Báo cáo khoa học: Characterization of surface n -alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills pot

Characterization of surface n -alkanes and fatty acids of the epiphytic lichen Xanthoria parietina , its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills A. Torres 1 ,I.Dor 2 , J. Rotem 3 , M. Srebnik 1 * and V. M. Dembitsky 1 1 Department of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065, The Hebrew University of Jerusalem, 91120, Israel; 2 Division of Environmental Sciences, Graduate School of Applied Science, The Hebrew University of Jerusalem, Israel; 3 Department of Plant Pathology, ARO, Ministry of Agriculture, Bet Dagan, Israel Surface alkanes and fatty acids from the thalli of the lichen Xanthoria parietina, its photobiont Trebouxia sp., and its mycobiont were analysed by GC-MS. The green alga Tre- bouxia sp. synthesized mainly unsaturated fatty acids such as (Z,Z,Z)-9,12,15-18 : 3 (Z,Z)-9,12-18 : 2 and (Z)-9-18 : 1, and light alkanes C 8 -C 15 (upto83%oftotaln-alkanes). However, the mycobiont contained mainly saturated fatty acids such as hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and also very long-chain n-alkanes C 22 –C 34 . Dehydroabietic acid was found in both lichen and mycobi- ont. The occurrence of different amounts of n-alkanes and fatty acids in the photobionts and mycobionts of X. parietina was shown for the first time. Lichens collected from different locations in the Jerusalem hills contained n-alkanes ranging in concentration from 187 to 211 mgÆ(g dry wt) )1 ; n-alkane concentrations in the photobiont and mycobiont were 17–24 and 215–262 mgÆ(g dry wt) )1 , respectively. Keywords: alkanes; lichen; mycobiont; photobiont Trebouxia; Xanthoria. Lichens have been described as Ôdual organismsÕ because they are symbiotic associations between two (or sometimes more) entirely different types of micro-organism: a fungus (termed the mycobiont) and a green alga or a cyanobacte- rium (termed the photobiont). These organisms have both algal and fungal properties [1,2] and produce n-alkane, unusual betaine ether glycerolipids [3,4], and saturated, unsaturated, branched, and halogenated fatty acids [5–20]. Many different bioactive secondary metabolites have also been isolated from lichen species [21,22], which have been used in pharmaceutical sciences [23]. One of main questions in lichen biology and chemistry is which compounds are synthesized by which symbiotic lichen partner? As fungi are often rich in secondary products [24,25,26],it is not surprising that many typical lichen substances are synthesized by the mycobiont. Culberson et al. [27] showed that experimentally produced combinations of a mycobiont with foreign photo- bionts generate the same lichen products as the mycobiont in a natural thallus with its usual partner. Fox & Huneck [28] showed that mycobionts could synthesize long-chain aliphatic acids, but they did not indicate which fatty acids were synthesized by the photobiont. In this study, we attempt to show which aliphatic hydrocarbons and fatty acids are produced by the photo- biont and which by the mycobiont isolated from the lichen Xanthoria parietina. Materials and methods Lichen samples Samples of X. parietina lichens were collected from the bark of trees in the Givat Ram Campus, Hebrew University, Abu Ghosh Village, Gilo Aleph and Ein Kerem (all on the outskirts of Jerusalem) during June to August 1998. X. parietina is widespread in Israel and also around the Jerusalem hills [29,30]. Isolation and cultivation of photobionts Photobiont was isolated by the micropipette method of Ahmadjian [31] and grown as described by Friedl [32]. The photobiont was examined both in the lichenized and cultured state by standard light microscopic techniques with modifications as described by Dor [33]. For identifi- cation, the isolated strains were compared with cultures of all known species of Trebouxia (Chlorophyta, Trebouxio- phyceae, Pleurastrophyceae) [34,35]. The lichen photobiont Trebouxia arboricola used in our study was isolated and grown in the Laboratory of Hydrobiology. Stock cultures of the alga were maintained on 3N Bold’s Basal Medium agar slants [36]. The medium contained per litre: 0.75 g NaNO 3 ; 0.175 g KH 2 PO 4 ;0.075gK 2 HPO 4 ; 0.075 g MgSO 4 Æ7H 2 O; Correspondence to V. M. Dembitsky, Department of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065, The Hebrew University of Jerusalem, 91120, Israel. Fax: + 972 2 675 8201, Tel.: + 972 2 675 7549, E-mail: dvalery@cc.huji.ac.il *AffiliatedwithTheDavidR.BloomCentreforPharmaceutical Research, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. Note: Parts of this paper were presented at the 68th Israeli Chemist’s Society, 27 January 2003, Tel Aviv. (Received 28 November 2002, revised 6 February 2003, accepted 5 March 2003) Eur. J. Biochem. 270, 2120–2125 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03556.x 0.025 g CaCl 2 ; and 0.025 g NaCl. In addition, 1 L medium contained the following micronutrients: 11.42 mg H 3 BO 3 ; 4.98 mg FeSO 4 Æ7H 2 O; 8.82 mg ZnSO 4 Æ7H 2 O, 1.44 mg MnCl 2 Æ4H 2 O; 0.71 mg MoO 3 ; 1.57 mg CuSO 4 Æ5H 2 O; 0.49 mg Co(NO 3 ) 2 Æ6H 2 O; 50 mg EDTA; and 31 mg KOH, with improved conditions as described previously [33]. Hydrocarbons and fatty acids from fresh centrifuged biomass (4.9 g) were extracted. Isolation and cultivation of mycobionts Mycobionts from X. parietina were obtained from the spores discharged from apothecia of a thallus, and were cultivated in test tubes containing 20 g malt extract (Difco), 4 g yeast extract (Difco), 100 g sucrose, 15 g agar, and water, pH 7.0, at 20 °C in the dark. After cultivation for 3 months, the colonies and slants with crystals were harvested. The mycobionts used in our study were cultivated by J. R. Extraction of hydrocarbons and fatty acids Lichen thalli from X. parietina and their photobiont Tre- bouxia sp. and mycobiont were extracted with pentane/ dichloromethane/methanol (40 : 30 : 30, by vol.) as des- cribed previously [37]. GC–MS analysis A Hewlett–Packard 5890 gas chromatograph (series II), modified for a glass capillary column coupled to a HP GC- mass selective detector (5971B MSD), was used. Hydrocar- bons and the methyl esters of fatty acids were analysed by GC on two capillary coupled columns as described previously [37], and also on an HP-5 column (10 m, internal diameter 0.32 mm, film thickness 0.25 mm) coupled with a second capillary column RTX-1701 (Restek, Boca Raton, 1 PA, USA; 30 m, internal diameter 0.32 mm · 0.25 lmfilm) and a third capillary column HP-FFAP (30 m, 0.32 mm · 0.25 lm film). The GC oven was programmed as follows: 2minat40°C; 2 °CÆmin )1 to 300 °C; 20 min at 300 °C. The injector temperature was kept at 180 °C (splitless). The flow rate of the carrier gas (helium) was 25 cmÆs )1 .TheMS detector was operated at 194 °C, with ionization energy set at 70 eV. The scan range was m/z 30–650, and scan rate 0.9 scans/second. Solvent delay was set at 10 min. Hydro- carbons and fatty acid methyl esters were identified by comparison with those found in the Wiley mass spectral library (7th edition). Results and discussion The complex hydrocarbons and fatty acids produced by the cultured photobiont green alga Trebouxia sp. and the mycobiont, and also by X. parietina, were separated by serially coupled capillary columns with consecutive non- polar and semipolar stationary phases [37]. GC-MS analysis of the hydrocarbons and fatty acids of X. parietina,its photobiont and mycobiont collected from four different locations indicated the presence of 27 n-alkanes and six major fatty acids. Quantitative data are shown in Table 1. The major constituents of lichen and mycobionts ranged from n-tricosaene (C 23 )ton-octacosane (C 28 ) in 10–16% of completely identified n-hydrocarbons. The major n-alkanes in the photobiont were