Planta (1989) 178:~76-183 Planta �9 Springer-Verlag 1989

Immunocytoehemical localization of patatin, the major glycoprotein in potato (Solarium tuberosum L.) tubers Uwe Sonnewald 1.*, Daniel Studer 2, Mario Rocha-Sosa ~*, and Lothar Willmitzer 1 1 IGF Berlin, Ihnestrasse 63, D-1000 Berlin 33, and ST2003 280633,01 19.04.89 K32 [0().2] L16 2 Eidgen6ssische Technische Hochschule, Mikrobiologisches Institut, CH-8092 Ztirich, Switzerland

Abstract. Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The pro- tein is synthesized as a pre-protein with a hydro- phobic signal sequence of 23 amino acids. Using different immunocytochemical methods we deter- mined the tissue-specific as well as subcellular lo- calization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-micro- scopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanurn tuber- osum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained de- tectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in pa- renchyma cells of potato tubers. The same distribu- tion was observed for the esterase activity in potato tubers.

Key words: Glycoprotein - Lipid acyl hydrolyse Patatin (immunocytochemical localization) - So-

lanum (patatin localization) - Vacuole

Introduction

Patatin is the trivial name for a family of immuno- logically identical glycoproteins with an apparent molecular weight of 40 kDa from potato (Solanum tuberosum L.) tubers. Patatin is present in all culti-

* Present address: Centro de Investigation sobre Fijacion de Nitrogeno, Cuernavaca, Morelos, Mexico ** To whom correspondence should be addressed

Abbreviations: PHA=phytohemagglutinin; TFMS=trif luoro- methanesulfonic acid

vars so far examined and acounts for up to 40% of the soluble protein (Racusen and Foote 1980). Under normal conditions the protein is expressed in tubers or stolons attached to developing tubers and to a much lower extent in roots (Mignery et al. 1988), but is not present in significant amounts under greenhouse and field conditions in leaves or stems (Paiva etal . 1983; Rosahl et al. 1986). However, it is possible to induce patatin in stems and petioles after removing the tubers and axillary buds (Paiva et al. 1983) or in leaves from tissue- culture-grown plants kept on high levels of sucrose, later referred to as: leaves induced for patatin ex- pression (Rocha-Sosa et al. 1989).

Since the protein is present in such high amounts in potato tubers, patatin is believed to serve as a storage protein. Unlike most other stor- age proteins however, an enzymatic function was found for patatin, i.e. a lipid-acyl-hydrolase activi- ty, polar lipids being used as substrates (Racusen 1984, 1986; Rosahl et al. 1987). These lipids are found in cellular membranes. The cellular function of patatin is unclear, but it is obvious that mem- branes of intact cells must be protected from this enzyme. Export out of the cell or compartmenta- tion of the protein within vacuoles could solve this problem.

Patatin is synthesized with a signal peptide (Kirschner and Hahn 1986), which allows the poly- peptide to enter the lumen of the endoplasmic re- ticulum (Blobel 1980). During transport to its final destination the signal sequence is cleaved (Park et al. 1983), the protein becomes N-glycosylated and the glycans are further modified to complex- glycans (data not shown). Antibodies raised against the glycosylated protein showed crossreac- tivity with other glycoproteins (phytohemagglutin- in isolated from Jack-bean; invertase from carrot cell walls). This crossreactivity is most likely me-

U. Sonnewald et al. : Localization of patatin 177

diated by antibodies reacting with a common gly- can epitope present on many plant glycoproteins, as for example Sophora japonica lectin, Erythrina cristagalli lectin, phaseolin from Phaseolus vulgaris and laccase from sycamore cells (Fournet et al. 1987; Ashford et al. 1987; Sturm et al. 1987; Taka- hashi et al. 1986). Since we are analyzing the glyco- sylation, stability and activity of the protein, we are also interested in the localization and function of the protein within the cell. Here we report the immunocytochemical localization of patatin in cells from potato tubers and leaves induced for patatin expression. Two different fixation methods, namely conventional chemical fixation and high- pressure freezing followed by freeze-substitution (Mfiller and Moor 1983) were used.

