Proc. Nati. Acad. Sci. USA Vol. 86, pp. 1529-1533, March 1989 Botany Specific localization of a plant cell wall glycine-rich protein in protoxylem cells of the vascular system (immunocytolocalization/lignin/Phaseolus vulgaris) BEAT KELLER, MATTHEW D. TEMPLETON, AND CHRISTOPHER J. LAMB'* Plant Biology Laboratory, Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138 Communicated by J. E. Varner, November 28, 1988 (receivedfor review September 27, 1988)

ABSTRACT An antibody against glycine-rich protein 1.8 amino acid; thus, it resembles the sequence of silk fibroin. of bean (Phaseolus vulgaris L.) was used for immunogold/ During normal development, the bean GRP 1.8 gene is only silver localization ofthe protein in different organs ofthe plant. expressed in young tissue, but it can be induced in older In hypocotyls, ovaries, and seed coats, the protein was found tissue by mechanical wounding. Antibodies against a fusion specifically in xylem cells of the vascular tissue. In hypocotyls, protein of 8-galactosidase and GRP 1.8 react with a protein only protoxylem cells were labeled with the antibody, which present in the cell wall fraction of developing ovaries (10). indicates a remarkable cell-type specificity for accumulation of The reaction pattern on tissue prints shows that this GRP is this cell wall protein. In mature hypocotyls, the protein was associated with the vascular system. A gene putatively restricted to the same subset of xylem cells but was no longer encoding a similar protein was first described in petunia (11). detected on tissue prints, where a positive antibody reaction Here we describe the localization of the bean GRP 1.8 depends on the transfer ofsoluble material from plant tissue to protein at the cellular level. By using immunocytochemistry, the nitrocellulose filter. This indicates that the glycine-rich the protein was found exclusively in tracheary elements of protein is insolubilized in the cell wall during development. In the protoxylem. The spatial pattern of GRP 1.8 in the cell longitudinal sections oftracheary elements ofyoung hypocotyls walls of these elements was similar to the distribution of and seed coats, the antibody stained a pattern very similar to lignin, which indicates a close interaction and possible that of the lignified secondary thickenings of the cell wall, functional relationship between the two components during which suggests a close functional relationship between glycine- formation of the cell wall. rich protein and lignin deposition during cell wall biogenesis in protoxylem cells. MATERIALS AND METHODS Plant Material. All plant material was from Phaseolus The polysaccharide portion of plant cell walls consists of vulgaris cv. Tendergreen. For seed coat proteins, we also cellulose, hemicelluloses, and pectic polysaccharides. De- analyzed the cultivar Greensleave. Plants from which hypo- pending on their function, certain cell types have specifically cotyls were obtained were grown in the dark. Fruits and seed adapted cell walls. In epidermal cells, cutin and suberin coats were from plants grown in the greenhouse or the field. reduce water loss. In xylem, the water-conducting elements Total seed coat protein for the study of developmental of the vascular system in higher plants, the cell walls are synthesis of GRP 1.8 was prepared by homogenizing seed lignified (1). The deposition oflignin, a polyphenolic polymer coats in SDS gel electrophoresis loading buffer (12). After a (2), reinforces the cell wall so that it can withstand the low speed centrifugation to remove insoluble components, pressure of the surrounding tissue and resist the tendency of the supernatant was directly loaded on the gel. the tracheary elements to collapse under tension when the Paraffin Embedding of Plant Tissue. Plant material was xylem cell has matured and contains the water column rather fixed in 2% (wt/vol) glutaraldehyde in 50 mM phosphate than living cytoplasm (3). Protoxylem is the first part of the buffer (pH 7.5) containing 150 mM NaCl for 2 hr at 40C. primary xylem to differentiate and mature. The tracheary Tissue was then dehydrated and embedded in paraffin es- elements of the protoxylem usually have a thin primary cell sentially as described by O'Brien and McCully (13). Sections wall on which rings or helices of secondary wall thickenings (10 ,um) were cut with an IEC microtome and mounted on are deposited (4). These wall thickenings contain cellulose polylysine-subbed slides (14). Paraffin was removed with two and lignin, but no protein has yet been shown to be a changes of xylene, and sections were hydrated in an structural component of this cell wall. ethanol/water series just prior to antibody treatment. To date, only two kinds of structural proteins have been Immunolocalization of GRP 1.8 and Tissue Prints. localized in plant cell walls. Hydroxyproline-rich glycopro- Immunogold/silver staining was performed as described by teins (HRGPs) are found in the walls of many cell types (5). Springall et al. (15). After rehydration, paraffin sections were In seed coats, an HRGP was localized in the hourglass cells treated with anti-GRP 1.8 antiserum diluted 1:3000 and and palisade epidermal cells (6). HRGPs are thought to incubated for 2 hr at room temperature. For immunogold strengthen the wall as they become covalently cross-linked staining and silver enhancement, a kit from Janssen Life into the wall network [reviewed by Fry (7)]. HRGPs may also Sciences Products was used, and the manufacturer's protocol be involved in the defense reaction against pathogens, as they was followed. After silver enhancement, the sections were are markedly induced after attempted infection (8, 9). Re- either stained with 0.05% toluidine blue in water or left cently, we have isolated a bean (Phaseolus vulgaris L.) gene, unstained. Pectic acid in cell walls stains reddish purple with GRP 1.8, which encodes a glycine-rich protein (GRP). The toluidine blue, whereas polyphenols and lignin give a blue- structure of the protein was deduced from the gene sequence green color (13). The sections were mounted in Permount and can be described by the formula (Gly-X)", where X is any (Fisher) and observed in a Nikon Diaphot TMD microscope.

