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Localization of Alkaloids in Sclerotia and Suspension Cultures of Claviceps purpurea (Fr.) Tul.
Biochem. Physiol. Pflanzen 174, 504-508 (1919)
Short Communication
Localization of Alkaloids in Sclerotia and Suspension Cultures of Claviceps purpurea (Fr.) Tul. D.
NEUMANN,
W.
LosECKE,
W.
MAIER
and D.
GROGER
Institute of Plant Biochemistry Academy of Sciences of the GDR, Halle (Smtle) Key Term Index: alkaloid storage, lipid droplets, ultrastructure; Claviceps purpurea.
Summary Cells of the outer layers of sclerotia
There is ample evidence that alkaloids are poisonous for the living cytoplasm. The exclusion of these particular secondary metabolites from the cytoplasm is essential for the normal function of cells. This principle is realized in plant systems in different ways: 1. Excretion from the cell and storage in dead parts of the plant [Xylem and sclerenchymatic cells of Berberis (CROliiWELL 1933)]. This type of alkaloid storage is very rarely distributed. In all cases an additional storage facility does occur in living parts of the plant. 2. Storage in living cells, whose cytoplasm is degenerating in later stages of development [Testa of different seeds, e.g. Datura stramonium (CLAUTRIAU 1894), Atropa belladonna (MoLLE 1895), Nigella damascena (MuNSCHE 1964); alkaloid cells of Sanguinaria canadensis (XEmLI.NX and Mt'LLER 1972), Chelidonium mc~jus (NEmiANN and MuLLER 1972), Corydalis cava (XEu:\lANN 1976), Macleaya c01·data (~EmrANN 1976)]. 3. Storage in vacuoles and other non-cytoplasmic compartments of living cells [Alkaloid cells in tissue cultures of Macleaya cordata (NEmiANN and MuLLER 1967), leaf parenchym cells of Nicotiana rusticct (KEmiA.NN 1975)]. Besides vacuoles and the endoplasmic reticulum also other cell compartments for the storage of alkaloids seem to exist. One of these compartments could be spherosomes or oil droplets. Histochemical methods which give clear-cut results for the localization of ergot alkaloids in cells are not yet available. Ultrastructural investigations of Claviceps purpurea have shown that sclerotia and saprophytically alkaloid producing strains contain remarkable amounts of lipid droplets but practically no aqueous non-cytoplasmic compartments. Therefore we have tried to correlate the alkaloid distribution with the ultrastructure.
Localization of Alkaloids in Sclerotia
505
The experiments were carried out with mature sclerotia of an ergotoxine rich strain of Claviceps purpurca (Fr.) Tul. and 5 days old submerged alkaloid producing cultures (strain Pepty 695). I solation of lipid droplets: The lipid droplets were isolated according to KLEINIG et al. (197 8). 2.5 g sclerotia were homogenized in 50 ml 0.1 M glycinefNaOH buffer pH 12 containing 0.4 M sucrose. After filtration through cotton cloth the larger cell fragments were removed by low speed centrifugatio11. The crude homogenate was overlayered with 5 ml buffer containing 0.3 M sucrose and 2 ml sucrose-free buffer in ultracentrifuge tubes. Centrifugation was performed at 28,000 rpm (max. 108,000 x g) for 2 h in a swing-out rotor. Under these conditions the lipid droplets floated into the sucrose-free buffer at the top of the tubes . •llkaloid determination: The lipid droplet fraction was extracted after alkalization with chloroform (3 times). The combined extracts were evaporated in vacuo to dryness and brought to
The outer layers of the mature sclerotia form a paraplectenchymatic tissue of single hyphae, which show a typical ultrastructural organisation. In the same layer the alkaloids arc localized (nioTHES and SILBER 1954). The cytoplasm of these cells gives a strong reaction with Os0 4 and no structures are visible excepting a large number of lipid droplets of different size (Fig. 1a). Similar pictures were obtained from cells of an alkaloid producing strain of Claviceps purpurec1. After a cultivation period of 5 days the cells show besides several nuclei a large number of lipid droplets (Fig. 1 b). Other organelles are not visible. After Os0 4-fixation and dehydration in acetone the lipid droplets show practically no contrast (Fig. 1 b) but in ultracryoscctions of Os04 -fixed cells the lipid droplets occur very electron dense (Fig. 1 c). Isolated lipid droplets showed a very similar picture compared with those which are located in whole cells. After Os04 -fixation and conventional embedding isolated lipid droplets had no contrast. They arc surrounded by a small border of cytoplasm, but not by a membrane (Fig. 1 d). In preparation procedures without dehydration in acetone or ethanol, e.g. glutaraldehyde/urea embedding, lipid droplets show after fixation with Os0 4 a very strong contrast (Fig.1e). Os0 4 react with the lipids, but the reaction products, which are soluble in acetone or ethanol, are lost during dehydration. Light and electron microscopical investigations revealed that the fraction, used for the alkaloid determination, consists of lipid droplets partially surrounded by small border of cytoplasm.
