Organ Specific Composition of Epicuticular Waxes of C is tus albidus L., Cistaceae

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1 Organ Specific Cmpsitin f Epicuticular Waxes f C is tus albidus L., Cistaceae Sabine Hennig, Paul-Gerhard Gülz Btanisches Institut der Universität zu Köln. Gyrhfstraße 15, D-5000 Köln 41, Bundesrepublik Deutschland and Kurt Hängst Museum für Ostasiatische Kunst der Stadt Köln, Universitätsstraße 100, D-5000 Köln 1, Bundesrepublik Deutschland Z. Naturfrsch. 43c, (1988); received August 25, 1988 Cistus albidus, Epicuticular Wax Cmpsitin, Organ Specific Patterns Epicuticular waxes frm varius rgans f Cistus albidus L. shwed always an rgan specific cmpsitin. Leaf wax cntained hmlgus series f very lng chained and saturated wax cmpnents, such as hydrcarbns, wax esters, fatty acids and alchls. Additinally in this wax extract triterpenids and great amunts f resin were fund. Sepal wax had nearly the same wax cmpsitin like that f leaves with a trend t shrter chain lengths and nt s great amunts f resin. Petal wax cntained the cmmn wax lipids, t, but the hmlgus series shwed a shift t shrter chain lengths, and great amunts f unsaturated lipids were fund in hydrcarbns, wax esters and fatty acids. In additin triterpenl esters were fund but n resin. Stamen wax was similar t that f petals. The shift t shrter chain lengths and the presence f great amunts f unsaturated lipids shwed a characteristic distributin fr stamens. Seed wax cntained nly saturated wax lipids. In cntrast t leaf waxes, the shrt chain cmpunds were dminating, and triterpenids and resin were missing. The chemical wax cmpsitin f the varius rgans f C. albidus was crrelated t the surface structures f these rgans by SEM pictures. Intrductin All aerial rgans f higher plants are cvered with a thin cntinuus wax layer. Often these wax layers are f fluid cnsistancy, fr example n petals and seeds. But n leaves this wax layer is mst superimpsed by crystalline structures f very different gemetrical frms [1 3]. The chemical cmpsitin f these epicuticular waxes is species specific [4, 5] and in additin several hints in literature shw als rgan specific wax patterns [6-16]. C. albidus L. [17] is a perennial evergreen scrub and a characteristic element f the macchias and garigues f the Mediterranean regin. This plant was used fr many studies cncerning epicuticular waxes [15, 16, 18 21] and flavnids [22 24]. Therefre, this Cistus species was selected t cmpare the wax cmpsitin f five different aerial rgans such as leaves, sepals, petals, stamens and seed cats frm the same plant in ne vegetatin perid under reprducible cnditins. Reprint requests t Dr. Paul-Gerhard Gülz. Verlag der Zeitschrift für Naturfrschung, D-7400 Tübingen /88/ $01.30/0 Materials and Methds Plant materials Plants f C. albidus were grwn frm seeds and cultivated under equal envirnmental cnditins in the fields f the Btanical Institute f the University f Clgne. Three plants f different rigin were used fr parallel studies. Quantitative and qualitative cmpsitins f the epicuticular waxes were analyzed frm leaves, sepals, petals, stamens, and seed cats. Methds Epicuticular waxes were extracted frm leaves, sepals, petals, and stamens with chlrfrm. They were dipped cnsecutively int three beakers f chlrfrm fr a ttal f 3 min. were treated with hexane in the same manner. The wax extractin was cmpletely, as shwn by SEM (see Fig. 9). The raw wax was redisslved in pentane and the sluble parts were chrmatgraphed n a silica gel clumn (Merck 60, Darmstadt) with the fllwing slvents f increasing plarity: 1. pentane t elute hydrcar- Dieses Werk wurde im Jahr 2013 vm Verlag Zeitschrift für Naturfrschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.v. digitalisiert und unter flgender Lizenz veröffentlicht: Creative Cmmns Namensnennung 4.0 Lizenz. This wrk has been digitalized and published in 2013 by Verlag Zeitschrift für Naturfrschung in cperatin with the Max Planck Sciety fr the Advancement f Science under a Creative Cmmns Attributin 4.0 Internatinal License.

