J. B. HARBORNE AND J. J. CORNER
Bridel, M. & Charaux, C. (1925). C.R. Acad. Sci., Paris, 180, 387. Cardini, C. E. & Yamaha, T. (1960). Arch. Biochem. Biophys. 86, 127. Cartwright, R. A., Roberts, E. A. H., Flood, A. E. & Williams, A. H. (1955). Chem. & Ind. p. 1062. Charaux, C. (1925). Bull. Soc. Chimn. biol., Paris, 7, 1053. Corner, J. J. & Harborne, J. B. (1960). Chem. & Ind. p. 76. Dodds, K. S. & Long, D. H. (1955). J. Genet. 53, 136. Dunlap, W. J. & Wender, S. H. (1960). J. Chromat. 3, 305. Gorter, K. (1908). Liebigs Ann. 358, 327; 359, 217. Griffiths, L. A. (1959). J. exp. Bot. 10, 437. Grisebach, H. & Ohis, W. D. (1961). Experientia, 17, 4. Harborne, J. B. (1960a). Biochem. J. 74, 262. Harborne, J. B. (1960b). Biochem. J. 74, 270. Harborne, J. B. & Corner, J. J. (1960). Biochem. J. 76, 53P. Harborne, J. B. & Corner, J. J. (1961). Arch. Biochem. Biophys. 92, 192. Harborne, J. B. & Sherratt, H. S. A. (1961). Biochem. J. 78, 298. Helferich, B. & Lutzmann, H. (1938). Liebigs Ann. 537, 11. Herrmann, K. (1958). Pharmazie, 5, 266. Hutchinson, A., Roy, C. & Towers, G. H. N. (1958). Nature, Lond., 181, 841.
Johnson, J. R. (1942). In Organic Reactions, vol. 1, p. 210. New York: John Wiley and Sons Inc. Kameswaramma, A. & Seshadri, T. R. (1947). Proc. Indian Acad. Sci. A, 25, 43. Karrer, W. (1958). Konstitution und Vorkommen der organischen Pflanzenstoffe, p. 379. Basle: Birkhauserverlag. Klosterman, H. J. & Muggli, R. (1959). J. Amer. chem. Soc. 81, 2188. Kosuge, T. & Conn, E. E. (1959). J. biol. Chem. 234, 2133. Lee, S. F. & Le Tourneau, D. (1958). Phytopathology, 48, 268. Levy, C. C. & Zucker, M. (1960). J. biol. Chem. 235, 2418. McCalla, D. R. & Neish, A. C. (1959). Canad. J. Biochem. Physiol. 37, 537. Payen, P. (1846). Liebigs Ann. 60, 286. Pridham, J. B. & Saltmarsh, J. B. (1960). Biochem. J. 74, 42P. Privat, G. (1959). C.R. Acad. Sci., Paris, 249, 456. Rabin, R. S. & Klein, R. M. (1957). Arch. Biochem. Biophys. 70, 11. Schutte, H. R. & Bohme, H. (1958). Qual. Plant. Mater. veg. 4, 474. Schwimmer, S. & Bevenue, A. (1956). Science, 123, 543. Sondheimer, E. (1957). Arch. Biochem. Biophys. 74, 131. Stoll, A., Renz, J. & Brack, A. (1950). Helv. chim. acta, 33, 1877.
Biochem. J. (1961) 81, 250
The Synthesis of mesolnositol in Germ-free Rats and Mice
BY N. FREINKEL AND R. M. C. DAWSON* Thorndike Memorial Laboratory, Second and Fourth Medical Service (Harvard), Boston City Hospital, Mass., U.S.A.

(Received 19 May 1961)

The precise role of dietary mesoinositol in mammalian nutrition remains controversial despite more than two decades of inquiry. Early reports linking inositol to the promotion of growth and prevention of alopecia in mice (e.g. Woolley, 1940, 1941) and rats (e.g. Pavcek & Baum, 1941; Cunha, Kirkwood, Phillips & Bohstedt, 1943) have not been uniformly reduplicated (e.g. Martin, 1941; Fenton, Cowgill, Stone & Justice, 1950; Ershoff & McWilliams, 1943; McCormick, Harris & Anderson, 1954). In addition, no characteristic syndrome of inositol deficiency has yet been described in man. However, the recent findings that added inositol is required for the propagation of human cells in tissue culture (Eagle, Oyama, Levy & Freeman, 1957), and unbound inositol is concentrated in most mammalian tissues (Dawson & Freinkel, 1961) prompted a re-examination of the endogenous biosynthesis of inositol. * Permanent address: Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge.
Excellent relevant data were available in this area before the inception of the present studies. Needham's (1924) observations of sustained inositoluria in rats maintained on low inositol diets had been re-enforced by the independent reports of Daughaday, Larner & Hartnett (1955) and Halliday & Anderson (1955) that [14C]inositol could be recovered from rats given repeated injections of ['4C]glucose. However, as was first suggested by Woolley (1942), such 'endogenous' inositol could have originated from alimentary tract micro-organisms which are known to profoundly affect the picture in other fields of mammalian metabolism, e.g. the [14C]urea catabolism of mammals is entirely due to associated microorganisms (Kornberg, Davies & Wood, 1954). To minimize intestinal contributions, Daughaday et al. (1955) excised the entire gut immediately before analysis of the carcass for inositol. The possibility of microbial biosynthesis and absorption of labelled inositol from the intestine during the 3 days

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INOSITOL IN GERM-FREE ANIMALS
preceding killing was not thereby excluded. Similarly, although Halliday & Anderson (1955) effected prolonged reduction of intestinal flora by feeding antibiotics and caecectomy, the potential for inositol synthesis by the residual intestinal yeasts was not eliminated. An alternative approach was employed by Luckey, Pleasants, Wagner, Gordon & Reyniers (1955), who maintained complete intestinal sterility in two germ-free rats during chronic balance studies. However, owing to 'the variation in inositol analyses of different lots of diet', they were unable to interpret their recovery of 74 and 40 % more inositol than had been fed as definite evidence for net biosynthesis. For the present experiments, the isotopic and germ-free techniques were combined. [1_14C]_ Glucose was acutely administered to germ-free mice and rats and recovery of [14C]inositol from the carcass was contrasted with values obtained under identical conditions in normal animals.