506
D.
NEuMANN
et
al.
Fig. 1. Eleclro11 1J,icruscopy of alkaloid producing Claviceps cells and of isolated lipid dropletes. a) Cells of the outer layer of a sclerotium of Cla1:iceps purpurea. Note the large area occupied by lipid droplets (l). Fixation: Glutaraldehydej0s0 4 • 8,000 x. b) Cells of a submerged alkaloid producing mltnre of Clariceps purpurcn. Besides several nuclei (n) and well contrasted n·toplasm a lot of lipid droplets (I) e;m be seen. Fix;:tion: Glutaraldehyde; Os0 4 • 4,000 x. c) Ultrathin cryoseetiun of a sderotial cell of Claviceps ]JUrpurea. Note the strong contn1st of the lipid droplets (I). Fixation: Glutaraldehyde/Os0 4 • 8,000 x.
Localization of Alkaloids in Sclerotia
507
The lipid droplets of the sclerotia contain after isolation in buffer of pH 12 90 flg alkaloidjml, compared with 8 flgfml in the lower phase of the gradient. In lipid droplets of submerged cultures we found 22 flgfml alkaloid and 10 flgfml in the lower phase of the gradient. Using an extraction buffer with pH 7 we found reversed results, this means the main part of alkaloids was located in the lower phase of the gradient. These experiments indicate that the ergot alkaloids in sclerotia and submerged cultures are apparently localized in the lipid droplets. We can not exclude that during homogenization and centrifugation a part of the alkaloid fraction especially ergometrine and clavines will be dissolved in the buffer phase. This assumption is supported also by the reversed distribution after isolation of lipid droplets with buffer of pH 7. Lipid droplets represent a metabolically inactive storage compartment. Alkaloids can be accumulated in this compartment without any influence on the metabolism. Storage of secondary plant products means not that the compounds remains for ever in the storage compartment. Lipid droplets of the sclerotia! cells are phagocytized by the developing vacuoles during germination and the lipids are metabolized inside the vacuoles (STREIBLOVA et al. 1978). The fate of the alkaloids is unknown, but one can speculate, that alkaloids are also metabolized by hydrolytic enzymes inside the vacuoles. This assumption is also supported by the results of GROGER (1958). In submerged cultures a similar pattern is observed. After transfer to a new medium the cells grow and develope vacuoles, which take up lipid droplets and metabolize their lipids (unpublished). Most of the microorganisms cultivated under submerged conditions are able to excrete overproduced metabolites into the surrounding medium. Some strains of Claviceps purpurea can store ergot alkaloids within the mycelium in submerged cultures. But there are also other strains known which excrete about 90% of the produced alkaloid, despite of having lipid droplets for the intracellular storage of the ergolines. Apparently an additional mechanism for the transport across the cytoplasm has been developed. Ultrastructural evidence for a compartmentated transport is still lacking. Another possibility could be the translocation in the endoplasmic reticulum similar to the alkaloid transport in some higher plants (NEUMANN 1975). References CLAUTRIAU, G.: Localisation et signification des alcaloides dans quelques graines. Ann. Soc. belg. Microscopie 18, 37 (1894). CROMWELL, B. T.: Experiments on the origin and function of berberine in Berberis darwinii. Biochem. z. 27, 860 (1933). GRi:iGER, D.: Weitere Untersuchungen iiber den Alkaloid-Stoffwechsel des Mutterkorns. Die Kulturpflanze 6, 243-257 (1958).
