as there was no demand for endogenously formed CO2, probably because the investigated plants used external CO2 for photosynthesis [3]. Under similar climatic conditions at night, the amount of malate in the morning is the same independent of the malate content in the preceding evening (Fig. 1). This suggests the existence of a limited capacity for malate accumulation. There is evidence that the water status of tiae plant is correlated with the accumulation ofmalate. Irrigation magnifies this accumulation capacity [4, 5]. Due to extremely low rainfall during winter 1978 preceding this measuring period, the plants had a considerable water stress compared to earlier measurements in March 1977. The water content of the leaves was about 89% in 1977 and about 84% in 1978. The lowered malate content in 1978 is consistent with other reports that the acidification decreases with the decline of soil and plant water potential [2, 6]. It is interesting to note that the citrate content of the leaves is very high. In some species of the Mesembryanthemaceae concentrations of 200 m M were measured which are four times higher than the malate content of these plants. Day-night variations of the citrate content in phase with malate were repeatedly observed. During the dry and hot conditions of Sept. 14
and 15, the citrate content in the leaves was high and a diurnal variation with an amplitude of 30 to 50% of the morning value was found in Prenia and Psilocaulon (Fig. 1). The diurnal variations as well as the total citrate content decreased when nights became cooler and moist again. The role of citrate in the acid metabolism of C A M plants and its relation to climatic conditions is still unknown. Summarizing, the acid metabolism of C A M plants in their natural environment responds rapidly to changes in the microclimate and it is obviously not correlated with the acid content of the preceding light or dark period. This investigation was supported by the Deutsche Forschungsgemeinschaft. Received July 24, 1979 1. KIuge, M., Ting, I.P.: Crassulacean Acid Metabolism (Ecological Studies 30). BerlinHeidelberg-New York: Springer 1978 2. Osmond, C.B. : Ann. Rev. Plant Physiol. 29, 379 (1978) 3. von Willert, D.J.: Bet. Dtsch. Bot. Ges. (in press) 4. Hanscom, Z., Ting, I.P.: Oecologia 33, 1 (1978) 5. von Willert, D.J., et al.: Naturwissenschaften (in press) 6. Szarek, S.R., Ting, I.P.: Plant Physiol. 54, 76 (1974)
A New Sensitizing Quinone from Lady Slipper (Cypripedium calceolus ) H. Schmalle Institut ffir Mineralogie und Petrographie der Universit~it, D-2000 Hamburg B.M. Hausen UniversitS.tshautklinik, D-2000 Hamburg Cases of dermatitis due to contact with orchids of the genus Cypripedium have been described since 1875 [1]. The last case was published in 1957 [2]. Most of the wild orchids are protected by law, however, contact with bred orchids may occur in commercial breeders, workers in botanical gardens, florists and home breeders, who handle them during nursing and delivery. Decreasing prices on the international flower markets as well as world-wide transportation facilities may be another reason for the possibility of an increasing number of persons who may come into contact Naturwissenschaften 66 (1979)
with these highly ornamental plants and who may develop an allergic contact dermatitis. Screening tests for quinones which are known for their sensitizing properties in Table 1. Crystal C16H1205) a = 7.446 (4) c~ = 92.18 (6) V = 1271.1 A 3 M=284.3 d = 1.486 g. cm 3
9 by Springer-Verlag 1979
data
of
cypripedin
other plants and woods [3] led to positive colour reactions with the Craven-test in different orchid species. In the European lady slipper Cypripedium calceolus L. as well as in the North-American showy lady slipper C. reginae Walt. which both have been incriminated for causing allergic skin lesions four resp. seven quinoid constituents could be detected by thin-layer chromatography (t.l.c.). Sensitizing experiments carried out in albino guinea pigs of the Pirbright-white strain with a raw short-ether extract (60 s) of both species (mainly leaves) were successful. The results revealed that only those fractions gave a positive eliciting reaction in the sensitized animals that contained the concentrated