Bot• Mag. Tokyo 79: 634-643 (Hattori Commemoration Number, 1966)

Chemical Nature of Major Cell Wall Constituents of and Dichotomosiphon with Special Reference to their Phylogenetic Positions

by Masaakira MAEDA*,Kazuko KURODA**, Yoshihiko IRIKI** *, Mitsuo CHIHARA*** )~, Kazutosi NIsIzAWA** and Tomoo MIWA**

ReceivedOctober 13, 1966

For the elucidation of phylogenetic relations of plants, morphological characters as well as the mode of life cycle, especially in lower plants, have thus far been considered essential. Besides these, the chemical nature of the substances constitut- ing the plant body or produced therein, though invisible directly, has recently been regarded as important and used in and phylogeny. The chemical nature of substances used for plant taxonomy is highly diversified and includes flavonoids, terpenes, fatty acids, hydrocarbons and others. Concerning these situations the book of Swains' may be consulted. In Japan the works of Kariyone2' on biflavones and leaf waxes of conifers, and those of Fu jita3>on essential oils are worth noting. Recent advances in biochemical genetics indicate that the chemical nature of plant products is controlled by genes in the same sense as are morphological characters, thus form- ing the basis for a chemical taxonomy. As for cell wall constituents, F. von Wettstein4' stated earlier that the presence or absence of chitin is important as a criterion for the classification of fungi. In the domain of algae, Miwa et al.5,6,7,studied the chemical nature of some siphonous in relation to their phylogenetic positions. They found that Siphonales can be divided sharply into two groups with respect to the nature of the polysac- charide which, in place of cellulose, constitutes the main cell wall substance and is obtainable in form of crude fiber. One group, including the genera, Codium and Derbesia, is characterized by the presence of mannan ; and the other group, to which Caulerpa, Bryopsis, Pseudodichotomosiphon,Halimeda, Udotea, Chlorodesmisand Avrainvillea belong, contains xylan in its place. Such a grouping is in accord with the classification by Feldmanng' of Siphonales based chiefly on the morphological characters, except for the position of Bryopsis. In the present study the chemical nature of the main cell wall material of Vaucheria and Dichotomosiphonwas investigated with a view to obtaining informa- tion concerning the phylogenetic position of these algae. The systematic position of Vaucheria in the system of algae seems not to be completely settled. Some phycologists have placed the Vaucheriaceae in the order Siphonales (Oltmanns 19229',Printz 1927'0', Okamura 193011',193612', Ijengar 1951'3', Fritsch 1935'40, and later Fritsch (1954)15'has proposed to establish an independent * Departmentof Biochemistry, Faculty of Engineeringand Science,Saitama University, Urawa,Japan. ** BotanicalInstitute , Faculty of Science,Tokyo Kyoiku University,Tokyo, Japan. ***Biological Laboratory , Departmentof Education,Shinshu University, Nagano, Japan. ****National Science Museum , Ueno,Tokyo, Japan. Oct.-Nov., 1966 MAEDA, M., et al. 635 order Vaucheriales in the siphonous green algae. Contrary to these views other workers would remove the Vaucheriaceae from and place them in the Xanthophyceae (Bohlin 190116, Feldmann 19458', Smith 195017), Papenfuss 1955180. As stated above, the main cell wall material of the siphonous green algae con sists either of xylan or of mannan and not cellulose. As to the cell wall constituents of members of the Xanthophyceae no definite information is available at present Cellulose has been considered to be generally lacking in this group of algae, though. its occurrence was reported in Tribonema as a result of X-ray study19). In Vaucheria the chemical nature of the main cell wall constituent has not beena thoroughly clarified. Thus, Nicolai and Prestonl9) concluded on the basis of physical investigation and staining reactions, that the skeletal substance of the cell wall of Vaucheria is not cellulose, however, others have reported that cellulose is present. (e. g., Printz 192710), Fritsch 195514) but Chadefaud (1945)20) separated this genus fromm the Vaucheriaceae and placed it in a new family . Since the fact that the main cell wall constituent of Dichotomosiphon was callose but not cel- lulose was considered as one of the most important properties distinguishing thiss genus from Vaucheria, the present study was undertaken to obtain some information about the systematic relation, if any, between Vaucheria and Dichotomosiphon by comparing chemical nature of main cell wall contituent.