The publication costs of this article were defrayed in part by page charge Abbreviations: GRP, glycine-rich protein; HRGP, hydroxyproline- payment. This article must therefore be hereby marked "advertisement" rich glycoprotein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

1529 Downloaded by guest on September 30, 2021 1530 Botany: Keller et al. Proc. Natl. Acad. Sci. USA 86 (1989) Photomicrographs were taken on Kodak Ektachrome 50 paraffin-embedded plant material were treated with anti-GRP tungsten or Kodak Technical Pan film 2415. 1.8 antiserum raised against a f3-galactosidase-GRP 1.8 Two antisera (4836 and 4837) from different rabbits were fusion protein (10). Two antisera from different rabbits were used. One (4836) we have described earlier (10). The other used and gave the same results in all experiments. Treatment antiserum (4837) against GRP 1.8 was also raised by immu- of the antisera with 8-galactosidase to avoid any possible nization with the jB-galactosidase-GRP 1.8 fusion protein and reaction ofanti-8-galactosidase antibodies present in the sera gave results that were indistinguishable from those obtained did not have any effect on the results. with antiserum 4836 in all the experiments. Antibodies In cross-sections of young hypocotyls, GRP 1.8 was found against the 8-galactosidase part of the fusion protein were to be localized specifically in tracheary elements of the absorbed by incubating the antiserum with a lysate of an protoxylem (Fig. la). The walls of these cells stained blue- Escherichia coli strain overexpressing B3-galactosidase green with toluidine blue, indicating the presence of lignin (pTRB1, ref. 16). The 100-fold concentrated bacterial lysate (13). In sections treated with a control serum, no such was centrifuged for 10 min at 12,000 x g in an SS34 rotor. The reaction was observed (Fig. lb). In the coats of green, supernatant contained high amounts of 83-galactosidase (>1 maximal mg/ml). One-half milliliter of the antiserum was incubated immature seeds that had almost reached their with 0.1 ml ofthe lysate for 2 hr at room temperature and was weight, GRP 1.8 was also specifically localized in tracheary then centrifuged. As controls we used two preimmune sera as elements of the vascular tissue (Fig. ic). The silver deposi- well as an antiserum raised against a minor protein of the tion indicating antibody binding was not uniform in these plant plasma membrane, which was kindly provided by N. tracheary elements and matched the pattern of lignin depo- Oleski (Salk Institute). None of these control sera gave any sition (Fig. ld). Again, no reaction was found with control positive reaction, and no reaction was observed when anti- serum (Fig. ld). serum was completely omitted. All these controls indicate If a protoxylem tracheary element in young hypocotyls (7 that the observed immunolabeling reactions are specific for days after germination) is sectioned longitudinally, the black GRP. silver enhancement deposit representing antibody binding Gel Electrophoresis and Immunoblotting. SDS/PAGE was sometimes followed a spiral-like pattern (Fig. le) very similar performed according to Laemmli (12). Subsequent immuno- to the pattern of lignification. Usually, the sections are not blotting was done as described by Towbin et al. (17), and the cut absolutely tangentially to the xylem and thus the surface immunoreactive bands were detected as described by DeBlas of the section cuts through the tracheary element at an and Cherwinski (18). oblique angle. Examination of such sections showed that GRP 1.8 was in nearly all cases localized in annular or helical patterns corresponding to the lignified secondary wall thick- RESULTS enings, although occasionally the protein appeared to cover Immunolocalization of GRP 1.8. We have previously found the complete area of the tracheary element walls within the by immunolabeling of tissue prints on nitrocellulose filters plane of the longitudinal section. Both cases are clearly seen that GRP 1.8 of bean is closely associated with the vascular in Fig. 2 a and b, in which longitudinal sections ofhypocotyls system. To localize the protein in specific cells, sections of were left unstained after immunogold/silver labeling. Again,

