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The lipids of a moss (Hypnum cupressiforme) and of the leaves of green holly (Ilex aquifolium)
~,196srVd.4.pp.769t0772.
Pu#unonPKuLtd.
RbltedinEal&8nd
SHORT COMMUNICATION
THE LIPIDS OF A MOSS (HYPNUM CUPRESSIFORME) AND OF THE LEAVES OF GREEN HOLLY (ILEX AQUIFOLIUM) B. W. NICHOU unikver-Laboratoly,c0lwortIlI3ouaE,sham~Bodtopd,England (Rswlved27 J-y EMS)
INTRODUCTION TEt3L.wlipids ofseveralhigherplants,namely~~‘redclover,**3~~bean,4~~~~ cask+ and lettuce6have now been partklly or wholly &rack&& and the close simiirity in lipid composition of all such deciduous tissues has been established. Thereis, however, a lack of comparableinformation for the evergreens,and lower plants such as the mosses. Mossesareofpartkukinterestsince GelkmanandSchlenk7haveshownthattheycontain arachidonicacid, an acid previouslymx&ctedinplanttissues. RESULTS
AND DISCUSSION
The major fatty acid-containinglipids of green holly leaves (Lkx @&an) and moss (Hyp?aumcupres.qorn4 are those glycolipids and phospholipids commonly found in the phomc tissue of higher plan& namely, the gakctosyl d@yceriM sulpholipid, cardiolipin,phosphatidyl-choline,-ethanolamine,4nositol and -glycerol. Only very minor qua&k of triglyceridesare present. Holly leaf extracts also contain a huge qumtity ofan acid- and alkali-stable lipid which gives a positive Lieberman-Burchardreackn, and migratesmore slowly than j3-sitosterolon thin layers of silica gel using hexane:diethyl ether(85:5v/v)am,mobilephase(Rfvalnecl:~~l~11;unknownlipid,~5). The fatty acid composition ofthe lipids moss and holly leavesis given in Tables 1 and 2 respectively. Previous studies on the fatty acid composition of various plant tissues’~2-6~s have indicateda preferentialconcentrationof the more highly unsaturatedfatty acids in the galactosyldiglycerides. Tables 1 and 2 show that this rule may also be exknded to the lipids
of
lCF.~,P.aoon,H.F.D~varandS.D.~~~~s.Rnr.Cormn.15,4a4(1964). *RO.Wem~1c,J.sei.Food&kl2,34(l%1). Biochim. my& ACM84,613 (196)). ~RO.WBEMNIC~~F.B.S~~~D, I)iocRsmistry3,1271(1%4). 4P.S.SmtryandM.Katw 5J.s.O~BMiAA.BHMION,J.~Rg.~~2(1%4). ~B.W.NmouandAT.Jmes,Unpubli&cdo~o= a4 426 (1964). 7J.L.GzusnwwandH.~,ExpsriarHQ 8 B. W. Nmiou and A. T. Mm, Ferte. Se#kn, hMchit~~66,1003 (1%4). 769
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of moss and holly. The gah&osyl diglyc&ies of the moss possess not only a high proportion of a-linolenic acid but they also contain most of the C, polyunsaturated acids, while the corresponding lipids of holly leaves possess a high linolenic acid content comparable to that in deciduous leave&. Inm~ofthespeciesofgrctnleafsofar~themonogalactosyl~~(MGfxi) fractioncontainarahigherproportionoflinolmicacidthanaayotherlipidfromtbe~etissue, including digalactosyl diglyceride (DGDG). There are a few green tissues in which the MGDG has a lower linolenic acid content than the DGDG, for ezample spinach l and moss, but the former lipid contains an increased quantity of7,10,13-hexadecatrienoic acid’ so that the total proportion of trienoic acids in the lipid is still higher than that in any other. SuperMally, it might seem permissible to argue that this hexadecatrienoic acid may be the immediate precursor of c&inolenic acid (9,X&1%ocMecatrienoic acid) by a process of chain elongation, but James9 and Harris and JameslOhave shown that in higher plants the Cls dienoic and trienoic acids are formed by desaturation of oleic acid. This 7,141~hexadecatrienoic acid may result from the desaturation of 7-e acid which G&llerman and Schlenk7 hsfvealso~o~~be~tintbr~hexadecenoicacidsofmoss. It~~~t~~ mechanism accom&? for the pmsence of the same hexadecatriex& acid in spinach lipids, although it has never been denionstrated that these contain 7-hez&ecenoic acid. There is no evidence that 9-hexadecenoic acid can be desaturated in a similar mnanner,a reaction which would result in the formation of 9,12,15&exadecatrienoic acid containing a krminal methylene group. An axulogous situation also exists in the lipids C&r& v&u& in which the dienoic acid content of the MGDG fraction is comprised of both Cis and Cls dienoic acids; whereas the DGDG fraction contains only a very small quantity of hexadecadienoic acid.” In the holly leaves and moss studied, trcu&-mc acid occurs only in the phosphatidyl glycerol fraction a specikity aheady observed in the leaf lipids of spinaCkl red clover,j castor,6and also in light grown ChZorek vz&zrkg Gellerman and Schlenk’ did not detect this acid in their moss extmcts, probably because of its sm&l overall concentration in the total lipids.
of
0A. T. JAMES, BQdlrlm. Bioplrys. ActaSr, 167 (I=). loRv.~BJ.RWoo~andA.T.J~~J.~22(1%5X ~~B.W.NKXHXS,B~~~~LIJ@~~~..AE~~~~~~~~.
I72
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Thus the specificities alreadyobservedregardingthe faw acid distributionin the lipids of C!hi&eZ& w&u& and in the leavesof deciduousplantsmay also be generallyapplicableto the lipids of the photosynthetic tissues of evergreens and mosses. EXPERIMENTAL
Tissues were macerated with isopropyl alcohol and the residue re-extracted with chloroform: methanol (2: 1 v/v) until devoid of pigment. No attempt was made to separate the photosymhetic tissue of the moss from the woody tissue. Extracts were dried in WCUO, redissolved in chloroform and the lipids fractionated by a combination of column chromatography on DEAE cellulose * and preparative thin-layer chromatography ‘on silica gel.* Fattyacidmethylesterswerepreparedbyrefiuxinglipidf~onswithmethanol:benzene: sulphuric acid (150: 75 : 10 v/v) and the esters analysed by gas-liquid chromatography in a Fye Argon chromatograph employing ethylene glycol adipate and Apiezon L as stationary phases. Confkmatory evidence for the identity of each acid was obtained by fractionating the methyl esters according to degree of unsaturation on thin layers of silica gel impregnated with silver nitrate, followed by further fractionation according to chain length by gas-liquid chromatography on Apiezon L. The location of double bonds in the C16monoenoic acids was de&mined by permanganate+eriodat.eoxidation of their methyl ester~‘~ (as isolated by preparative gas-liquid chromatography), followed by GLC of the methyl esters ofthe reaction products. The double bondlocationofthehexadecatrienoic~dinthemoss~~kr*lsumedonthe~ofthe evidence obtained by Gellerman and !khlenk’ on a variety of mosses and on the observations . of other workers (see %orland’3) that this is the only hexadccatrrenoic acid found in plant leaves. The major CZOtetraenoicacid presumablypossews the 5,8,11,14-con@ration by analogywiththefwingsof Gclkman and Scblenk’ for other mosscs;themiuor~acids of the same degreeof unsaturation but of slightly different retention volumes on polyester columns are probably positional isomers of arachidonic acid. SUMMARY
Thefattyacidcompositionofthelipidsofa moss (Hypramr tz4pra@fnne) andthegreen leaves of a holly (Ilcx agafoolium) is similar to that of the green leaves of deciduous higher plants. JHmo~~1li~~,thC~lyllflO(LtUTatedC2gacidSare~ywncentrstedintbe~osyl diglycerides, and in both moss and holly lipids trua.v-3-hexadecenoicacid occurs only in the phosphatidyl glycerol fraction.