Materials and Methods

Vaucheria : Two species were used. V. sessilis was harvested at the end of March in a shallow stream near Tokyo ; V. longicaulis, a marine species, was obtainedd from Okinawa. Dichotomosiphon : D. tuberoses was collected in Okinawa. All of the materials were carefully freed from attached impurities, washedd thoroughly with fresh water, air-dried and finally dried at 105° to constant weight and ground to coarse powder. A part of fresh-water Vaucheria was preserved in ethanol for use in histochemical tests. Ash.-The sample was heated at about 550 ° in an electric muffle furnace for 12 hr. The residue was dissolved in 6 N nitric acid and the insoluble matter filtered off. The filtrate was evaporated and dried to constant weight. Nitrogen.-Total nitrogen was determined by micro-Kjeldahl method. Pentosan and methylpentosan.-These were estimated by the method of Kruger- Tollens-Krober2l) and Elett-Tollens-Mayer22), respectively. Reducing sugars.--In most cases Somogyi's method23) and for pentose Tracey's method24' were applied. In the presence of xylose, glucose was estimated by the use of glucose oxidase, the enzyme preparation employed (" Deoxin ", Nagase Co., Ltd.) had almost negligible action upon xylose. Paper chromatography.-Ascending chromatography was carried out on Toya filter paper No. 52 with n-butanol-pyridine-water (6: 4 : 3, v/v) as developer. For the detection of sugars, either acetone-silver nitrate or benzidine-trichloroacetic acid was sprayed. Infrared spectra.-A Nihon Bunko, type iR-S instrument was used. Measure- ment was made with the sample in a potassium bromide disk. X636 Bot. Mag. Tokyo Vol. 79

Results

Vaucheria sessilis and V. longicaulis. Histochemical tests.-Staining reactions for cellulose were carried out with the fronds either preserved in alcohol or dried in air. Both the freshwater and marine species gave similar results. The cell wall stained pale blue with iodine and satu- rated zinc chloride and blue with iodine and concentrated sulfuric acid. Upon pre- treatment of the fronds with dilute hypochlorite solution, the color intensity increased significantly with both reagents. Iodine and concentrated calcium chloride stained the cell wall red. Analysis of whole fronds.-The ash, nitrogen, pentosan and methylpentosan contents of dried fronds were determined. The results are presented in Table 1.

Table 1. Analysis of whole frond (Numerals on dry weight basis).

Preparation of crude fiber.-Samples of both species were extracted respectively twice with hot 80% ethanol each time for 1 hr., then treated with 1% hydrochloric acid at 18-20° overnight. After washing with water the residue was heated with 1.25% sodium hydroxide at 100° for 30 min. The residue was bleached with dilute sodium hypochlorite (1 available chlorine) at room temperature for 1 hr. and freed from excess oxidant by washing with a dilute solution of sodium sulfite. The product was then heated with 1.25% sulfuric acid at 100° for 30 min. and washed thoroughly with water until free of sulfuric acid. Yield, 23% of dried frond. The crude fiber thus obtained showed, when examined under the microscope, an almost unchanged structure. The previously mentioned staining reactions for cellulose were clearer with those crude fiber preparation than with the original fronds. The fiber preparations showed distinct double refraction. Acid hydrolysis of crude fiber.-100 mg of each crude fiber preparation was mixed with 2 ml of 72% sulfuric acid and kept at 0° for 24 hr. The dark brown product obtained was diluted with water to 40 ml and heated at 100° for 2 hr. The hydro- lyzate was neutralized with barium carbonate and filtered. The filtrate was concen- trated in vacuo to a syrup which was then dissolved in hot ethanol. After filtration the ethanolic solution was evaporated and the product finally dissolved in 3 ml water. Paper chromatography showed the presence of glucose only. Enzymatic hydrolysis of crude fiber.-A commercial praparation of" Trichoderma- Cellulase " (Mei ji-Seika Co., Ltd.) was dissolved in water, dialyzed free of reducing substances and lyophilized. Each of the crude Vaucheria fiber preparations was ground to fine powder and 50 mg thereof was suspended in 1.5 ml of 0.1 M acetate buffer, pH 5.2, and incubated with 0.5 ml of 3% cellulase solution at 30° . After 24 hr. the reaction mixture was centrifuged and the clear supernatant fluid was examined chromatographically. Besides the major spot of glucose , several smaller spots of lower migration rates were found. The latter seemed to be oligosaccharides com- posed of glucose, since the extracts from two of these spots yielded upon acid hyro- lysis a single spot corresponding to glucose. Oct.-Nov., 1966 MAEDA, M., e t al. 637