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FIG. 1. Immunolocalization of GRP 1.8 in different tissues. Paraffin-embedded sections were treated with either anti-GRP 1.8 antiserum (a, c, and e) or preimmune serum (b and d). (a and b) Cross-sections of young (7 days after germination) hypocotyls. (c and d) Sections of seed coats of green seeds, which have almost reached their maximal weight. (e) Longitudinal section of young hypocotyl. The sections were stained with 0.05% toluidine blue in water. Lignified structures appear in blue-green, whereas the positive immunoreaction is indicated by the black silver deposit. (Bars = 50 ,um.) ., Downloaded by guest on September 30, 2021 ns;vt A. Botany: Kefler et aL Proc. Natl. Acad. Sci. USA 86 (1989) 1531 Table 1. Loss of GRP 1.8 immunoreactivity in tissue prints a during development Age of hypocotyls, Average Number of days after length of immunoreactive germination hypocotyls, cm regions per section* 7 3.5 7.2 ± 1.7 I 8 6.0 3.6 ± 3.2 .,P. 9 8.5 4.4 ± 4.3 11 14.0 0.0 ± 0.0 Tissue sections 2-mm thick were cut from the middle part of hypocotyls, and tissue prints were performed as described by Cassab and Varner (6). b *Each immunoreactive region corresponds to the protoxylem ele- ments of one vascular bundle. Values given are the means ± SD. amounts below the threshold level of detection are present, but the results clearly demonstrate a quantitative loss ofGRP

-A.- . immunoreactivity on tissue prints. -2. . It ... .., -,&. V. v To distinguish between insolubilization or loss of the antigen, we embedded 13-day-old hypocotyls, which did not show any positive reaction on tissue prints. Sections of this tissue were treated with anti-GRP 1.8 antiserum. There is still strong immunoreactivity in embedded sections of this tissue (Fig. 3 a and b). Interestingly, in this older tissue, the protein c is likewise only detected in protoxylem elements. No reac- tion was observed in sections treated with control antisera 4 (Fig. 3c). q, , . a

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FIG. 2. Immunolocalization of GRP 1.8 in longitudinal sections *tk0 of young hypocotyls. Sections were treated with anti-GRP 1.8 antiserum (a and b) or with preimmune serum (c). The sections cut through the tissue at an oblique angle to the xylem. (Bar = 100 S&m.)

no staining was observed in control sections treated with preimmune serum (Fig. 2c). In cross-sections of bean ovaries, GRP 1.8 was also found b in the regularly arranged tracheary elements in the pod wall (results not shown). Insolubilization ofGRP 1.8 During Hypocotyl Development.

= It is well established that certain cell wall HRGPs become I . m insolubilized in the wall during development [reviewed by Fry (7)]. To study the possible insolubilization of GRP, hypocotyls of different ages were analyzed by the tissue printing technique (6). In this technique, soluble cellular material is transferred to nitrocellulose by capillary action to leave an imprint image of the tissue sample. The nitrocellu- lose is then treated with the antibody. Thus, detection of the C antigen depends on its solubility and provides a rapid assay for possible insolubilization or loss of the protein. Reactivity with anti-GRP 1.8 antiserum was analyzed on tissue prints of hypocotyls 7 to 11 days after germination. We excised a 2-mm section from the middle part of 10 individual hypocotyls on each day. These tissue sections were then printed on nitrocellulose, and the number ofimmunoreactive regions were counted. Each immunoreactive area corre- sponds to the protoxylem elements of one vascular bundle. The average numbers of immunoreactive regions per section are shown in Table 1. In young hypocotyls (7 days), there is FIG. 3. Immunolocalization of GRP 1.8 in cross-sections of old a in tissue or 9 maximum of 8 such regions, slightly older (8 bean hypocotyls (13 days after germination). Sections were treated days), we have observed up to 12 regions, corresponding to with anti-GRP 1.8 (a and b) or with control antiserum (c). The the number of vascular bundles in such hypocotyls. How- sections were left unstained after immunolabeling. As in young ever, by day 11 no immunoreactive areas could be found hypocotyls (Fig. la), only protoxylem elements react with the anymore. We cannot exclude by this method that GRP antibody. (Bar = 100 s.m.) Downloaded by guest on September 30, 2021 1532 Botany: Keller et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1 2 3 4 5 6 strength combined with flexibility (19), GRP might well be involved in the provision of an extensible cell wall. These properties would