d) Isolated lipid droplets of sclerotia of Claviceps purpurea after embedding in Durcupan ACM. The lipid droplets (1) show no surrounding membrane, they are covered by a thin layer of cytoplasm. Fixation: Os0 4 • 40,000 x. e) Isolated lipid droplets after embedding in glutaraldehyde/urea. The lipid droplets show a strong contrast. Fixation: Os0 4 • 24,000 x . 33 Biochem. Physiol. Pflanzen, Bd. 17 4
508
D. NEUM\NN eta!., Localization of Alkaloids in Sclerotia
KLEINIG, H., STEINI{U, C., KoPP, C., and ZAAR, K.: Oleosomes (spherosomes) from Daucus carota suspension culture cells. Planta 140, 233-237 (1978). LuFT, J. H.: Permanganate- a new fixative for electron microscopy. J. biophys. biochem. Cytol. 2, 799 (1956). MoLLE, PH.: La localisation des alcaloides dans solanacees. Ann. Soc. belg. Microscopie 21, 8-20 (1895). MOTHES, K., and SILBER, A.: Die Konstanz des Alkaloidgehaltes bei verschiedenen Rassen von Mutterkorn. Forschungen und Fortschritt 28, 46-61 (1954). MuNSCHE, D.: Zytologische Untersuchungen zur Biosynthese des Damascenins in Nigella damascena. Flora 154, 317-324 (1964). NEUMANN, D.: Beitriige zur Physiologic der Alkaloide. VI. Ultrastruktur der Parenchymzellen und Speicherung von Alkaloid in Blattstecklingen von N1:cotiana rustica L. Biochem. Physiol. Pflanzen 168, 511-518 (1975). - Interrelationship between morphology of alkaloid storage cells and the possible mechanism of alkaloid storage. Nova Acta Leopoldina, Suppl. 7, 77-81 (1976). - and MuLLER, E.: Intrazelluliirer Nachweis von Alkaloiden in Pflanzenzellen im Iicht- und elektronenmikroskopischen MaBstab. Flora lii8, 479-492 (1967). - - Beitriige zur Physiologie der Alkaloide. III. Chelidonium majus und Sanguinaria canadensis. Ultrastruktur der Alkaloidbehiilter, Alkaloidaufnahme und -verteilung. Biochem. Physiol. Pflanzen 163, 375-391 (1972). PEASE, D. C., and PETERSON, R. G.: Polymerisable glutaraldehyde- urea mixtures as polar, watercontaining embedding media. J. Ultrastr. Res. 41, 133-139 (1972). STREIBLOVA, E., Rue, llf., and KYBAL, J.: Fine structure of imbibed sclerotia! cells of Calviceps purpurea (Fr.) Tul. revealed by freeze-etching. Zeitschr. Allgem. Mikrobiol. 18, 123-134 (1978). Received February 20, 1979.
Author's address: Herrn Dr. DIETER NEUMANN, Institut fiir Biochemic der Pflanzen der Ad W der DDR, DDR- 402 Halle (Saale), Weinbergweg.
Short Communication
Localization of Alkaloids in Sclerotia and Suspension Cultures of Claviceps purpurea (Fr.) Tul. D.
NEUMANN,
W.
LosECKE,
W.
