Isolation, histochemistry and monosaccharide composition of the walls of root hairs from Heterozostera tasmanica (Martens ex Aschers.) den Hartog

Aquatic Botany, 47 (1994) 29-37 0304-3770/94/$07.00 © 1994 - Elsevier Science B.V. All fights reserved

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Isolation, histochemistry and monosaccharide composition of the walls of root hairs from Heterozostera tasmanica (Martens ex Aschers. ) den Hartog J u d i t h Webster ~, Bruce A. Stone* Department of Biochemistry, La Trobe University, Bundoora, Vic. 3083, Australia (Accepted 18 August 1993)

Abstract The walls of root hairs of the marine monocotyledon Heterozostera tasmanica (Martens ex Aschers. ) den Hartog were isolated after disruption of the root hairs by sonication. Histologicalexamination using a range of stains indicated the presence of polyanionic molecules in the walls. After treatment with the detergent cetyltrimethylammonium bromide (CTAB), the staining with Alcian Blue was abolished. Protein staining was observed only after CTAB treatment. The root hairs stained with Auramine O before and after CTAB treatment suggesting the presence of lipid and/or cutin. Both Congo Red and Calcofluor White M2R induced intense ultraviolet fluorescenceafter detergent treatment, indicating the presence of fl-glucans. Callose ( ( 1~3)-~-gluean) deposits were revealed, after CTABtreatment, by the aniline blue tluorochrome. Carbohydrates constituted 83% (w/w) of the root hair wall. No lignin was detected, but a significant amount ( 17%, w/w) of the wall preparation was not recovered. The main monosaecharides present were apiose, 40 mol %, uronic acids 33 mol % and glucose, 10 mol %, together with smaller amounts of rhamnose, fucose, arabinose, xylose, mannose and galactose totalling 17 mol %. It is likely that the major wall polysaccharideis an apiogalacturonan and that most of the glucosearises from cellulose.

Introduction R o o t hairs are s p e c i a l i z e d a b s o r b i n g s t r u c t u r e s arising b y d i f f e r e n t i a t i o n o f t r i c h o b l a s t cells in the r o o t e p i d e r m i s in a r e g i o n b e h i n d the z o n e o f active cell division. T h e y arise f r o m small papillae o n the t r i c h o b l a s t surface a n d like p o l l e n t u b e s e x t e n d b y tip g r o w t h . R o o t hairs are generally p r e s e n t o n the r o o t s o f terrestrial plants, a l t h o u g h t h e i r d e n s i t y a n d length is q u i t e v a r i a b l e *Corresponding author. Ipresent Address: Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Vic. 3052, Australia.

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(Clarkson, 1985). Functionally, they are water and nutrient absorbing organs, their existence greatly extends the absorptive surface area of the root and its zone of 'penetration' into the substrate. Root hair development and factors influencing their initiation have been discussed by Cormack (1962), Smith and Robertson ( 1971 ), and Bittner and Buschmann ( 1983 ). The appearance of root hair walls has been studied by light and electron microscopy (Scott, 1950; Scott et al., 1956, 1958). The walls are covered by a mucilagenous sheath. The outer, a-layer, which is confined to the tip, is composed of cellulosic microfibrils that are orientated randomly (Frey-Wyssling and Miihlethaler, 1949), whereas in the inner layer, which is continuous over the whole length of the hair, the microfibrils are ordered (Pluymaekers, 1982; Sassen et al., 1985; Lloyd and Wells, 1985; Emons, 1986; Emons and van Maaren, 1987). The a-layer is also reported to contain pectins and other non-cellulosic polysaccharides. Proteins have been found in the root hair walls of white clover (Trifolium repens L.) (Gerhold et al., 1985), peas (Pisum sativum L.) (Rohm and Werner, 1987) and soyabeans (Glycine max L.) (Werner and Wolff, 1987 ). The organization of the microfibrillar component of root hairs has been examined by Belford et al. ( 1958), and Belford and Preston ( 1961 ), but the only comprehensive chemical analysis of root hair walls was made by Mort and Grover ( 1988 ). They analyzed root hair walls of two terrestrial monocotyledons, maize (Zea mays L. ), wheat ( Triticum aestivum L. ) and six dicotyledons, soyabean ( Glycine max), pea (Pisum sativum), alfalfa (Medicago sativa L.), spinach ( Spinacea oleracea L. ), radish ( Rhaphanus sativus L.) and cotton (Gossypium hirsutum L.); their monosaccharide compositions were shown to be similar. Non-carbohydrate, non-protein material was also found to contribute to the structural integrity of the root hair wall. As part of a study of the walls of vegetative parts of the marine monocotyledon Heterozostera tasmanica (Martens ex Aschers. ) den Hartog and its pollen, we describe in this paper the histochemistry and monosaccharide composition of walls of the root hairs. This first description of the composition of root hair walls for a marine monocotyledon is compared with the published compositions of root hair walls from terrestrial mono- and dicotyledons. Materials and methods