probably be changed towards a more rigid structure after the observed insolubilization of GRP 1.8. Longitudinal sections of conducting elements reveal some details about the subcellular localization ofGRP in xylem cell I*.. walls. Whereas sometimes only the rings of the secondary thickened wall are stained with the antibody (Fig. le), in rare AG.Ibw sections we find a big area stained (see Fig. 2a). This indicates GRP1.8-_ that GRP is not only present in secondary thickenings but also possibly in the primary wall, where it might be more uniformly distributed. However, these large areas may also represent the secondary walls of obliterated protoxylem tracheary elements. Further studies should clarify the struc- tural, temporal, and functional aspects of the interaction between lignin and GRP during development. The vascular localization of GRP also explains the abun- dance of a GRP in soybean seed coats found by Varner and Cassab (20), since this organ is extremely rich in vascular tissue. We exploited the abundance of GRP in seed coats to study the kinetics ofGRP accumulation during development. FIG. 4. Analysis of GRP 1.8 accumulation during seed coat The marked accumulation of GRP during the phase of rapid development. Total seed coat proteins (60 ,ug) were separated on a increase in weight probably reflects the formation and dif- 10o SDS/polyacrylamide gel, blotted onto nitrocellulose, and then ferentiation of vascular tissue at this stage of development. reacted with anti-GRP 1.8 antiserum. Lanes 1-6 represent different GRP becomes insoluble during development, as it was no stages of seed development, which were defined by the total fresh longer detected in tissue prints of older hypocotyls but was weight ofthe seed: 1, 70 mg; 2, 170 mg; 3, 370 mg; 4, 1000 mg; 5, 1000 still present when embedded and sectioned material was mg; 6, 1100 mg. In stages 1-4, the pod is still green; in stages 5 and examined. GRP 1.8 is rich in tyrosine, which accounts for 7% 6, it is partially yellow. The arrow points to the band of GRP 1.8. ofthe amino acid residues (10). Tyrosine has been implicated Accumulation of GRP 1.8 During Seed Development. Seed in the intramolecular cross-linking of cell wall HRGPs coats were chosen to examine the accumulation of GRP 1.8 through isodityrosine linkages, and similar intermolecular during development. This tissue is very rich in vascular tissue linkages may be involved in the insolubilization of these and gives a strong signal with the antibody on sections (Fig. proteins (7). Such a mechanism might also be involved in lc). Total seed coat protein was extracted, and 60 ug of GRP cross-linking. Our data indicate that in hypocotyls GRP protein was analyzed by immunoblotting after separation of insolubilization is complete only when the major phase of the proteins on a SDS/polyacrylamide gel. GRP 1.8 was elongation growth of the hypocotyl is over, about 11 days detectable at all stages of seed coat development (Fig. 4). after germination, which is consistent with the involvement However, there was a strong increase of GRP 1.8 in the of GRP insolubilization in the termination of extension period of rapid weight accumulation. When the seed weight growth. was 370 mg, very little GRP 1.8 was found (Fig. 4, lane 3), The high tyrosine content of GRP 1.8, the potential for a whereas at a weight of 1000 mg, large amounts of GRP 1.8 highly regular arrangement of these residues in the molecule were observed (Fig. 4, lanes 4-6). The apparent molecular (10), and the close correlation of GRP accumulation with the mass of GRP 1.8 in seed coats is 53 kDa, which is identical pattern of lignification might suggest that these tyrosine with that of the protein found in cell walls of bean pods (10). residues are the substratum for initiation of the oxidative Concomitant with the marked increase of GRP 1.8 during the polymerization chain reaction for lignin synthesis. There are phase of rapid growth, additional immunoreactive bands a number ofadvantages in having such a reaction initiate from appear on the immunoblot. Most of these are of higher a protein rather than a low molecular weight metabolite: molecular mass than the GRP 1.8 species. These might and reduction of random represent GRP 1.8 molecules cross-linked to each other or to spatial temporal control, diffusion, other cell wall components. and control of the properties of the lignin (for example, different GRPs