Journal of General Microbiology (1975), 89,245-255
245
Printed in Great Britain
The Lipids of Agaricus bisporus
By P A T R I C I A F. S. B Y R N E A N D P. J. B R E N N A N
Department of Biochemistry, University College, Dublin 4, Ireland
(Received
10December
1974; revised
10 March
1975)
SUMMARY
A comparison of the lipid composition of the vegetative and reproductive stages
of Agaricus bisporus revealed no major qualitative differences, although quantitative divergences exist. The glycolipids consisted of acylglucoses, acylmannitol,
acyltrehalose and a glucosyloxyfatty acid. Two of the acylglucoses corresponded
to a tetra-acylglucose and to either a di- or a triacylglucose. The phospholipids were
distinctive in that phosphatidylcholine could not be detected. Phosphatidylethanolamine and phosphatidylserine were the major phosphoglycerides. Examination of the neutral lipids revealed the expected array of acylglycerols, free and
esterified sterols, and free fatty acids. A substantial amount (26 to 33 %) of the
fatty acids of the neutral lipids from both sporophore and mycelium were
apparently of chain length greater than CIS. Linoleic acid was a minor component
of the total neutral-lipid fatty acids but comprised about one-half of the total
free fatty acids.
INTRODUCTION
Little is as yet understood of the metabolic and physiological processes. which are involved in the changes from vegetative growth and stranding to the initiation of sporophores.
Schisler (1967) and Wardle & Schisler (1969) proposed a possible involvement of lipids in
sporophore initiation in the cultivated mushroom. It was at first suspected that sterols had
a stimulatory effect, but later it was shown that growth of the mycelium was increased by the
addition of esters of oleic and linoleic acids to the basal medium and this in turn resulted
in an increase in sporophore production. Very little was known at this stage about the lipid
composition of the cultivated mushroom, apart from a preliminary survey (Hughes, I 962).
It was therefore considered that a comparative analysis of the lipid composition of the
vegetative and reproductive growth stages of Agaricus bisporus would clarify the metabolic
processes underlying sporophore initiation. For similar reasons, Holtz & Schisler (I 971)
analysed the lipid composition of four mushroom strains. Such studies are also important
in the context of fungal lipid chemistry, a relatively unexplored field (Brennan et al. 1974).
Several fungi have been examined to determine if there is a role for soluble carbohydrate
in dormancy breaking in fungal spores (Brennan et al. 1974). We have examined such components in A . bisporus. We have also sought the presence in these fungi of acylated forms
of the soluble carbohydrates since there could be a reciprocal relationship between free
and acylated polyols during germination of some fungi (Brennan et al. 1974).
METHODS
Organisms. Cultures of Agaricus bisporus were derived from sporophore tissue and maintained on 2 % (w/v) malt agar slopes. Mature sporophores of a commercial variety of
A . bisporus were supplied by An Foras Taluntais (Agriculture Research Institute), Kinsealy,
County Dublin, Ireland.
Culture conditions. Agaricus bisporus was grown in 2 % (v/v) liquid malt buffered to
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P. F. S. B Y R N E A N D P. J. B R E N N A N
pH 6.8 with 0.1% (w/v) calcium carbonate. Flasks containing 500 ml or I 1 of medium
were inoculated with mycelial discs, 3 mm in diameter, cut from Petri dish cultures of A .
bisporus on 2 "/o (w/v) malt agar. Cultures were incubated a t 27 "C for 8 to 1 2 weeks. The
mycelium was harvested by filtration and washed in 0.9 % (w/v) NaCl.
Extraction of carbohydrates and lipids from mycelium and sporophore. Harvested, fullywashed mycelium of A . bisporus was ground lightly with acid-washed sand by using a
mortar and pestle, and extracted with chloroform-methanol (2: I , v/v). The solid matter
was removed by filtration and re-extracted twice. The combined liquid extracts were dried
on a rotary evaporator and washed by the system of Folch, Lees & Sloane-Stanley (1957).
