New Ways to Look at the Architecture of Plant Cell Walls1
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Plant Physiol. (1989) 91, 31-33 0032-0889/89/91/0031 /03/$01 .00/0 Received for publication March 19, 1989 and in revised form May 30, 1989 Communication New Ways to Look at the Architecture of Plant Cell Walls1 Localization of Polygalacturonate Blocks in Plant Tissues Joseph E. Varner*2 and Rosannah Taylor 3 Institute of Molecular Biology, Academia Sinica, Nankang Taipei, 11529 Republic of China (J.E.V.); and Biology Department, Washington University, St. Louis, Missouri (R.T.) ABSTRACT We report the use of Ni2" and Co2l on free-hand sections of soybean (Glycine max L.) and Bidens sp to localize polygalactu- ronates. In soybean only the hourglass cells of the seedcoat stain intensely. In the pod the epidermis of the outer pod wall and a few layers of subepidermal cells stain lightly, while that part of the funiculus adjacent to the seedcoat palisade epidermal cells stains heavily and the neck of the funiculus close to the pod also stains. In Bidens stem sections, the walls of the collenchyma stain most intensely. We are interested in the architecture of the cell walls of dicotyledonous plants. The general plan of a typical wall involves cellulose microfibrils embedded in a more or less amorphous matrix; the microfibrils resist tension, the matrix resists compression. The matrix includes hemicelluloses and pectins. Growing cells produce walls appropriate to their place in the cell body by controlling the relative rates of synthesis and secretion of the various wall components. In the case of pectin, because it is secreted with its uronic acid residues largely in the methyl ester form, dramatic changes in physical properties can be achieved in muro. Secretion of pectin methyl esterase (PME) or perhaps activation of PME already in place generates uronic acids with pKs in the range of 3.5 to 5. Thus protons are generated in muro. These lower the pH and raise the osmotic potential of the wall. PME preferentially hydro- lyzes uronic acid esters in blocks, thereby generating blocks of polygalacturonate residues that can bind Ca2" to form egg box structures with a high linear charge density (2). These structures consist of dimers and larger aggregates (6). The gelling ability of partially deesterified pectins in dilute solu- tions is remarkably sensitive to degree of esterification and concentration of Ca2" (and Ba2 , Sr", Cu2", Cd2", and Co2" but not K+ or Mg2+; Ni2" was not tried) (8). 'This project was supported by U.S. Department of Energy grant No. DE-FG0284ER13255 to J. E. V. and was conducted largely at the Institute of Molecular Biology of Academia Sinica, ROC, under the support of the National Science Council of ROC. 2 Present address: Department of Biology, Washington University, St. Louis, MO 63130. 3 National Science Foundation postdoctoral fellow in plant biology. We have wondered if these negatively charged pectate mac- romolecules might interact with the positively charged exten- sins. Immunocytochemical methods have been used to local- ize extensin (1, 7) and rhamnogalacturonan I (5) in higher plant cell walls, and immunocytochemical methods have been used to localize alginic acid in brown algae (9). Alginates have also been localized with the delightfully ingenious oligogulu- ronate hybridization probes. In this procedure isolated blocks of guluronate sequences were labeled with a fluorochrome and added to sections of Fucus along with Ca2" or EDTA. Binding of the probe occurred only in the presence of Ca2", and the binding was specific for distinct regions of specific cell types (10). The success of Vreeland et al. (10) in replacing in situ a gel component with an externally applied probe encouraged us to try an analogous experiment. We reasoned that Ni2" and Co2" should bind to uronic acids just as Ca2" does because (a) Thibault and Rinaudo (8) showed in model in vitro studies that Co2" binds to polygacturonate as strongly as Ca2" and (b) Ni2" and Co2" are similar in many respects. The walls which bound Ni2" and Co2" were visualized by addition of Na2S after treatment of the sections with Ni2+ or Co2+. MATERIALS AND METHODS Nickel or Cobalt Staining Plant materials were collected from a vacant lot near the Institute of Molecular Biology. All sections were made free hand with Gillette Blue Blade double-edge razor blades. The sections were washed (a) in 0.5% Nonidet P-40 (NP-40) for 30 min, (b) in NaOCl (bleach purchased at a local grocery and diluted fivefold) for 30 min (step b hydrolyzes pectin methyl esters and is omitted as desired), and (c) in 0.05% NP- 40 for 30 min. These sections were placed in 10 mm NiCl2 or 2 mm Co2" for 30 min and were rinsed in 0.05% NP-40 for 30 min. After placing the prepared sections in distilled water, Na2S was added to a final concentration of 0. 1 to 1 %. A dark brown to black color develops in seconds. A wide latitude is tolerable in each of these steps. The results were easily observable with a high quality lOx hand lens or a dissection microscope. For photography we used Nikon and Olympus dissection scopes with top lighting or transmitted light. 31 Downloaded from https://academic.oup.com/plphys/article/91/1/31/6085438 by guest on 25 December 2021
Transcript of New Ways to Look at the Architecture of Plant Cell Walls1
Plant Physiol. (1989) 91, 31-330032-0889/89/91/0031 /03/$01 .00/0
Received for publication March 19, 1989and in revised form May 30, 1989
Communication
New Ways to Look at the Architecture of Plant Cell Walls1
Localization of Polygalacturonate Blocks in Plant Tissues
Joseph E. Varner*2 and Rosannah Taylor 3
Institute of Molecular Biology, Academia Sinica, Nankang Taipei, 11529 Republic of China (J.E.V.); andBiology Department, Washington University, St. Louis, Missouri (R.T.)