EXPERIMENTAL

Animals. Five days before the studies with radioactive glucose on 26 October 1959, germ-free rats (born 21 September 1959), germ-free mice (born 13 September 1959) and normal rats and mice (born 10 September 1959) were sent by air-express from the Lobund Institute, Notre Dame, Ind., U.S.A., to the Charles River Laboratories, Dover, Mass., U.S.A. A germ-free isolator (Reyniers, 1959) containing food, water, cages, dissection tools and cotton swabs was employed for the transport and subsequent maintenance of the germ-free animals. At the end of the [I4C]glucose experiments, swabbings were obtained from the inside walls of the isolator and from the surfaces and freshly expressed faeces of the germ-free animals. Sterile cultures from these swabbings in thioglycollate broth and on blood-agar plates indicated that contamination had not occurred during transport and maintenance of the animals at the Charles River Laboratories. All animals were generously donated by Professor P. C. Trexler of the Lobund Institute; maintenance of isolators and assistance with germ-free technique were generously provided by Dr H. L. Foster and his associates of the Charles River Laboratories. Injection solutions. Chromatographically pure [1-14C]glucose (Nuclear-Chicago Corp., Ill.) was combined with unlabelled glucose (National Bureau of Standards) to prepare an injection solution containing 9-8 mg. of glucose and 30,c of '4C/ml. The material was sterilized by autoclaving for 20 min. with 16 lb./in.2 pressure. An ampouled portion together with appropriate syringes and needles was introduced into the isolator by passage through the supply lock, by using cold sterilization with a peracetic acid spray. To establish that the [1-_4C]glucose was not contaminated with radioactive inositol, any contamination was concentrated by growing the inositol-dependent yeast Kloeckera brevis in the presence of the [14C]glucose. A sample (1 ml.) of the glucose injection solution was added to the growth medium of Campling & Nixon (1954). Carrier inositol (4-8 pg.) was added and the mixture inoculated with K. brevis. The yield of K. brevis cells obtained after 3 days'
growth at 250 was normal as judged by turbidity readings (4-9 jug.). The cells, which previous experiments had shown contain all the inositol in combined form (R. M. C. Dawson & N. Freinkel, unpublished work), were hydrolysed with 6N-HC1 for 40 hr. at 105. To the hydrolysate was added 50,ug. each of inositol and glucose as carriers and it was then dried in vacuo. The residue was dissolved in water and passed through a column of mixed-bed ion-exchange resin, Amberlite MB-1 (8 cm. long x cm. diameter). The effluent and washings were evaporated to low volume and applied as spots to two paper-strip chromatograms 2-5 cm. wide. These were developed with the solvents and conditions described below, and, after drying, passed through an automatic micromil-window strip scanner (NuclearChicago Actigraph). The strips were treated with acetoneAgNO3 and ethanolic NaOH reagents to detect the inositol and glucose spots (Trevelyan, Procter & Harrison, 1950). All of the radioactivity was confined to the glucose area. Under the conditions of the test % of the radioactivity occurring as mesoinositol would readily have been detected.

METHODS

Experimental work on germ-free and normal animals. All studies were performed with male paired germ-free and normal animals. Samples of the glucose injection solution were administered at hourly intervals intraperitoneally as follows: one germ-free and one control rat were each given three 1 ml. injections; one germ-free and one control rat each received six ml. injections; and one germ-free and one control mouse were each treated with three 0-5 ml. injections. The germ-free animals were injected by using the rubber manipulating gloves in the isolator. All animals were allowed free access to food throughout the experimental period. The animals were killed by a blow on the head 1 hr. after the last injection of each pair. Immediately thereafter, the animals were homogenized with 11N-H2SO4. Mice were introduced directly into 50 ml. of 11 N-H2SO4 contained in a Waring Blendor. Separate blenders were employed for each animal. Rats were preliminarily sectioned into smaller pieces and ground with a meat grinder into 100 ml. of 11 N-H2S04/100g. body wt. contained in Waring Blendors. Subsequent processing was by a modification of the method of Halliday & Anderson (1955). The suspensions (animals homogenized with 11 N-H2S04) were hydrolysed in sealed bottles at 16 lb./in.2 pressure for 12-3 hr. at 1220. The black hydrolysates were cooled and to each was added 25 mg. of carrier inositol. The hydrolysates were diluted with an equal volume of water and solid CaC03 was added in small portions until they were at about pH 5. The precipitate of CaSO4 and other insoluble matter was filtered off under pressure and the precipitate washed well with water. The filtrate and washings were combined and decolorized with Darco G60 charcoal. This was most effectively carried out in the cold with successive small portions of charcoal, and filtering before using the next portion. Samples of the clear filtrate were retained for inositol assay (Campling & Nixon, 1954) as described bv Freinkel, Dawson, Ingbar & White (1959). The remainder of the filtrates were passed through columns of mixed-bed ion-exchange resin, Amberlite MB-1 (2 in. diameter, 12 in. long for mouse experiments, 14 in. long for germ-free rat filtrates, and 17 in. long for control rats). The effluents and

N. FREINKEL AND R. M. C. DAWSON
washings were free of SO2- ions when tested with Ba2+ ions. They were evaporated to low volume (about 0-5 ml.) and 4 ml. of ethanol was added to induce crystallization. The separated crystals were dissolved in 2 ml. of water. The mixture was centrifuged, and the supernatant separated from debris, evaporated to low volume (0-25-0.5 ml.) and treated with 2-3 ml. of ethanol. After the inositol had been allowed to crystallize for 1 hr. at 00, it was collected and recrystallized from aqueous ethanol. The crystals were dried (e.g. for animal no. 6: melting point, 222-5 uncorr.). The samples of crystalline inositol were dissolved in 5 ml. of water. Samples (1 ml.) were evaporated on planchets for radioactive assay with an automatic micromil-window flow counter (Nuclear-Chicago Corp.) and a further sample (0.1 ml.) was used for microbiological inositol assay (Campling & Nixon, 1954). Chromatography of inositol samples. Samples of the above inositol solutions equivalent to 1-2 mg. were run on paper chromatogram strips 5 in. wide. Two solvent systems were used: (1) propanol-ethyl acetate-water (24:13:7, by vol.) and (2) propan-2-ol-acetic acid-water (3: 1:1, by vol.), both of which give a good separation of inositol from sugars such as glucose. After the chromatograms had been run (40 hr., descending) they were dried and a central strip i in. wide was cut out and treated with the acetone-AgNO, and ethanolic NaOH reagents for detecting polyols (Trevelyan et al. 1950). Only an inositol spot was rendered visible, and the strips on either side of this were cut out of the untreated paper and extracted twice over 10 hr., each time with 20 ml. of water. A sample (0.05 ml.) was taken for inositol assay and the remainder (ml.) transferred to a planchet for counting. For radioactive assay, sufficient counts were observed to reduce the probable error of the measurement to less than 3%. All planchets were appropriately corrected for selfabsorption to a mass of 5 mg.
RESULTS Results are summarized in Table 1. In the rats, total recovered inositol, after cprrection for added inositol carrier, averaged 27.7/ng./100 g. body wt. in the two germ-free, and 30-1 Tg./100 g. body wt.