The upper phase of the Folch wash, which contained most of the water-soluble components
of the mycelium, was concentrated on the rotary evaporator and stored at -20 "C. The
lower organic phase, containing the lipid, was evaporated to dryness and stored in the cold
under nitrogen. Sometimes this lipid was extracted with acetone (Brennan & Ballou, 1967)
and centrifuged to yield acetone-soluble and acetone-insoluble lipid fractions.
Sporophores of A . bisporus (3 Ib) were macerated with acid-washed sand in a mortar.
Lipids were extracted with chloroform-methanol (2: I and I :2, v/v) as described for the
mycelium. An emulsion of the lipids was prepared (Brennan & Ballou, 1967) and freezedried.
Chromatography of carbohydrates and lipids. The aqueous carbohydrate-containing fraction obtained by the procedure of Folch et al. ( I 957) was applied to a column of Sephadex
G-25 (240 x 3 cm) and eluted with water. The pooled carbohydrate-containing fractions
were concentrated and then chromatographed on preparative sheets of pre-washed Whatman
3MM paper in ethyl acetate-acetic acid-formic acid-water ( I 8 :3 :I :4, by vol. ; solvent A).
Other chromatographic solvent systems used for identification of the carbohydrates were :
ethyl acetate-pyridine-water (8 :2: I, by vol.; solvent B); butan-I-01-pyridine-water (3 : I : I ,
by vol.; solvent C); butan-I-01-acetic acid-water (3: I :I , by vol.; solvent D).
Silicic acid (Mallinckrodt, St Louis, Missouri, U.S.A.) was used for the fractionation of
total soluble and acetone-soluble lipids. Up to 3 g of lipid extract of mycelium in chloroform were applied to columns (35 x 4 cm) and lipid classes eluted by a modification of the
procedure of Vorbeck & Marinetti (1965). Neutral lipids were eluted with chloroform, and
generally accounted for about 50 % of the total lipid mass. A glycolipid-containing fraction
was removed with 50 % (v/v) acetone in chloroform followed by absolute acetone. Phospholipids were finally eluted with 50 % (v/v) methanol in chloroform. Columns in which silica
gel G (Merck) replaced silicic acid were also used for the separation of the major lipid
classes. Neutral lipid and glycolipid classes were also obtained from the acetone-soluble
lipids by a modification of the silicic-acid column procedure of Hirsch & Ahrens (1958).
Lipid was'applied to a column (30 x 2 cm) equilibrated with light petroleum (b.p. 40 to
60 "C) and elution of lipids progressed with successive 300 ml applications of light petroleum, 30 % (v/v) diethyl ether in light petroleum, chloroform, 10% (v/v) methanol in
chloroform, and 50 % (v/v) methanol in chloroform. The combined ether fractions contained 36 % of the recovered lipid and the 10 % (v/v) methanol in chloroform eluate
contained 48 %.
DEAE-cellulose chromatography (Brennan & Ballou, I 967) was also utilized to separate
neutral polar and neutral non-polar lipid classes from the phospholipids of mycelium.
While phospholipids were retained on the column, the neutral non-polar lipids were eluted
with chloroform-methanol-water (20 :9 : I , by vol.) and glycolipids with 50 % (v/v) acetone
in chloroform.
Individual lipid species were separated by t.1.c. Neutral lipids were resolved on plates of
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Lipids of Agaricus bisporus
247
silica gel H or silica gel G in light petroleum-diethyl ether-acetic acid (85 : 15 :2, by vol. ;
solvent E). Glycolipids were resolved in chloroform-methanol (9 : I , v/v; solvent F),
chloroform-methanol (10:I , vJv; solvent G), chloroform-methanol-water (65 :25 :4, by
vol. ; solvent H), and chloroform-methanol-water (50: 21 :3, by vol. ; solvent J). Solvents
H and J were also used for resolution of phospholipids, as was chloroform-methanol-28 %
(v/v) NH, (65:25:5, by vol.; solvent K).
Silica gel H was used for preparative t.1.c. and lipids were located by the use of iodine
vapour or a water spray. Bands were outlined and excised with a razor blade. The gel was
transferred to sintered-glass funnels and lipids eluted with chloroform-methanol (2 : I , v/v)
followed by chloroform-methanol (I :2, v/v). The eluted lipids were washed (Folch et al.
