ON THE CONTENT OF CELL-WALL CONSTITUENTS IN VARIOUS PLANT MATERIALS Maija-Liisa Salo with technical assistance from Terttu Mäkinen Department of Animal Husbandry, University of Helsinki Received January 14, 1965 The cell-walls of plants are composed principally of carbohydrates and lignin. The cell-wall carbohydrates are polysaccharides made up of neutral and acidic sugar components. The most common of these polysaccharides is cellulose, while the others are classified under the general term of hemicelluloses. Very few of the studies on cell-wall carbohydrates have included quantitative determinations of various sugar anhydrides. Such numerical data have been pre- sented by Gaillard (3, 4), Waite and Gorrod (12) and Jarrige (5) chiefly on certain grasses and legumes. Sternkopf (11) determined some hemicellulose con- stituents in green maize, while Andrews et al. (1) studied, mainly quantitatively, the polysaccharide composition of various plant leaves. The purpose of the present investigation was to determine the quantitative composition of the cell-walls in different plants and in some cases in different parts of the same plant. The results obtained by the procedures used were compared with the amounts of membrane substances determined by the method of Paloheimo and Paloheimo (7) and with crude fibre. Material and methods The plant materials investigated included both feeding stuffs as well as plant material not intended for animal consumption. The analysis procedures have been described by the author in another paper (10). It can be mentioned, however, that fructosan in the grasses was extracted with cold water, and that also in this in- vestigation all of the uronic anhydrides were included in the hemicellulose, since they were not separated into different fractions. https://www.c-info.fi/en/info/?token=x_-UGSctCuPJ02l9._QKlypBWTKqtEu5RKrnlcA.s_8yAHvTaGRVwblsCZeti4yPAqXWODHqveOebjK4Nfhkj0_LOz2SFrYaGthatMmvDdYkQajCy2g7562gFt72g2EhylCAP63tcRR01xx1LOkFdKiTApTqmZOgI4gdlfo2secODMXj3yDnH14LckVb1HC83C_V9hms88jMHW5pkYwLh0VE74Wo_4BKmULqKisgVuydTDZeWNSRzu1YoQuh9DXQ20qFzpI9-HkekbFDSNPj Table 1. Analytical data on the content of cell-wall constituents in various plant materials. % of dry weight %of total cell-wall substances % of total neutral sugar anhydrides of Ja hemicellulose S Hemicellulose Hemicellulose £ '* Timothy, leaf stage 12.4 4.7 16.1 3.5 36.7 33.8 12.8 43.9 9.5 10 15 25 50 20.0 6.9 Cocksfoot, pasture stage 16.2 4.8 22.2 4.4 45.6 35.5 10.5 44.3 9.6 10 20 20 50 17.2 11.3 Meadow fescue, » 15.1 5.1 21.1 4.9 46.2 32.7 11.0 45.7 10.6 10 20 20 50 18.8 13.0 Meadow grass, • 18.4 4224 6 5.4 ' 52.6 35.0 8.0 46.8 10 2101020 60 14.2 7.9 Cocksfoot, early heading stage 20.1 4.8 22.7 7.0 54.6 36.8 8.8 41.6 12.8 5 10 10 75 12.6 7.0 Meadow fescue, early head- ing stage 19.9 4.7 26.1 7.7 58.4 34.1 8.0 44.7 13.2 5 10 10 75 12.1 6.0 Timothy, blooming 20.1 4.2 28.6 9.3 62.2 32.3 6.8 46.0 14.9 5 10 10 75 8.6 5.6 » straw 23.0 4.0 29.0 12.2 68.2 33.7 5.9 42.5 17.9 5 10 10 75 4.4 4.9 Rye, straw 23.6 3.6 34.0 14.2 75.4 31.3 4.8 45.1 18.8 + + +5 10 85 2.8 6.0 Common reed (Phragmites communis) June 21 19.4 4.0 27.4 9.3 60.1 32.3 6.6 45.6 15.5 + + + 5 10 85 16.3 9.4 Rush (Scirpus lacuster), Aug. 22 15.8 6.0 26.5 8.6 56.9 27.8 10.5 46 6 15.1 10 ++ 15 75 10.3 7 8 Sedge (Carex gracilis), June 22 18.0 5.8 23.9 10.6 58.3 30.9 10.0 41.0 18.1 5 20 75 17.5 6.1 Oat hulls 32.9 4.6 30.2 13.8 81.5 40.4 5.6 37.1 16.9 + + +5 10 85 1.4 6.3 Wheat bran 24.6 3.0 7.5 6.6 41.7 59.0 7.2 18.0 