Collection of root hairs Roots ofH. tasmanica were dissected from whole plants collected from the sea-bed in Corio Bay near Geelong, Victoria, Australia (Webster and Stone, 1994). The entire root surface was covered by abundant root hairs entrapping sand grains and organic detritus. These contaminating particles were removed by gentle sonication in ice-cold 80°/0 ( v / v ) aqueous ethanol without

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significantly breaking the root hairs. The dense detritus was sedimented by low speed centdfugation. The cleaned roots were air-dried and comminuted in a rotating blade mill (Wiley mill, Arthur H. Thomas, Philadelphia, PA, USA) over a 60 mm mesh sieve. By increasing the sonication time, root hairs could be broken from the root, although some fragmentation occurred at this step. Free root hairs were collected as a layer over the pelleted roots by low speed centrifugation. This layer was carefully removed with a Pasteur pipette and air-dried. Small portions of root epidermis were still attached to some of the root hairs. The dissociated root hairs were washed extensively with 80% (v/v) aqueous ethanol to remove residual cytoplasmic and extracellular mucilagenous material. Staining with iodine in potassium iodide failed to reveal any starch contaminants. The walls were dried by solvent exchange (ethanol, methanol and n-pentane ) and then in vacuo and stored over silica gel.

Neutral monosaccharide analysis Wall preparations were hydrolyzed with sulphuric acid under the conditions described by Saeman et al. (1954). The monosaccharides released were reduced and acetylated, and the alditol acetates separated and quantified by gas chromatography according to the procedure ofBlakeney et al. ( 1983 ). All analyses were in duplicate.

Uronic acids analysis The uronic acid content of duplicate wall samples was determined by a colorimetric method (Blumenkrantz and Asboe-Hansen, 1973) with galactutonic acid as a standard.

Microscopical methods For bright field microscopy, a Carl Zeiss Universal microscope (Carl Zeiss, Germany) fitted with a 50 W tungsten lamp was used. For fluorescence microscopy, the microscope was equipped with a Zeiss III RS epi-illuminating condenser, and an HBO 200W illuminator, with appropriate fluorescence filter combinations (FC I or FC II). FC I (blue excitation 2m~ 365 nm) was used for both autofluorescence and Calcofluor fluorescence; FC II (green excitation 2m~ 546 rim) was used for Congo red, Aniline blue and Auramine O fluorescence. Alcian Blue 8GX: (C.I. 74240) BDH, Peele, UK; 1% (w/v) in 3% (v/v) acetic acid, pH 2.5. Ruthenium Red: BDH, Peele, UK; 0.02% (w/v) in 1% (w/v) ammonium acetate.

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Toluidine Blue: (C.I. 52040) BDH, Poole, UK; 0.05% (w/v) in 0.02 M sodium benzoate buffer, pH 4.4. Ponceau 2R: (C.I. 16150) Chroma-Gesellschaft, Schmid and Co., Stuttgart, Germany; 0.2% (w/v) in water with two drops of 18 M H2S04 100 ml- 1. Phloroglucinol-HCl: No. P-3502 Sigma Chemical, St. Louis, MO, USA; 20 mg phloroglucinol in 0.95 ml ethanol and 0.05 ml water plus 2 ml 10 M HC1. Auramine O: (C.I. 41000) Sigma Chemical, St. Louis, MO, USA; 0.01% (w/v) in 0.05 M tris-HC1 buffer, pH 7.2. Calcofluor White M2R New: (C.I. 40622) American Cyanamid, Bound Brook, NJ, USA; 0.01% (w/v) in water. Congo Red: (C.I. 22120) Canadian Laboratory Supplies, Canada; 0.01% (w/v) in Na-phosphate buffer pH 7.8, iodine, 0.2 in 50% (v/v) ethanol. Aniline Blue Fluorochrome: (Sodium, 4, 4'-[carbonylbis(benzene-4, 1diyl)bis (imino) ] bisbenzene-sulphonate) Biosupplies Australia, Melbourne, Australia; 0.25% (w/v) in water. Cetyltrimethylammonium bromide (CTAB) was used at 50/0 (w/w) and treatments were for I 0 min with shaking at room temperature. Results and discussion