in the two normal, animals. The findings are in substantial agreement with the average values for carcass inositol of 28-1 mg./100 g. which Halliday & Anderson (1955) obtained in three normal rats by a similar fractionation procedure, and slightly greater than the 17-3 mg./100 g. (Daughaday et al. 1955) and 23-.3 mg./100 g. (McCormick et al. 1954) recovered by somewhat different techniques. Incomplete recovery of inositol precluded estimation of total carcass inositol in the germ-free mouse. The value of 27-8 mg./100 g. in the control mouse is somewhat less than the average of 39-2 mg./100 g., which Woolley (1942) observed in six mice fed on a stock diet for 4 weeks after weaning. Significant quantities of radioactivity were present in the inositol crystallized from the carcasses of all mice and rats after the administration of [1-14C]glucose. That this constituted newly synthesized [14C]inositol was established for each animal by the demonstration of constant inositol specific radioactivity during chromatographic resolution in two solvent systems. In this determination of specific radioactivity the microbiological assay of inositol gave values within about + 6 % and the radioactivity was determined within + 3 %. Further, the [14C]inositol isolated from one animal was used to support the growth of K. brevis. The yeast was then recovered from the culture medium, it was hydrolysed with acid and inositol was then isolated from the hydrolysate by strip chromatography. The specific radioactivity of the isolated and original inositol were identical (315 and 314 counts/min./mg. of inositol), confirming that the 14C had been incorporated into mesoinositol. Similar experiments reported in the Experimental section had already shown that the [14C]glucose injected was free of meso[14C]inositol. In the paired rats and mice killed 3 hr. after the first injection of [1-14C]glucose, the total counts
Table 1. The formation of radioactive inositot from [1-14C]glucose in vivo Recovery of [14C]inositol

Animal Rats Germ-free

Weight experiment (g.) (hr.)*

108 180

Duration of Recovered inositol
Counts/mg. of inositol/min.t

(mg.)t

51-5 75-5 57-3 83-0

Average

132 132-5 335

164 - 3c.

Total counts 13 612
Per cent of [1-_4C]glucose administered
0-0279 0-0410 0-0787 0-0558
Control Germ-free Control
Mice 3 22-259 0-0478 Germ-free 35-592 245-5 0-0704 Control * Interval elapsing between the first of a series of injections of [1-14C]glucose and killing. For details of administration schedules, see text. t Before fractionation 25-0 mg. of carrier inositol was added to each carcass'. t Specific radioactivities of inositol (counts/min./mg.) isolated after paper chromatography of crystalline material int (A) propanol-ethyl acetate-water, and (B) propan-2-ol-acetic acid-water. Average chromatographic specific activities were employed to derive minimal estimates of 'total counts' recovered as radioactive inositol from each animal.
recovered as radioactive inositol were greater in the control than in the germ-free animals. In the paired rats killed at 6 hr., total recovered radioactive inositol was greater in the germ-free animal. Paired values did not differ by more than 50 % in any instance, thus suggesting that intestinal microflora do not appreciably influence the fractional rates of endogenous inositol biosynthesis from administered [14C]glucose in these species.
INOSITOL IN GERM-FREE ANIMALS 253 therefore that 0-05 % represents an approximate
minimal figure for the conversion at equilibrium. Since the half-time for the turnover of glucose in the rat is less than 1 hr. (Feller et al. 1950), and since not enough glucose was injected at any time to disrupt equilibrium conditions, it might be anticipated that most of the labelled glucose in all of the studies had equilibrated with the exchange, able glucose pool. In the rat, turnover of body glucose amounts to 100 mg./hr./100 g. body wt. (Feller et al. 1950). Thus, taking 0-05 % as a minimal figure for the conversion at equilibration, this may be extrapolated for 24 hr. to estimate that a minimum of about 1-2 mg. of glucose/100 g. of rat is converted into inositol per day. Moreover, unless the metabolism and distribution of glucose are altered by intestinal sterility, the fact that fractional conversions of [1-14C]glucose were of similar magnitude in the germ-free rats (i.e. 0-03 and 0-08 % at 3 and 6 hr. respectively) would suggest that the minimal estimate of 1-2 mg. may equally obtain in these animals. Unfortunately, translation of the values for glucose into actual quantities of inositol is precluded by the lack of information about pathways of inositol biosynthesis. If conversion of glucose into inositol were equimolar, then the results would be compatible with a minimum synthesis from glucose of 1-2 mg. of inositol/100 g. of rat/day. However, at least in yeast, such an equimolar transformation does not obtain (Charalampous, 1957), and consequently in this species the cyclization theory of inositol formation from glucose (Fischer, 1945) does not appear to be tenable. Thus, although the present studies constitute clear evidence of endogenous inositol biosynthesis, they do not resolve the question of the nutritional adequacy of this process. SUMMARY

1. The biosynthesis of inositol from glucose has been examined in normal and germ-free rats and mice. 2. After the acute intraperitoneal administration of [1 -14C]glucose, [14C]inositol could be isolated from all animals. In paired germ-free and control rats and mice, the recoveries of radioactive inositol were of the same order of magnitude. 3. The findings would indicate that inositol can be formed from glucose in these species and that endogenous biosynthesis does not require the presence of alimentary canal micro-organisms. This investigation was supported in part by Research Grant A-1571 and Training Grant 2A-5060, National
Institute of Arthritis and Metabolic Diseases, United States Health Service, Bethesda, Md., U.S.A., and by the Capps Fund of HIarvard University, and the Wellcome Trust, London.
DISCUSSION In the present experiments it has been established that the [14C]glucose injected contains no contaminating me8o[14C]inositol. On the other hand, the crystalline mesoinositol of the correct melting point isolated from the carcasses of both germ-free and normal animals after [14C]glucose injection contained appreciable radioactivity. That this did not represent contamination of the isolated inositol with another metabolic product formed from the [14C]glucose relies on the findings: (a) that the specific activity of the [14C]inositol remained constant on paper chromatography in two solvent systems; (b) that it remained constant after a growth cycle through K. brevis and reisolation of the inositol from the yeast. These results leave little doubt that normal and germ-free animals can biosynthesize labelled inositol, after the injection of [1-14C]glucose. Since the completion of these studies, Eagle, Agranoff & Snell (1960) have reported the occurrence of definite, albeit limited, synthesis of inositol from radioactive glucose in isolated human tissue culture. The combined observations would indicate that mammalian tissues are capable of inositol production even without microbial synergy, and that all of the mammalian requirements for inositol need not be met exogenously. It cannot, however, be assumed without qualification that the unknown metabolic processes which synthesize [14C]inositol result in a net formation of inositol which is available for the animal economy, for it is conceivable that if such processes are reversible, catabolism of inositol will outstrip synthesis. Kinetics of glucose metabolism have been sufficiently documented in the rat (Feller, Strisower & Chaikoff, 1950) to justify efforts at more quantitative treatment of the present results. In control rats, 0-041 and 0-056 % of the counts in the administered [1-14C]glucose could be recovered as radioactive inositol after experimental periods of 3 and 6 hr. respectively. The figures are in the same neighbourhood as the values of 0-05 and 0-07 % obtained by Halliday & Anderson (1955) when normal rats were killed 2 hr. after the completion of a series of ten injections of [1-14C]glucose at 1 hr. intervals. It seems reasonable to assume

