Planta
Planta (1989) 178:~76-183
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 protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization 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-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanurn tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers. Key words: Glycoprotein - Lipid acyl hydrolyse Patatin (immunocytochemical localization) - Solanum (patatin localization) - Vacuole
Introduction Patatin is the trivial name for a family of immunologically 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=trifluoro-
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 tissueculture-grown plants kept on high levels of sucrose, later referred to as: leaves induced for patatin expression (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 storage proteins however, an enzymatic function was found for patatin, i.e. a lipid-acyl-hydrolase activity, 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 membranes of intact cells must be protected from this enzyme. Export out of the cell or compartmentation of the protein within vacuoles could solve this problem. Patatin is synthesized with a signal peptide (Kirschner and Hahn 1986), which allows the polypeptide to enter the lumen of the endoplasmic reticulum (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 complexglycans (data not shown). Antibodies raised against the glycosylated protein showed crossreactivity with other glycoproteins (phytohemagglutinin isolated from Jack-bean; invertase from carrot cell walls). This crossreactivity is most likely me-
U. Sonnewald et al. : Localization of patatin
diated by antibodies reacting with a c o m m o n glycan 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; Takahashi et al. 1986). Since we are analyzing the glycosylation, 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 highpressure 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, F R G or Simga (Miinchen, FRG), respectively.
177
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). Chemical 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 chromatography, 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 specific for the protein portion of patatin. The specificity of the antibodies was tested by immunoblotting. Proteins were separated on 12.5% sodium dodecyl sulfate (SDS) potyacrylamide gels, prepared by the method of Laemmli (1970). Immunoblot analysis 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 containing 2% uranyl acetate (diluted 20% uranyl acetate solution in methanol) and 0.2% glutaraldehyde (dilution of 50% glutaraldehyde 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 fixation was done as follows: samples were fixed by vacuum infil-
Fig. 2. Hand-cut section of a potato tuber fixed with 2% formaldehyde and incubated with a-Naphtyl-acetate. The esterase activity is visible because of the colour reaction within the parenchyma cells. Pa, parenchyma; Pe, periderm; x 60; bar= 500 lam
178
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 enhancement. Specific label is only obtained with the specific antibody, 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.
U. Sonnewald et al. : Localization of patatin
Immunolocalization. Silver-colored thin sections were cut with a diamond knife and mounted on nickel grids for electron microscopy, or 1-gin-thick sections were mounted on poly-L-lysine-coated slides for light microscopy. To overcome the masking of antigenic sites by osmium tetroxide (Bendayan and Zollinger 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 distilled 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-(hydroxymethyl)-/,3-propanediol (Tris)-HC1, pH 7.5, 500 m M NaC1) for 10 min at room temperature. Labelling was done with affinitypurified patatin-protein-specific antibodies in TBS with 1% BSA and 0.02% azide (or only TBS) for I h at room temperature 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
Fig. 4. Potato tuber chemically fixed. Sections were labeled with the patatin-protein-specific antibody, followed by goat anti-rabbit 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 intercellular (Is) space are not labeled, x 1000; bar= 10 [tm
anti rabbit (GAR) IgG-colloidal gold. The incubation was followed 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-microscopic 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. Fixation was carried out for 30 min on ice and the slices were extensively washed with phosphate buffer afterwards, e-Naphtyl-acerate staining of esterases was done according to the protocol of Sigma Diagnostics.
Results
Antiserum specificity. Antisera raised against glycoproteins often lack specificity since they react not only with the protein portion, b u t also with the glycan p o r t i o n o f the glycoprotein ( G r e e n w o o d
179
and Chrispeels 1985). Oligosaccharide sidechains, so far f o u n d on plant glycoproteins, are not specific for a particular protein, but can be f o u n d on m a n y glyocproteins. T h e crude antiserum raised against the glycosylated patatin protein reacts not only with patatin but also with other tuber proteins, i.e. invertase f r o m c a r r o t cell walls and P H A isolated f r o m beans (data n o t shown). Since this antiserum was n o t specific e n o u g h for the i m m u n o cytochemical localization o f patatin, we decided to separate the glycan-specific f r o m the proteinspecific antibodies as described in materials and methods. The result o f the two-step affinity chrom a t o g r a p h y is shown in Fig. 1. The glycan-specific antibodies recognize the glycosylated patatin as well as the other plant glycoproteins, b u t do not recognize patatin after chemical deglycosylation with T F M S (Edge et al. 1981). The protein-specific antibodies, on the other hand, specifically react only with patatin in b o t h its glycosylated and deglycosylated form.