Purification of crude fiber.-A sample of the crude fiber from V. sessilis was mixed well with ten times its weight of 17.5% sodium hydroxide; after 30 min. the mixture was diluted with an equal volume of water and centrifuged. The residue was washed successively with dilute acetic acid and with water, and dried. Yield 98.9%. This preparation showed also marked staining reactions for cellulose as well as distinct double refraction. Moreover, a sample was subjected to acetolysis25', and the product obtained was purified by repeated recrystallization from a mixture of chloro- form and methanol. Its melting point, 222°, and infrared spectrum agreed well with those of a-octaacetylcellobiose.

Dichotomosiphon tuberoses. Analysis of whole fronds.-Quantitative determination of some of the constitu- ents are shown in Table 2.

Table 2. Analysis of whole frond of Dichotomosiphon tuberosus (Numerals on dry weight basis).

The ash content was found to be high, with major cations of Ca, K, and Na. The content of SO4 shown in this table includes both inorganic and ester form. Especially worthy of note is the high content of pentosan which as will be shown, represents the major cell wall constituent. Preparation of crude fiber.-A sample of dried Dichotomosiphon fronds was treated with 30 times its weight of 1% hydrochloric acid at room temperature. After 24 hr. the fronds were washed with water. The alkaline extract was neutralized and dialyzed against running tap water for 3 days. Upon acid hydrolysis of the dialysis residue and examination by paper chromatography, the presence of f ucose (RG* 1.13), xylose (1.02), glucose (1.00), an unidentified hexose (0.85) and an uronic acid (0.23) was made propable. The alkali extracted algal substance was heated with 30 parts of 1.25; sulfuric acid at 100° for 30 min. After filtration the residue was washed with water and bleached with hypochlorite. The crude fiber thus obtained was washed successively with dilute acetic acid, sodium sulfite and water, and dried. Yield, 30% of the whole frond. Staining reactions of the crude fiber for cellulose with iodine reagents were negative, and no double refraction was demonstrated . The pentosan content was found to be 81.4%. Acid hydrolysis of the crude fiber yielded a large amount of D-xylose together with a small amount of D-glucose, as detected by paper chromato- graphy. With this hydrolyzate the ratio of xylose (determined by Tracey's method) to glucose (glucose oxidase method) was approximately 80:20. Preparation of xylan from crude fiber.-As the crude fiber of Dibhotomosiphon seemed to be of polysaccharide nature, with xylose as major constituent, the isolation of xylan was attempted. Crude fiber was extracted under mechanical stirring with 20 parts of 10 sodium hydroxide at 10° for 1 hr.; the mixture was centrifuged, and the pale yellow supernatant fluid was poured into an equal volume of 95 ethanol