1957) before drying and further analysis. The periodate-Schiff reagent (Shaw, 1968) was
used to distinguish glycolipids from other lipid-solublecomponents. A solution of 0-2 % (w/v)
anthrone in concentrated sulphuric acid was used for the same purpose. The molybdenumblue reagent (Dittmer & Lester, 1964) was used to locate phospholipids.
Chromatography of lipid hydrolysis products. Intact lipids were hydrolysed with 2 M-HCl
at IOO "C for 3 h. Fatty acids were extracted with light petroleum (b.p. 40 to 60 "C) and
with diethyl ether. The aqueous layer was evaporated to dryness and HCl was removed
in vacuo over KOH pellets before paper chromatography. Deacylation of lipid was accomplished by treatment with 4 M-NaOH (Ballou, Vilkas & Lederer, 1963). The water-soluble
products of deacylated lipids were resolved by chromatography in propan-2-01-aqueous
NH,-water (7 : I :2, by vol. ; solvent L). Carbohydrates on paper chromatograms were'
located with the AgN0,-NaOH reagent (Anet & Reynolds, 1954) or with the periodatebenzidine dip reagent (Gordon, Thornburg & Werum, 1956). A I % (w/v) solution of
p-anisidine-HC1 in butan-1-01-methanol (9 :2, v/v) was used specifically to locate reducing
sugars. Amino acids, glycerylphosphorylethanolamine and glycerylphosphorylserine were
located with a spray composed of 0.25 % (w/v) ninhydrin in acetone. The Dragendorff
reagent (Kates, 1972)was used for the detection of choline and glycerylphosphorylcholine.
Gas-liquid chromatography of fatty acids. Methyl esters of fatty acids were obtained by
transmethylation or by acid hydrolysis followed by methylation with diazomethane. For
transmethylation the lipid was incubated with 4-5 % (w/v) methanolic-HC1 in a sealed tube
at 60 "C for 24 h. Alternatively, lipids were hydrolysed with 2 M-HCland the free fatty acids
extracted with diethyl ether and dried. Diazomethane in diethyl ether (2 ml) was added and
the solution incubated at room temperature for 12 h and applied to gas-liquid chromatograms. Methylated fatty acids were separated on a column (5 ft x in.) of 10 % polyethyleneglycoladipate (PEGA) on Chromasorb W (I 00/200 mesh; Wilkins Instrument and Research
Ltd, Manchester). The column was operated isothermally between 140 and 160 "C and the
carrier gas (N,) flow rate was 50 ml/h. Assignments for each peak were obtained from a
semi-logarithmic plot of relative retention times against chain length and degree of unsaturation of standard mixtures of fatty acid methyl esters. Peak areas were estimated by
weighing the peak tracings, and the percentage of each acid was calculated from the ratio
of the area of its peak to the total area of all peaks.
Synthesis of stearyl-0-glucoses. The procedure described by Asselineau ( I 955) for the
synthesis of palmityl-0-glucoses was modified as follows. First, stearyl chloride was synthesized by adding thionyl chloride (SOCI,) dropwise to solid stearic acid (28 g ) at 70 "C
until all of the stearic acid had dissolved. This mixture was refluxed for I h with continuous
evacuation to remove SO2, SOCl, and HCl. The recovered stearyl chloride (22 g ) was a pale
yellow solid with a m.p. of 26 "C. Stearyl chloride (10 g) was dissolved in anhydrous benzene (20mI) and added dropwise to 20g of anhydrous glucose partially dissolved in
+
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248
anhydrous pyridine at o "C. The mixture was left for 20 h at room temperature, stirred for
3 h and acidified to pH I by the addition of cold 2 M-H,SO, (70 ml). Lipid was removed
by repeated extraction of the mixture with cold chloroform. The chloroform phase was
washed several times with water, dried over anhydrous sodium sulphate and weighed. The
yield was 3-6g. The fluff lying between the chloroform and water phases was recrystallized
three times from dioxane and retained as a rich source of monostearyl-0-glucose (Asselineau,
1955). The average number of acyl groups per glucose unit for three different assays on
this material was 1.3. Thin-layer chromatography in solvents F and G showed one anthronepositive spot with an R, value between 0.1 and 0.2. This evidence of poor solubility in
chloroform and water, low mobility on t.1.c. and the presence of approximately one acyl
group, indicated that this compound was a monostearyl-0-glucose.