Histochemical and immunocytochemical localization ofpatatin. The tissue-specific expression o f patatin was examined by two different m e t h o d s on the light-microscopic level for p o t a t o tubers. On the one hand, histochemical localization was achieved using h a n d sections o f p o t a t o tubers and c~-naphthyl acetate as substrate for the esterase activity. As shown in Fig. 2 the esterase activity is restricted to the p a r e n c h y m a cells and is not detectable in
180
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 antibody (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. Immunogold staining was done on LR-White-embedded thin sections of potato tubers using patatinprotein-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 preimmune serum as shown for chemically fixed tissue (Fig. 3a). One rather peculiar observation, how-
U. Sonnewald et al. : Localization of patatin
ever, is that the patatin-specific label is not equally distributed in the vacuoles, but seems to be concentrated 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 patatin is not associated with any of these structures.
Subcellular localization of patatin. Ultrathin sections of potato tubers or leaves induced for patatin expression were used for the subcellular localization of patatin protein. In both cases specific labelling 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 chloroplasts, mitochondria or nuclei of potato leaves induced for patatin expression also showed no label
U. Sonnewald et al. : Localization of patatin
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 sections 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.
181
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 approach was done using highly purified antibodies specific for the protein portion of patatin. The purification was necessary since the crude serum, raised against the glycosylated protein, showed crossreactivity with other plant glycoproteins. Thus, in earlier immunofluorescence studies, using the nonpurified patatin serum, an unspecific cell-wall fluorescence was obtained (data not shown). Greenwood and Chrispeels (1985) made a similar observation with antibodies raised against phaseolin, using tissue from wild-type tobacco as well as from transgenic tobacco expressing phaseolin; unspecific cell-wall fluorescence was observed in both tissues. Having purified the antibody we analyzed first the localization of patatin in potato tuber cells.
182 Patatin was found to be localized mainly in parenchyma 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 esterase 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 (Rosahl et al. 1987), and, as is evident from Fig. 2, the distribution of the esterase activity closely follows that found for patatin. Patatin is present in very high amounts in parenchyma cells of potato tubers. Its enzymatic activity could destroy cellular membranes, which might be an explanation for the observed instability 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 immunocytochemistry we found the protein to be localized in vacuoles of parenchyma cells from potato tubers. There was also some label found in the cytosol, but this could be explained by patatin being transiently present in the endoplasmic reticulum and Golgi apparatus during synthesis and processing. The structural preservation does not allow us to distinguish between the endoplasmic reticulum, Golgi apparatus and cytosol, but since patatin 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 indicate 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 formation 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 response, since it is present in such high amounts.
U. Sonnewaldet al. : Localizationof patatin 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 eicosapentaenoic acid are present in membranes of Phytophthora infestans but not in potatoes. These compounds serve as elicitors and are responsible for the hypersensitive 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 function 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 Botany, C-016 Universityof California, San Diego, La Jolla, Calif., USA) for the help during the purificationand further characterization of the patatin antibodies.We also thank R. Lurz (MaxPlanck-Institut ffir molekulareGenetik, Berlin, WestGermany) for the opportunityto use the electron microscopeand 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 Bundesministeriumffir Forschungund Technologie.