REFERENCES

Campling, J. D. & Nixon, D. A. (1954). J. Physiol. 126, 71. Charalampous, F. C. (1957). J. biol. Chem. 225, 595. Cunha, T. J., Kirkwood, S., Phillips, P. H. & Bohstedt, G. (1943). Proc. Soc. exp. Biol., N. Y., 54, 236. Daughaday, W. H., Lamer, J. & Hartnett, C. (1955). J. biol. Chem. 212, 869. Dawson, R. M. C. & Freinkel, N. (1961). Biochem. J. 78, 606. Eagle, H., Agranoff, B. W. & Snell, E. E. (1960). J. biol. Chem. 235, 1891. Eagle, H., Oyama, V. I., Levy, M. & Freeman, A. E. (1957). J. biol. Chem. 226, 191. Ershoff, B. H. & McWilliams, H. B. (1943). Proc. Soc. exp. Biol., N.Y., 54, 227. Feller, D. D., Strisower, E. HI. & Chaikoff, I. L. (1950). J. biol. Chem. 187, 571. Fenton, P. F., Cowgill, G. R., Stone, M. A. & Justice, D. H. (1950). J. Nutr. 42, 257.
Fischer, H. 0. L. (1945). Harvey Lect. 40, 156. Freinkel, N., Dawson, R. M. C., Ingbar, S. H. & White, R. W. (1959). Proc. Soc. exp. Biol., N. Y., 100, 549. Halliday, J. W. & Anderson, L. (1955). J. biol. Chem. 217, 797. Kornberg, H. L., Davies, R. E. & Wood, D. R. (1954). Biochem. J. 56, 355, 363. Luckey, T. D., Pleasants, J. R., Wagner, M. W., Gordon, H. A. & Reyniers, J. A. (1955). J. Nutr. 57, 169. McCormick, M. H., Harris, P. N. & Anderson, C. A. (1954). J. Nutr. 52, 337. Martin, G. J. (1941). Science, 93, 422. Needham, J. (1924). Biochem. J. 18, 891. Pavcek, P. L. & Baum, H. M. (1941). Science, 93, 502. Reyniers, J. A. (1959). Ann. N.Y. Acad. Sci. 78, 47. Trevelyan, W. E., Procter, D. P. & Harrison, J. S. (1950). Nature, Lond., 166, 444. Woolley, D. W. (1940). Science, 92, 384. Woolley, D. W. (1941). J. biol. Chem. 139, 29. Woolley, D. W. (1942). J. exp. Med. 75, 277.
Biochem. J. (1961) 81, 254
Changes in Microsomal Components Accompanying Cell Differentiation of Pea-Seedling Roots
BY U. E. LOENING Department of Botany, University of Edinburgh

(Received 17 March 1961)

This paper describes some changes found in the microsomes of pea roots during growth (see Whaley, Mollenhauer & Leech, 1960, for a review on plant microsomes). The growing-plant root provides a convenient source of material, because growth is largely in one direction and cell division is confined to the apex, so that it is possible to obtain batches of cells in progressive stages of development by cutting serial transverse segments. Small segments of the root tips were used, so that differences between the meristem and the first stages of differentiation could be studied. The experiments depend on the chance observation that the sedimented microsomal pellet may be readily separated into two components, one particulate and the other largely membranous. These components were examined by their protein and nucleic acid contents, by electrophoresis and by electron microscopy.

A preliminary communication of these results has been published (Loening, 1960).

MATERIALS AND METHODS

Preparation of root-tip segment8. Pea seeds of the variety Meteor (Sutton and Sons, Reading) were sown in horticultural vermiculite-soft tap water (3:1, v/v) and ger-
minated in the dark at 250 for 48 hr. The roots were then 3-4 cm. long; exceptionally long or short ones were rejected. The roots were cut into three serial segments, the first tip segment being 1-6 mm. long, the second 1-8 mm. and the third basal segment mm. To facilitate the cutting of large numbers of tips, a block of four Perspex sheets of thicknesses 1-6, 1-8, 3-0 and 6-0 mm. was drilled with 55 holes of suitable diameter to hold the roots. The block was placed, thinnest sheet downwards, flat on a sheet of glass and the roots were inserted into the holes so that the tips touched the glass. The protruding older parts of the roots were sliced off flush with the Perspex with a microtome blade and the block was inverted. The Perspex sheets were then removed one by one and the exposed segments sliced off. The whole operation took 10-15 min. The lengths of the segments are chosen so that the first includes the meristem (potentially dividing cells) and the root cap, but few developed or elongating cells. The segment consists therefore of largely undifferentiated tissue. The second segment is a zone of increased metabolic activity, including protein synthesis (see Heyes & Brown, 1956). There is some cell enlargement and cell vacuoles appear. This segment therefore shows the beginnings of differentiation. The oldest part of the second segment and the youngest of the third is the zone with the most rapid rate of cell elongation. Cells of the third segment are very much larger and vacuolated, although the final cell volume is not reached until some 10-11 mm. from the tip. The average weight and number of cells per segment is shown in Table 1.

B. A. COOKE AND W. TAYLOR
Kamil, I. A., Smith, J. N. & Williams, R. T. (1953b). Biochem. J. 54, 390. McGuire, J. S. & Tomkins, G. M. (1960). J. biol. Chem. 235, 1634. Miura, S. (1911). Biochem. Z. 36, 25. Neubauer, 0. (1901). Arch. exp. Path. Pharmak. 46, 133. Ofner, P. (1955). Biochem. J. 61, 287. Roy, A. B. (1956). Biochem. J. 63, 294. Sie, H. & Fishman, W. H. (1957). J. biol. Chem. 225, 453.
Taylor, W. (1954). Biochem. J. 56, 463. Taylor, W. & Scratcherd, T. (1961). Biochem. J. 81, 398. Vestermark, A. & Bostrom, H. (1959). Exp. Cell Re8. 18, 174. Vestermark, A. & Bostrom, H. (1960). Acta chem. 8cand. 13, 2133. Wiest, W. G. (1959). Endocrinology, 65, 825. Wotiz, H. H. & Lemon, H. M. (1954). J. biol. Chem. 206, 525.
Biochem. J. (1963) 87, 218
The Biosynthesis of Phenolic Glucosides in Plants
BY J. B. PRIDHAM AND MARGARET J. SALTMARSH Department of Chemistry, Royal Holloway College (University of London), Englefteld Green, Surrey
(Received 12 October 1962)
The first claim that phenolic glucosides were formed when phenols were fed to plants was made by Ciamician & Ravenna (1916). These workers reported that saligenin (o-hydroxybenzyl alcohol) was converted into salicin (o-hydroxymethylphenyl ,B-D-glucopyranoside) by maize seedlings. Other feeding experiments with a variety of phenols have since been carried out and it is now clear that glucosylation of phenols is a common reaction in plant tissues. These studies, leading to the implication of uridine diphosphate glucose in the reaction, have been reviewed by Pridham (1960a). More recent advances have included feeding coumarin and o-coumaric acid to white clover with the formation of melilotyl- and o-coumaryl-glucosides respectively (Kosuge & Conn, 1959) and allowing the leaves of various plant species to take up cinnamic acid derivatives (Harborne & Corner, 1961). In this latter case the corresponding glucose esters were the main products, but with caffeic acid the 3- and 4-f-glucosides were also formed. Yamaha & Cardini (1960 a) have now studied in some detail the enzyme from wheat germ which is specific for the formation of glucosides from uridine diphosphate glucose and phenols with 1,4-dihydroxy groupings (cf. Cardini & Leloir, 1957; Pridham & Saltmarsh, 1960) and the enzyme which transfers glucose from uridine diphosphate glucose to arbutin with the formation of a f-gentiobioside (Yamaha & Cardini, 1960b; cf. Pridham, 1957, 1960b; Anderson, Hough & Pridham, 1960). The wheat-germ enzyme will also transfer glucose from adenosine diphosphate glucose to quinol (Trivelloni, Recondo & Cardini, 1962). Marsh (1960) has transferred glucuronic acid from uridine diphosphate glucuronic acid to quercetin using an enzyme from French beans and Barber (1962) has converted this flavonol into the glycoside rutin with an enzyme preparation from
mung beans in the presence of uridine diphosphate glucose (or thymidine diphosphate glucose) and thymidine diphosphate rhamnose. The formation of phenolic a-glucosides in the absence of nucleotides has been observed with A8pergiltu8 niger extracts, maltose being used as a glucose donor (Pridham, 1961). Acidic derivatives of phenolic glucosides are also produced when broad-bean seeds are treated with simple phenols (Pridham & Saltmarsh, 1962). This study has been undertaken to establish the nature of the glucosides formed by feeding simple phenols to the broad bean (Viciafaba) and to compare these products with those obtained from experiments in vitro. All possible steps have been taken to determine accurately the structures of the phenolic glucosides produced, but in some cases low yields or instability of the compounds, or both, have prevented a complete chemical and physical identification. We consider that inadequate characterization may have led to error in some previous publications. For example, some doubt exists about the product of glucosylation of saligenin, where isomeric monoglucosides could be formed by reaction with the phenolic or the primary hydroxyl