The other stearyl-0-glucoses were present in the chloroform extract. This was applied to
a silicic-acid column and the anthrone-positive material, eluted with a linear gradient of
acetone in chloroform, was collected and subjected to preparative t.1.c. in solvent G. Three
lipid bands with R, values of 0.90, 0.80 and 0.60 were located with the water spray. The
average numbers of acyl groups per unit of glucose in the first and last of these three were
3-9 and 2 . 1 , respectively, suggesting that these were a tetrastearyl-0-glucose and a distearyl0-glucose.
RESULTS
Soluble carbohydrates of A . bisporus
Soluble carbohydrate was present in the aqueous phase after partitioning the chloroformmethanol extract of the mycelium. Treatment of this fraction with equal parts of Amberlite
IRA-400 (OH-) (Rohm and Haas Co., Philadelphia, U.S.A.) and Dowex-go (H+) (Sigma)
removed most of the pigment and only 10 % of the carbohydrate, indicating that the bulk
of the carbohydrate was neutral. Chromatography of the soluble carbohydrate on Sephadex
G-25 yielded only one included peak, indicating that the mycelium contained little polysaccharide. Preparative paper chromatography in solvent A of the eluate from the Sephadex
columns yielded three well-separated components with chromatographic properties identical
to those of glucose, mannitol and trehalose. These identifications were confirmed by comparative chromatography of the isolated individual carbohydrates in three other solvent
systems (solvents B, C and D). Further evidence for these identifications was obtained when
a chromatogram, developed with p-anisidine-HCI, showed that the material corresponding
to glucose was reducing while those with RF values equivalent to mannitol and trehalose
did not react with the reagent. In addition, the trehalose after acid hydrolysis and paper
chromatography yielded only glucose. From these results it was concluded that glucose,
mannitol and trehalose were the major water-soluble carbohydrates in the mycelium of
A . bisporus. The major carbohydrate constituents of the malt used in the growth of the
mycelium were shown to be maltose and glucose by paper chromatography in solvent A.
There were traces of other carbohydrates, none of which appeared to be mannitol or
trehalose. It would therefore seem that the soluble carbohydrates of A . bisporus are not
taken directly from the growth medium but are synthesized by the fungus.
Lipids of A . bisporus
This analysis was prompted by a search for acylated forms of the free carbohydrates
described above. In the course of this investigation neutral lipids and phospholipids were
also examined. The total extractable lipid or the acetone-soluble and insoluble lipid fractions
from A . bisporus mycelium and sporophore were washed and divided into three classes,
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neutral lipids, glycolipids and phospholipids, by DEAE-cellulose, silicic acid or silica gel G
chromatography.
Neutral lipids of A . bisporus. Regardless of the isolation procedure, neutral lipids were
found to represent about 50 % of the total mycelial and sporophore lipid. Thin-layer chromatography of the neutral lipids showed a mixture of acylglycerols and free fatty acids.
A further component with an R F of 0.90 was probably a steryl ester. There was one additional lipid present in the neutral lipid fractions with an R F of 0.20, similar to that of
cholesterol. This material reacted positively to a resorcinol-sulphuric acid spray (Lisboa,
1964) and produced a blue-black colour, typical of sterols. The identification of the acylglycerols was confirmed by their isolation, degradation with acid and alkali, and recognition
of glycerol as the only water-soluble product. Triacylglycerols and free fatty acids comprised the major neutral lipids of both mycelium and sporophore and there were no obvious
quantitative or qualitative differences between the products from the two sources.
From the results of analysis of the neutral lipids of A . bisporus we conclude that in both
the vegetative and reproductive growth stages mono-, di- and triacylglycerols, free fatty
acids and sterols comprised 40 to 50 % of the total lipid. These results are in close agreement
with those obtained by Hughes (1962) and by Holtz & Schisler (1971) for A . bisporus
sporophore lipid. However, contrary to the present report, Holtz & Schisler (1971) could
not detect free sterols in mycelial extracts of A . bisporus, though they suggested that free
sterols were the end products of synthetic events during the fruiting process.