15 8 + + +5 40 55 15.7 6.7 Red clover, leaf stage ..11.0 10.0 9.2 4.8 35.0 31.4 28.6 26.3 13.7 25 30 30 15 + + 28.0 10.6 » blooming ..12.6 10.2 16.9 10.4 50.1 25.1 20.4 33.7 20.8 20 15 25 40 + + 15.2 7.2 » heads 10.8 11.0 12.8 11.4 46.0 23.5 23.9 27.8 24.8 30 20 30 20 + + 20.0 6.4 » leaves 10.6 10.9 9.5 5.2 36.2 29.3 30.1 26.2 14.4 20 40 25 .15 + + 28.7 8.5 » blooming, up- per halves of stalks 12.6 11.1 22.5 8.2 54.4 23.1 20.4 41.4 16.1 20 10 20 50 + + 8.9 5.9 Red clover, blooming, lower halves of stalks 15.0 9.3 26.0 10.1 60.4 24.8 15.4 43.0 16.8 15 20 10 55 + 4- 7.8 5.3 Lucerne, later leaf stage .. 11.4 10.4 12.8 5.9 40.5 28.1 25.7 31.6 14.6 25 25 25 25 + 4- 27.4 11.5 128 129 Marrow-stem kale, leaves 10.2 12.9 7.8 3.6 34.5 29.6 37.4 22.6 10.4 30 30 30 10 + + + 20.2 15.2 Marrow-stem kale, stalk rind 11.4 16.9 14.0 1.8 44.1 25.9 38.3 31.7 4.1 30 10 40 20 + + + 7.5 11.1 Marrow-stem kale, vascular bundles 16.2 6.9 29.3 15.5 67.9 23.9 10.2 43.1 22.8 10 5 5 80 + 4.1 3.9 Swede, tops 10.5 12.6 8.2 3.8 35.1 29.9 35.9 23.4 10.8 30 30 30 10 + + + + 25.4 10.5 » peeled 10.7 9.9 7.5 0.8 28.9 37.0 34.3 25.9 2.8 20 40 30 10 ++ 10.7 5.8 » rind 11.0 15.2 10.3 2.7 39.2 28.0 38.8 26.3 6.9 30 15 35 20 + + + + 17.3 7.5 Sugar beet tops 7.7 8.3 6.6 3.6 26.2 29.4 317 25.2 13.7 30 + 60 10 ++ 16.0 14.2 » pulp 20.2 16.2 23.1 6.9 66.4 30.4 24.4 34.8 10.4 20 10 65 5 ++ 14.2 5.1 Chickweed (Stellaria media) 8.9 9.1 11.1 1.9 31.0 28.7 29.4 35.8 6.1 30 20 30 10 10 + + 28.4 21.7 Birch leaves (Betula verru- cosa) 11.3 9.0 7.2 14.4 41.9 27.0 21.5 17.2 34.3 25 25 25 25 + + + 19.8 3.9 Alder leaves (Alnus incana) 10.4 8.5 5.9 8.5 33.3 31.2 25.5 17.8 25.5 25 30 30 15 + + 26.2 4.3 Aspen leaves (Populus tre- mulus) 10.7 8.8 9.3 10.1 38.9 27.5 22.6 23.9 26.0 20 20 30 30 + + 18.7 4.3 Equisetum praiense, June 30 13.4 15.0 17.0 4.1 49.5 27.1 30.3 34.3 8.3 30 30 10 + + + 30 + 20.7 15.4 Equisetum limosum, July 16 14.2 15.9 20.5 4.8 55.4 25.6 28.7 37.0 8.7 30 30 10 + 30 + 11.0 15.8 Dryopteris Unnaeana, leaves, July 1 9.6 8.7 10.6 19.2 48.1 20.0 18.1 22.0 39.9 35 45 10 10 + + 20.1 7.6 Sphagnum recurvum 22.4 13.8 19.8 3.4 59.4 37.7 23.2 33.4 5.7 40 25 5 25 5 + + 6.5 3.9 Polytrichum commune 29.3 6.1 18.5 21.8 75.7 38.7 8.1 24.4 28.8 30 30 5 5 30 + 6.2 2.7 Cladonia rangiferina 64.5 2.8 10.1 3.0 80.4 80.2 3.5 12.6 3.7 35 25 40 3.9 2.6 Spruce wood (Picea ex- celsa) 17.6 3.3 46 0 24 1 910 19 4 3 6 50 4 26 5 10 10 10 30 40 0.3 0.2 Pine wood (Pmus silvestris) 14.7 3 4 39.6 25.1 83.0 17.7 4.3 47.7 30.3 10 10 10 30 40 0.6 0.2 Birch wood (Betula verru- cosa) 21.5 4.2 40.0 15.7 81.4 26.4 5.2 49.1 19.3 ++ ++ + 100 + 0.2 0.2 Alder wood (Alnus incana) 18.3 4.8 35.7 19.0 77.8 23.5 6.2 45.9 24.4 5 10 + + + 85 + 1-6 0.3 130 Results and discussion Table 1 presents the contents of cell-wall carbohydrates and crude lignin as determined by the author’s analysis scheme. In order to characterize the nature of the samples, the crude protein and ash contents are also shown. The first plant group consists of various grasses as well as two sedge plants. Also oat hulls and wheat bran are listed there. A characteristic feature of the grasses is the high content of cellulose and hemicellulose, which, with the exception of uronic anhydrides, increases with age. The composition of the hemicellulose in the different grass species of the same growth stage is rather similar. Xylose anhydride is the dominating component already at the early leaf stage, and its proportion increases in older plants. Anhydrides of galactose, glucose