group.

A preliminary account of this work has been published (Pridham & Saltmarsh, 1960).
METHODS General method8. M.p. values are uncorrected. Ultraviolet-absorption spectra were measured with a Unicam SP.500 spectrophotometer (1 cm. cell). Free phenolic
hydroxyl groups were detected by a bathochromic shift in the presence of base (Mansfield, Swain & Nordstrom, 1953) and ene-diol groupings were revealed by a hypsochromic shift on subsequent addition of borate ions to the alkaline

Vol. 87

PHENOLIC GLUCOSIDES IN PLANTS
solution (Swain, 1954). Infrared measurements were made with a Unicam SP. 100 double-beam spectrophotometer. In the enzyme-catalysed reactions control incubations with boiled-enzyme preparations were always carried out. Paper chromatography. Phenolic compounds and sugars were examined on Whatman no. 1 and no. 3 papers respectively by the descending technique. The solvent systems were: A, butan-l-ol-ethanol-water (40:11:19, by vol.); B, ethyl acetate-acetic acid-water (9:2:2, by vol.); C, NN-dimethylformamide-benzene (4:1, v/v; stationary phase), light petroleum (b.p. 60-80; mobile phase) (Wickberg, 1958); D, NN-dimethyl sulphoxide-benzene (4: 1, v/v; stationary phase), di-isopropyl ether (mobile phase) (Wickberg, 1958). Molybdate-buffered papers were prepared as described by Pridham (1959). The stability of glucosides to oxidation was investigated by spotting these compounds on the starting lines of chromatograms, spraying with an aqueous M-FeCl3 solution and allowing them to dry at room temperature. These chromatograms were then developed with solvent A and treated with spray A. Paper electrophoresis (see Pridham, 1959). Whatman no. 3 paper was used with the following buffer solutions: A, 0-2 Msodium borate, pH 10-0; B, 0-05M-glycine, pH 10-0; C, 8 mM-ammonium molybdate, pH 5-2; D, 0-2M-sodium acetate, pH 5-2. Location of compounds. Phenolic compounds were detected on paper chromatograms and electrophoretograms by the use of diazotized p-nitroaniline-NaOH (spray A; Swain, 1953) and by examination under u.v. light, a Chromatolite (Hanovia Ltd., Slough, Bucks.) being used. p-Anisidine hydrochloride (spray B; Hough, Jones & Wadman, 1950) was used for reducing sugars and urea hydrochloride (spray C; Isherwood, 1954) for ketoses. Feeding experiments. Viciafaba (var. Johnson's Longpod) seeds were steeped in water (24 hr.) and then placed between layers of cotton wool soaked in an aqueoas solution (1 %, w/v) of the phenol for 3 days. Treatment of 6-day-old maize seedlings (Zea mays, var. Golden Harvest) with saligenin was carried out by a similar procedure. With willow (Salix daphnoides), cut ends of shoots were placed in aqueous solutions of saligenin (1%, w/v) for 48 hr. before examination. Isolation of phenolic glucosides. The testas were removed from the treated bean seeds and the cotyledons and embryos macerated with aqueous (90%, v/v) methanol. The resulting slurries were centrifuged (3000 rev./min. for 15 min. at 50) and the supernatants were then concentrated and examined on paper chromatograms (solvents A and B) for the presence of phenolic glucosides (u.v. light and spray A). Maize seedlings and willow shoots were extracted by a similar method. Fractionations of the extracts were achieved on cellulose columns or on Whatman no. 3 paper (solvent A). Acidic hydrolysis. The glucosides were heated with 1-5 N-HCI at 1000 for 4 hr. and the solutions then evaporated to dryness over NaOH pellets in a vacuum desiccat6r. The hydrolysates were examined on paper chromatograms with solvents A and B (sprays A and B). Enzymic hydrolysis. The glucosides were treated with almond f-glucosidase (Sigma Chemical Co.; 1%, w/v, in 0-02M-sodium acetate buffer, pH 5-5) at 270 for 48 hr. and the digests then examined chromatographicaily in the same way as the acidic hydrolyses. Complete hydrolysis of compound (XV) was effected with yeast invertase (British Drug