n-octane (C 8 ), that varied from 21% to 41%, n-decane (C 10 ), n-undecane (C 11 ), and n-dodecane (C 12 ). The complete GC-MS separation of hydrocarbons and fatty acid methyl esters identified from the lichen, photobiont and mycobiont is shown in Fig. 1. Peak 5 [scan 7805; 117.7 min; m/z:M + 314(19), 239(100), 240(24), 299(18), 141(9), 197(8)] was found to be the methyl ester of dehydroabietic acid. This is the first time that dehydro- abietic acid has been found in both lichen and mycobiont. This acid (of the triterpenoid resin family) which occurs widely in higher plants [38] is a typical constituent of coniferous resins and is highly reactive, participating in the protection of wounded trees against microbial attacks [39]. Dehydroabietic acid has been found in traditional Chinese medicines, and is used in many Asian countries [40]. It has also been found as a major effluent component in the paper and pulp industry [41] and is one of the toxic compounds accumulated in fish and other aquatic organisms [42]. Ten major fatty acids were identified in the lichen, photobiont and mycobiont (Table 1). The mycobiont and lichen do not contain the (Z,Z,Z)-9,12,15-18 : 3 fatty acid, but this fatty acid is found in green alga only to a concentration of 9.8–17% (Fig. 2). According to Bychek- Guschina [43], photobionts of Trebouxia erici and Trebouxia impressa (cultures from the Botanical Institute, St Petersburg, Russia) do not contain the 18 : 3 and 18 : 2 fatty acids, and produce only two fatty acids: 16 : 0 (30% and 51%, respectively) and 18 : 1 (69% and 42%, respect- ively). It is probably an artefact of the GC analysis. Mycobionts produce saturated fatty acids such as tetra- decanoic acid (5–9%), hexadecanoic acid (34–37%) and octadecanoic acid (23–30%) (Table 1). Three isomers, cis- 9-18 : 1, cis-10-18 : 1 and cis-12-18 : 1, have been identified from the lichen, photobiont, and mycobiont. Lichens, symbiotic organisms of fungi and algae, syn- thesize numerous metabolites, Ôlichen substancesÕ,which comprise aliphatic, cycloaliphatic, aromatic, and terpenic compounds. Lichens and their metabolites have manifold biological activities such as antiviral, antibiotic, antitumor, allergenic, plant growth inhibitory, antiherbivore, and enzyme inhibitory [44–47]. Usnic acid, a very active lichen substance, is used in pharmaceutical preparations [48]. The leaves of higher plants contain waxy alkanes and have been shown to be useful for protection against UV-B radiation, as well as photoinhibition [49]. According to Piervittori et al. [50], in X. parietina, this layer is made up of a relatively high amount of n-alkanes ranging from C 18 to C 34 . The main compounds detected in the Abu Ghosh Valley and other places near the Jerusalem hills were C 24 , C 25 ,andC 26 . The total content of the n-alkane fraction did not show any statistical difference between the populations growing in the four different places (Table 1). On analysis of hydrocarbons from the lichen Lobaria pulmonaria [51] the major hydrocarbons found were C 27 (21.5%), C 28 (9.7%), C 29 (32.6%), and C 31 (7.9%). Light aliphatic hydrocarbons C 9 ,C 10 ,C 11 ,C 12 and C 13 were found in the lichen Evernia prunastri [52]. Zygadlo et al. [53] studied 14 species of Argentinian lichens for the presence of alkanes, and found that the main ones were C 27 ,C 29 and C 31 . The alkanediene C 17 H 32 and normal alkanes in the C 9 –C 17 range, with the C 17 alkadiene as the major hydrocarbon, were detected Ó FEBS 2003 Alkanes and fatty acids of Xanthoria and symbionts (Eur. J. Biochem. 