and arabinose occur to some extent in plants of all ages. The cell-wall carbohydrate composition of rush and sedge resembles very closely that of the grasses. The quantitative nature of their hemi- cellulose is also similar, except that they contain slightly more uronic anhydrides. The composition of oats hulls is comparable to that of straw, whereas wheat bran dif- fers appreciably from the other plant materials included in this group. The composition of leguminous cell-walls diverges widely from that of the grasses. The former contain less cellulose and also less neutral sugar anhydrides of hemicellulose, while their content of uronic anhydrides is more than double that of the grasses. The cellulose content of clovers increases as the plant ages, but the in- crease in hemicellulose is quite small and the uronic anhydride content is independent of the growth stage, just as is the case with the grasses. The composition of clover leaves at the flowering stage of the plant is approximately the same as that of young clover plants in the spring at the leafy stage; the flowers and buds contain more cell-wall substances than the leaves, and the stalks are especially rich in cel- lulose and lignin. All the analysis results from lucerne agree with what would be expected from clover at the same growth stage. The composition of hemicellulose in legumes differs greatly from that in grasses and segde plants. The principal neutral sugar anh} rdrides are the same as in grasses and in angiosperms as a whole, but the xylose content of clover is relatively low. The legume samples also showed traces of mannose and rhamnose, which are lacking in the grasses. A distinguishing feature of root crops and their tops is the high sugar content and the low cell-wall content. Of the latter group of substances, uronic anhydrides are abundant and xylose anhydrides scanty. The hemicellulose composition in tops of the same genus is quite similar, a feature noted also in other related plants. This appears to be a suitable characteristic for the chemical taxonomy of plants. The cell-wall composition of the leaves and stem cortex of marrow-stem kale is similar, while that of the vascular bundles of the stem resembles wood and straw. The rind of swedes is richer in cell-wall substances, especially xylose, than the leaves of the plant, while the pulp is very low in such substances, as was to be excepted. The con- tent of uronic anhydrides in this group of plant materials is high, particularly in the rind; this is also evident from the composition of sugar beet pulp. The compound involved is evidently galacturonic anhydride, characteristic of pectic substances. Stellaria media, a common weed in fields of root crops, resembles 131 in its cell-wall composition the tops of root crops. The hemicellulose composition of this plant is unusual, since it is the only one of the angiosperms studied by the author in which there were more than traces of mannose anhydride. The leaves of trees are found to contain surprisingly small amounts of cell- wall polysaccharides. Their total amount, together with crude lignin, make up only about 40 % of the dry matter. It is also notable that such leaves contain 9.5— 12 % sugars and even 9.5—12 % crude fat (according to Paloheimo et ah, 8), so that only 15 % is undetermined substances, and these include organic acids other than uronic acids. Although the analysis procedure is intended for animal feeding stuffs, it is apparently suitable also for cryptogams