2 S. Hennig et al. Organ Specific Cmpsitin f Epicuticular Waxes frm Cistus albidus bns, 2. 2-chlrprpane t elute wax esters and 3. methanl t elute alchls and fatty acids. These fractins were checked by T L C, G C and chemical reactins as described previusly [25 27]. Fr T L C, precated silica gel plates (Merck 60, Darmstadt) were used. The slvent system was tluene fr Rfl, dichlrmethane : ethyl acetate (24:1) fr Rn- Plates, impregnated with silver nitrate in acetnitrile, were run in dichlrmethane : ethyl acetate (24:1) fr Rfi. As spray reagents were taken brmthyml blue and carbazle fr triterpenids and sterls [28], Fr gas chrmatgraphy a Hewlett Packard mdel 5710 A with F I D and integratr 3380 S was used fr mst substances. A 25 m glass capillary clumn D U H T - O V 101 was prgrammed frm C with 4 C/min advance. A 10 m glass capillary clumn F F A P was used fr fatty acid methyl esters, temp C, rate 4 C/min. The surface f leaves, sepals, petals, stamens and seed cats were studied with a scanning electrn micrscpe, type Philips P S M E 500. The fresh plant material was sputtered with gld. Pictures were taken with a plarid camera. Results Epicuticular waxes were extracted frm the varius rgans f C. albidus in very different amunts. Leaves resulted in 3.6% epicuticular extract/dry weight, sepals in similar amunts, but petals cntained nly 0.6% wax/dry weight, stamens 1.0% and seeds nly 0.1%. Fig. 1 demnstrates these crrelatins and shws als the relatin f wax layer t the surface f these rgans. and petals have the thinnest wax layers, whereas sepals and especially % Wax/dry weight 807 leaves shw multiple values, because their extracts cntained nt nly the cmmn epicuticular wax cmpnents but als great amunts f resin (abut 50 60%). This is a characteristic bservatin f mst Cistus leaves [24] and demnstrated in Fig. 1. Leaves had an extractable epicuticular layer f abut 200 pg x cm - 2, petals f 5 pg x cm - 2, and seeds f 13 pg x cm - 2. Plants f arid regins shwed ften similar leaf wax layers including great amunts f resin. Thick depsits f pg x cm - 2 have been fund n leaves f the Mediterranean plants Ceratnia siliqua, eurpaea [5]. Pistacia lentiscus, and Olea A l l waxes f the C. albidus rgans studied cntained the fllwing cmpnents: hydrcarbns, wax esters, fatty acids, alchls and, with the exceptin f seeds, triterpenids. Leaves and sepals shwed in additin great amunts f resin, see Table I. The waxes frm all rgans f C. albidus shwed a cmpsitin f lng chained lipids with characteristic and specific patterns fr each rgan. Hydrcarbns Hydrcarbns were fund in all rgan waxes in frm f hmlgus series with prevailing dd carbn numbers. The cmpsitin f these hydrcarbns was different in their chain length maxima and the ccurrence f unsaturated cmpunds, see Fig. 2. Leaves cntained alkanes, with chain lengths ranging frm Q 9 t C 37 with ne dminating cmpund C29 f mre than 50% [15]. had nearly the same alkane cmpsitin, but their distributin pattern is nt s steep as frm leaves, and unsaturated hydrcarbns were fund in amunts f 3%. shwed a quite different distributin pattern. Wax pg cm - 2 U Leaves n Leaves JZL Fig. 1. Yield f epicuticular waxes frm varius rgans f C. albidus: A) in relatin t the dry weight (% dry wt.) and B) in relatin t the surface (pgx cm" 2 ).