ABSTRACT
We report the use of Ni2" and Co2l on free-hand sections ofsoybean (Glycine max L.) and Bidens sp to localize polygalactu-ronates. In soybean only the hourglass cells of the seedcoat stainintensely. In the pod the epidermis of the outer pod wall and afew layers of subepidermal cells stain lightly, while that part ofthe funiculus adjacent to the seedcoat palisade epidermal cellsstains heavily and the neck of the funiculus close to the pod alsostains. In Bidens stem sections, the walls of the collenchymastain most intensely.
We are interested in the architecture of the cell walls ofdicotyledonous plants. The general plan of a typical wallinvolves cellulose microfibrils embedded in a more or lessamorphous matrix; the microfibrils resist tension, the matrixresists compression. The matrix includes hemicelluloses andpectins. Growing cells produce walls appropriate to their placein the cell body by controlling the relative rates of synthesisand secretion of the various wall components. In the case ofpectin, because it is secreted with its uronic acid residueslargely in the methyl ester form, dramatic changes in physicalproperties can be achieved in muro. Secretion ofpectin methylesterase (PME) or perhaps activation ofPME already in placegenerates uronic acids with pKs in the range of 3.5 to 5. Thusprotons are generated in muro. These lower the pH and raisethe osmotic potential of the wall. PME preferentially hydro-lyzes uronic acid esters in blocks, thereby generating blocksof polygalacturonate residues that can bind Ca2" to form eggbox structures with a high linear charge density (2). Thesestructures consist of dimers and larger aggregates (6). Thegelling ability of partially deesterified pectins in dilute solu-tions is remarkably sensitive to degree of esterification andconcentration of Ca2" (and Ba2 , Sr", Cu2", Cd2", and Co2"but not K+ or Mg2+; Ni2" was not tried) (8).
'This project was supported by U.S. Department of Energy grantNo. DE-FG0284ER13255 to J. E. V. and was conducted largely atthe Institute of Molecular Biology of Academia Sinica, ROC, underthe support of the National Science Council of ROC.
2 Present address: Department of Biology, Washington University,St. Louis, MO 63130.
3 National Science Foundation postdoctoral fellow in plant biology.
We have wondered if these negatively charged pectate mac-romolecules might interact with the positively charged exten-sins. Immunocytochemical methods have been used to local-ize extensin (1, 7) and rhamnogalacturonan I (5) in higherplant cell walls, and immunocytochemical methods have beenused to localize alginic acid in brown algae (9). Alginates havealso been localized with the delightfully ingenious oligogulu-ronate hybridization probes. In this procedure isolated blocksof guluronate sequences were labeled with a fluorochromeand added to sections of Fucus along with Ca2" or EDTA.Binding of the probe occurred only in the presence of Ca2",and the binding was specific for distinct regions of specificcell types (10).The success of Vreeland et al. (10) in replacing in situ a gel
component with an externally applied probe encouraged usto try an analogous experiment. We reasoned that Ni2" andCo2" should bind to uronic acids just as Ca2" does because(a) Thibault and Rinaudo (8) showed in model in vitro studiesthat Co2" binds to polygacturonate as strongly as Ca2" and(b) Ni2" and Co2" are similar in many respects. The wallswhich bound Ni2" and Co2" were visualized by addition ofNa2S after treatment of the sections with Ni2+ or Co2+.