HousesLtd. concentrate, 0-2 ml.) in 0-1 M-sodium phosphate buffer, pH 7-5 (0-2 ml.), at 250 for 30 min. Methylation of compound (VI). Diazomethane (10 ml.) prepared by Nierenstein's (1930) method was added to freeze-dried (VI) (10 mg.) in dry methanol (10 ml.) and kept at 00 for 18 hr. Acetic acid was then added dropwise until the yellow colour of the solution disappeared. After concentration to dryness the methylated product was heated with methanolic (7 %, w/v) HCI and evaporated to dryness again over NaOH pellets in a vacuum desiccator. The hydrolysate was examined on paper chromatograms with solvents C and D and compared with authentic 2,4and 2,5-dimethoxyphenols. Preparation of 2,5-dimethoxyphenol sulphonate. Conc. H2SO4 (3 ml.) was added to quinol dimethyl ether (6 g.) and the mixture heated on an oil bath at 110-120 for 1 hr. The reaction mixture, after cooling, was poured into ice-water and NaHCO3 added to give pH 6-7. The solution was then heated to boiling point and saturated with NaCl and filtered. On cooling, white crystals ofsodium 2,5-dimethoxybenzenesulphonate were obtained The p-toluidine derivative had m.p. 203 [Gallent (1958) found m.p. 202-2030]. Preparation of 2,5-dimethoxyphenol. Sodium 2,5dimethoxybenzenesulphonate (6 g.) was fused with NaOH (10 g.) and KOH (4 g.) in an iron crucible. The fusion product was dissolved in water, the pH adjusted to 5-6 with conc. H2S04 and the solution extracted with ether. Concentration of the ethereal extract gave an oil in low yield. This compound behaved as a typical dimethoxyphenol in solvents A, C and D and gave a blue azo dye with spray A. Separation of compounds (IX) and (X). Initial purification of the mixed glucosides was achieved in thick-paper chromatograms with solvent A. The resulting mixed fraction (IX and X) was then resolved by paper electrophoresis with buffer A, compound (IX) having M-U4 (rate of movement relative to salicylic acid; Pridham, 1959) 0-29 and (X) MSA 0. The bands were eluted from the electrophoretograms with water. o-Hydroxybenzyl fi-glucoside. This was prepared by the method of Anderson et al. (1960). p-Hydroxy-and m-hydroxy-benzyl fi-glucosides. These were synthesized from glucose and the corresponding phenol by the biochemical method of Bourquelot & Herissey (1913). Wheat-germ enzyme. This was prepared by Yamaha & Cardini's (1960a) method and the fraction I obtained by these workers used for the synthesis of glucosides. Broad-bean enzyme. Dormant bean seeds with testas removed (35 g.) were macerated with 0-05M-sodium phosphate buffer, pH (105 ml.), at 0. The slurry was left to stand for 2 hr. at 50, centrifuged (g at 00 for 20 min.) and the supernatant was dialysed against 0-05M-sodium phosphate buffer, pH 7 0, at 5. Glucose transfer to phenols in vitro. The phenol (10 mg.) was incubated with UDP-glucose (6 mg.), enzyme (wheat germ or broad bean; 0-1 ml.) and 0-05M-tris-HCl buffer (0-1 ml., pH 7-4) at 37 under toluene. The final pH of the reaction mixture was 7-2. Paper-chromatographic examination (solvents A and B) was carried out after incubation for 5 hr. Transfer of glucose to phenols was also achieved by incubating (at 370) wheat-germ enzyme (0-1 ml.) with 0-05Mtris-HCl buffer (0-1 ml., pH 7-4), ATP (2-4 mg.), UTP (1-4 mg.), x-D-glucose 1-phosphate (2-1 mg.) and the phenol (1-3 mg.) in the presence of MgCl2 (0-5 mg.) and NaF

acetate was identical with that of authentic p-hydroxyphenyl f-D-glucopyranoside penta-acetate. The unequivocal identification of (I) supports the structures proposed for the other glucosides, which could only be examined by less rigorous methods. The mono f-glucoside (II), obtained by feeding with resorcinol, was isolated as a freeze-dried powder and had paper-chromatographic and electrophoresis properties identical with those of authentic mhydroxyphenyl ,B-glucoside. Similarly, compound (III), which was derived from catechol, was electrophoretically and chromatographically indistinguishable from o-hydroxyphenyl ,B-glucoside. Two mono fl-glucosides, compounds (IV) and (V), in the approximate proportions of 3:1 (as judged visually from paper chromatograms) were produced in low yields by seeds fed with pyrogallol. Compound (IV) gave a red-brown colour on molybdatetreated paper chromatograms and on molybdate electrophoretograms (buffer C). This reaction, in conjunction with the RF and MsA values (Table 1) is highly characteristic of compounds containing an ene-diol grouping (Pridham, 1959). The spectrum of (IV) is also characteristic of an ene-diol and the structure of this compound must therefore be 2,3dihydroxyphenyl fl-glucoside. Compound (V) gave negative ene-diol reactions and is presumably 2,6dihydroxyphenyl P-glucoside. Extracts prepared after feeding 1,2,4-trihydroxybenzene to bean seeds contained three compounds, RESULTS (VI), (VII) and (VIII), in the approximate proporFeeding experiments tions of 6:2: 1; which behaved as monoglucosides Preliminary experiments showed that broad- on paper chromatograms and electrophoretograms. bean seeds that had been allowed to germinate for Insufficient amounts of (VIII) could be obtained for 24 hr. before feeding with phenols gave the highest hydrolysis studies and this compound seemed to be yields of glucosides, in comparison with organs of much more unstable than (VI) or (VII). Compound the mature plant. In addition, seed extracts con- (VII) behaved as an ene-diol in the presence of tained fewer interfering phenolic substances than molybdate ions, unlike compound (VI). Attempts extracts of the green parts of the plant and this to detect the ene-diol grouping in (VII) by spectrofacilitated the isolation of the glucosylated pro- photometry failed owing to the instability of the ducts. The main properties of the glucosides formed compound. Treatment of all three glucosides with in the feeding experiments are summarized in dilute aqueous ferric chloride solution showed that Table 1. Quinol gave a higher yield of the cor- only (VI) was not oxidized, and therefore preresponding monoglucoside, arbutin (I; 300mg. sumably did not possess a potential quinonoid from 110 g. dry wt. of seeds) than any other phenol structure. Methylation followed by hydrolysis of that was used for feeding. Arbutin, unlike the other (VI) yielded a compound that co-chromatographed glucosides, was obtained in a crystalline form with 2,4-dimethoxyphenol and gave an azo dye of from water: m.p. and mixed m.p. 1990, [ac] -620 the same colour with spray A. This methylated (c 1-8 in water) (Found: C, 49-7; H, 6-3. Calc. for phenol could be easily distinguished from 2,5C12H1607,H20: C, 49-7; H, 6.2%), and was also dimethoxyphenol by the use of 'two organic-phase' characterized by paper chromatography and chromatography (solvents C and D). The evidence electrophoresis against authentic arbutin and by strongly suggests that compounds (VI) and (VII) comparative u.v. and i.r. spectrophotometry. The are 2,4- and 3,4-dihydroxyphenyl f-glucosides penta-acetate of (I) had m.p. and mixed m.p. 146-50 respectively. Compound (VIII) may well be the (Found: C, 54-3; H, 5-6. Calc. for C22H26012: C, 2,5 isomer.