Material and methods

Materials. Potato (SoIanum tuberosum L.) cultivars Berolina and D6sir6e were used for the localization of patatin in tubers or potato leaves induced for patatin expression. Immunological reagents were obtained from Janssen, Beerse, Belgium. Material for chromatography was obtained from Pharmacia, Freiburg, FRG or Simga (Miinchen, FRG), respectively.

Fig. 1. Immunoblot analysis of potato tuber proteins and PHA isolated from beans using glycan-specific antibodies (lanes l ~ ) or protein-specific antibodies (lanes 4-6). The glycan-specific antibodies react with the glycosylated forms of patatin (lane 1), with an other tuber protein (lane 1) and with PHA (lane 3 ) but not with chemically deglycosylated tuber proteins including patatin (lane 2). The protein-specific antibodies only react with patatin in the glycosylated forms (lane 4) and the deglycosylated form (lane 5) but not with PHA (lane 6)

Isolation and chemical deglycosylation of patatin. The protein was isolated as described by Racusen and Foote (1980). Chemi- cal delycosylation with trifluoromethanesulfonic acids (TFMS) was done according to Edge et al. (1981).

Preparation of antibodies. The preparation of antiserum against the glycosylated protein was done as described by Rosahl et al. (1986). The serum was further purified by affinity chromatogra- phy, first using phytohemagglutinin (PHA)-Sepharose (kindly provided by Arnd Sturm and Maarten Chrispeels, San Diego, Calif., USA) as an affinity gel, as described by Smith et al. (1978) to isolate antibodies specific for the complex glycans found on patatin and other plant glycoproteins. The serum, which did not bind to the PHA-Sepharose was used for a second affinity chromatography with patatin-Sepharose as an affinity gel. The obtained immunoglobulin G (IgG) fraction was specif- ic for the protein portion of patatin. The specificity of the anti- bodies was tested by immunoblotting. Proteins were separated on 12.5% sodium dodecyl sulfate (SDS) potyacrylamide gels, prepared by the method of Laemmli (1970). Immunoblot analy- sis was carried out according to the procedure detailed in the BioRad manual (Bio Rad, Richmond, Calif., USA).

Fixation and embedding. Cryofixation of thick specimens by high-pressure freezing was done as described by Studer et al. (1989). Freeze substitution (slightly modified according to Mfiller et al. 1980) was performed in anhydrous acetone con- taining 2% uranyl acetate (diluted 20% uranyl acetate solution in methanol) and 0.2% glutaraldehyde (dilution of 50% glutar- aldehyde in water) or 2% osmium tetroxide. The frozen samples were kept at - 9 0 ~ C, - 6 0 ~ C and - 3 0 ~ C for 8 h each step and finally brought to 0 ~ C in a cryostage (FSU 010, Balzer, Lichtenstein). Finally the samples were washed three times in 100% ethanol and embedded in LR-White resin (Polyscience, Miinchen, FRG) as chemically fixed samples. Chemical fixa- tion was done as follows: samples were fixed by vacuum infil-

Fig. 2. Hand-cut section of a potato tuber fixed with 2% form- aldehyde and incubated with a-Naphtyl-acetate. The esterase activity is visible because of the colour reaction within the pa- renchyma cells. Pa, parenchyma; Pe, periderm; x 60; bar= 500 lam

178 U. Sonnewald et al. : Localization of patatin

Fig. 3a-d. Potato tuber chemically fixed (a, b, d) or cryofixed (2% OsO4-acetone; c) and embedded in LR-White resin. The sections were labeled with the patatin-protein-specific antibody (b-d) or with preimmune serum (a), followed by goat anti-rabbit IgG antibody coupled to 10-nm colloidal gold and silver en- hancement. Specific label is only obtained with the specific anti- body, but not with the preimmune serum. Pa, parenchyma; Pe, periderm; x 100 (a, b); x400 (c, d); bars=400 gm (a, b); 40 gm (c, d)

tration of 1.25% glutaraldehyde in 0.1 M Na-phosphate buffer (pH 7.0) and 1 mM ethylenediaminetetraacetic acid (EDTA) overnight at 4 ~ C. The tissue was washed, dehydrated with 70% and 100% ethanol on ice and embedded in LR-White resin. Polymerization was done at 55 ~ C.