and woody materials, even though some cases give erroneous values for crude lignin. For instance, the unusually high lignin content of the hair-cap moss (Polytrichum) is due to the fact that the core of its stem does not dissolve in the polysaccharide hydrolysis used. On the other hand, the low percentage of lignin in hard woods as compared with soft woods arises from the fact that certain materials in hard wood, which some investigators regard as lignin, dissolve in diluted acid, in this case in the hemicellulose hydrolysing solution (cf. 9). The different families of cryptogamous plants investigated in the present studies differ greatly from one another in their cell-wall composition. Common to all of them are their abundance of mannose anhydride and their deficiency of pentose anhydrides. Large amounts of uronic anhydrides are found in Equisetum and Sphagnum. Lichen (Gladonia) is exceptional, since no pentose anhydrides are to be found in it. The hemicellulose polysaccharides in wood are readily hydrolysable in the conventional acidic solution, since the combined amounts of mannose and xylose in the cellulose hydrolysate make up only 10—15 % of the total sugars. There is more mannose than xylose. Distinct differences are observed in the hemicellulose composition between hard and soft wood. In the former, hemicellulose is composed principally of xylose anhydride, while in the latter mannose anhydride is the pri- mary component. In Table 2 the sum of the cell-wall constituents is compared with Paloheimo’s membrane substances and crude fibre. In column c it can be seen that the membrane substances amount to 35—115 % of the above-mentioned sum. In grasses there are only small amounts of cell-wall materials which are soluble in dilute acid. The leaves and flowers of red clover are more readily soluble than grasses, while the stems are less easily dissolved. Root crops and their tops, as well as the leaves of trees, are also readily soluble. In wood, clover stalks and certain other plant materials the mem- brane substance percentage was found to be higher than the sum of the cell-wall frac- tions. This may be due to several reasons: to analytical errors, to the presence in the cell-walls of organic components other than carbohydrates and lignin, or to the fact that lignin dissolves more readily in the concentrated acid used in the polysaccharide hydrolysis than in the dilute acid used for determining the membrane substances. This latter dilute solution consisted of 0.05 N hydrochloric acid. It is somewhat Table 2. Cell-wall contents of the materials in Table 1 compared to the membrane substances (Palo- heimo’s method) and crude fibre. abed e f Total Membrane sub- Crude fibre Ratio cell-wall stances cellu- sub- (Paloheimo’s lose: stances method) crude from % % % % fibre Table 1. of dry wt. of item a ofdrywt. of item a Timothy, leaf stage 36.7 Cocksfoot, pasture stage 45.6 Meadow fescue, pasture stage 46.2 Meadow grass, pasture stage 52.6 Cocksfoot, early heading stage 54.6 Meadow fescue, early heading stage .... 58.4 Timothy, blooming 62.2 » straw 68.2 Rye, straw 75.4 Common reed (Phragmites communis) .. 60.1 Rush (Scirpus lacuster) 56.9 Sedge (Carex gracilis) 58.3 Oat hulls 81.5 Wheat bran 41.7 Red clover, leaf stage 35.0 » blooming 50.1 » » heads 46.0 * leaves 36.2» » » upper halves of stalks 54.4 » » lower » » 60.4 Lucerne, later leaf stage 40.5 Marrow-stem kale, leaves 34.5 » » stalk rind 44.1 » n vascular bundles .... 