The fatty-acid composition of the mycelium and sporophore neutral lipids are shown in
Table I . A wide variety of both saturated and unsaturated fatty acids is evident. A substantial proportion of the fatty acids from both sources appeared to be of carbon chain
length greater than C1,, in contrast to the observations of Holtz & Schisler (I971), who
reported no significant levels of fatty acids greater than C,, :2. Moreover, in the present work
linoleic acid (C18:2)was a minor component (4 to 7 %) of the neutral lipids, yet Hughes
(1962) and Holtz & Schisler (1971) reported that linoleic acid comprised between 50 and
80 % of the fatty acids of the neutral lipids of sporophore. In an effort to resolve this
apparent discrepancy, the dried acetone-soluble lipids of A . bisporus sporophore were
extracted with hexane, preferentially to remove free fatty acids. This fraction was treated
directly with diazomethane, thereby avoiding cleavage of the fatty acids of complex lipids,
and the methylated free fatty acids were analysed (Table 2). It was found that linoleic acid
was the major free fatty acid in sporophores, comprising almost 50 % of the total free fatty
acids, and suggests that it is therefore not a prominent component of the acylglycerols of
A . bisporus.
There are some differences between the neutral-lipid fatty-acid composition of mycelium
and sporophore (Table I). For instance, fatty acids with chain lengths greater than CIS:
represented 33 % of all mycelial neutral-lipid fatty acids, compared with 26 % for sporophore neutral lipids. There was much more of the CI6 fatty acids, particularly palmitoleic
acid, in the sporophore. However, the differences were small and may be unimportant,
since sporophore and mycelium were grown under widely different conditions and fattyacid composition can vary considerably with altered conditions, such as changes in medium
composition, pH, temperature, age of culture and degree of aeration (Hilditch & Williams,
1964).
Glycolipids of A . bisporus. The first indication of the presence of glycolipids in A . bisporus
was obtained when the products of acid hydrolysis of the total mycelial lipids were examined
by paper chromatography in solvent A. Substantial amounts of glucose and mannitol were
observed. Since these two carbohydrates were also present in the free form, they could have
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Table
I.
Fatty-acid composition of the neutral lipids of A . bisporus
mycelium and sporophore
Identifications were based on a comparison with the relative retention ratios of
standard mixtures of fatty acid esters.
Percentage (w/w) of fatty acids
Growthstage C,,;, C,,.,
Mycelium
Sporophore
*
I
2
3
3
C15:l C16:O
ClCZ1
ci6:2
II
21
2
3
8
18
<I
2
CI,:I c 1 8 : 0
c17:0
8
2
3
<I
I4
12
C18:l c 1 8 . 2
9
6
7
4
CZO:O
CZO:IOthers*
17
5
3
< I
I3
21
This mostly consisted of two fatty acid esters with retention times indicative of CZ0:,and Cz1:,.
been contaminants of the lipid fraction. However, the lipid fractions had been thoroughly
washed by the method of Folch et al. (1957), and when a sample of the intact lipid was
chromatographed on paper no free sugars were detectable. It was therefore concluded that
the glucose and mannitol originated from lipid. A carbohydrate analysis on the acetonesoluble lipids of A . bisporus mycelium revealed that the bulk (80 %) of the lipid-soluble
carbohydrate was present in this fraction. Therefore most of the glycolipids of A . bisporus
mycelium exhibited solubility properties typical of non-ionic polar lipids (Shaw, I 970).
When the carbohydrate contents of the water-soluble material from both acid and mildalkaline hydrolysed lipid were compared, it was found that 87 % of the glycolipids were
alkaline-labile and therefore of the acylated-sugar type. Since only 1 3 % of the glycolipids
were alkaline-resistant, the glycosylceramides reported by Weiss & Stiller (I 972) appear to
represent only a small proportion of the total glycolipids of A . bisporus.