References
Ashford, D., Dwek, R.A., Welply, J.K., Hormans, S.W., Lis, H., Taylor, G.N., Radeemacher, T.W. (1987) E1 ~2-D-Xylose and ~1 -~ 3-L-fucosesubstituted N-linked oligosaccharides from Erythrina cristagalli lectin. Eur. J. Biochem. 166, 311-320 Bendayan,M., Zollinger,M. (1983) Ultrastructural localization of antigenic sites on osmium-fixedtissues applyingthe protein A-goldtechnique.J. Histochem.Cytochem.31,101-109 Blobel, G. (1980) Intracellularprotein topogenesis. Proc. Natl. Acad. Sci. USA 77, 1496-1500 Bostock, R.M., Kuc, J., Laine, R.A. (1981) Eicosapentaenoic and arachidonicacids from Phytophtora infestans elicit fungitoxic sesquiterpenesin the potato. Science212, 67 69 Craig, S., Goodchild, D.J. (1984) Periodate-acid traetment of sections permits on-grid immunogold localization of pea seed vicilinin ER and Golgi. Protoplasma 122, 3544 Dennis, S., Galliard, T. (1974) Wax ester formation catalysed by isoenzymesof lipolytic acyl hydrolase. Phytochemistry 13, 2469-2473 Doman, D.C., Trelease, R.N. (1985) Protein A-goldimmunocytochemistryof isocitrate lyasein cotton seeds. Protoplasma 124, 157-167 Edge, A.S.B., Faltynek, C.R., Hof, L., Reichert, L.E., Jr., Weber, P. (1981) Deglycosylationof glycoproteins by trifluoromethanesulfonicacid. Anal. Biochem. 118, 131-137 Fournet, B., Leroy, Y., Wieruszeski,J.-M., Montreuil, J., Poretz, R.D., Goldberg, R. (1987) Primary structure of an N-
U. Sonnewald et al. : Localization of patatin glycosidic carbohydrate unit derived from Sophorajaponica lectin. Eur. J. Biochem. 166, 321 324 Greenwood, J.S., Chrispeels, M.J. (1985) Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco. Plant Physiol. 79, 65-71 Kirschner, B., Hahn, H. (1986) Patatin, a major soluble protein of the potato (Solanum tuberosum L.) tuber is synthesized as a larger precursor. Planta 168, 386-389 Laernmli, U.K. (1970) Cleavage of strcutural proteins during assembly of the head of bacteriophage T4. Nature 227, 680685 Mignery, G.A., Pikaard, C.S., Park, W.D. (1988) Molecular characterization of patatin multigene family of potato. Gene 62, 27~44 Mfiller, M., Martin, T., Kriz, S. (1980) Improved structural preservation by: freeze-substitution. In: Electron microscopy 1980, vol 2, pp 720--721, Brederoo, T., De Priester, W., eds. 7th european congress on electron microscopy foundation, Leiden Mfiller, M., Moor, H. (1983) Cryofixation of thick specimes by high pressure freezing. In : The science of Biological specimen preparation for microscopy and microanalysis. Proc. 2nd Pfefferkorn Conference, pp. 131-138. Revel, J.-P., Haggis, G.H., Barnard, T. eds. SEM Inc., AMF O'Hare Nishimura, M. (1982) pH in vacuoles isolated from castor bean endosperm. Plant Physiol. 70, 742-744 Paiva, E., Lister, R.M., Park, W.D. (1983) Induction and accumulation of major tuber proteins of potato in stems and petioles. Plant Physiol. 71, 161-168 Park, W.D., Blackwood, C., Mignery, G.A., Hermodson, M.A., Lister, R.M. (1983) Analysis of the heterogeneity of the 40,000 molecular weight tuber glycoprotein of potatoes by immunological methods and by NHz-terminal sequence analysis. Plant Physiol. 71, 156-160 Racusen, D. (1984) Lipid acyl hydrolase of patatin. Can. J. Bot. 62, 1640-1644
183 Racusen, D. (1986) Esterase specificity of patatin from two potato cultivars. Can. J. Bot. 64, 2104-2106 Racusen, D., Foote, M. (1980) A major soluble glycoprotein of potato tubers. J. Food Bioehem. 4, 43--52 Rocha-Sosa, M., Sonnewald, U., Frommer, W., Stratmann, M., Schell, J., Willmitzer, L. (1989) Both developmental and metabolic signals activate the promoter of a class I patatin gene. EMBO J. 8, 23-29 Rosahl, S., Eekes, P., Schell, J., Willmitzer, L. (1986) Organspecific gene expression in potato: isolation and characterization of tuber-specific cDNA sequences. Mol. Gen. Genet. 202, 368 373 Rosahl, S., Schell, J., Willmitzer, L. (1987) Expression of a tuber specific storage protein in transgenic tobacco plants: demonstration of an esterase activity. EMBO J. 6, 1155 1159 Smith, J.A., Hurrell, J.G.R., Leach, S.J. (1978) Elemination of nonspecific adsorption of serum proteins by sepharosebound antigens. Anal. Biochem. 87, 299-305 Studer, D., Michel, M., Mfiller, M. (1989) Cryofixation of plant tissue by high pressure freezing in: Proceedings of 9th European Congress om Electron Microscopy, York, England, in press Sturm, A., Van Kuik, J.A., Vliegenthart, J.F.G., Chrispeels, M.J. (1987) Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin. J. Biol. Chem. 262, 13392-13403 Takahashi, N., Hotta, T., Ishihara, H., Mori, M., Tejima, S., Bligny, R., Akazawa, T., Endo, S., Arata, Y. (1986) Xylosecontaining common structural unit in N-linked oligosaccharides of laccase from sycamore cells. Biochemistry 25, 388 395 Received 22 August; accepted 29 November 1988