67.9 Swede, tops 35.1 » peeled 28.9 » rind 39.2 Sugar beet tops 26.2 » pnlp 66.4 Chickweed (Stellaria media) 31.0 Birch leaves (Betula verrucosa) 41.9 Alder » (Alnus incana) 33.3 Aspen » (Populus tremula) 38.9 Equisetum pratense 49.5 » limosum 55.4 Dryopteris linnaeana, leaves 48.1 Sphagnum recurvum 59.4 Polytrichum commune 75.7 Cladonia rangi/erina 80.4 Spruce wood (Picea excelsa) 91.0 Pine * (Pinus silvestris) 83.0 Birch * (Betula verrucosa) 81.4 Alder » (Alnus incana) 77.8 30.9 84.2 18.3 34.8 76.3 24.6 34.6 74.9 23.6 45.0 85.6 28.2 47.8 87.5 28.4 52.0 89.0 32.0 56.2 90.4 35.2 61.3 89.9 37.4 79.1 104.9 47.5 57.8 96.2 34.0 53.6 94.2 31.3 52.4 89.9 29.1 79.1 97.1 40.1 20.0 48.0 10.5 18.0 51.4 11.3 38.9 77.6 27.8 29.5 64.1 21.6 16.6 45.9 11.2 59.8 110.0 42.2 64.5 106.8 47.8 26.7 65.9 18,9 21.4 62.0 11.3 25.9 58.7 17.0 74.2 109.3 53.8 18.2 51.9 10.6 10.2 35.3 7.6 19.0 48.5 13.3 15.5 59.2 9.4 37.8 56.9 24.4 21.0 67.7 12.6 23.5 56.1 15.6 15.9 47.7 12.8 24.6 63.2 18.3 32.2 65.1 20.0 40.9 73.8 24.7 28.2 58.6 15.6 67.3 113.3 45.9 69.0 91.1 33.8 69.1 85.9 30.7 92.2 101,3 75.0 88.2 106.3 70.4 91.6 112.5 63.2 89.7 115.3 63.6 49.9 1: 1.14 53.9 1:1.11 51.1 1: 1.12 53.6 1:1.15 52.0 1: 1.25 54.8 1: 1.23 56.6 1: 1.23 54.8 1: 1.29 63.0 1: 1.40 56.6 1: 1.24 55.0 1: 1.18 49.9 1: 1.22 49.2 1: 1.33 25.2 1: 1.40 32.3 1: 1.23 55.5 1: 1.84 47.0 1; 1.69 30.9 1; 1.18 77.6 1: 1.88 79.1 1: 1.84 46.7 1: 1.48 32.8 1: 1.45 38.5 1: 1.22 79.2 1: 1.84 30.2 1: 1.29 26.3 1: 1.01 33.9 1: 1.29 35.9 1: 1.42 36.7 1: 1.06 40.6 1: 1.14 37.2 1:2.17 38.4 1:2.17 47.0 1: 1.97 40.4 1; 1.18 44.6 1: 1.20 32.4 1: 1.47 77.3 1: 2.32 44.6 1: 1.83 38.2 1. 3.04 82.4 1: 1.63 84.8 1; 1.78 77.6 1: 1.58 81.7 1: 1.78 133 surprising to observe that even with such mild hydrolysis, more than one-half of the total cell-wall substances in certain plant materials dissolved. According to the data in the last column in Table 2, crude fibre makes up 25 85 % of the sum of the cell-wall constituents. Crude fibre is regarded as containing only a part of the cell-wall substances, and the composition of this part has been found by many investigators to vary in different plants materials (e.g. 2,6). In the present experiments a definite correlation is to be seen between the solubilities in the membrane and crude fibre determination processes. Another observation which can be made is that in all the materials in which the crude fibre exceeded 60 % of the sum of the cell-wall components, the membrane percentage was higher than this sum. Since crude fibre is considered as roughly corresponding to the amount of cel- lulose, comparisons are made in the last column between the cellulose and fibre contents. In general, the amount of crude fibre is I—2 times that of cellulose. In a couple of the cryptogams and in the leaves of trees the figure is above 2. In this respect there are differences between different plant species. At an early growth stage the difference between the cellulose and crude fibre percentages is smaller than at later stages, and in some delicate plant tissues there is practically no difference between the amounts of cellulose and crude fibre. Summary In this investigation, analyses were made of the contents of hemicellulose, cellulose, crude lignin, crude protein and ash in 43 different plant materials. In addition, the proportions of various sugar anhydrides in the hemicellulose fraction were determined. The carbohydrate analyses were made by hydrolysing to mono- saccharides, except for the uronic anhydrides, which were