To determine the nature of the acetone-soluble acylated carbohydrates of A. bisporus,
a portion of the mild-alkaline hydrolysate was chromatographed in solvent A. Mannitol,
glucose, trehalose and glycerol were readily recognized among the products. The conditions
for deacylation do not result in glycosidic cleavage and it is therefore inferred that each of
these sugars and sugar alcohols originated from their acylated counterparts.
Chromatography of total chloroform-methanol-soluble lipids on silicic acid, and successive elution with chloroform, 50 % (v/v) acetone in chloroform, acetone and 50 % (v/v)
methanol in chloroform, showed that the acylated sugars were confined to the acetonecontaining fractions. Thin-layer chromatography in solvent G of the combined glycolipidcontaining fractions showed five individual components. All produced a blue colour with
the periodate-Schiff reagent, indicative of glycolipids with a number of acyl groups (Shaw,
1968). Phosphorus was always absent. Two of the glycolipids, with R, values of 0.25 and
0.65 in solvent G, produced only glucose on deacylation and yielded an initial typical green
and later a blue colour with the periodate-Schiff reagent. These two acylglucoses were
compared with the synthesized stearylglucoses by t.1.c. in solvents G and H. One corresponded to the tetrastearyl-0-glucose in both solvents. However, with the other (R,0.70
in solvent H) it was impossible to distinguish between a di- and a triacylglucose. The
acylated forms of mannitol and trehalose have not yet been isolated in a pure form.
Table
2.
Free fatty acids of A. bisporus sporophore
Fatty acid
Percentage (w/w) of fatty acids
37'5
14.0
48.5
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25 I
Fig. I . (a) Thin-layer chromatograph in solvent H of the major glycolipid of A. bisporus sporophore.
The plate was sprayed with the periodate-Schiff reagent. (6) Thin-layer chromatograph in solvent
K of the phospholipids of A. bisporus sporophore. The plate was sprayed with the molybdenumblue reagent. I , Phosphatidylcholine; 2, total phospholipid fraction (chloroform-methanol eluate
from a silicic acid column) from A . bisporus; 3, phosphatidylethanolamine.
When the approach described above was extended to the glycolipid fraction from A .
bisporus sporophore it was obvious that some of the lipid glucose and mannitol originated
in their acylated counterparts. There was insufficient of these glycolipids present for further
characterization. However, the bulk of the lipid glucose from the sporophore could not be
attributed to acylglucoses and a glycolipid was detected in the extracts which was nondeacylatable, since it exhibited the same chromatographic mobility before and after mildalkali treatment. This glycolipid was purified by preparative t.1.c. in solvent H (Fig. ~ a ) .
It displayed the double band and chromatographic properties typical of the monoglucosyloxyoctadecenoic acid from Aspergillus niger (Laine et al. 1972). Acid hydrolysis and
paper chromatography of this glycolipid showed only glucose. An i.r. spectrum showed the
following characteristics : (i) the presence of -OH group absorption at 3380 cm-l, pointing
to a number of unsubstituted glucose -OH groups; (ii) strong -CH, group absorption at
2880 cm-l and 2820 cm-l; (iii) strong -COOH absorption at 1710cm-l, indicating that the
carboxylic group of the fatty acid was free. Thus the glycolipid corresponded in most
respects to the glucosyloxyfatty acid of A . niger (Laine et al. 1972).
A search for this alkali-stable glycolipid in mycelial extracts showed small amounts of it
on thin-layer plates. It was purified by applying acetone-soluble lipid to a column of silicic
acid and by eluting the glycolipid fraction with chloroform-methanol (9: I , v/v). This
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P. F. S . B Y R N E A N D P. J. B R E N N A N
fraction was applied to a column of Sephadex LH-20 in chloroform. The early eluted fractions contained a considerable amount of pigment and were discarded. The subsequent
pigment-free chloroform eluates were combined and chromatographed in solvent F. A
double-band lipid with R, of 0.30 was identical to the glucosyloxyfatty acid from A . niger
in all respects : it yielded a purple-blue colour with the anthrone spray, glucose was the only
carbohydrate present, and it was unaltered by alkali treatment. Examination of the fatty
acids of the glycolipid by t.1.c. in benzene showed mainly hydroxy fatty acids with an Rp
of 0-16and little of the non-hydroxylated fatty acid type with an R, of 0.70. An i.r. spectrum
of the intact glycolipid again showed absorption peaks in the -OH, -CH, and -COOH
group areas.
