Histochemical and Chemical Studies on the Cell Wall of Prasiola Japonica

Bot. Meg. Tokyo 80: 109-117 (March 25, 1967) Histochemical and Chemical Studies on the Cell Wall of Prasiola japonica by Hiroshi TAKEDA*, Kazutosi NISIzAWA* and Tomoo MIWA* Received October 26, 1966 Abstract Cell wall constituents of Prasiola japonica Yatabe were histochemically and chemically examined with special reference to phylogenetic position. Cell wall of Prasiola japonica is composed largely of a xylomannan consisting approximately of 93% mannose and 7% xylose. The layers C and D were found to be composed of cellulose and polyuronide-like polysaccharide, respectively. Besides these, the presence of rhamnose containing polysaccharide fractions was demon- strated, though they could not be obtained pure. However, no cell wall components containing galactose residue were detected in Prasiola. All these results exclude the possibility of its relation to Porphyra, but no defi- nite conclusion can be drawn as to the phylogenetic position of Prasiola. The chemical nature of the cell wall of this alga was rather unique and no algae with comparable cell wall properties have thus far been known among Ulvales. Hence, it may probable to support the view that the genus Prasiola should be separated from Urotrichales to establish an independent Order Schizogoniales in Chlorophyta. The chemical nature of plant constituents has been often regarded as a basis of classification of plants, and especially in some groups of algae it can be an important clue to the elucidation of their phylogeny. Thus, Miwa et al.1'2,3) have shown that siphonous green algae may be classified into two groups in respect of the chemical nature of the polysaccharides which constitute a bulk of cell wall. Each group has either xylan or mannan in place of cellulose. The phylogenetic position of the genus Prasiola still remains unsettled. The family, Prasiolaceae has been often placed in Urotrichales4°5> and regarded as close to Ulvaceae. There has been, however, another opinion that Prasiolaceae should be separated to form a distinct Order, Schizogoniales6'7'8>. In addition, a quite different view was presented by Okamura9~ and Ishikawa10> who claimed that Prasiola might have some relation to a lower red alga, Porphyra (Bangiales), for following reasons : 1) both Prasiola and Porphyra form monolayered thallus, 2) they have stellate plastid with a pyrenoid at the center, 3) the structure of their nuclei is primitive with little differentiation and 4) they have no motile sexual cells. However, the last point lost its validity at present, since motile gamate has been found for Prasiola japonica by Yabe11>, Ueda12~, Fu jiyamal3~ and recently for P. stipitata by Friedmann14.15~. In this respect, therefore, it appears likely that Prasiola belongs to green algae rather than to Bangiales. * Botanical Institute, Tokyo Kyoiku University, Otsuka, Tokyo, Japan. Present address of H. Takeda : Biological Institute, Department of General Education, Niigata University, Niigata, Japan. 110 Bot. Mag. Tokyo Vol. 80 From phylogenetic point of view, Miwa16> studied the chemical nature of cell wall components of Prasiola japonica and compared it with that of Porphyra tenera and Enteromorpha Linza. It was shown that the bulk of constituents of cell wall of Prasiola japonica was mannan, while galactan formed majar cell wall constituent of Porphyra and methylpentosan together with cellulose formed those of Enteromorpha. It was at that time also by Miwa that cellulose was proved to be present in Prasiola, though it was not necessarily an a-cellulose. In Enteromorpha the presence of cellulose was made probable by the isolation of octaacetylcellobiose upon acetolysis, but no such compound was obtained from Porphyra . Thus, the chemical nature of Pra- siola japonica seemed unique, and it may be needed to discuss the phylogeny of Prasiola on the basis of not only its morphological characters but also the chemical nature of cell wall constituents. At that time, however, Miwa could not make detailed study on the cell wall constituents of this alga due to shortage of experi- mental material. The present authors therefore attempted to reexamine and extend the previous work, since more material has become available at present. Materials and Methods Material: Prasiola japonica Yatabe (Kawanori in Japanese) was collected in the Ohchi river in Saitama-ken, in the Nippara river in Nishitama-gun, Tokyo and in the Fuji river in Shizuoka-ken. Paper chromatography : The separation and identification of sugars were carried out by ascending technique using Toyo filter paper No. 50. As solvent systems were used, (1) butanol : pyridine : water (6 : 4 : 3, v/v)17', (2) pyridine : ethylacetate : acetic acid : water (5 : 5: 1 : 3, v/v)18' and water saturated phenoll9>. As developers, (1) alka- line silver nitrate20>, (2) benzidine trichloroacetic