins, we identified two proteins as being plasmodesmatal associated and termed them PAP26 and PAP27 (Yahalom et al. 1991). Subsequently we developed a procedure for isolating clean cell walls containing embedded plasmodesmata. About 20 pro- teins were associated with the isolated wall fraction. Some of these proteins, including the 41 kDa protein, were enriched during the isolation procedure and it

B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls 75

Fig. 6 a-d. immunolocalization of the 41 kDa protein in mesocotyl sections by immunogold electron microscopy. Gold particles are specifical- ly localized over plasmodesmata-containing cell wall segments and in particular to the plasmodesmal structure (p and/or arrowheads). Gold label is also observed over the Golgi apparatus (G). W Wall. Bar: 200 nm

76 B.L. Epel et al.: 41 kDa protein from mesocotyl cell walls

Fig. 7 a~ b. Immunolocalization of the 41 kDa protein in root sections by immunogold electron microscopy. Root tissue from about 0.5 mm above the root tip was fixed and embedded, a Gold particles are specifically localized over the plasmodesmata (P) and Golgi apparatus (G). b Also the 41 kDa protein was localized in fibrous material (F and/or alTowheads) present in the intercellular space (S). W Wail. Bar: 200 nm

was suggested that some of these proteins may be plasmodesmatal associated (Kotlizky et al. 1992). The possibility existed, however, that some of these might be tightly-bound wall-associated non-plasmo- desmatal proteins that remained bound even follow- ing rigorous treatments to rid them from the fraction. Thus, a definitive identity as a PAP required immu- nolocalization. Silver-enhanced immunogold light microscopy showed a trans-wall and punctate immuno-labeling pattern. Electron microscopic immunogold labeling localized the 41 kDa protein to plasmodesmata-con- taining cell wall segments and, more specifically, in many occasions to the plasmodesmatal structure. The trans-wall localization of the protein observed in light microscopy and the electron microscopical observa- tion of gold particles over all parts of the plasmodes- mata suggest that the protein is located over the whole length of the structure. Similar results were obtained for root plasmodesmata which do not contain PAP26 (Fig. 2 A), confirming that the antibody labeled the 41 kDa protein. The broad punctate trans-wall stain- ing patterns observed in silver-enhanced immunogold

light microscopy probably represent large aggregates of plasmodesmata associated with pit fields. The presence of unmarked plasmodesmata or the presence of gold particles on the cell wall without a clear asso- ciation to the plasmodesmatal structure can be explained by the thickness of the resin section (approx. 70-80 nm) and the fact that in these sections gold labeling occurs only on those antigens that are located at the section surface. Relatively small struc- tures like plasmodesmata (diam. 40-50 nm) may be completely embedded in the section and antigens are therefore not available for labeling. Otherwise, frag- ments of plasmodesmata retained in the section may not be identified in the electron microscope as a clear visible structure, but merely by gold labeling of the antigens present. The finding that the 41 kDa protein was present in the soluble fraction and could be released from the wall/plasmodesmatal fraction by 100 mM Na2CO3 pH 11 suggests that this protein is probably a periph- eral protein (Findley 1987). It may be located within the pore of the plasmodesma, possibly associated with the plasmalemma or the desmotubule. Ding et al.

B. L. Epel et al.: 41 kDa protein from mesocotyl cell walls 77

(1992) and Botha et al. (1993) using image enhancing techniques have shown the presence of electron dense globular structures associated with plasmamembrane of the plasmodesma, with the desmotubule and with- in the plasmodesma sleeve. Furthermore, filamentous structures were also suggested to be present. Immu- nogold labeling does not have high enough resolution to determine if or whether plasmodesmatal associated protein PAP41 is congruent with any of these globular structures or with the filaments. The association of the 41 kDa protein with Golgi apparatus suggests that this protein is probably trans- ported to plasmodesmata via Golgi membranes. Pri- mary plasmodesmata are apparently formed in part as the result of fusion of Golgi at the phragmoplast on the laying down of the primary cell wall (Hepler 1982). The region of the mesocotyl sectioned and probed was from the 5 mm region below the coleop- tile node, a region undergoing rapid elongation and secondary plasmodesmata formation is occurring (Epel et al. 1992). Apparently Golgi are also involved in the formation of secondary plasmodesmata (Kollmann and Glock- mann 1991). The identity of the fibrous material in the intercellular spaces which is immunostained with S-41 is unknown. It is possible that this material rep- resents remnants of plasmodesmata left behind fol- lowing the digestion of the middle lamella.

Acknowledgements This research was supported by The Israeli Science Foundation administered by the Israeli Academy of Sciences and Humanities (project no. 361/88), by the United States-Israel Binational Agricul- tural Research and Development Fund (project no. US-1384-87), by the Joint German-Israeli Research Program, MOST, NCRD (project no. DISNAT 1036), by the Joint Dutch-Israeli Agricultural Research Program (DIARP), and by The Karse-Epel Fund for Botanical Research at Tel Aviv University.

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