Planta (1989) 178:~76-183
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 protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization 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-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanurn tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers. Key words: Glycoprotein - Lipid acyl hydrolyse Patatin (immunocytochemical localization) - Solanum (patatin localization) - Vacuole
Introduction Patatin is the trivial name for a family of immunologically 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=trifluoro-
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 tissueculture-grown plants kept on high levels of sucrose, later referred to as: leaves induced for patatin expression (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 storage proteins however, an enzymatic function was found for patatin, i.e. a lipid-acyl-hydrolase activity, 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 membranes of intact cells must be protected from this enzyme. Export out of the cell or compartmentation of the protein within vacuoles could solve this problem. Patatin is synthesized with a signal peptide (Kirschner and Hahn 1986), which allows the polypeptide to enter the lumen of the endoplasmic reticulum (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 complexglycans (data not shown). Antibodies raised against the glycosylated protein showed crossreactivity with other glycoproteins (phytohemagglutinin isolated from Jack-bean; invertase from carrot cell walls). This crossreactivity is most likely me-
U. Sonnewald et al. : Localization of patatin
diated by antibodies reacting with a c o m m o n glycan 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; Takahashi et al. 1986). Since we are analyzing the glycosylation, 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 highpressure 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, F R G or Simga (Miinchen, FRG), respectively.
177
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). Chemical 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 chromatography, 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 specific for the protein portion of patatin. The specificity of the antibodies was tested by immunoblotting. Proteins were separated on 12.5% sodium dodecyl sulfate (SDS) potyacrylamide gels, prepared by the method of Laemmli (1970). Immunoblot analysis 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 containing 2% uranyl acetate (diluted 20% uranyl acetate solution in methanol) and 0.2% glutaraldehyde (dilution of 50% glutaraldehyde 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 fixation was done as follows: samples were fixed by vacuum infil-
Fig. 2. Hand-cut section of a potato tuber fixed with 2% formaldehyde and incubated with a-Naphtyl-acetate. The esterase activity is visible because of the colour reaction within the parenchyma cells. Pa, parenchyma; Pe, periderm; x 60; bar= 500 lam
178
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 enhancement. Specific label is only obtained with the specific antibody, 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.
U. Sonnewald et al. : Localization of patatin
Immunolocalization. Silver-colored thin sections were cut with a diamond knife and mounted on nickel grids for electron microscopy, or 1-gin-thick sections were mounted on poly-L-lysine-coated slides for light microscopy. To overcome the masking of antigenic sites by osmium tetroxide (Bendayan and Zollinger 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 distilled 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-(hydroxymethyl)-/,3-propanediol (Tris)-HC1, pH 7.5, 500 m M NaC1) for 10 min at room temperature. Labelling was done with affinitypurified patatin-protein-specific antibodies in TBS with 1% BSA and 0.02% azide (or only TBS) for I h at room temperature 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
Fig. 4. Potato tuber chemically fixed. Sections were labeled with the patatin-protein-specific antibody, followed by goat anti-rabbit 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 intercellular (Is) space are not labeled, x 1000; bar= 10 [tm
anti rabbit (GAR) IgG-colloidal gold. The incubation was followed 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-microscopic 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. Fixation was carried out for 30 min on ice and the slices were extensively washed with phosphate buffer afterwards, e-Naphtyl-acerate staining of esterases was done according to the protocol of Sigma Diagnostics.