Histochemistry of root hair walls Root hair walls were examined both before and after treatment with CTAB. The staining properties of the walls are summarized in Table 1. The cationic stains, Alcian Blue, Ruthenium Red and Toluidine Blue, all stained the root hair walls before CTAB treatment. These stains react with polyanionic polymers, e.g. pectins, however, only Ruthenium Red and Toluidine Blue gave positive reactions after CTAB treatment. On the other hand, Ponceau 2R staining for protein was apparent only after CTAB treatment. After CTAB treatment, the walls of the hairs fluoresced strongly with Calcofluor and Congo Red, especially near their point of attachment to the epidermal cell, indicating they were rich in a fl-glucan, presumably cellulose. After CTAB washing, the Aniline Blue fluorochrome induced small patches of yellow fluorescence that were distributed randomly along the hairs. This is consistent with the presence of callose deposits and similar to the pattern of callose deposition in root hairs of other species (Lerch, 1960; Kumarasinghe and Nutman, 1977; Cooper, 1982; Berry and McCuUy, 1989). Callose deposition may be a result of wounding during plant collection. Treatment with the amphipathic detergent, CTAB, may have two effects. One could be by the formation of insoluble complexes with polyanionic polymers through its cationic group, that would block staining. Thus, the observation that Alcian Blue staining is abolished by CTAB treatment may be explained if CTAB, like cetylpyridinium chloride, blocks Alcian Blue binding

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Z Webster,B.A. Stone~Aquatic Botany 47 (1994) 29-37

Table 1 Staining properties of root epidermal cells and root hairs ofH. tasmanica (Martens ex Aschers. ) den Hartog Staining reagent

Stain

CTAB/Stain

Root

Hair

Root

Hair

Alcian blue

Brown

Blue

Brown

Clear

Ruthenium red

Brown

Red

Brown

Red

Toluidine blue

Brown

Red

Brown

Red

Ponceau 2R

Red

Clear

Red

Red

Phloroglucinol/HC1

-ve

--

- v e

-

Auramine O

- ve

+ve

-ve

+ve

--

ve

-ve

-re

+ve

-ve

-ve

-ve

+ ve

-ve

-ve

+ve

+ve

Calcofluor White M2R New Congo red Aniline blue fluorochrome

ve

ve

Specificity

Polyanions Scott et al. ( 1964); Benes (1968) Polyanions Luft (1971) Stains walls Lignin green/blue Polyanions pink/purple O'Brien and McCully ( 1981 ) Protein Blakeney and Stone ( 1981 ) Lignin Harris et al. (1982) Sporopollenin, cutin and lipid Heslop-Harrison (1977) Chitin and fl-glucans Wood et al. (1983) Cellulose Wood et al. (1983) (l~3)-and ( 1~ 3,1 -, 4 ) -fl-D-glucans Evans et al. ( 1984); Stone et al. (1984)

Abbreviations: - v e , negative; + ve, positive.

by competing for anionic binding sites on wall polymers (Scott et al., 1964). This cannot, however, account for the results with Ruthenium Red and Toluidine Blue which are also cationic dyes. It may be that their binding to the polyanionic polysaccharides is tighter than Alcian Blue and that they are not displaced by CTAB. The second action could be the removal, by solution, of some lipid material whose presence is indicated by Auramine O staining, to expose underlying wall structures. This would explain the staining of the root hair walls by Ponceau 2R, Calcofluor White M2R, Congo Red and the Aniline Blue fluorochrome after CTAB treatment. These observations are comparable with those made for the walls of white mustard (Sinapis alba L. ) root hairs (Belford and Preston, 1961 ) where amorphous polysaccharide material had to be removed by successive extraction with H202-glacial acetic acid ( 1:1, v/v), 2% H2SO4 and 2% KOH, before cellulose microfibrils in the a-layer could be detected. Auramine O stained the walls both before and after treatment with CTAB.