270) 2121 Table 1. Comparative n-alkane and fatty acid compositions of X. parietina, the photobiont Treb ouxia sp., and the mycobiont (% of total n-alkanes) collected in the places indicated. Values in parentheses are n-alkanes [mgÆ(g dry wt) )1 ]. Abu Ghosh Gilo Aleph Givat Ram Ein Kerem Lichen (192.31) Photobiont (23.89) Mycobiont (247.87) Lichen (211.45) Photobiont (17.28) Mycobiont (262.12) Lichen (206.52) Photobiont (19.34) Mycobiont (238.79) Lichen (187.94) Photobiont (21.67) Mycobiont (215.16) n-Octane (C 8 ) 0.56 26.47 1.12 21.23 0.44 37.18 1.14 41.34 n-Nonane (C 9 ) 0.32 6.81 0.33 3.81 0.26 5.42 0.43 7.63 n-Decane (C 10 ) 0.23 14.88 0.47 17.72 0.21 10.88 0.53 7.35 n-Undecane (C 11 ) 0.48 14.04 0.42 18.88 0.29 16.24 0.38 14.44 n-Dodecane (C 12 ) 0.18 9.34 0.53 14.29 0.14 4.26 0.26 5.14 n-Tridecane (C 13 ) 0.12 2.64 0.13 2.48 1.96 0.28 2.63 n-Tetradecane (C 14 ) 0.58 0.14 4.94 0.71 n-Pentadecane (C 15 ) 0.15 0.71 0.32 0.28 0.63 0.88 0.53 n-Hexadecane (C 16 ) 0.28 0.94 0.59 1.17 0.45 1.08 0.27 0.92 n-Heptadecane (C 17 ) 0.64 1.61 0.82 1.87 0.71 1.48 0.59 1.53 n-Octadecane (C 18 ) 1.13 0.93 0.95 1.56 1.02 0.83 0.84 1.04 n-Nonadecane (C 19 ) 1.61 3.05 1.72 3.17 1.72 2.88 1.84 2.86 n-Icosane (C 20 ) 2.04 2.42 2.37 2.44 1.91 2.02 2.34 2.58 n-Heneicosane (C 21 ) 2.97 3.85 3.02 4.47 2.91 4.09 2.81 4.22 n-Docosane (C 22 ) 9.58 2.61 7.44 8.06 2.21 7.77 8.79 2.91 7.63 7.93 2.35 5.96 n-Tricosane (C 23 ) 11.23 4.09 9.08 10.84 3.66 9.21 11.23 4.34 9.79 10.21 2.84 8.54 n-Tetracosane (C 24 ) 13.81 5.58 10.76 11.77 4.19 10.48 12.77 5.76 10.83 11.57 4.83 10.06 n-Pentacosane (C 25 ) 13.71 5.42 14.23 15.57 3.37 12.95 15.01 4.16 12.02 13.01 4.44 11.16 n-Hexacosane (C 26 ) 13.32 4.22 15.55 13.01 2.53 14.42 14.81 3.49 16.51 15.35 3.89 16.20 n-Heptacosane (C 27 ) 10.23 2.16 11.21 10.85 1.36 10.91 11.51 1.57 10.87 11.48 2.51 13.62 n-Octacosane (C 28 ) 7.66 0.86 6.77 7.84 6.49 7.33 1.12 6.47 7.63 0.56 7.49 n-Nonacosane (C 29 ) 5.29 0.24 4.44 4.58 4.87 4.34 4.61 5.33 4.91 n-Triacontane (C 30 ) 2.25 3.02 2.58 2.78 2.64 2.81 2.91 3.01 n-Hentriacontane (C 31 ) 1.05 1.61 0.86 2.02 0.71 1.59 1.73 2.98 n-Docotriacontane (C 32 ) 0.56 1.24 0.58 1.14 0.44 1.31 0.82 1.28 n-Tritriacontane (C 33 ) 0.16 0.97 0.32 0.86 0.18 1.15 0.23 0.94 n-Tetratriacontane (C 34 ) 0.21 0.76 0.15 1.01 0.13 Major fatty acids, as methyl ester (% of total fatty acids) 1. Hexane-1,6-dioic acid 6.12 0.99 8.36 5.11 2.17 7.66 6.53 7.82 8.12 4.21 5.33 6.34 2. Tetradecanoic (14 : 0) 4.89 5.18 9.23 4.45 3.18 8.16 5.63 2.67 6.43 3.29 2.98 4.96 3. Hexadecanoic (16 : 0) 34.56 31.82 37.46 39.97 29.16 38.11 41.39 23.63 36.15 44.89 28.29 33.96 4. Octadecanoic (18 : 0) 12.34 1.17 25.75 11.34 2.21 22.85 9.76 4.91 28.86 6.87 3.12 30.02 6. (Z,Z,Z)-9,12,15-18 : 3 3.42 16.93 0.24 1.95 17.11 1.02 9.81 Trace 2.13 14.22 7. (Z,Z)-9,12-18 : 2 19.22 22.68 3.26 18.45 25.91 2.77 10.48 17.43 1.97 13.21 19.17 2.07 8. (Z)-9-18 : 1 8.31 14.37 3.89 9.14 9.22 4.18 8.31 19.42 3.59 7.94 21.97 4.12 9. (Z)-10-18 : 1 4.11 4.57 4.76 3.55 3.11 6.27 5.13 5.34 4.87 4.64 2.11 5.14 10. (Z)-12-18 : 1 0.73 0.88 1.94 0.46 0.11 2.16 0.87 0.83 0.75 0.79 Other fatty acids 6.30 0.79 7.05 4.10 7.03 9.89 9.59 8.10 9.18 12.07 2.02 13.39 2122 A. Torres et al.(Eur. J. Biochem. 270) Ó FEBS 2003 in E. prunastri [54]. These were the first reports of the occurrence of light hydrocarbons such as C 9 –C 14 in lichen species. Gaskell et al. [55] reported hydrocarbons with chain lengths of C 17 –C 33 in three species: Cetraria nivalis, Cetraria crispa,andSiphula ceratites. Two collections of each lichen were made several years apart but at the same locations. The main alkanes in the two species of Cetraria were C 27 ,C 29 , and C 31 . The most abundant alkanes were those in S. ceratites: 18 alkanes. The fractions of alkanes from S. ceratites samples included a component with a mass spectrum that indicated it to be an equimolar mixture of 7-methyl and 8-methyl heptadecanes. The same component was found in trace amounts in the first sample of the two Cetraria species. Also observed in the fraction of hydro- carbons of the second sample of S. ceratites were a series of anteiso-alkanes. Anteiso-C 26 was the most prevalent, with smaller amounts of anteiso-C 24 , anteiso-C 23 and anteiso-C 22 . Twenty-one hydrocarbons were identified in