with different tyrosine arrays may lead to different lignin structures in terms of density and three- DISCUSSION dimensional pattern). GRP 1.8 was found to be a cell wall protein that is highly We have previously described a second GRP gene (GRP specific for tracheary elements. In cross-sections of hypo- 1.0) in bean (10), which has a similar, yet clearly distinguish- cotyls, it was possible to specifically localize GRP 1.8 in able, primary structure compared to GRP 1.8. It will be very protoxylem elements. These are the first tracheary elements interesting to determine whether this protein is also localized formed during development. No antibody labeling was found in the vascular system. As the promoter structures are rather in metaxylem elements. GRP 1.8 epitopes might not be different, but the protein structure quite similar, this protein exposed in the walls of these elements due to a completely might well accumulate in a different cell type. different wall structure in metaxylem. However, the pres- The protoxylem-specific synthesis of GRP 1.8 provides a ence of GRP 1.8 in the cell walls of protoxylem elements marker for the formation of the first xylem elements. Future might also reflect the functional difference between proto- analysis of the GRP 1.8 promoter in transgenic plants should xylem and metaxylem. Protoxylem, unlike metaxylem, needs enable us to define the cis- and trans-acting elements and to be very extensible during development (4). Moreover, factors involved in the differentiation ofearly vascular tissue. metaxylem typically has continuous regions of secondary Further studies on the regulation and function of the gene wall thickenings. GRP is proposed to have a structure similar encoding protoxylem-specific cell wall GRP hopefully will to silk fibroin (10, 11). Considering, for example, the prop- improve our understanding of wall biogenesis and the differ- erties of spider silk, which has an extraordinary tensile entiation of woody tissue in dicotyledonous plants. Downloaded by guest on September 30, 2021 Botany: KeHer et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1533 We thank T. Voelker for seed coat proteins and N. A. Oleski for 9. Corbin, D. R., Sauer, N. & Lamb, C. J. (1987) Mol. Cell. Biol. providing the antiserum against a plasma membrane protein. B.K. 7, 4337-4344. was the recipient of a postdoctoral fellowship from the European 10. Keller, B., Sauer, N. & Lamb, C. J. (1988) EMBO J. 7, Molecular Biology Organization. M.D.T. was a visiting scientist 3625-3633. from the Plant Disease Division, Department of Scientific and 11. Condit, C. M.,& Meagher, R. B. (1986) Nature (London) 323, Industrial 178-181. Research, Auckland, New Zealand. This work was sup- 12. Laemmli, U. K. (1970) Nature (London) 227, 680-685. ported by a grant from the Samuel Roberts Noble Foundation to 13. O'Brien, T. P. & McCully, M. F. (1981) The Study of Plant C.J.L. Structure: Principles and Selected Methods (Termacarphi Pty., Melbourne, Australia). 1. Esau, K. (1977) Anatomy ofSeed Plants (Wiley, New York). 14. Angerer, R. C., Stoler, M. & Angerer, L. M. (1987) in In Situ 2. Higuchi, T. (1985) Biosynthesis and Biodegradation of Wood Hybridization: Applications in Neurobiology, eds. Valentino, Components (Academic, New York). K., Eberwine, J. & Barchai, J. (Oxford Univ. Press, London), 3. Aloni, R. (1987) Annu. Rev. Plant Physiol. 38, 179-204. pp. 42-69. 4. Cutter, E. (1969) Plant Anatomy: Experiment and Interpreta- 15. Springall, D. R., Hacker, G. H., Grimelius, L. & Polak, J. M. tion, Part!. Cells and Tissues (Addison-Wesley, Reading, MA). (1984) Histochemistry 81, 603-608. 16. Buerglin, T. R. & DeRobertis, E. M. (1987) EMBO J. 6, 2617- 5. Cassab, G. I. & Varner, J. E. (1988) Annu. Rev. Plant Physiol. 2625. Plant Mol. Biol. 39, 321-353. 17. Towbin, H., Staehelin, T. & Gurdon, J. (1979) Proc. NatI. 6. Cassab, G. 1. & Varner, J. E. (1987) J. Cell Biol. 105, 2581- Acad. Sci. USA 76, 4350-4353. 2588. 18. DeBlas, A. L. & Cherwinski, H. M. (1983) Anal. Biochem. 133, 7. Fry, S. C. (1986) Annu. Rev. Plant Physiol. 37, 165-186. 214-219. 8. Showalter, A. M., Bell, J. N., Cramer, C. L., Bailey, J. A., 19. Gosline, J. M., DeMont, M. E. & Denny, M. W. (1986) En- Varner, J. E. & Lamb, C. J. (1985) Proc. Natl. Acad. Sci. USA deavour 10, 37-43. 82, 6551-6555. 20. Varner, J. E. & Cassab, G. I. (1986) Nature (London) 323, 110. 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