Phospholipids of A . bisporus. Phospholipids represented about 17 % of the total mycelial
lipids. They were obtained as either the acetone-insoluble lipid fraction or as the chloroformmethanol (I : I , v/v) eluate when total mycelial lipid was applied to silicic acid columns.
Thin-layer chromatography in solvent H showed only two major phospholipids, both of
which reacted with the molybdenum blue and ninhydrin reagents and had R, values equivalent to phosphatidylethanolamine and phosphatidylserine (Fig. I b). In several t.1.c. systems
lipids with R, values similar to phosphatidylcholine were obvious, but were devoid of
phosphorus and choline since they did not react with the molybdenum blue or Dragendorff
reagents. When the phospholipid fractions from a species of Alternaria and Saccharomyces
cerevisiae were chromatographed under these same conditions the phosphatidylcholine of
both fungi reacted with the Dragendorff spray to yield a typical orange colour. The conclusion is that phosphatidylcholine is not a major component of A . bisporus mycelium. Acid
hydrolysis of the phospholipid fraction, chromatography of the water-soluble products in
solvent L, and exposure of the chromatogram to ninhydrin showed ethanolamine and serine
with trace amounts of other amino acids. Choline could not be detected on these chromatograms with the Dragendorff reagent. Chromatography of the acid hydrolysate in solvent A
and treatment of the chromatogram with AgN0,-NaOH showed glycerol from the phosphoglycerides and small amounts of inositol from phosphatidylinositol.
A sample of the mycelial phospholipid fraction was also deacylated and the products
chromatographed in solvent L. Glycerylphosphorylethanolamine,glycerylphosphorylserine
and glycerylphosphorylinositol were obvious with the AgN0,-NaOH reagent, whereas
glycerylphosphorylcholine was not detectable.
A preparation of A . bisporus sphorophore phospholipid was obtained by DEAE-cellulose
chromatography. The experimental approach described above for mycelial phospholipid
was employed, and there was no apparent difference between the phospholipid composition
of the mycelial and sporophore extracts ; phosphatidylethanolamine and phosphatidylserine
were predominant in sporophores and phosphatidylcholine was again undetected.
DISCUSSION
The conclusion that the soluble carbohydrates of A . bisporus - glucose, mannitol and
trehalose - are not directly taken up from the medium but are synthesized by the fungus, is
supported by evidence of certain similarities between the free polyol pattern of the fruiting
stages (reported by Hughes, Lynch & Somers, 1958), and of the mycelium (reported here).
Hughes et al. (1958) used mushrooms taken from commercial compost beds and subjected
the extracted carbohydrates to hydrolysis with I M-H,SO, for 24 h. Therefore, trehalose
was not detectable and among the seventeen chromatographically distinguishable carbohydrates there were probably some monosaccharide degradation products. However,
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Lipids of Agaricus bisporus
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glucose, galactose and mannitol were present in relatively large quantities. In the present work
galactose was not identified in A . bisporus mycelium. Mannitol has been known as a major
mushroom carbohydrate since I 947 (McConnell & Esselen, I 947). Mannitol and trehalose
were also previously recognized in the fruiting bodies of A . bisporus by Rast (1965). Carbohydrate and acyclic polyols of the type reported here also occur in other fungi. For instance,
Le Tourneau (I 966) isolated trehalose, mannitol and arabitol from Sclerotinia sclerotiorum
and showed that they comprised 5 to 10 % of the dry weight of the sclerotia. This is to be
compared to the 3 to 4 % for A . bisporus found in the present work. Similar levels of endogenous trehalose and polyols occur in the uredospores of Puccinia graminis (Reisener et al.
1962) and in the conidia of Penicillium chrysogenum (Ballio, Di Vittorio & Russi, 1964).