Cetraria islan- dica [56]. The most abundant was heptadecadiene-1,8 (69.5%). Cultivated mycobionts isolated from lichens collected in different places in the Jerusalem hills had the same hydrocarbon content as the lichen (Table 1). It is known that some lichen species contain both n-alkanes and branched alkanes [5], but our studies and others [50,53] have shown that X. parietina contains only n-alkanes. More interesting experimental data were obtained dur- ing studies of the green alga Trebouxia sp. It was surprising to find that the major n-alkanes produced by the photo- biont were n-octane (C 8 , varying from 21% to 41% of total hydrocarbons), n-decane (C 10 ,7–8%),n-undecane (C 11 , 14–9%), and n-dodecane (C 12 , 5–14%) (Table 1). These light hydrocarbons have also been found as minor components in some lichen species, varying from 0.12% to 1.12% [51,52]. GC separation of light hydrocarbons isolated from green alga Trebouxia sp.isshowninFig.1. This species is widespread in many lichens, including Xanthoria aureola [57], and more than 10 algal species belonging to this genus have been isolated from lichens; however, their hydrocarbons and fatty acids have not been analysed. Fig. 1. Gas chromatographic separation on serially capillary coupled columns of n-alkanes and methyl esters of fatty acids from Xa nthoria parietina (I), photobiont Trebouxia sp. (II) and mycobiont (III). Lichen X. parietina was collected in Givat Ram (Table 1). Major identified peaks show on three parts on GC-MS, peaks: 1, hexane-1,6-dioic acid, dimethyl ester; 2, tetradecanoic acid, methyl ester; 3, hexadecanoic acid, methyl ester; 4, octadecanoic acid, methyl ester; 5, dehydroabietic acid, methyl ester. Ó FEBS 2003 Alkanes and fatty acids of Xanthoria and symbionts (Eur. J. Biochem. 270) 2123 Successful separation of hydrocarbons and fatty acids from the lichen, photobiont and mycobiont was achieved by using serially coupled capillary GC columns with consecu- tive nonpolar and semipolar stationary phases as described previously [37]. We have also shown that the cultivated photobiont Nostoc sp. isolated from Collema sp. lichen can synthesize more than 130 metabolites including, cyclo- hexane, cyclopentane, aliphatic saturated hydrocarbons (C 7 –C 30 ) and fatty acids. It has been shown that photobionts and mycobionts use different biosynthetic pathways, for example, in the biosyn- thesis of sterols. Thus, Lenton et al. [58] showed that the photobionts Trebouxia sp. and Trebouxia decolorans and also the mycobiont isolated from X. parietina synthesized different sterols. X. parietina and its mycobiont were found to contain ergosterol and lichesterol as the major consti- tuents together with lower levels of three other C 28 sterols: ergosta-7,22-dien-3b-ol, ergosta-7,24(28)-dien-3b-ol, and ergosta-7,22,24(28)-trien-3b-ol. The photobionts Trebouxia sp. and T. decolorans contained predominantly porifera- sterol, with lower levels of cliosterol, ergost-5-en-3b-ol, brassicasterol and cholesterol. These results indicate the presence of separate biosynthetic pathways for some secondary metabolites in the photobionts and mycobionts of lichens. Our results show that Trebouxia sp. predominantly produces the following unsaturated fatty acids: 9,12,15- 18:3,9,12-18:2,and9-18:1,aswellaslightn-alkanes C 8 –C 15 . The major saturated fatty acids obtained from the mycobiont are: tetradecanoic (14 : 0), hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and very long-chain n-alkanes C 22 –C 34 . References 1. Galun, M., ed. 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Biochem. 270) 2125 . Characterization of surface n -alkanes and fatty acids of the epiphytic lichen Xanthoria parietina , its photobiont a green alga Trebouxia sp. , and its. Israel Surface alkanes and fatty acids from the thalli of the lichen Xanthoria parietina, its photobiont Trebouxia sp. , and its mycobiont were analysed by GC-MS. The