determined by the de- carboxylation method. The sum of the cell-wall constituents thus determined was subsequently compared with Paloheimo ’s membrane substances and crude fibre. Large variations in the cell-wall composition were found between different plant materials. Furthermore, there were large differences in the solubility of the cell-wall substances of different plant materials in the hydrolysing procedures used in determinations of membrane substances and crude fibre. The hemicellulose composition of different species in the same genus and even in the same family was found to be similar in definite plant parts and at de- finite growth stages. This appears to be a generic characteristic in the chemical taxonomy of plants. REFERENCES (1) Andrews, P. & Hough, L, & Stacey B. 1960. Polysaccharide composition of leaves. Nature 185: 166-167. (2) Armstrong, D. G. & Cook, H. & Thomas, B. 1950. The lignin and cellulose contents of certain grassland species at different stages of growth. J. Agric. Sci. 40; 93 99. 134 (3) Gaillard, B. D. E. 1958. A detailed summative analysis of the crude fibre and nitrogen-free extractive fractions of roughages. 11. The analysis of straw, hay, grass and mangold. J. Sci. Food Agric. 9: 346 353. (4) —»— 1962. The relationship between the cell-wall constituents of roughagesand the digestibility of the organic matter. J. Agric. Sci. 59: 369 373. (5) Jarrige, R. 1960. The membrane constituents of herbage. Proc. 8. Intern. Grassl. Congr. 1960, p. 628-634. (6) Paloheimo, L. 1953. Some persistant misconceptions concerning the crude fibre and the nitrogen- free extract. J. Sci. Agric. Soc. Finl. 25: 16 22. (7) —* & Paloheimo, I. 1949. On the estimation of the total of vegetable membrane substances. Ibid. 21; 1-16. (8) & Vainio, K. A. & Kero, M.-L. & Herkola, E. 1961. Analyses of plant products in greater detail. Ibid 33: 51 56. (9) Salo, M.-L. 1957. Lignin studies. I. Investigation concerning lignin determination. Ibid. 29: 185-193. (10) — »— 1965. Determination of carbohydrate fractions in animal foods and faeces. Acta agr. fenn. 105 11) Sternkopf, G. 1964. Untersuchungen tiber die chemisch-analytische Auftrennung des stickstoff- freien Extraktstoff (NFE) in griinem Pflanzenmaterial. Arch. Tierernähr. 14: 59 66. (12) Waite, R. & Gorrod, R. N. 1959. The comprehensive analysis of grasses. J. Sei. Food Agric. 10: 317-326. SELOSTUS: SOLUSEINÄMIEN KOOSTUMUKSESTA ERILAISISSA KASVIAINEISSA Maija-Liisa Salo Yliopiston kotieläintieteen laitos, Helsinki Kasvien soluseinämät ovat koostuneet pääasiassa hiilihydraateista ja ligniinistä. Soluseinämä- hiilihydraatit ovat neutraaleista ja happamista sokeriyksiköistä rakentuneita polysakkarideja. Näistä on selluloosa yleisin. Muista käytetään tavallisesti yhteisnimitystä hemiselluloosa. Tutkimuksessa esitetään 43 eri kasvimateriaalin hemiselluloosa-, selluloosa-, raakaligniini-, raaka- proteiini- ja tuhkapitoisuussekä hemiselluloosafraktiosta lisäksi eri sokerianhydridien osuudet. Edelleen siinä verrataan soluseinämäaineiden summaa Paloheimon menetelmällä määritettyyn kettoaineeseen sekä raakakuituun. Tutkimuksessa todettiin suuria eroja eri kasviaineiden soluseinämäainekoostumuksessa ja samoin soluseinämäaineiden liukoisuudessa kettoaine- ja raakakuitukeitoissa. Saman kasvisuvun ja kasviheimonkin eri lajien hemiselluloosan koostumus todettiin hyvin saman- laiseksi tietyssä kasvuvaiheessa ja tietyssä kasvinosassa. Se näyttää olevan eräs kasvien kemialliseen taksonomiaan sopiva lajituntomerkki.