3 808 S. Hennig et al. Organ Spécifié Cmpsitin f Epicuticular Waxes frm Cistus albidus Table I. Cmpsitin and yield f epicuticular waxes frm varius rgans f C. albidus. Leaves mg wax % wax mg wax % wax mg wax % wax mg wax % wax mg wax % wax Hydrcarbns Wax esters Triterpenl esters Unidentified Fatty acids Alchls Resin + triterpenids Lst n clumn Raw wax O O a> GL u> c -O I -O X nlln Leaves C-atms n l-l II M C-atms XI n n C-atms r, n n C-atms n C-atms n n il Fig. 2. Distributin patterns f hydrcarbns frm waxes f varius rgans f C. albidus. Saturated, S unsaturated. The hydrcarbn maximum shifted t C 2 s and alkenes were fund f abut 8% [15, 20]. Stamen hydrcarbns cntained alkenes f abut 80%, and the dminating alkene had the chain length C 31. Seed waxes again cntained mst saturated alkanes, but their distributin pattern was very flat and the main cmpnent C 29 was present with less than 30%. Alkenes were fund nly in traces [16]. Wax hydrcarbns f C. albidus shwed always rgan specific cmpsitins and distributin patterns with distinct maxima. Wax esters Wax esters with chain lengths ranging frm C 32 t C54 were present in all waxes. But the hmlgus series f prevailing even numbered C-atms differed in chain length maxima and the presence f unsaturated esters, see Fig. 3. Leaf waxes cntained nly saturated wax esters. The distributin pattern had a maximum f C44 with abut 34% [15]. The saturated alchls and fatty acids f these esters were checked by ethanlysis. Sepal wax had nearly the same pattern with identical maximum f C44 (30%). shwed a quite different cmpsitin f wax esters. Ethanlysis f these esters resulted in the presence f many unsaturated fatty acids [15]. The esters with chain length C 36 (abut 27%) were predminant and cntained als unsaturated alchls. In additin t the wax esters triterpenl esters were fund with abut 17%. Stamen wax esters shwed similar results

4 809 S. Hennig et al. Organ Spécifié Cmpsitin f Epicuticular Waxes frm Cistus albidus Û N J* 0 a> Q. a) a> in 0) X 1 Leaves a M C-atms r~i n nf1n C-atms PI n PI C-atms n fi n n JZL C-atms saturated. Sepal waxes cntained nearly the same fatty acid cmpsitin, but leic acid was present in 2%. In petal waxes mst unsaturated fatty acids culd be detected with ne dminating chain length f C J8. Oleic acid, linleic acid and als linlenic acid were fund in these waxes. Stamen waxes shwed similar cmpsitins f fatty acids as petals. Only the prprtins f the unsaturated acids were different. The seed waxes cntained nly saturated free fatty acids. But in this case the shrt chain fatty acid C 16 (90%) was dminating. Alchls In all waxes f the varius rgans f C. albidus primary alchls with chain lengths frm C 16 t C 30 were fund. The chain lengths C 2, C 2 2, C 24, and C 26 Leaves C-atms n n n ri n n C-atms Fig. 3. Distributin patterns f wax esters frm waxes f varius rgans f C. albidus. Saturated, S unsaturated a C-atms t thse f petals. They als had great amunts f unsaturated fatty acids. The maximum f the wax esters distributin pattern was fund with C 38 in this rgan. Triterpenl esters were als detected in amunts f abut 13%. Seed waxes cntained hmlgus series f saturated wax esters in a flat distributin pattern. Triterpenl esters culd nt be detected [16]. And again wax esters f C. albidus shwed rgan specific cmpsitins and distributin patterns with distinct and in this case flat maxima. Fatty acids 6 «n TJ Ö a >s H U :1-3 II :1-3 C-atms C-atms Free fatty acids were fund in all C. albidus waxes in hmlgus series with chain lengths frm C 14 t C 3, see Fig. 4. In leaf waxes free fatty acids with the maxima f C 22 (50%) and C 24 (38%) were fund nly JZL U C-atms Fig. 2. Distributin patterns f hydrcarbns frm waxes f varius rgans f C. albidus. Saturated, S unsaturated.