and separated quinones. In C. calceolus a red quinone running on t.l.c, plates with the highest RF value gave the strongest eliciting reaction. This quinone was purified by further preparative t.l.c, and obtained in pure form by crystallization from acetone. The quinone was recrystallized from a solution of acetone and benzine (40/60) (v/v 1:1); a dark-red crystal of the dimensions 0.20 x 0.60 • 0.70 mm was mounted along the a-axis. Preliminary crystal data were collected by rotation and Weissenberg photographs. The intensities for all reflections with 2 ~ < 4 4 ~ were obtained at r o o m temperature on a Syntex P2I fourcircle computer-controlled diffractometer with graphite-monochromated MoKe radiation (2=0.7107A) using the 0 / 2 ~ scan technique. 3104 reflections were measured, of which 2120 had the intensities I > 34 (/) based on counting statistics. LP corrections were applied, but none for absorption, because of the small value of Ix(MoKc0 and the crystal size. The crystal data are given in Table 1. Initially, the density of the specimen was estimated to be 1.47 g . c m -3 (a value which is often found in crystalline quinoid substances), while the corresponding chemical formula was supposed to be, e.g., C30H4o010 and Z = 2 for the space group P1. The distribution of E values by parity
(2,8-dimethoxy-7-hydroxy- 1,4-phenanthraquinone,
b = 16.921 (9) /3= 107.03 (5) space group P1 m.p.=252-253 ~ (uncorr.) F(000)= 592
c = 10.6t9 (6)A y = 95.17 (5)~ z=4
527
groups showed a partial superlattice problem:
EEE
EEO
EOE
EO0
1.41
1.60
0.56
0.61
OEE
OEO
OOE
000
1.65
1.51
0.52
0.50
Thus, about 75% of atomic positions (x, y, z) should be found after translation by +b/2 in (x, 1/2+y, z), while 25% should have any other position. The structure was solved by direct methods with M U L T A N 78 [4]. Centrosymmetric trials gave no solution. After some acentric cycles of trial-and-error calculations, one model with four distinct molecules gave an R1 value of 37%. Further cycles of refinements and calculations of difference maps with the SHEL-X program [5] showed subsequently the correct positions of four times 21 atoms. The centre of symmetry was found and the atomic positions and isotropic temperature parameters of the superlattice were refined to an R1 value of 25%. Chemical aspects (bonding distances and angles) led to the structure of a p-quinone with one methoxy group, another one was situated at the third ring system, which contained a hydroxy group too. The use of the correct scattering factors [6] as well as further cycles of isotropic refinement led to an R1 value of 13.2%. The consecutive calculations of the difference maps [5] now showed all of the 24 expected H atoms. After introduction of anisotropic temperature factors and refinement of all positional parameters the calculation was stopped at an R1 value of 6.02% (Fig. 1). The obtained sensitizing quinone was named cypripedin. Further crystallographic descriptions of the structure, e.g., hydrogen bonding, packing of molecules and the superlattice problem will be reported later in Acta Crystallographica. Cypripedin belongs to the group of rare non-terpenoid phenanthraquinones chemically termed as 2,8-dimethoxy-7-hydroxy1,4-phenanthraquinone. As far as we could exhaust from the literature this quinone has not been reported from natural sources previously. Phenolic phenanthrenes, which might be considered as precursors of cypripedin and the other still unknown Cypripedium quinones and which commonly are called phy528
glossum hircinum (L.) Koch (loroglossol, hircinol) [7, 8]. They are produced as defensive substances against fungal attacks in the corms of the orchids. It might be imagined hypothetically that these phytoalexins are enzymically or by other influences oxidized to the corresponding phenanthraquinones like cypripedin (Fig. 2). Further investigations on the structures of the other quinones are in progress. Fig. 1. ORTEP drawing [9] of the two independent molecules of cypripedin with atom numbering and vibrational ellipsoids at the 50% probability level without H atoms
We thank the Deutsche Forschungsgemeinschaft for financial support, Miss F. Gach for her technical assistance and Dr. G. Adiwidjaja for the physical measurements.