Yeasts also produce large amounts of free polyols such as erythritol, arabitol, glycerol and
mannitol during fermentation processes (Spencer & Sallans, I 956).
The possible functions of these large stores of free carbohydrate have been explored by
a number of workers and it appears as if they are endogenous reserves of carbon in spores.
Such a role has been proposed for mannitol and trehalose during germination of sclerotia
of S. sclerotiorum (Le Tourneau, 1966). In dormant sclerotia of Claviceps purpurea the levels
of mannitol and trehalose are very low but increase considerably during activation and
finally fall off again during germination (Mitchell & Cooke, 1968). Apparently the mannitol
is translocated via the stipe to the stroma and there It probably provides the energy source
for sexual reproduction (Cooke & Mitchell, 1969). The pattern followed by both the lipid
and carbohydrate fractions suggests that the major substrate utilized is the acylated form
of the sugars. Sussman (1961) reported that in Neurospora the carbohydrate fraction, composed mainly of trehalose, is not utilized in the ascospores until after dormancy has been
broken and then the trehalose gradually disappears. He proposed that the activation process
in Neurospora ascospores involves the induction of an enzyme system through which
trehalose is metabolized, whereas endogenous lipid is the principal fuel used by the dormant
spore.
The glycolipids of fungi in general, and the Basidiomycetes in particular, have received
scant attention, which is regrettable since they should yield useful indications about the
function of the glycolipids of higher organisms. From the present work and that of others
(Brennan et al. 1974) it now appears that there are three classes of glycolipid in fungi.
Simple acylated sugars and sugar alcohols of the type described here have also been found
in Rhodotorula species (Tulloch & Spencer, 1964). We have now presented evidence for the
presence in A . bisporus of a representative of the glycosyloxyfatty acid class of glycolipid.
It has not been fully established whether this belongs to the monoglucosyloxyfatty acid
variety, as found in Aspergillus niger (Laine et al. 1972) or to the sophoroside variety present
in the yeasts Candida (Tulloch, Spencer & Deinema, 1968) and Torulopsis (Stodola,
Deinema & Spencer, 1967). The possibility that representatives of the third class of fungal
glycolipids, the mycosphingoglycolipids (Brennan et al. 1g74), exist in A . bisporus has not
been investigated, although simple monoglycosylceramides have been found (Weiss &
Stiller, 1972).
The most noteworthy aspect of the phospholipid composition of A . bisporus described
here is the apparent absence of phosphatidylcholine. However, contrary to our evidence,
Holtz & Schisler (1971) found that phosphatidylethanolamine and phosphatidylcholine
were the most prominent individual phospholipids in both mycelium and sporophore, and
small amounts of phosphatidylserine and phosphatidylinositol were tentatively identified
only in the sporophore. It is known that the phospholipid composition of some eukaryotic
micro-organisms is subject to variation depending on the genetic and nutritional state of
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P. F. S . B Y R N E A N D P. J. B R E N N A N
the organism (Margnall & Getz, 1973). However, it does not seem that different growth
conditions are responsible for the altered phospholipid composition in Agaricus, since we,
like Holtz & Schisler (I 971) obtained sporophores directly from commercial mushroom
beds. A methodological comparison of both works offers little elucidation; Holtz &
Schisler (1971) did not use the Dragendorff reagent, which is specific for the choline, but
instead identified phosphatidylcholine by i.r. spectroscopy.
While it is generally considered that the principal phospholipids in eukaryotic organisms
are phosphatidylethanolamine and phosphatidylcholine this is apparently not so. Merdinger
(1969) was unable to identify any choline or inositol in lipid extracts of the yeast-like fungus,
Pullularia pullulans; phosphatidylethanolamine and phosphatidylserine were the principal
phospholipids. In agreement with previous reports (Nichols, Harris & James, 1965; Echlin
& Morris, 1965), Nichols (1968) was unable to find any phosphatidylcholine, phosphatidylinositol or phosphatidylethanolamine in extracts of blue-green algae.
This work was supported by a grant from the National Science Council (Ireland). We
thank Miss Patricia Flynn for the preparation and characterization of the stearyl-0-glucoses.
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