Pu#unonPKuLtd.
RbltedinEal&8nd
SHORT COMMUNICATION
THE LIPIDS OF A MOSS (HYPNUM CUPRESSIFORME) AND OF THE LEAVES OF GREEN HOLLY (ILEX AQUIFOLIUM) B. W. NICHOU unikver-Laboratoly,c0lwortIlI3ouaE,sham~Bodtopd,England (Rswlved27 J-y EMS)
INTRODUCTION TEt3L.wlipids ofseveralhigherplants,namely~~‘redclover,**3~~bean,4~~~~ cask+ and lettuce6have now been partklly or wholly &rack&& and the close simiirity in lipid composition of all such deciduous tissues has been established. Thereis, however, a lack of comparableinformation for the evergreens,and lower plants such as the mosses. Mossesareofpartkukinterestsince GelkmanandSchlenk7haveshownthattheycontain arachidonicacid, an acid previouslymx&ctedinplanttissues. RESULTS
AND DISCUSSION
The major fatty acid-containinglipids of green holly leaves (Lkx @&an) and moss (Hyp?aumcupres.qorn4 are those glycolipids and phospholipids commonly found in the phomc tissue of higher plan& namely, the gakctosyl d@yceriM sulpholipid, cardiolipin,phosphatidyl-choline,-ethanolamine,4nositol and -glycerol. Only very minor qua&k of triglyceridesare present. Holly leaf extracts also contain a huge qumtity ofan acid- and alkali-stable lipid which gives a positive Lieberman-Burchardreackn, and migratesmore slowly than j3-sitosterolon thin layers of silica gel using hexane:diethyl ether(85:5v/v)am,mobilephase(Rfvalnecl:~~l~11;unknownlipid,~5). The fatty acid composition ofthe lipids moss and holly leavesis given in Tables 1 and 2 respectively. Previous studies on the fatty acid composition of various plant tissues’~2-6~s have indicateda preferentialconcentrationof the more highly unsaturatedfatty acids in the galactosyldiglycerides. Tables 1 and 2 show that this rule may also be exknded to the lipids
of
lCF.~,P.aoon,H.F.D~varandS.D.~~~~s.Rnr.Cormn.15,4a4(1964). *RO.Wem~1c,J.sei.Food&kl2,34(l%1). Biochim. my& ACM84,613 (196)). ~RO.WBEMNIC~~F.B.S~~~D, I)iocRsmistry3,1271(1%4). 4P.S.SmtryandM.Katw 5J.s.O~BMiAA.BHMION,J.~Rg.~~2(1%4). ~B.W.NmouandAT.Jmes,Unpubli&cdo~o= a4 426 (1964). 7J.L.GzusnwwandH.~,ExpsriarHQ 8 B. W. Nmiou and A. T. Mm, Ferte. Se#kn, hMchit~~66,1003 (1%4). 769
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46 31 26 t
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of moss and holly. The gah&osyl diglyc&ies of the moss possess not only a high proportion of a-linolenic acid but they also contain most of the C, polyunsaturated acids, while the corresponding lipids of holly leaves possess a high linolenic acid content comparable to that in deciduous leave&. Inm~ofthespeciesofgrctnleafsofar~themonogalactosyl~~(MGfxi) fractioncontainarahigherproportionoflinolmicacidthanaayotherlipidfromtbe~etissue, including digalactosyl diglyceride (DGDG). There are a few green tissues in which the MGDG has a lower linolenic acid content than the DGDG, for ezample spinach l and moss, but the former lipid contains an increased quantity of7,10,13-hexadecatrienoic acid’ so that the total proportion of trienoic acids in the lipid is still higher than that in any other. SuperMally, it might seem permissible to argue that this hexadecatrienoic acid may be the immediate precursor of c&inolenic acid (9,X&1%ocMecatrienoic acid) by a process of chain elongation, but James9 and Harris and JameslOhave shown that in higher plants the Cls dienoic and trienoic acids are formed by desaturation of oleic acid. This 7,141~hexadecatrienoic acid may result from the desaturation of 7-e acid which G&llerman and Schlenk7 hsfvealso~o~~be~tintbr~hexadecenoicacidsofmoss. It~~~t~~ mechanism accom&? for the pmsence of the same hexadecatriex& acid in spinach lipids, although it has never been denionstrated that these contain 7-hez&ecenoic acid. There is no evidence that 9-hexadecenoic acid can be desaturated in a similar mnanner,a reaction which would result in the formation of 9,12,15&exadecatrienoic acid containing a krminal methylene group. An axulogous situation also exists in the lipids C&r& v&u& in which the dienoic acid content of the MGDG fraction is comprised of both Cis and Cls dienoic acids; whereas the DGDG fraction contains only a very small quantity of hexadecadienoic acid.” In the holly leaves and moss studied, trcu&-mc acid occurs only in the phosphatidyl glycerol fraction a specikity aheady observed in the leaf lipids of spinaCkl red clover,j castor,6and also in light grown ChZorek vz&zrkg Gellerman and Schlenk’ did not detect this acid in their moss extmcts, probably because of its sm&l overall concentration in the total lipids.