acid21 and (3~ resorcinol-HC121' were adopted. Determination of sugar: The method of Somogyi22> was used. Hydrolysis of polysaccharides: A given amount of sample was heated with 100 part of 1 N sulfuric acid in a boilling water bath for 3 hr. and the hydrolyzate was neutralized with barium hydroxide. Experiments and Results (I) Histochemical observations. Staining tests. In Figs. 1 and 2 a photomicrograph and a diagrammatic illustra- tion of a cross section of this alga are shown respectively. Layer C, the innermost layer of cell wall was stained deep blue with zinc chloride-iodine and readily dissolved in a cuprammonium solution. Layer D, corresponding to the middle lamella of the wall was colored deep red with ruthenium red. This reagent stained layer B pale red to pink but not layer C, as shown in Fig. 3. Since the wall C together with the protoplast can be sharply separated from layer B by a slight press (empty parts in Fig. 3), layer B and C seem to be only loosely connected. Upon iodine-potassium iodide staining after treatment with 60°o sulfuric acid, layer C was colored deep blue and layer B pale blue. Staining tests after alkali treatment. Upon treatment of the thallus with 4% sodium hydroxide at 90° for 10 min. and washing with water, layer D completely disappeared and a layer, which seemed to be layer B and stained pink with ruthenium red, remained at the outermost part (Fig. 4-A). This reagent did not stain the March, 1967 TAKEDA, H., et al. 111 layer C which, however, was stained deep blue with zinc chloride-iodine (Fig. 4-B). The outermost layer A was not stained by the latter reagent. On the basis of their behavior toward alkali and staining reagents layer D seemed to be an acidic polysaccharide and layer C may be cellulose. Autodigestion of thallus. As already shown by Miwa, the frond of this alga is very liable to autolyze. Thus, air-dried fronds were added to about ten fold weight of water and a small amount of Fig. 1. Cross section of adult thallus. x460 toluene and allowed to stand at about 25° for 6 days. Most of cell walls were disintegrated and cells were separated from each other. At this stage of autolysis, layer B disappeared whilst layers A and C remained undissolved, as shown in Fig. 5. Such disintegration was not observed when the fronds had been pre- viously heated at about 100 ° for 30 min., but when the autolyzate from non-heated fronds had been added, it occurred at the parts of the fronds with which the autolyzate came into contact. Treatment with Takadiastase. Air dried fronds were heated in water at about 100 ° for 30 min. to inactivate the enzyme for autolysis and then incubated with 1% commercial Takadiastase in Fig. 2. Diagram of a proposed structure acetate buffer of pH 4.5 at 30° for 20 hr. of cell wall. By this treatment layer B was attacked and reducing sugar was produced. Treatment with Trichoderma-cellulase. Air-dried fronds were incubated with 1 commercial preparation of Trichoderma- cellulase (manufactured by Meiji Seika Kaisha Ltd.) under the same conditions as with Takadiastase treatment. After :20 hr. most of the innermost wall together with cell contents were lost leaving the framework of most part of cell wall which was stained pink with ruthenium red but not with zinc chloride-iodine (Fig. 6). Thus, layer C seemed to be Fig. 3.. Surface view of the thallus, removed by the action of cellulase pre- stained with ruthenium red. Layer D is paration. From this finding and staining stained red. x1058. behavior toward zinc chloride-iodine, 112 Lot. Mag. Tokyo Vol. 80 Fig. 4. Surface view of thallus after alkali treatment. A : Stained with ruthenium red. Layer D is dissolved, layer B is stained pink and layer C is not stained. B : Stained with zinc chloride-iodine. Layer D is dissolved, layer C is stained deep blue and layer B is not stained. Fig. 5. Disintegration of cell wall by Fig. 6. A part of frond acted upon by autolysis. Layer B and D disappeared Trichoderma-cellulase. In vacant cells layer completely; layer A and C remained al- C was dissolved and cell contents were ex- most intact. x427. truded, leaving layers B and D almost intact. In some cells layer C remained still intact. layer C seems to consist of cellulose.. As can be seen in Fig. 6, layer D also appeared to be affected by this cellulase treatment. Electron microscopic observation. With an orninary optical microscope it became probable that the cell wall of Prasiola japonica was composed of four layers. To confirm this a cross section of the cell wall was examined by electron microscope. As shown in Fig. 7, Fig. 7. Electron micrograph of the cross the existence of three layers correspond- section of cell wall. ing to B, C and D was ascertained. March, 1967 TAKEDA,H., et al. 113 (II) Preparation of polysaccharides from three layers A, B and C and some of their properties.

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