5 S. Hennig et al. Organ Specific Cmpsitin f Epicuticular Waxes frm Cistus albidus 810 were predminant. The alchls were saturated and acids series. In additin triterpenl esters were fund shwed n rgan specific cmpsitins. In additin in this wax, but n resin. Stamen wax was similar t triterpenids culd be identified in the waxes f that f petals cncerning the shrter chain lengths leaves, sepals, petals and stamens but nt in seeds. and the presence f unsaturated cmpunds. But this These triterpenids cnsisted f a very cmplex mix- wax shwed an riginally rgan specific cmpsitin. ture f triterpenls which culd nt be identified in Seed cat wax shwed a special lipid cmpsitin. detail up t date. A l l cmmn wax lipids were fund t be saturated. The distributin patterns f alkanes and wax esters shwed a flat figure and shrt chain cmpunds were Discussin dminating in cntrast t leaf wax. The epicuticular waxes f the varius rgans f C. albidus shwed characteristic rgan specific cm- These epicuticular wax cmpsitins f varius rgans f C. albidus shuld be nw crrelated t the psitins. Leaf wax cnsisted f hmlgus series surface structures f these rgans with pictures pr- f very lng chained and saturated lipids with distinct duced by the scanning electrn micrscpy. The maxima. In additin t the cmmn wax cmpnents S E M pictures f a leaf surface (Fig. 5) shw a very the leaf extract included great amunts f resinus dense cver f hairs and glandular trichmes. S it terpenes. Sepal wax had nearly the same wax cm- was very difficult t have a lk at the bttm f the psitins like thse f leaves. But the maxima f the leaf surface in rder t see the structure f the sur- patterns were nt s strngly marked, unsaturated face wax. Only with a magnificatin f mre than cmpunds culd be detected in small amunts and 3200 x we were able t have a lk int the under- the prprtin f resin was essentially lwer. Petal grund f the trichmes and culd see a waxy r wax was quite different frm that f leaves r sepals. liquid wax layer with n crystalline structures, see The distributin patterns shwed a shift t shrter Fig. 6. The very lng chained and saturated lipid chain lengths and especially unsaturated lipids culd cmpsitin f the leaf wax may suggest a crystalline be fund in the hydrcarbns, wax esters and fatty structure accrding t ther wax studies [3, 29]. Fig SEM frm the surface f a C. albidus leaf. Fig. 5: Bar = 100 jam; Fig. 6: Bar = 1 un.

6 811 S. Hennig et al. Organ Specific Cmpsitin f Epicuticular Waxes frm Cistus albidus But, because f the great amunts f resin n these leaves, the wax lipids may be redisslved in these resinus substances. S E M pictures f the surface f a sepal shw identical waxy layer and therefre are nt dcumented here. The S E M pictures f the surface f a petal (Fig. 7) demnstrate a thin cntinuus wax layer f fluid cnsistancy. The predminance f shrt chained and unsaturated lipids in this wax crrbrated these results. The surface f the petals in additin shws parallel series f papillae. They were fund in all Cistus petals in similar arrangements. The seed cat surface appears in the S E M picture (Fig. 8) as a flded cntinuus wax layer f fluid cnsistency. The wax cmpsitin f seed cats has nly saturated lipids. But in cntrast t the leaf wax, these lipids shw a shift t shrter chain lengths and in additin n dminating main cmpnent was bserved. This wax culd be extracted cmpletely with hexane as demnstrated in Fig. 9, accrding t the cmpsitin f wax lipids mst with shrt chain length cmpnents. In this picture the strngly flded epidermal cell walls f the seed cat are nw visible. The rgan specific wax cmpsitins and surface structures may prbably be crrelated t their rgan Fig. 7. SEM frm the surface f a C. albidus petal. Bar = 10 ^m. Fig SEM frm the surface f a C. albidus seed. Fig. 8: Bar = 10 im; Fig. 9: Waxes were extracted with hexane. Bar = 1 (im.