of
0A. T. JAMES, BQdlrlm. Bioplrys. ActaSr, 167 (I=). loRv.~BJ.RWoo~andA.T.J~~J.~22(1%5X ~~B.W.NKXHXS,B~~~~LIJ@~~~..AE~~~~~~~~.
I72
B.W.Nm
Thus the specificities alreadyobservedregardingthe faw acid distributionin the lipids of C!hi&eZ& w&u& and in the leavesof deciduousplantsmay also be generallyapplicableto the lipids of the photosynthetic tissues of evergreens and mosses. EXPERIMENTAL
Tissues were macerated with isopropyl alcohol and the residue re-extracted with chloroform: methanol (2: 1 v/v) until devoid of pigment. No attempt was made to separate the photosymhetic tissue of the moss from the woody tissue. Extracts were dried in WCUO, redissolved in chloroform and the lipids fractionated by a combination of column chromatography on DEAE cellulose * and preparative thin-layer chromatography ‘on silica gel.* Fattyacidmethylesterswerepreparedbyrefiuxinglipidf~onswithmethanol:benzene: sulphuric acid (150: 75 : 10 v/v) and the esters analysed by gas-liquid chromatography in a Fye Argon chromatograph employing ethylene glycol adipate and Apiezon L as stationary phases. Confkmatory evidence for the identity of each acid was obtained by fractionating the methyl esters according to degree of unsaturation on thin layers of silica gel impregnated with silver nitrate, followed by further fractionation according to chain length by gas-liquid chromatography on Apiezon L. The location of double bonds in the C16monoenoic acids was de&mined by permanganate+eriodat.eoxidation of their methyl ester~‘~ (as isolated by preparative gas-liquid chromatography), followed by GLC of the methyl esters ofthe reaction products. The double bondlocationofthehexadecatrienoic~dinthemoss~~kr*lsumedonthe~ofthe evidence obtained by Gellerman and !khlenk’ on a variety of mosses and on the observations . of other workers (see %orland’3) that this is the only hexadccatrrenoic acid found in plant leaves. The major CZOtetraenoicacid presumablypossews the 5,8,11,14-con@ration by analogywiththefwingsof Gclkman and Scblenk’ for other mosscs;themiuor~acids of the same degreeof unsaturation but of slightly different retention volumes on polyester columns are probably positional isomers of arachidonic acid. SUMMARY
Thefattyacidcompositionofthelipidsofa moss (Hypramr tz4pra@fnne) andthegreen leaves of a holly (Ilcx agafoolium) is similar to that of the green leaves of deciduous higher plants. JHmo~~1li~~,thC~lyllflO(LtUTatedC2gacidSare~ywncentrstedintbe~osyl diglycerides, and in both moss and holly lipids trua.v-3-hexadecenoicacid occurs only in the phosphatidyl glycerol fraction.