* RG : migration rate relative to glucose . 638 Bot. Mag. Tokyo Vol. 79 with stirring, whereby a white precipitate formed. After refrigeration overnight, the precipitate was collected, washed successively with an ethanol-acetic aid mixture (1: 1), ethanol and ether, and dried in vacuo. For further purification (removal of silica) the product was stirred in distilled water for 24 hr., in 50°o ethanol containing 1% hydrochloric acid for 24 hr., then treated with 2°o solution of ammonium molyb date in N sulfuric acid at 20° for 2 hr.2G>. The product was finally thoroughly washed with water and dried. Yield purified cell wall substance was 75°0 of the crude fiber. The purified polysaccharide is white powder of somewhat fibrous consistency with an ash content of less than 0.1%. It is insoluble in water, dilute acid and organic solvents but soluble in dilute alkali. Elementary analysis : found : C, 44.09; (ash 0.09) H, 6.12 calculated for C5H8O4: C, 45.45; H, 6.06 Specific rotation : Ca=-2S.8° (c 1.20, in N NaOH) When hydrolyzed by heating with N sulfuric acid at 100° for 2 hr., it yielded 97.5% reducing sugar (Somogyi's method, calculated as xylose). Determination of xylose by Tracey's method showed the xylose content to be 92°0, while the glucose content as estimated by glucose oxidase method gave 9.2°0. Paper chromatography revealed the presence of abundant xylose together with a small amount of glucose; no other sugars were found. The infrared absorption spectrum showed nearly the same pattern as that of the xylans of Bryopsis, Udotea and Avrainvillea with type 2b absorption at 896 cm-1, which indicates the existance of units of j3-configaration (Fig. 1). This finding taken together with the negative value of the specific rotation indicates that this poly- saccharide is a kind of J3-xylan. To obtain direct confirmation of the identity of the main sugar component as D-xylose, 0.5 g of xylan was suspended in 20 ml of 3% nitric acid and heated to boiling for 90 min. The hydrolyzate was neutralized with barium hydroxide and deionized by treatment with ion-exchangers. Glucose was next remoxed by the use of brewer's yeast and the residual solution concentrated in vacuo to a syrup. The product was dissolved in hot ethanol, filtered and concentrated to a syrup. Upon storage in the refrigerator this solidified to a crystalline mass. When recrystallized

Fig. 1. Infrared spectra of xylans from 1. Dichotomosiphon 2. Avrainvillea 3. Udotea 4. Bryopsis Oct.-Nov., 1966 MAEDA, M., et al. 639 from ethanol the product melted at 144° and showed (aJij=+19.1° (c 2.36 in H20), in agreement with the corresponding values of D-xylose. The extraction of xylan from the crude fiber by alkali left a small amount of grey powdery substance. This residue again showed no optical anisotropy and none of the staining reactions for cellulose. When treated with 72°o sulfuric acid under the condition used for the hydrolysis of cellulose, and examined by paper chromatography, a small amount of xylose (Rc 1.02) together with a trace each of arabinose (RG 1.04) and glucose (RG 1.00) was found. All these results may be taken to indicate the absence of cellulose in the crude fiber of Dichotomosiphon. Determination of molecular weight.-This was carried out both chemically by hypoiodite procedure27' and physically by viscosity measurement28'. In the first method, 46.4 mg of xylan consumed 0.70 ml 0.1 N iodine; assuming one reducing end per molecule, the average molecular weight was calculated as Mn=5800 (DP=44). The specific viscosity was measured with xylan solutions of differing concentra- tion in 0.1N sodium hydroxide at 20°. The results are presented in Table 3. From

Table 3. Viscosity measurement of Dichotom osiphon xylan.

thesed ata the ~Sp/c value at the concentration of 0.340 g/ml was obtained graphically as 3.74. By putting this value into the Staudinger formula, ~7Sp/c= Km x M, the molecular weight was calculated to be My=5300 (DP=40). The value Km=7.2x 10-4, obtained with Caulerpa xylan by direct sedimentation measurement, was applied to Dichotomosiphon xylan7'. Thus the values of molecular weight of Dichotomosiphon xylan obtained by hypoiodite method (5800) and by viscosity method (5300) agreed well. Periodate oxidation of xylan.-From the viewpoint of phylogenetic relation, the mode of linkage of the constituent sugars in the polysaccharide seems to be im- portant. To elucidate this point periodate oxidation of the xylan from Dichoto- mosiphon was carried out. A sample of 100.0 mg was dissolved in 5 ml of N NaOH, treated with 30 ml of 0.1 M potassium metaperiodate and diluted with water to 100 ml (pH 9.6). The mix- ture was kept at 7-10° in the dark with occassional stirring. At appropriate inter- vals samples were withdrawn and the amounts of periodate consumed and of formic acid produced were determined. A blank test was made without xylan. As a re- ference for the progress of xylan oxidation, the periodate oxidation of sucrose in alkali was carried out in parallel. The results are presented in Table 4. Thus, the consumption of periodate by Dichotomosiphon xylan was very small, suggesting that most or all of its linkages are 1: 3. Assuming a single chain struc- ture and made up solely of 1,3-linked sugar residues, the xylan would consume two 640 Bot. Mag. Tokyo Vol. 79