7 812 S. Hennig et al. Organ Specific Cmpsitin f Epicuticular Waxes frm Cistus albidus specific physilgical functins. Leaves and sepals f characterized by high amunts f unsaturated and C. albidus shrt chain cmpunds. The S E M pictures (Fig. 7) in are very lng living rgans, leaves abut ne year. Their surface had t adapt themselves t agreement t these results shw a fluid wax layer n the demands f lng living rgans in a semi arid cli- the surface f petals and als stamens. mate. They shuld be steadfast against envirnmen- The surface f the seed cats had t adapt itself t tal factrs such as wind, rain, sun r gaseus, liquid the prcess f shrinking and f swelling by germina- and slid particals. The S E M pictures 5 and 6 dem- tin. Accrding t the dminance f shrt chain but nstrate that the leaf surface f C. albidus is cvered with dense standing trichmes and a wax layer f saturated lipids, the surface f seed cats has a fluid wax layer, t, as shwn in Fig. 8. fluid cnsistency. The chemical analysis f this wax The differences in the chemical cmpsitin f extract shws in additin t the cmmn saturated epicuticular waxes frm the varius rgans f C. albi- and lng chained lipids great amunts f resin, dus may be primarily cnditined by enzymes, reg- 5 times f the wax. The secretin f resin is bserved ulating the chain length and the desaturatin f the ften in xermrphic plants f arid and semi arid lipids. Each rgan has enzymes specific fr the bi- species [24]. synthesis f distinct chain lengths fr different lipids, Thus, the high amunts f resin may be the reasn and desaturatin enzymes, which are activated in regins and fund in nearly all Cistus fr the fluid r waxy surface layer. In cntrast t leaves and sepals, the petals and stamens f C. albidus petals and stamens, but nt in leaves and seed cats, r they are missing in the last rgans. are very shrt living rgans f nly ne day. They must develpe themselves in a few minutes ut f their buds. Therefre, the surface wax layer must fllw these mvements and has t Acknwledgement The authrs wish t thank Mrs. E. Müller fr very be f elastic prperties. The waxes f bth rgans are excellent technical assistance. [1] C. A. Jeffree, E. A. Baker, and P. J. Hllway, in: Micrbilgy f Aerial Plant Surfaces (C. H. Dickinsn and T. F. Preese, eds.), pp , Academic Press, New Yrk [2] W. Barthltt and E. Wllenweber. Trpische und subtrpische Pflanzenwelt 32, 33 97, Akad. Wiss. Lit. Mainz, F. Steiner, Wiesbaden [3] P.-G. Gülz, Ber. dtsch. Bt. Ges. 99, (1986). [4] A. P. Tullch, in: Chemistry and Bichemistry f Natural Waxes, (P. E. Klattukudy, ed.), pp , Elsevier, Amsterdam [5] E. A. Baker, in: The Plant Cuticle (D. F. Cutler, K. L. Alvin, and G. E. Price, eds.), Academic Press, New Yrk [6] V. Wllrab, Cllect. Czech. Chem. Cmmun. 32, (1967). [7] K. Stransky, M. Streibl, and V. Herut, Cllect. Czech. Chem. Cmmun. 32, (1967). [8] V. Wllrab, Cllect. Czech. Chem. Cmmun. 33, (1968). [9] V. Wllrab, M. Streibl, and F. Srm, Cllect. Czech. Chem. Cmmun. 33, (1968). [10] K. Stransky and M. Streibl, Cllect. Czech. Chem. Cmmun. 34, (1969). [11] G. A. Herbin and P. A. Rbins, Phytchemistry 8, (1969). [12] P. J. Hllway and E. A. Baker, Ann. appl. Bil. 66, (1970). [13] E. A. Baker, J. Prcpiu, and G. M. Hunt, J. Sei. Fd. Agric. 26, (1975). [14] S. Nagy, H. E. Nrdby, and J. C. Lastinger, Phytchemistry 14, (1975). [15] P.-G. Gülz, P. Prksch, and D. Schwarz, Z. Pflanzenphysil. 92, (1979). [16] P. Krllmann, C. Eich, and P.-G. Gülz, Z. Naturfrsch. 39 c, (1984). [17] E. F. Warburg, Cistus L., in: Flra Eurpaea, Vl. 2 (T. G. Tutin et al., eds.), p. 282, Cambridge University Press, Cambridge, Mass [18] P.-G. Gülz, E. Latza, L. Köhlen, and R. Schütz, Z. Pflanzenphysil. 89, (1978). [19] P. Prksch, R. Engel, and P.-G. Gülz, Z. Pflanzenphysil. 89, (1978). [20] P.-G. Gülz, E. Amigni, B. Schmitz, and H. Egge, Z. Pflanzenphysil. 94, (1979). [21] P.-G. Gülz and G. Dielmann, Z. Pflanzenphysil. 100, (1980). [22] J. D e Pascual Teresa, J. G. Urnes, P. Basabe, and J. A. Pint Del Rey, An. Quirn. 74, (1978). [23] E. Wllenweber and K. Mann, Z. Naturfrsch. 39c, (1984). [24] T. Vgt, P. Prksch, and P.-G. Gülz, J. Plant Physil. 131, (1987). [25] P.-G. Gülz, H. Hemmers, J. Bdden, and F.-J. Marner, Z. Naturfrsch. 42c, (1987). [26] P.-G. Gülz, R. W. Scra, E. Müller, and F.-J. Marner, J. Agric. Fd Chem. 35, (1987). [27] P.-G. Gülz, J. Bdden, E. Müller, and F.-J. Marner, Z. Naturfrsch. 43c, (1988). [28] P. Gsh and S. Thakur, J. Chrmatgr. 240, (1982). [29] H. Hemmers, P.-G. Gülz, and K. Hängst, Z. Naturfrsch. 41c, (1986).