Immunolocalization. Silver-colored thin sections were cut with a diamond knife and mounted on nickel grids for electron mi- croscopy, or 1-gin-thick sections were mounted on poly-L-ly- sine-coated slides for light microscopy. To overcome the mask- ing of antigenic sites by osmium tetroxide (Bendayan and Zoll- inger 1983; Craig and Goodchild 1984; Doman and Trelease 1985), osmium-tetroxide-postfixed sections were treated with aqueous 0.56 M Na-metaperiodate for 20 rain, washed with dis- tilled water, incubated with 0.1 N HCI for 10 rain, washed again with distilled water and blocked with 3 % bovine serum albumin (BSA) in Tris-buffered saline (20raM 2-amino-2-(hydroxy- methyl)-/,3-propanediol (Tris)-HC1, pH 7.5, 500 mM NaC1) for 10 min at room temperature. Labelling was done with affinity- purified patatin-protein-specific antibodies in TBS with 1% BSA and 0.02% azide (or only TBS) for I h at room tempera- ture or overnight at 4 ~ C. Controls were done in parallel using preimmune serum or TBS with 1% BSA and 0.02% azide. The grids or slides were then washed with TBS, TTBS (TBS with 0.1% Tween 20) and TBS, each step for 5 min and labeled indirectly for 1 h at room temperature with 10- or 15-nm goat

U. Sonnewald et al. : Localization of patatin 179

Fig. 4. Potato tuber chemically fixed. Sections were labeled with the patatin-protein-specific antibody, followed by goat anti-rab- bit IgG antibody coupled to 10-nm colloidal gold and silver enhancement. Specific label is seen within the parenchyma cell; starch (s), cell walls (Cw), protein crystals (pc) and the intercel- lular (Is) space are not labeled, x 1000; bar= 10 [tm

anti rabbit (GAR) IgG-colloidal gold. The incubation was fol- lowed by three wash steps with; TBS and the samples were then treated with a continuous stream of distilled water. Grids were stained with 2% aqueous uranyl acetate for 20 rain. The colloidal-gold immunolabelling was enhanced for light-micro- scopic visualisation. Silver enhancing was performed according to the protocol of Janssen Life Sciences. Electron micrographs were taken with a Philips (Eindhoven, The Netherlands) 400 electron microscope (at 80 kV).

Histochemical localization. Potato tubers were cut by hand and the slices directly fixed with freshly prepared 2% formaldehyde in 100 mM Na-phosphate buffer, pH 7.0, 1 mM EDTA. Fixa- tion was carried out for 30 min on ice and the slices were exten- sively washed with phosphate buffer afterwards, e-Naphtyl-ace- rate staining of esterases was done according to the protocol of Sigma Diagnostics.

Results

Antiserum specificity. Antisera raised against gly- coproteins of ten lack specificity since they react not only with the prote in por t ion, bu t also with the glycan por t ion o f the glycoprote in (Greenwood

and Chrispeels 1985). Oligosaccharide sidechains, so far found on plant glycoproteins, are not specif- ic for a par t icular protein, but can be found on many glyocproteins. The crude ant iserum raised against the glycosylated pata t in prote in reacts not only with patat in but also with other tuber pro- teins, i.e. invertase f rom car ro t cell walls and P H A isolated f rom beans (data no t shown). Since this ant iserum was no t specific enough for the immuno- cytochemical localizat ion o f patat in, we decided to separate the glycan-specific f rom the protein- specific antibodies as described in materials and methods. The result o f the two-step affinity chro- ma tog raphy is shown in Fig. 1. The glycan-specific antibodies recognize the glycosylated pata t in as well as the other plant glycoproteins, bu t do not recognize patat in after chemical deglycosylat ion with T F M S (Edge et al. 1981). The protein-specific antibodies, on the other hand, specifically react only with pata t in in bo th its glycosylated and deg- lycosylated form.