H3C~o
Received July 10, 1979
H
Orchinol
O
Fig. 2. Structure of orchinol as an example for a phytoalexin hypothetically considered as a precursor of a Cypripedium quinone, e.g., cypripedin toalexins have already been found in different orchid species, as for example in Orchis militaris L. (orchinol) and Himanto-
1. Babcock, H.H.: Pharmacist 8, 1 (1875) 2. Beierlein, H. : Orchidee 8, 95 (1957) 3. Hausen, B.M.: Arbeitsmed., Sozialmed., Prfiventivmed. 13, 161 (1978) 4. Main, P., et al. : MULTAN 78 : a modified program-system (1978), after Germain, G., Main, P., Woolfson, M.M.: Acta Cryst. A 27, 368 (1971) 5. Sheldrick, G. : SHEL-X : program for crystal structure determination, Cambridge 1975 6. International tables for C-ray crystallography; Vol. III, p. 202. Birmingham: Kynoch Press 1969 7. Hardegger, E., Schellenbaum, M., Corrodi, H.: Helv. Chim. Acta 46, 1171 (1963) 8. Urech, J., et al.: ibid. 46, 2758 (1963) 9. Johnson, C.K.: ORTEP:ORNL-3794, revised. Oak Ridge National Laboratory 1971
Synergism between Chemical and Physical Stimuli in Host Colonization by an Ambrosia Beetle J.P. Vit6 Forstzoologisches Institut der Universit~t, D-7800 Freiburg i. Br.
A. Bakke Norsk Institutt for Skogsforskning, 1432-A_s-NLH, Norway We propose a hypothesis in which chemical and physical stimuli synergize host recognition and colonization by the ambrosia beetle Trypodendron ( = Xytowrus) lineatum O1. (Coleoptera: Scolytidae). In a sequence of behavioral events ("Reizkette"), ~-pinene, a monoterpene common to the coniferous host trees of the ambrosia beetle, appears responsible for host re-
cognition. Ethanol synergizes the response [1], as it develops under anaerobic metabolism in tree trunks suitable for breeding [2] ; together with the ct-pinene, the ethanol achieves host acceptance and induces attack. Invasion of the host's bark and sapwood triggers release of the aggregating pheromone 3,3,7-trimethyl-2,9-dioxatricyclo[3.3.1.04'7]nonane called "lineatin" [3]
Naturwissenschaften 66 (1979) 9 by Springer-Verlag 1979
and 15, the citrate content in the leaves was high and a diurnal variation with an amplitude of 30 to 50% of the morning value was found in Prenia and Psilocaulon (Fig. 1). The diurnal variations as well as the total citrate content decreased when nights became cooler and moist again. The role of citrate in the acid metabolism of C A M plants and its relation to climatic conditions is still unknown. Summarizing, the acid metabolism of C A M plants in their natural environment responds rapidly to changes in the microclimate and it is obviously not correlated with the acid content of the preceding light or dark period. This investigation was supported by the Deutsche Forschungsgemeinschaft. Received July 24, 1979 1. KIuge, M., Ting, I.P.: Crassulacean Acid Metabolism (Ecological Studies 30). BerlinHeidelberg-New York: Springer 1978 2. Osmond, C.B. : Ann. Rev. Plant Physiol. 29, 379 (1978) 3. von Willert, D.J.: Bet. Dtsch. Bot. Ges. (in press) 4. Hanscom, Z., Ting, I.P.: Oecologia 33, 1 (1978) 5. von Willert, D.J., et al.: Naturwissenschaften (in press) 6. Szarek, S.R., Ting, I.P.: Plant Physiol. 54, 76 (1974)
A New Sensitizing Quinone from Lady Slipper (Cypripedium calceolus ) H. Schmalle Institut ffir Mineralogie und Petrographie der Universit~it, D-2000 Hamburg B.M. Hausen UniversitS.tshautklinik, D-2000 Hamburg Cases of dermatitis due to contact with orchids of the genus Cypripedium have been described since 1875 [1]. The last case was published in 1957 [2]. Most of the wild orchids are protected by law, however, contact with bred orchids may occur in commercial breeders, workers in botanical gardens, florists and home breeders, who handle them during nursing and delivery. Decreasing prices on the international flower markets as well as world-wide transportation facilities may be another reason for the possibility of an increasing number of persons who may come into contact Naturwissenschaften 66 (1979)