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This stain is said to be specific for mycolic acid (Mote et al., 1975 ) and staining has previously been taken to indicate the presence of lipid and cutin (Heslop-Harrison, 1977). Other workers have identified material thought to be lipid or cutin in root hair walls by staining with Sudan III for cutin and saturated lipids, and dimethylaminoazobenzeneand 50% HC1 for cutin (Scott et al., 1956, 1958; Dawes and Bowler, 1959 ). Phloroglucinol staining for lignin was negative. Composition of root hair walls

Carbohydrates (neutral monosaccharides, 52% w/w and uronic acids, 31% w/w) are the predominant components of the root hair walls. No acid-insoluble residue was found, suggesting that acid-insoluble lignin or inorganic compounds were not present. A significant fraction of the wall (17%) was unaccounted for in the analyses. Some of this fraction could be protein which is present in amounts between 5 and 8% in the walls of dicotyledon root hairs (Mort and Grover, 1988 ) and in walls of other parts of/-/, tasmanica plants (Webster and Stone, 1994). The nature of the remaining material is uncertain. Mort and Grover ( 1988 ) reported the presence of a presumptive polyphenolic material, either as a surface coating or an integral part of the root hair walls of a number of terrestrial species, and suggested that this material could form part of their 'unaccounted' fraction. In other experiments Hamilton and Mort (cited in Knirel et al. (1989)), report material retaining the root hair wall shape after hydrogen fluoride solvolysis and trypsin treatment. Such treatments would remove polysaccharides and proteins, respectively, but leave lignin and lipids. Auramine O staining for lipids and cutin was positive for the H. tasmanica root hairs (Table 1 ), and they have been shown to be components of other root hairs (Scott et al., 1956, 1958; Newcomb and Bonnett, 1965 ). However, in the case of H. tasmanica root hairs the histochemical data (Table 1) showed a negative response for lignin (phloroglucinol-HC1). The H. tasmanica root hair walls are rich in apiose and uronic acid (Table 2 ), which gives a presumptive indication of the presence of an apiogalacturonan, a pectic polymer characteristic of walls of seagrass from the Zosteraceae (Ovodova et al., 1968) and the aquatic duckweed (Lemna minor L.) (Beck, 1967; Hart and Kindel, 1970). The proportion of these two sugars is even higher than found in the walls of other parts of H. tasmanica plants (Webster and Stone, 1994). The other sugars present: rhamnose, fucose, xylose, arabinose, mannose and galactose are presumably components of the apiogalacturonan or other non-cellulosic polysaccharides. If all the glucose was from cellulose this would represent ~ 5% of the wall. This is a much lower value than found in the walls of other parts of H. tasmanica (Webster and Stone, 1994) or in root hairs of the terrestrial plants analyzed by Mort and

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Table 2 Monosaecharide and uronic acid composition (mole %) of H. tasmanica root hair walls Monosaccharide or Uronic acid

Composition (%)

Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Apiose Uronic acid

3 1 1 6 4 2 10 40 33

Grover ( 1988 ) among which spinach had the lowest (11%) cellulose content, and maize and wheat the highest, 45% and 38% respectively. The low level of cellulose and the abundant pectic apiogalacturonan make the composition of root hair walls of H. tasmanica unusual and distinguish them from the walls of root hairs from those terrestrial plants so far examined. However, their composition resembles that recently reported (Shedletzky et al., 1992) for walls of the suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) that had been adapted to grow on the cellulose-synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB). Their cell wall composition was quite different from nonadapted cells. The cellulose and xyloglucan content was markedly reduced (less than 10%), whereas the level of pectic polymers was increased, although their tensile strength was reduced to only about 30% of non-adapted cells. It appears that these cell wails, which are not subjected to compressive stresses, can withstand (osmotic) turgor forces. This situation would be comparable with the seagrass root hairs that are in a permanently aqueous environment and whose walls must withstand only the turgor pressure of the protoplast. This may not necessitate the strengthening of the walls with the rich network of cellulose microfibrils present in terrestrial root hairs (Mort and Grover, 1988).