Table 4. Periodate oxidation. A. Dichotomosiphon xylan.

moles of oxidant at the non-reducing end and one mole at the reducing end, and release one mole of formic acid at the non-reducing end. On the basis of this con- sideration the degree of polymerization (DP) was calculated as 46 from periodate consumption and as 41 from formic acid production. These values are in accord with those obtained by hypoiodite oxidation (44) and by viscosity (40). All 1,3-linked xylose and glucose residues occupying the interior part of the xylan chain should resist periodate oxidation. Actually the existence of intact xylose and glucose was demonstrated in the periodate oxidized polysaccharide. Thus, after termination of the oxidation reaction, the mixture was treated with an excess of ethylene glycol to decompose the remaining periodate, then dialyzed for 3 days. The product was hydrolyzed and examined by paper chromatography. A large amount of xylose (RG 1.02) together with a small amount of glucose (1.00) was detected.

Discussion

Concerning the chemical nature of the cell wall of Vaucheria Mirande29> reported, on the basis of staining reactions and solubility tests, that the cell wall consisted of cellulose and pectic substance and this view was taken over by some phycologists'0"4,3°,. On the other hand Nicolai and Prestonstated, as a result of physical studies includ- ing X-ray analysis and the use of polarization microscopy as well as staining reac- tions, that two species of Vaucheria did not contain any native cellulose. In the present study a so-called crude fiber fraction of the cell wall, resistant to hot dilute alkali and acid, and hence regarded as skeletal wall material, was isolated from two species of Vaucheria. In each case the substance was soluble in cupram- monium, was optically anisotropic and was stained bluish purple by iodine and con- centrated zinc chloride, and blue by iodine and concentrated sulfuric acid. Hydro- lysis with sulfuric acid under relatively drastic conditions produced glucose as the sole monosaccharide detected; moreover, on acetolysis, octaacetylcellobiose was obtain- ed. These results indicate the cellulose nature of this cell wall substance. Even if Oct.-Nov., 1966 MAEDA, M., et al. 641 not a " native " cellulose it is clearly a polysaccharide consisting of 1,4-linked j3-D glucose residues. The chemical nature of the main cell wall substance of Dichotomosiphon hass remained obscure up to the present. Ernst3l' and Virieux32' regarded the cell wall as composed of cellulose, while Mirande29' and Puymaly32~ reported that it consists of callose and pectic substances. At that time " callose " and " pectic substances " were not chemically defined, having been known solely by staining reactions. The present study revealed that the main cell wall constituent of Dichotomosiphon tuberosus is a kind of xylan. No trace of cellulose was detected. The purified xylan showed an approximate molecular weight of 5300-6000 (DP 40-46) and consisted of about 91-92 D-xylose and 8-9% D-glucose. All of the sugar residues appeared to be of fl-configuration and connected by 1,3-linkages. These properties of Dichoto- mosiphon xylan are astonishingly concordant with those of the xylans of Caulerpa, Udotea, Halimeda, Chlorodesmis and Bryopsis7'. Earlier, Vaucheria and Dichotomosiphon were included in the same family, Vau- cheriaceae31' 32),and this system was followed by Fritschl4'. Later, however, in spite of the similarity in external morphology the difference in various other essential properties between these genera led Dichotomosiphon to be removed from the Vau- cheriaceae to an independent family Dichotomosiphonaceae20'. The present study demonstrated that a sharp distinction exists also on the chemical nature of the cell wall between these two genera, supporting the latter view on the systematic position of Dichotomosiphon. Formely Vaucheria was regarded as a member of Shiponales9-14), while some workers considered that this genus should be included in Xanthophyceae16'34). In recent years most phycologists have seemed to support the latter opinion17'18' 35-39> Again, in respect of the chemical nature of the cell wall Vaucheria has been shown to be entirely different from Siphonales. In the former the major cell wall substance is cellulose, whereas in the latter it is either xylan or mannan7'. Thus, the view of the withdrawal of the Vaucheriaceae from Siphonales seems to be corroborated by the present work. Whether or not those xanthophyceous algae which are closely related to Vaucheria also contain cellulose as major cell wall constituent remains to be elucidated. No reliable information is available at present.