with these highly ornamental plants and who may develop an allergic contact dermatitis. Screening tests for quinones which are known for their sensitizing properties in Table 1. Crystal C16H1205) a = 7.446 (4) c~ = 92.18 (6) V = 1271.1 A 3 M=284.3 d = 1.486 g. cm 3
9 by Springer-Verlag 1979
data
of
cypripedin
other plants and woods [3] led to positive colour reactions with the Craven-test in different orchid species. In the European lady slipper Cypripedium calceolus L. as well as in the North-American showy lady slipper C. reginae Walt. which both have been incriminated for causing allergic skin lesions four resp. seven quinoid constituents could be detected by thin-layer chromatography (t.l.c.). Sensitizing experiments carried out in albino guinea pigs of the Pirbright-white strain with a raw short-ether extract (60 s) of both species (mainly leaves) were successful. The results revealed that only those fractions gave a positive eliciting reaction in the sensitized animals that contained the concentrated and separated quinones. In C. calceolus a red quinone running on t.l.c, plates with the highest RF value gave the strongest eliciting reaction. This quinone was purified by further preparative t.l.c, and obtained in pure form by crystallization from acetone. The quinone was recrystallized from a solution of acetone and benzine (40/60) (v/v 1:1); a dark-red crystal of the dimensions 0.20 x 0.60 • 0.70 mm was mounted along the a-axis. Preliminary crystal data were collected by rotation and Weissenberg photographs. The intensities for all reflections with 2 ~ < 4 4 ~ were obtained at r o o m temperature on a Syntex P2I fourcircle computer-controlled diffractometer with graphite-monochromated MoKe radiation (2=0.7107A) using the 0 / 2 ~ scan technique. 3104 reflections were measured, of which 2120 had the intensities I > 34 (/) based on counting statistics. LP corrections were applied, but none for absorption, because of the small value of Ix(MoKc0 and the crystal size. The crystal data are given in Table 1. Initially, the density of the specimen was estimated to be 1.47 g . c m -3 (a value which is often found in crystalline quinoid substances), while the corresponding chemical formula was supposed to be, e.g., C30H4o010 and Z = 2 for the space group P1. The distribution of E values by parity
(2,8-dimethoxy-7-hydroxy- 1,4-phenanthraquinone,
b = 16.921 (9) /3= 107.03 (5) space group P1 m.p.=252-253 ~ (uncorr.) F(000)= 592
c = 10.6t9 (6)A y = 95.17 (5)~ z=4
527
groups showed a partial superlattice problem:
EEE
EEO
EOE
EO0
1.41
1.60
0.56
0.61
OEE
OEO
OOE
000
1.65
1.51
0.52
0.50
Thus, about 75% of atomic positions (x, y, z) should be found after translation by +b/2 in (x, 1/2+y, z), while 25% should have any other position. The structure was solved by direct methods with M U L T A N 78 [4]. Centrosymmetric trials gave no solution. After some acentric cycles of trial-and-error calculations, one model with four distinct molecules gave an R1 value of 37%. Further cycles of refinements and calculations of difference maps with the SHEL-X program [5] showed subsequently the correct positions of four times 21 atoms. The centre of symmetry was found and the atomic positions and isotropic temperature parameters of the superlattice were refined to an R1 value of 25%. Chemical aspects (bonding distances and angles) led to the structure of a p-quinone with one methoxy group, another one was situated at the third ring system, which contained a hydroxy group too. The use of the correct scattering factors [6] as well as further cycles of isotropic refinement led to an R1 value of 13.2%. The consecutive calculations of the difference maps [5] now showed all of the 24 expected H atoms. After introduction of anisotropic temperature factors and refinement of all positional parameters the calculation was stopped at an R1 value of 6.02% (Fig. 1). The obtained sensitizing quinone was named cypripedin. Further crystallographic descriptions of the structure, e.g., hydrogen bonding, packing of molecules and the superlattice problem will be reported later in Acta Crystallographica. Cypripedin belongs to the group of rare non-terpenoid phenanthraquinones chemically termed as 2,8-dimethoxy-7-hydroxy1,4-phenanthraquinone. As far as we could exhaust from the literature this quinone has not been reported from natural sources previously. Phenolic phenanthrenes, which might be considered as precursors of cypripedin and the other still unknown Cypripedium quinones and which commonly are called phy528
glossum hircinum (L.) Koch (loroglossol, hircinol) [7, 8]. They are produced as defensive substances against fungal attacks in the corms of the orchids. It might be imagined hypothetically that these phytoalexins are enzymically or by other influences oxidized to the corresponding phenanthraquinones like cypripedin (Fig. 2). Further investigations on the structures of the other quinones are in progress. Fig. 1. ORTEP drawing [9] of the two independent molecules of cypripedin with atom numbering and vibrational ellipsoids at the 50% probability level without H atoms
We thank the Deutsche Forschungsgemeinschaft for financial support, Miss F. Gach for her technical assistance and Dr. G. Adiwidjaja for the physical measurements.
H3C~o
Received July 10, 1979
H
Orchinol
O
Fig. 2. Structure of orchinol as an example for a phytoalexin hypothetically considered as a precursor of a Cypripedium quinone, e.g., cypripedin toalexins have already been found in different orchid species, as for example in Orchis militaris L. (orchinol) and Himanto-
1. Babcock, H.H.: Pharmacist 8, 1 (1875) 2. Beierlein, H. : Orchidee 8, 95 (1957) 3. Hausen, B.M.: Arbeitsmed., Sozialmed., Prfiventivmed. 13, 161 (1978) 4. Main, P., et al. : MULTAN 78 : a modified program-system (1978), after Germain, G., Main, P., Woolfson, M.M.: Acta Cryst. A 27, 368 (1971) 5. Sheldrick, G. : SHEL-X : program for crystal structure determination, Cambridge 1975 6. International tables for C-ray crystallography; Vol. III, p. 202. Birmingham: Kynoch Press 1969 7. Hardegger, E., Schellenbaum, M., Corrodi, H.: Helv. Chim. Acta 46, 1171 (1963) 8. Urech, J., et al.: ibid. 46, 2758 (1963) 9. Johnson, C.K.: ORTEP:ORNL-3794, revised. Oak Ridge National Laboratory 1971
Synergism between Chemical and Physical Stimuli in Host Colonization by an Ambrosia Beetle J.P. Vit6 Forstzoologisches Institut der Universit~t, D-7800 Freiburg i. Br.
A. Bakke Norsk Institutt for Skogsforskning, 1432-A_s-NLH, Norway We propose a hypothesis in which chemical and physical stimuli synergize host recognition and colonization by the ambrosia beetle Trypodendron ( = Xytowrus) lineatum O1. (Coleoptera: Scolytidae). In a sequence of behavioral events ("Reizkette"), ~-pinene, a monoterpene common to the coniferous host trees of the ambrosia beetle, appears responsible for host re-
cognition. Ethanol synergizes the response [1], as it develops under anaerobic metabolism in tree trunks suitable for breeding [2] ; together with the ct-pinene, the ethanol achieves host acceptance and induces attack. Invasion of the host's bark and sapwood triggers release of the aggregating pheromone 3,3,7-trimethyl-2,9-dioxatricyclo[3.3.1.04'7]nonane called "lineatin" [3]
Naturwissenschaften 66 (1979) 9 by Springer-Verlag 1979