(2-0 mg.). The reaction mixtures were again examined on paper chromatograms (solvents A and B) after 5 hr. In one experiment a-D-glucose 1-phosphate in the above reaction mixture was replaced by o-D-galactose 1-phosphate in an attempt to transfer galactose to quinol and resorcinol. Resorcinol (10 mg.) was also incubated (at 370) separately with OC-D-glucose 1-phosphate, methyl ac-D-glucoside, maltose and cellobiose (6 mg. each) in the presence ofwheatgerm enzyme (0-1 ml.) and 0-05M-tris-HCl buffer (0-1 ml., pH 7-4) and the digests were examined chromatographically (solvents A and B) after 5 hr. Attempted transfer of glucose to phenols with almond f3-glucosidase. f-Glucosidase (Sigma Chemical Co.; 0-2 ml.; 1 %, w/v, in 0-05M-sodium acetate buffer, pH 5-6) was incubated separately with arbutin (14 mg.), cellobiose (16 mg.), methyl fl-D-glucoside (10 mg.) and D-glucose (9 mg.) in the presence of quinol, resorcinol or 1,2,4-trihydroxybenzene (1-4 mg. each) at 270. The digests were examined on paper chromatograms after 8, 24 and 72 hr. (solvents A and B). Attempted transfer of fructose to phenols with yeast invertase. The phenol (10 mg.), sucrose (20 mg.), invertase (British Drug Houses Ltd. concentrate; 0-2 ml.) and 0-1 Msodium phosphate buffer, pH 7-5 (2 ml.), were incubated at 25 and samples taken and examined chromatographically (solvents A and B) after 5 min., 3 hr. and 20 hr.; a high pH was chosen in the hope that increased ionization of the phenolic hydroxyl groups would facilitate the transfer reaction. Only with saligenin was a glycosylated product (XV) obtained and this was isolated from the reaction mixture by chromatography on thick paper (solvent A). The hydrolysis products obtained on heating (XV) with 0-01N-H2SO4 at 1000 for 10 min. were examined on paper chromatograms (solvents A and B).
J. B. PRIDHAM AND M. J. SALTMARSH 1963 54-8; H, 5-4 %). The i.r. spectrum of the penta-
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222 J. B. PRIDHAM AND M. J. SALTMARSH 1963 o-Hydroxybenzyl f-glucoside (IX) was the main be detected on paper with a spray reagent for product formed on feeding saligenin to germinating nitro compounds (Maddy, 1959). This evidence, beans. Compound (IX) and authentic o-hydroxy- together with hydrolysis studies, suggests that benzyl P-glucoside (Anderson et al. 1960) behaved (XIV) is p-nitrophenyl p9-glucoside. 2,4-Dinitroidentically on paper chromatograms and electro- phenol-treated seeds did not produce detectable phoretograms and gave the same red coloration glucosides. with the diazonium spray reagent (A). A trace of salicin (X) was also detected as a u.v.-absorbing Glucosylation by wheat-germ and bean enzymes with spot on a paper electrophoretogram (buffer A) of uridine diphosphate glucose and other donors the seed extract. Failure of compound (X) to react A wheat-germ enzyme (cf. Yamaha & Cardini, with spray A indicated the absence of a free 1960a) with UDP-glucose produced the correspondphenolic hydroxyl group, as did u.v. spectrophoto- ing mono when incubated with quinol, metry, which on the other hand showed the pre- resorcinol P,-glucosides and catechol. With saligenin, and msence of a free phenolic hydroxyl group in the major alcohol, only the alcoholic groups product (IX) (cf. Rabat6 & Ramart-Lucas, 1935). hydroxybenzyl were glucosylated, giving o-hydroxy-(IX) and mThe electrophoresis and chromatographic behaviour hydroxy-(XI)-benzyl p-glucoside. No glucoside of compound (X) was identical with that of authen- formation could be detected with p-hydroxybenzyl tic salicin. o-Hydroxybenzyl P-glucoside was also gave one glucoside that was the major glucoside formed when willow shoots and alcohol. Pyrog4llol identical with 2,3-dihydroxyphenyl p-glucoside maize seedlings were allowed to take up saligenin. (IV); 2,4-dihydroxyphenyl (VI) was the In the latter case no salicin was detected; with the only compound obtained P-glucoside from 1,2,4-trihydroxywillow, salicin was present but was, of course, also benzene. When UDP-glucose was replaced by found in the control shoots. as Oc-D-glucose 1-phosm-Hydroxybenzyl alcohol was likewise converted potential glucose donors such methyl CX-D-glucoside, into the corresponding alcoholic glucoside (m- phate, maltose, cellobiose orbe demonstrated with hydroxybenzyl P-glucoside, XI) by the bean. No glucose transfer could not When ATP, UTP and definite evidence was obtained for the presence of resorcinol as an acceptor. used to replace UDPa-D-glucose 1-phosphate were the isomeric phenolic glucoside in the seed extracts. glucose, however, mono-p-glucoside formation was The glucoside obtained by incubating m-hydroxy- observed with quinol, resorcinol, catechol and salibenzyl alcohol with excess of D-glucose and f- genin as acceptor substrates. (With the last-named, oglucosidase (Bourquelot & Herissey, 1913) gave the hydroxybenzyl p9-glucoside, IX, was again formed.) same colour with spray reagent A and had identical to synthesize phenolic galactosides by chromatographic, electrophoresis and spectral Attempts O-D-glucose 1-phosphate by OC-D-galactose replacing properties with compound (XI). Hydrolysis of 1-phosphate failed. A phosphate buffer extract of both compounds gave glucose and m-hydroxybenzyl dormant broad-bean seeds also glucosylated quinol, alcohol. in the presence of In contrast, p-hydroxybenzyl alcohol was con- resorcinol, catechol and saligenin UDP-glucose, giving the same products as the verted largely into p-hydroxymethylphenyl P- wheat-germ enzyme. This preparation was, howglucoside (XII). The absence of a free phenolic hydroxyl group in this compound was evident from ever, less active than the wheat-germ enzyme. electrophoresis and spectrophotometric studies. Experiments with emulsin and invertase A small amount of a compound (XIII) that gave a Incubation of quinol, resorcinol and 1,2,4-tripink-purple colour with spray A also accompanied (XII). Insufficient quantities of (XIII) were pre- hydroxybenzene with cellobiose, arbutin, methyl sent for detailed studies but it behaved in an identi- P-D-glucoside or high concentrations of D-glucose in cal manner with the glucoside obtained from the presence of P-glucosidase (cf. Bourquelot & p-hydroxybenzyl alcohol (by Bourquelot & Heris- Herissey, 1913) did not produce glucosides, and sey's, 1913, method) on paper chromatograms and fructose could not be transferred from sucrose to electrophoretograms. The synthetic compound had quinol or 1,2,4-trihydroxybenzene by yeast invertAmax 279 m,u shifting to 290 mp. on addition of ase. This latter enzyme did, however, produce a alkali, and on hydrolysis with acid and P-glucosidase fructoside (XV) with saligenin, which gave a pink yielded glucose and p-hydroxybenzyl alcohol. It is colour with the diazonium spray A, thus indicating therefore assumed that this glucoside and com- the presence of a free phenolic hydroxyl group. This was supported by the spectrum, A,m. 274 mp, pound (XIII) are p-hydroxybenzyl P-glucoside. A compound (XIV) with 'Amaxm,u was found changing to 291 mpu with alkali. The compound in extracts of beans that had been fed with p-nitro- could also be located on chromatograms as a blue phenol. The compound was electrophoretically spot [RF in solvent A, cf. compound (IX), immobile with all the buffers used, and could Table 1] with urea hydrochloride, a specific reagent