Summary

1. From Vaucheria and Dichotomosiphon cell walls, substances considered to form the skeletal cell wall were isolated as "crude fiber" and their chemical nature was examined. 2. The major cell wall substance of Vaucheria was found to be cellulose as confirmed chiefly by chemical means including acetolysis. 3. In Dichotomosiphon the major cell wall substance is represented by xylan. Its chemical structure was quite the same as the xylan forming the skeletal cell wall of Caulerpa, Bryopsis, Halimeda and Chlorodesmis. 4. These findings seem to substantiate the view that Dichotomosiphon, which was initially placed in the Vaucheriaceae, should be removed from this family. 5. The existence of cellulose in Vaucheria and its absence in Siphonales is additional evidence for the separation of Vaucheria from Siphonales.

The writers wish to express their gratitude to Dr. Yamagishi of the College of 642 Bot. Mag. Tokyo Vol. 79

Agriculture and Veterinary Medicine, Nihon University and to Dr. Kamura of the Ryukyu University for their kindness shown in the collection of algal materials. Sincere thanks are also due to Professor E. J. Hehre of the Albert Einstein College of Medicine, Yeshiva University, for his reading of the manuscript.

References 1) Swain, T., Ed., Chemical Plant Taxonomy (Academic Press, New York, 1963). 2) Kari- yone, T., J. Pharmacogn. Soc. Japan 16 : 1 (1962). 3) Fu jita, Y., Biological Science (Tokyo) 10 : 145 (1958). 4) Wettstein, Fr. von, Sitzungsber. Akad. Wiss. Wien, Math.-naturw. Kl. Abt. I, 130: 3 (1921). 5) Iriki, Y., and Miwa, T., Nature, 185 : 178 (1960). 6) Iriki, Y., Suzuki, T., Nisizawa, K. and Miwa, T., Nature, 187 : 82 (1960). 7) Miwa, T., Iriki, Y., and Suzuki, T., Colloques internationaux de C. N. R. S. No. 103 : 135 (1961) . 8) Feldmann, J., Compt. rend. Acad. Sci., Paris 222 : 752 (1946). 9) Oltmanns, F., Morphologie and Biologie der Algen, 2 Aufl. I (Gustav Fischer, Jena, 1922). 10) Printz, H., in Engler-Prantl's die nati rlichen Pflanzenfamilien, Bd. III (1927). 11) Okamura, K., Sorui-Keitogaku, (Uchida-rokakuho, Tokyo, 1930). 12) , Nippon Kaisoshi, (Uchida-rokakuho, Tokyo, 1936). 13) I jengar, M. O. P., Manual of Phycology, Smith, G. M., Ed. (Chronica Botanica, Waltham, Massachusetts, 1951). 14) Fritsch, E., The Structure and Reproduction of the Algae, I (Cambridge University Press, London, 1935). 15) , 8 eme Congres internat. Bot. Rapp. comm. sect. 17: 83 (1954), Paris. 16) Bohlin, K., Cited in Papenfuss (18). 17) Smith, G. M., The Fresh-Water Algae of the United States, 2nd. Ed. (McGraw-Hill, New York, 1950). 18) Papenfuss, G. F., Classification of the Algae, in A Century of Progress in the Natural Sciences (California Academy of Science, San Francisco, 1955). 19) Nicolai, E., and Preston, R. D., Proc. Roy. Soc. London, B, 140: 244 (1952). 20) Chadefaud, M., Bull. Soc. Bot. France 92: 47 (1945). 21) van der Haar, A. W., Anleitung zum Nachweis, zur Bestimmung der Monosaccharide and Alde- hydsauren, 63 (Borntrager, Berlin, 1920). 22) , ibid., 67. 23) Somogyi, M., J. Biol. Chem., 160: 61 (1945). 24) Tracey, M. V., Biochem. J. 47: 433 (1950). 25) Organic Synthesis 17: 36 (1947). 26) Feigl, F., and Krumholz, P., Mikrochemie 7 : 82 (1929). 27) Willstatter, R., and Schudel, G., Ber. deut. chem. Ges. 51: 780 (1918). 28) Horton, D., and Wolfrom, M. L., in Comprehensive Biochemistry, Florkin and Stoz, Ed. Vol. 5, 185 (Elsevier Publishing Co., New York, 1963). 29) Mirande, R., Ann. Sci. Nat. Bot. ix, 18: 147 (1913). 30) Wurdach, M. E., Ohio J. Sci., 23: 181 (1923). 31) Ernst, A., Beih. hot. Centralbl. 13: 115 (1902). 32) Virieux, J., Proc.-Verb. Soc. Hist. Nat. Doubs 19: 36 (1911). 33) Puymaly, A., Bull. Soc. Bot. Geneve II. 9 : 120 (1917). 34) Pascher, A., Hedwigia 53: 6 (1912). 35) Maekawa, F., J. Fac. Sci. Univ. Tokyo, Sec., Bot. 7 : 543 (1960). 36) Feldmann, J., Uppsala Universitets Arskrif t 6 : 59 (1958). 37) Silva, T. C., Physiology and Biochemistry of Algae, Lewin, Ed. (Academic Press, New York, 1962). 38) Ueda, S., Iwamoto, K., and Miura, A., Suisan-shokubutsugaku (Koseikaku, Tokyo, 1963). 39) Hirose, H., General Phycology (Uchida- rokakuho, Tokyo, 1965).