Acknowledgments We are grateful to Dr D.A. Bulthuis, Marine Science Laboratories, Queenscliff, Victoria for his advice regarding choice of seagrass for this study. The work was supported by the Australian Marine Sciences and Technology Grants Scheme.

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Lloyd, C.W. and Wells, B., 1985. Microtubules are at the tips of root hairs and form helical patterns corresponding to inner wall fibrils. J. Cell. Sci., 75: 225-238. Luft, J.H., 197 I. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec., 171: 347-368. Mort, A.J. and Grover, P.B., 1988. Characterization of root hair cell walls as potential barriers to the infection of plants by rhizobia. Plant Physiol., 86:638-641. Mote, R.F., Muhm, R.L. and Gigstad, D.C., 1975. A staining method using acridine orange and auramine O for fungi and mycobacteria in bovine tissue. Stain Technol., 50: 5-9. Newcomb, E.H. and Bonnett, H.T., 1965. Cytoplasmic microtubule and wall microfibril orientation in root hairs of radish. J. Cell Biol., 27: 575-589. O'Brien, T.P. and McCully, M.E., 1981. The study of plant structure: principles and selected methods. Termarcarphi Pry., Melbourne, section 6.90. Ovodova, R.G., Vaskovsky, V.E. and Ovodov, Y.S., 1968. The pectic substances of Zosteraceae. Carbohydr. Res., 6: 328-332. Pluymaekers, H.J., 1982. A helicoidal cell wall texture in root hairs ofLimnobium stoloniferum. Protoplasma, 112:107-116. Rohm, M. and Werner, D., 1987. Isolation of root hairs from seedlings ofPisum sativum. Identification of root hair specific proteins by in situ labeling. Physiol. Plant., 69:129-136. Saeman, J.F., Moore, W.E., Mitchell, R.L. and Millet, M.A., 1954. Techniques for the determination of pulp constituents by quantitative paper chromatography. Tappi, 37: 336-343. Sassen, M.M.A., Wolters-Arts, A.M.C. and Traas, J.A., 1985. Deposition of cellulose microfibrils in cell walls of root hairs. Eur. J. Cell. Biol., 37: 21-26. Scott, E.M., 1950. Internal suberization of tissues. Bot. Gaz., 11 l: 378-394. Scott, E.M., Hamner, K.C., Baker, E. and Bowler, E., 1956. Electron microscope studies of cell wall growth in the onion root. Am. J. Bot., 43:313-324. Scott, E.M., Hamner, K.C., Baker, E. and Bowler, E., 1958. Electron microscope studies of the epidermis ofAllium cepa. Am. J. Bot., 45:449-46 I. Scott, J.E., Quintarelli, G. and Dellovo, M.C., 1964. The chemical and histochemical properties of Alcian Blue. I. The mechanism of Alcian Blue staining. Histochemie 4: 73-85. Shedletzky, E., Shmuel, M., Trainin, T., Kalman, S. and Delmer, D., 1992. Cell wall structure in cells adapted to growth on the cellulose-synthesis inhibitor 2, 6-dichlorobenzonitrile. Plant Physiol., 100: 1201-1230. Smith, K.A. and Robertson, P.D., 1971. Effect of ethylene on root extension of cereals. Nature, 234: 148-149. Stone, B.A., Evans, N.A., Bonig, I. and Clarke, A.E., 1984. The application ofSirofluor, a chemically defined fluorochrome from aniline blue for the histochemical detection of callose. Protoplasma, 122: 191-195. Webster, J. and Stone, B.A., 1994. Isolation, structure and monosaccharide composition of the walls of the vegetative parts of Heterozostera tasmaniea (Martens ex Aschers. ) den Hanog. Aquat. Bot., 47: 39-52. Werner, D. and Wolff, A.B., 1987. Root hair specific proteins in Glycine max. Z. Naturforsch, 42: 537-541. Wood, P.J., Fulcher, R.G. and Stone, B.A., 1983. Studies on the specificity of interaction of cereal cell wall components with Congo Red and Calcofluor. Specific detection and histochemistry of ( 13 ), ( 14 )-~Doglucans. J. Cereal Sci., l: 95-100.