MATERIALS AND METHODS
Nickel or Cobalt Staining
Plant materials were collected from a vacant lot near theInstitute of Molecular Biology. All sections were made freehand with Gillette Blue Blade double-edge razor blades. Thesections were washed (a) in 0.5% Nonidet P-40 (NP-40) for30 min, (b) in NaOCl (bleach purchased at a local groceryand diluted fivefold) for 30 min (step b hydrolyzes pectinmethyl esters and is omitted as desired), and (c) in 0.05% NP-40 for 30 min. These sections were placed in 10 mm NiCl2 or2 mm Co2" for 30 min and were rinsed in 0.05% NP-40 for30 min. After placing the prepared sections in distilled water,Na2S was added to a final concentration of 0.1 to 1%. A darkbrown to black color develops in seconds. A wide latitude istolerable in each of these steps.The results were easily observable with a high quality lOx
hand lens or a dissection microscope. For photography weused Nikon and Olympus dissection scopes with top lightingor transmitted light.
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Plant Physiol. Vol. 91,1989
Physical Printing
Sections were cut from fresh tissue at thicknesses of 300 to400 ,tm and placed in 0.05 M CaCl2 or 0.05 M EGTA bufferedat pH 5.9 with 0.1 M potassium acetate. After 1 h some of thesections from EGTA were rinsed briefly in water and placedin 0.05 M CaCl2 or 0.05 M NiCl2 buffered at pH 5.9 with 0.1M potassium acetate. After 1 h two sections were selectedfrom each ofthe four treatments for printing on nitrocellulosemembrane (Schleicher and Shuell NCBA85). The sectionswere picked out ofthe washing dish with a piece of filter paper3 or 4 cm long and 4 or 5 mm wide. In a few seconds theblotting capacity of the paper removes the free water from thesection. The sections were then placed on a small piece (1 x
5 cm is convenient) of nitrocellulose membrane placed on
one thickness of Whatman No. 1 filter paper. A 2 x 2 cm
piece ofthe hard paper that is used to protect the nitrocellulosemembrane during shipment is placed over the section. Thenthe section surface is printed onto the nitrocellulose by press-
ing firmly on the hard paper with an index finger for 2 or 3s. The print can be observed immediately although its ap-
pearance changes somewhat as the moisture evaporates fromthe print. The physical prints are stable at least for months.The prints should be observed both by transmitted light andlow angle unilateral lighting in order to get the most possibleanatomical information from the print.
RESULTS AND DISCUSSION
Nickel or Cobalt Staining
In preliminary experiments we visualized bound nickel byadding diluted Clorox to the Ni2+ treated sections to form a
black nickelic oxide. However, the color fades in minutes to
C
1i~ 2mm
Figure 1. Free hand cross-section of Bidens stem. Nickel binding isgreater in epidermis and collenchyma cell walls with some binding inprimary xylem, cambium, and phloem cell walls. C, collenchyma; Xy,xylem.
Figure 2. Bidens cross-section physical prints. A, Treatment withwater for 30 min before printing. This print shows clear impressionsof the epidermis and collenchyma bundles. B, Treated with EGTA for30 min and printed. Note reduced impressions of the epidermis(arrow) and collenchyma bundles. E, epidermis; C, collenchyma.
hours because nickelic oxide is a powerful oxidant, and itslowly oxidizes the wall components to regenerate the color-less nickelous ion. Nonetheless, that procedure allowed us tosee that epidermal and collenchyma cell walls of the stems ofgrape, celery, and several other common species selectivelybound much more Ni2" than the walls of other cell types.
Visualization of bound Ni2" or Co2" with Na2S is more
satisfactory. Bidens pilosa stem sections bind Ni2+ most abun-dantly in the epidermal and collenchyma cell walls (Fig. 1).The primary xylem and the cambium and phloem cell wallsstain lightly, and the walls of other types show little or no
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32 VARNER AND TAYLOR
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NEW WAYS TO LOOK AT THE ARCHITECTURE OF PLANT CELL WALLS
Figure 3. Freehand cross-section of soybean and pod stained withNi2+. In the seed coat only the hourglass cells stain intensely. In thepod the epidermis of the outer pod wall and a few layers of subepi-dermal cells stain lightly. The portion of the funiculus (arrow) that isadjacent to the seedcoat palisade epidermal cells stains heavily asdoes the neck of the funiculus. Sc, seed coat; P, pod; Hg, hourglass;F, funiculus; Pe, palisade epidermis.