Histochemical and immunocytochemical localization ofpatatin. The tissue-specific expression o f pata t in was examined by two different methods on the l ight-microscopic level for po ta to tubers. On the one hand, his tochemical localization was achieved using hand sections o f po ta to tubers and c~-naph- thyl acetate as substrate for the esterase activity. As shown in Fig. 2 the esterase activity is restricted to the pa renchyma cells and is not detectable in

180 U. Sonnewald et al. : Localization of patatin

Fig. 5a-d. Immunochemical localization of patatin in vacuoles of chemically fixed potato tubers. Sections were incubated with preimmune serum (a) or with the patatin-protein-specific anti- body (b-d), followed by goat anti-rabbit IgG antibody coupled to 15-nm colloidal gold. Mainly vacuoles are labeled. Cw, cell wall; Is, intercellular space; C, cytoplasma; V, vacuoles; vpc, vacuolar protein cluster-like structure; x 17000 (a); x 11000 (b); x 13000 (e, d); bars=0.5 ~tm (a, d) or 1 ~tm (b, e)

periderm cells. These results are consistent with the silver-enhanced immunogold localization. Im- munogold staining was done on LR-White-embed- ded thin sections of potato tubers using patatin- protein-specific antibodies. Specific label was found within parenchyma cells using chemically fixed (Fig. 3 b, d; 4) or cryofixed (Fig. 3 c) tissue, but there was no detectable labelling in periderm cells (Fig. 3 b) or on sections incubated with preim- mune serum as shown for chemically fixed tissue (Fig. 3a). One rather peculiar observation, how-

ever, is that the patatin-specific label is not equally distributed in the vacuoles, but seems to be concen- trated in the cluster-like structures (Fig. 4). So far it is not possible to rule out that these structures are artefacts caused by the fixation procedure, but a possible biological function will be discussed later. Protein crystals, the cell wall and starch grains are not labeled (Fig. 4), indicating that pata- tin is not associated with any of these structures.

Subcellular localization of patatin. Ultrathin sec- tions of potato tubers or leaves induced for patatin expression were used for the subcellular localiza- tion of patatin protein. In both cases specific label- ling with the patatin-protein-specific antibody was observed mainly in vacuoles, but also, though to a much lower extent, in the cytosol (Fig. 5b-d; 6 b-d). Only sparse labelling was found on cell wails and the intercellular space of potato tubers (Fig. 5b-d). Other compartments such as chloro- plasts, mitochondria or nuclei of potato leaves in- duced for patatin expression also showed no label

U. Sonnewald et al. : Localization of patatin 181

Fig. 6 a-d. Immunochemical localization of patatin in vacuoles of cryofixed (2% uranyl acetate-acetone) potato leaves induced for patatin synthesis. Sections were labeled with preimmune serum (a) or with the patatin-protein-specific antibody (b-d), followed by goat anti-rabbit IgG antibody coupled to 15-nm colloidal gold. C, cytoplasm; Chl, chloroplast; Cw, cell wall; M, mitochondrion; V, vacuole; x l0000 (a); x13000 (b); x 10000 (c); x 6000 (d); bars= 1 pm

above background (Fig. 6 b-d). Incubation of sec- tions with preimmune serum again only gave rise to a few gold particles dispersed over the whole section, but there was no specific labelling of any cellular structure (Fig. 5a; 6a), thus indicating the specificity of the antibody used. Again on both, chemically fixed and cryofixed tissue, patatin seems to be present in cluster-like structures within the vacuole as shown for chemically fixed potato tubers in Fig. 5 b.

Discussion

In this study, we used immunocytochemistry to localize patatin, the major glycoprotein of potato tubers, in vacuoles of potato tubers as well as in leaves induced for patatin expression. This ap- proach was done using highly purified antibodies specific for the protein portion of patatin. The pu- rification was necessary since the crude serum, ra- ised against the glycosylated protein, showed cross- reactivity with other plant glycoproteins. Thus, in earlier immunofluorescence studies, using the non- purified patatin serum, an unspecific cell-wall fluo- rescence was obtained (data not shown). Green- wood and Chrispeels (1985) made a similar obser- vation with antibodies raised against phaseolin, us- ing tissue from wild-type tobacco as well as from transgenic tobacco expressing phaseolin; unspecif- ic cell-wall fluorescence was observed in both tis- sues.