摘 要

前 田 昌 徹*・ 黒 田 和 子**・ 入 来 義 彦***・ 千 原 光 雄***㌔ 西 沢 十 俊**・ 三 輪 知 雄 轡: yα 鷹 加 γ∫α お よ びD∫choオ0襯OSゆhoπ の 細 胞 膜 主 成 分 の 化 学 的 性 質 と そ の 分 類:系統 と の 関 連

1)γ απc加 γ∫α(フ シ ナ シ ミ ド ロ)属2種 お よ びD∫cんoオo初osゆhoπ 如 加 γosπsチ ヨ ウ チ ン ミ ド ロ か ら ・ そ れ ぞ れ 細 胞 膜 の 骨 組 み を な す と 考 え ら れ る 物 質 を 「粗 繊 維 」 と し て 分 離 し,そ の 化 学 的 性 質 を 調 べ た ・ 2)フ シ ナ シ ミ ドロ の 主 細 胞 膜 物 質 が セ ル ロ ー ス で あ る こ と を 酢 化 分 解 な ど,主 と し て 化 学 的 手 段 に よ っ て 確 認 し た. 3)チ ヨ ウ チ ン ミ ドロ で は,主 細 胞 膜 を 構 成 す る 物 質 は キ シ ラ ン で あ っ た.こ の キ シ ラ ン は,化 学 的 構 造 の 点 で イ ワ ヅ タ,ハ ネ モ,サ ボ テ ン グ サ,マ ユ ハ キ モ の 主 細 胞 膜 を つ く っ て い る キ シ ラ ン と 同 一 で あ っ た ・ Oct.-Nov., 1966 MAEDA, M., et al. 643

4)こ れ らの 結 果 は,は じめ フ シ ナ シ ミ ドロ 科 に入 れ られ て い た チ ヨ ウチ ン ミ ドロ を,こ の 科 か らは ず す べ き で あ る とす る見 解 を 証 拠 だ て る もの と思 わ れ る. 5)主 細 胞 膜 物 質 と して,フ シ ナ シ ミ ドロに は セル ロー スが 存 在 し,ク ダ モ 目(Siphonales)に これ が 欠 けて い る こ と は,フ シ ナ シ ミ ドロ を ク ダ モ 目か らは ず す 考 え方 に対 す る証 拠 を さ ら につ け加 え る も ので あ る. (*埼 玉 大 学 理 工 学 部 生 化 学 教 室 ・**東 京 教 育 大 学 理 学 部 植 物 学教 室 ・***信 州 大 学 教 育 学 部 生 物 学 研 究 室 ****国 立 科 学 博 物 館)