staining. Celery and ageratum stems show the same kind ofspecificity for Ni2+ binding (data not shown), that is stainingof epidermis and collenchyma, as Bidens.What is the evidence that Ni2+ and Co2` ions occupy the
positions normally filled by Ca2+? First, Ca2+ competes withNi2" and Co21 for those positions. When sections are soakedin solutions containing Ni2+ and Ca2+ or Co2" and Ca2+, theconcentration of Ca2+ must be 5 to 10 times that of Ni2` orCo21 to prevent entirely their binding to the cell walls. Mg2+and K+ do not compete with either Ni2` or Co2+. Second,treatment of the sections with EGTA softens the epidermaland collenchyma tissue so that they no longer leave a physicalprint on nitrocellulose membrane (Fig. 2). Third, readditionof Ni2l or Co2+ to sections that have been treated with EGTAfirms up the epidermis and collenchyma as effectively as Ca2+(data not shown). It must be said that none of the ions willbring the tissues all the way back to their original firmness.This may be due to loss of polygalacturonates to solutionduring the EGTA treatment or to an irreversible shrinkage ofthese tissues due to loss of Ca2+ (3).
Because we have some immunocytochemical data on thedistribution of extensin in the bean seed coat and pod ofsoybeans (1, 4), we have tried the Ni2+ stain on soybean (Fig.3). In the seed coat only the hourglass cells stain intensely.
Extensin is abundant in both hourglass cells and palisadeepidermal cells. In the pod, the epidermis of the outer podwall and a few layers of subepidermal cells stain lightly whilethe conducting cells of the phloem are intensely stained. Inthe funiculus, a single layer of cells adjacent to the seedcoatpalisade epidermal cells stains heavily, and the neck of thefuniculus close to the pod also stains.
It is clear from the results reported here that the use of Ni2"and/or Co2" to study the role of polygalacturonates in wallarchitecture will be rewarding. In those cell walls where exten-sin and polygalacturonates colocalize there is an opportunityto find if and how interaction occurs.
ACKNOWLEDGMENTS
We thank Fang Guang-Chen for running through the proceduresdescribed in "Materials and Methods" to make certain that theprocedures worked as written. The initial experiments that demon-strated the feasibility of using Ni2" as a pectin stain were by J. E. V.in the laboratory of Prof. Liliana Cardemil, University of Chile,Faculty of Sciences, Department of Biology, Santiago, in connectionwith work supported by National Science Foundation grant INT-8502512.
LITERATURE CITED
1. Cassab GI, Varner JE (1987) Immunocytolocalization of exten-sin in developing soybean seedcoats by immunogold-silverstaining and by tissue printing on nitrocellulose paper. J CellBiol 105: 2581-2588
2. Grant GT, Morris ES, Reese DA, Smith PJC, Thom D (1973)Biological interactions between polysaccharides and divalentcations: the egg-box model. FEBS Lett 32: 195-198
3. Kutschera U, Briggs WR (1988) Growth, in vivo extensibility,and tissue tension in developing pea internodes. Plant Physiol86: 306-311
4. Meyer DJ, Afonso CL, Galbraith DW (1988) Isolation andcharacterization of monoclonal antibodies directed againstplant plasma membrane and cell wall epitopes: identificationofa monoclonal antibody that recognizes extensin and analysisof the process of epitope biosynthesis in plant tissue and cellcultures. J Cell Biol 107: 163-178
5. Moore P, Darvill AG, Albersheim P, Staehelin LA (1986) Im-munogold localization ofxyloglucan and rhamnogalacturonanI in the cell walls of suspension-cultured sycamore cells. PlantPhysiol 82: 787-794
6. Powell DA, Morris ER, Gidley MG, Reese DA (1982) Confor-mations and interactions of pectins. II. Influence of residuesequence on chain association in calcium pectate gels. J MolBiol 15: 517-531
7. Stafstrom JP, Staehelin LA (1988) Antibody localization ofextensin in carrot cell walls. Planta 174: 321-332
8. Thibault JF, Rinaudo M (1986) Chain association of pecticmolecules during calcium-induced gelation. Biopolymers 25:455-468
9. Vreeland V, Zablackis E, Doboszewski B, Laetsch WM (1987)Molecular markers for marine algal polysaccharides. Hydro-biologia 151/152: 155-160
10. Vreeland V, Morse SR, Robichaux RH, Miller KL, Hua S-ST,Laetsch WM (1989) Pectate distribution and esterification inDubeutia leaves and soybean nodules studies with a fluorescenthybridization probe. Planta 177: 435-446
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