Having purified the antibody we analyzed first the localization of patatin in potato tuber cells.

182 U. Sonnewald et al. : Localization of patatin

Patatin was found to be localized mainly in paren- chyma cells having a high amount of accumulated starch and was not detectable in periderm cells.

It has been shown that patatin not only serves a storage function, but also displays an enzymatic function, i.e. an esterase activity (Racusen 1984, 1986; Rosahl et al. 1987). To test whether this es- terase activity found in potato tubers has the same tissue specificity as patatin, we performed in situ esterase staining with hand-cut sections of tubers using e-naphtyl acetate as substrate, c~-Naphtyl acetate is known to be a substrate for patatin (Ro- sahl et al. 1987), and, as is evident from Fig. 2, the distribution of the esterase activity closely fol- lows that found for patatin.

Patatin is present in very high amounts in pa- renchyma cells of potato tubers. Its enzymatic ac- tivity could destroy cellular membranes, which might be an explanation for the observed instabili- ty and rapid degradation of protoplasts isolated from tubers (Racusen 1986). Export out of the cell compartmentation, or inactivation could prevent the destruction of intact cells by patatin. By im- munocytochemistry we found the protein to be lo- calized in vacuoles of parenchyma cells from pota- to tubers. There was also some label found in the cytosol, but this could be explained by patatin be- ing transiently present in the endoplasmic reticu- lum and Golgi apparatus during synthesis and pro- cessing. The structural preservation does not allow us to distinguish between the endoplasmic reticu- lum, Golgi apparatus and cytosol, but since pata- tin is glycosylated and contains a signal peptide (Krischner and Hahn 1986), it is unlikely that a cytosolic form exists. Storage of patatin in vacuoles may lead to the inactivation of the esterase activity, since the pH of vacuoles is known to be rather acidic. The intravacuolar pH of isolated vacuoles from castor-bean endosperm was determined to be 5.7-5.9 (Nishimura 1982). The isoelectric points for three known patatin proteins are 5.33, 4.79 and 5.2, respectively. Solubility of proteins next to their isoelectric point is minimized and the observation that patatin seems to cluster in vacuoles could indi- cate that it is aggregated and if so, presumably inactive.

The cellular function of patatin is not known, but esterases are responsible for the major lipid breakdown in disrupted cells and the wax-ester for- mation after wounding (Dennis and Galliard 1974). This could indicate that patatin is stored inactively in vacuoles as a storage protein. Upon wounding, patatin is released, becomes activated and could be involved in the rapid wound re- sponse, since it is present in such high amounts.

Another presently still speculative possibility may be the involvement of patatin in the response to pathogens, via releasing polyunsaturated fatty acids, i.e. arachidonic acid and eicosapentaenoic acid. Esters of arachidonic acid and eicosapentaen- oic acid are present in membranes of Phytophthora infestans but not in potatoes. These compounds serve as elicitors and are responsible for the hyper- sensitive response in potatoes (Bostock et al. 1981).

In order to test whether the compartmentation of patatin is necessary for cells to survive we are constructing chimeric genes which should allow the protein to be expressed in the cytosol. In addition patatin-coding regions of different members of the patatin gene family will be expressed in transgenic plants (i.e. tobacco) using heterologous promoters. This should allow us to study the enzymatic func- tion of single patatin genes which might give us a clue about the biological function of patatin in higher-plant cells.

We thank Arnd Sturm and Maarten Chrispeels (Dept. of Bota- ny, C-016 University of California, San Diego, La Jolla, Calif., USA) for the help during the purification and further character- ization of the patatin antibodies. We also thank R. Lurz (Max- Planck-Institut ffir molekulare Genetik, Berlin, West Germany) for the opportunity to use the electron microscope and G. Wurz (Institut fiir Pflanzenphysiologie, Berlin, West Germany) for introducing one of the authors (U. Sonnewald) in the technique of electron microscopy. This work was supported by a grant from the Bundesministerium ffir Forschung und Technologie.

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Received 22 August; accepted 29 November 1988