for ketoses, and on hydrolysis with yeast invertase it yielded saligenin and fructose (the compound was not hydrolysed by P-glucosidase). These hydrolysis products were also rapidly released when (XV) was heated with 0-01 N-sulphuric acid (o-hydroxybenzyl ,-glucoside is stable under these conditions). This evidence strongly suggests that (XV) is o-hydroxybenzyl #-fructofuranoside.
DISCUSSION This study has shown that the major primary products formed by feeding broad-bean seeds with
mono-, di- and tri-hydric phenols are the corresponding mono-p-glucosides. The formation of oligoglucosides or glucosylation of more than one hydroxyl group cannot be altogether excluded in view of the difficulty in detecting small amounts of these higher-molecular-weight compounds in the complex extracts. With enzyme preparations from wheat-germ and broad-bean seeds with UDP-glucose as the glucose donor, phenols were also glucosylated in vitro and the resulting products closely resembled those obtained from the experiments in vivo. The reaction with the di- and tri-hydric phenols in vitro did, however, appear to be more specific and only one isomer in each case resulted, this corresponding to the major component produced in the feeding experiments. Glucosylation of phenols has also been achieved with UTP, CX-D-glucose 1-phosphate and a wheat-germ extract, i.e. utilizing the UDP-glucosepyrophosphorylase of the germ. Attempts to synthesize phenolic glucosides by simple transferase reactions involving almond P-glucosidase and 'low energy'-potential glucose donors failed. Similar unsuccessful experiments have been recorded in the past with glucose-containing disaccharides, aryland alkyl-glucosides and glucose as donors (Cardini & Yamaha, 1958; Pridham, 1960b; Anderson et al. 1960). Such systems will, of course, glucosylate aromatic and aliphatic alcohols (Rabate, 1935; Anderson et al. 1960; Dedonder, 1961; Jermyn, 1961). Fructosylation of phenolic hydroxyl groups also could not be effected with sucrose and yeast invertase although here again this enzyme readily transfers fructose to other sugar molecules and to simple aliphatic alcohols (Edelman, 1956; Dedonder, 1961) and now, as we have shown, to saligenin, forming o-hydroxybenzyl fl-fructofuranoside. UDP-glucose occurs widely in plant tissues (Ginsburg, Stumpf & Hassid, 1956; Rowan, 1959; Ziegler, 1960; Dutton, Carruthers & Oldfield, 1961), and Abdel-Wahab & El-Kinawi (1959, 1960) have presented evidence which suggests that it occurs in Vicia faba. We also have chromatographic evidence for the existence of this nucleotide in the

broad bean. It is probable therefore that UDPglucose is a donor molecule for the glucosylation of phenols in the bean, and indeed in all higher plant tissues although other nucleotide derivatives, such as thymidine diphosphate glucose (Barber, 1962), may also be involved in this reaction. Many different types of phenols can be glucosylated by plant tissues. This may be due to the presence in the cells of a number of different enzymes (or different active sites on one enzyme) with relatively high acceptor specificities or to a single enzyme of low specificity. The former hypothesis at present seems more likely in view of the isolation from wheat germ of an enzyme which is specific for the glucosylation of phenols with 1,4-dihydroxy groupings. Crude wheat-germ extracts with UDPglucose will glucosylate phenols with other hydroxyl arrangements (Yamaha & Cardini, 1960a). A multienzyme system in which the components have different activities could also account for the different amounts of the isomeric monoglucosides produced on feeding di- and tri-hydric phenols and phenolic alcohols to bean seeds. With a single enzyme these results might be explained by stereospecificity or by the relative chemical reactivity of hydroxyl groups in the polyhydric compound. Hydroxyl reactivity and stereospecificity would presumably also play important roles with a multienzyme system. Preliminary feeding experiments with pyrogallol, saligenin and p-hydroxybenzyl alcohol first led us to believe that the most strongly dissociated hydroxyl group was most readily glucosylated but this theory became unsatisfactory when a number of other phenols were tested in vivo and in vitro. (With saligenin, hydrogen-bond formation between the hydrogen of the phenolic hydroxyl group and the alcoholic oxygen atom strongly activates the alcohol group, and one would expect this on purely chemical grounds to be readily glucosylated, as was the case.) It should also be remembered that results from feeding experiments and experiments with crude enzymes in vitro could be misleading owing to the instability of the phenolic glucosides to other enzyme systems such as the hydrolases and phenol oxidases. It is of interest to consider the biosynthesis of salicin as our results suggest that saligenin is not the precursor. Ciamician & Ravenna (1916) claimed that salicin was formed by maize seedlings that had been treated with saligenin. Our own experiments with maize and Salix daphnoides strongly suggest that o-hydroxybenzyl fl-glucoside is the major product formed from saligenin. With maize, no salicin could be detected. Salicin occurs naturally in Salix daphnoides, but here no marked increase was observed. These differences in results may be due to the fact that Ciamician & Ravenna (1916)

J. B. PRIDHAM AND M. J. SALTMARSH

REFERENCES

used a method that did not clearly distinguish between the two isomeric glucosides of saligenin. According to Yamaha & Cardini (1960a) wheatgerm extracts incubated with UDP-glucose and saligenin produce a compound with similar chromatographic properties to salicin. Here again our own experiments with wheat-germ and broad-bean enzymes showed clearly that the major product is the isomeric glucoside and that little, if any, salicin is produced. It is possible therefore that in vivo saligenin is not the precursor of salicin, always assuming that our feeding experiments allowed the phenol to reach the correct site for salicin synthesis in the plant. Ibrahim & Towers (1959) suggest that salicylic acid may be a common metabolite of plants, and Klambt (1962) has shown that benzoic acid can be converted into salicylic acid and salicylic acid f-glucoside by several species of plants. It is conceivable therefore that salicin biosynthesis proceeds via the corresponding glucoside followed by reduction of the carboxyl group to a primary alcohol group. In this connexion, it can be noted that helicin (o-formylphenyl g-D-glucoside), gaultherin (o-carboxymethylphenyl 6-O-g-D-XylOSyl-fSD-glucoside) and violutin (o-carboxymethylphenyl 6-0-/1-L-arabinosyl-/J-D-glucoside) occur naturally in plants (Mcllroy, 195 1); a glycoside of salicylic acid has so far not been found, however.
SUMMARY 1. The structures of the mono /-glucosides formed by germinating broad-bean seeds in the presence of various phenols have been studied. 2. Enzyme preparations from wheat-germ and broad-bean seeds will glucosylate phenolic and alcoholic hydroxyl groups in the presence of uridine diphosphate glucose (or for wheat-germ enzyme, with uridine triphosphate and a-D-glucose 1phosphate). The products obtained with these enzymes closely resemble those produced in the experiments in vivo. 3. The nature of the phenol-glucosylating system in plants is considered. 4. Saligenin in vivo and in vitro mainly gives rise to o-hydroxybenzyl P-glucoside, and not salicin. The biosynthesis of this latter compound is discussed. We are greatly indebted to Spillers Ltd. for a generous gift of wheat germ and to Professor E. Adler for specimens of methylated phenols. The paper-electrophoresis apparatus was built with the aid of a grant from The Royal Society. Professor E. J. Bourne is thanked for his interest and encouragement and M.J.S. acknowledges the receipt of a postgraduate studentship from the Department of Scientific and Industrial Research.

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