Results
Antiserum specificity. Antisera raised against glycoproteins often lack specificity since they react not only with the protein portion, b u t also with the glycan p o r t i o n o f the glycoprotein ( G r e e n w o o d
179
and Chrispeels 1985). Oligosaccharide sidechains, so far f o u n d on plant glycoproteins, are not specific for a particular protein, but can be f o u n d on m a n y glyocproteins. T h e crude antiserum raised against the glycosylated patatin protein reacts not only with patatin but also with other tuber proteins, i.e. invertase f r o m c a r r o t cell walls and P H A isolated f r o m beans (data n o t shown). Since this antiserum was n o t specific e n o u g h for the i m m u n o cytochemical localization o f patatin, we decided to separate the glycan-specific f r o m the proteinspecific antibodies as described in materials and methods. The result o f the two-step affinity chrom a t o g r a p h y is shown in Fig. 1. The glycan-specific antibodies recognize the glycosylated patatin as well as the other plant glycoproteins, b u t do not recognize patatin after chemical deglycosylation with T F M S (Edge et al. 1981). The protein-specific antibodies, on the other hand, specifically react only with patatin in b o t h its glycosylated and deglycosylated form.
Histochemical and immunocytochemical localization ofpatatin. The tissue-specific expression o f patatin was examined by two different m e t h o d s on the light-microscopic level for p o t a t o tubers. On the one hand, histochemical localization was achieved using h a n d sections o f p o t a t o tubers and c~-naphthyl acetate as substrate for the esterase activity. As shown in Fig. 2 the esterase activity is restricted to the p a r e n c h y m a cells and is not detectable in
180
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 antibody (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. Immunogold staining was done on LR-White-embedded thin sections of potato tubers using patatinprotein-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 preimmune serum as shown for chemically fixed tissue (Fig. 3a). One rather peculiar observation, how-
U. Sonnewald et al. : Localization of patatin
ever, is that the patatin-specific label is not equally distributed in the vacuoles, but seems to be concentrated 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 patatin is not associated with any of these structures.
Subcellular localization of patatin. Ultrathin sections of potato tubers or leaves induced for patatin expression were used for the subcellular localization of patatin protein. In both cases specific labelling 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 chloroplasts, mitochondria or nuclei of potato leaves induced for patatin expression also showed no label
U. Sonnewald et al. : Localization of patatin
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 sections 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.
181
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 approach was done using highly purified antibodies specific for the protein portion of patatin. The purification was necessary since the crude serum, raised against the glycosylated protein, showed crossreactivity with other plant glycoproteins. Thus, in earlier immunofluorescence studies, using the nonpurified patatin serum, an unspecific cell-wall fluorescence was obtained (data not shown). Greenwood and Chrispeels (1985) made a similar observation with antibodies raised against phaseolin, using tissue from wild-type tobacco as well as from transgenic tobacco expressing phaseolin; unspecific cell-wall fluorescence was observed in both tissues. Having purified the antibody we analyzed first the localization of patatin in potato tuber cells.
182 Patatin was found to be localized mainly in parenchyma 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 esterase 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 (Rosahl et al. 1987), and, as is evident from Fig. 2, the distribution of the esterase activity closely follows that found for patatin. Patatin is present in very high amounts in parenchyma cells of potato tubers. Its enzymatic activity could destroy cellular membranes, which might be an explanation for the observed instability 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 immunocytochemistry we found the protein to be localized in vacuoles of parenchyma cells from potato tubers. There was also some label found in the cytosol, but this could be explained by patatin being transiently present in the endoplasmic reticulum and Golgi apparatus during synthesis and processing. The structural preservation does not allow us to distinguish between the endoplasmic reticulum, Golgi apparatus and cytosol, but since patatin 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 indicate 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 formation 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 response, since it is present in such high amounts.
U. Sonnewaldet al. : Localizationof patatin 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 eicosapentaenoic acid are present in membranes of Phytophthora infestans but not in potatoes. These compounds serve as elicitors and are responsible for the hypersensitive 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 function 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 Botany, C-016 Universityof California, San Diego, La Jolla, Calif., USA) for the help during the purificationand further characterization of the patatin antibodies.We also thank R. Lurz (MaxPlanck-Institut ffir molekulareGenetik, Berlin, WestGermany) for the opportunityto use the electron microscopeand 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 Bundesministeriumffir Forschungund Technologie.
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