MAIER
and D.
GROGER
Institute of Plant Biochemistry Academy of Sciences of the GDR, Halle (Smtle) Key Term Index: alkaloid storage, lipid droplets, ultrastructure; Claviceps purpurea.
Summary Cells of the outer layers of sclerotia
There is ample evidence that alkaloids are poisonous for the living cytoplasm. The exclusion of these particular secondary metabolites from the cytoplasm is essential for the normal function of cells. This principle is realized in plant systems in different ways: 1. Excretion from the cell and storage in dead parts of the plant [Xylem and sclerenchymatic cells of Berberis (CROliiWELL 1933)]. This type of alkaloid storage is very rarely distributed. In all cases an additional storage facility does occur in living parts of the plant. 2. Storage in living cells, whose cytoplasm is degenerating in later stages of development [Testa of different seeds, e.g. Datura stramonium (CLAUTRIAU 1894), Atropa belladonna (MoLLE 1895), Nigella damascena (MuNSCHE 1964); alkaloid cells of Sanguinaria canadensis (XEmLI.NX and Mt'LLER 1972), Chelidonium mc~jus (NEmiANN and MuLLER 1972), Corydalis cava (XEu:\lANN 1976), Macleaya c01·data (~EmrANN 1976)]. 3. Storage in vacuoles and other non-cytoplasmic compartments of living cells [Alkaloid cells in tissue cultures of Macleaya cordata (NEmiANN and MuLLER 1967), leaf parenchym cells of Nicotiana rusticct (KEmiA.NN 1975)]. Besides vacuoles and the endoplasmic reticulum also other cell compartments for the storage of alkaloids seem to exist. One of these compartments could be spherosomes or oil droplets. Histochemical methods which give clear-cut results for the localization of ergot alkaloids in cells are not yet available. Ultrastructural investigations of Claviceps purpurea have shown that sclerotia and saprophytically alkaloid producing strains contain remarkable amounts of lipid droplets but practically no aqueous non-cytoplasmic compartments. Therefore we have tried to correlate the alkaloid distribution with the ultrastructure.
Localization of Alkaloids in Sclerotia
505
The experiments were carried out with mature sclerotia of an ergotoxine rich strain of Claviceps purpurca (Fr.) Tul. and 5 days old submerged alkaloid producing cultures (strain Pepty 695). I solation of lipid droplets: The lipid droplets were isolated according to KLEINIG et al. (197 8). 2.5 g sclerotia were homogenized in 50 ml 0.1 M glycinefNaOH buffer pH 12 containing 0.4 M sucrose. After filtration through cotton cloth the larger cell fragments were removed by low speed centrifugatio11. The crude homogenate was overlayered with 5 ml buffer containing 0.3 M sucrose and 2 ml sucrose-free buffer in ultracentrifuge tubes. Centrifugation was performed at 28,000 rpm (max. 108,000 x g) for 2 h in a swing-out rotor. Under these conditions the lipid droplets floated into the sucrose-free buffer at the top of the tubes . •llkaloid determination: The lipid droplet fraction was extracted after alkalization with chloroform (3 times). The combined extracts were evaporated in vacuo to dryness and brought to
The outer layers of the mature sclerotia form a paraplectenchymatic tissue of single hyphae, which show a typical ultrastructural organisation. In the same layer the alkaloids arc localized (nioTHES and SILBER 1954). The cytoplasm of these cells gives a strong reaction with Os0 4 and no structures are visible excepting a large number of lipid droplets of different size (Fig. 1a). Similar pictures were obtained from cells of an alkaloid producing strain of Claviceps purpurec1. After a cultivation period of 5 days the cells show besides several nuclei a large number of lipid droplets (Fig. 1 b). Other organelles are not visible. After Os0 4-fixation and dehydration in acetone the lipid droplets show practically no contrast (Fig. 1 b) but in ultracryoscctions of Os04 -fixed cells the lipid droplets occur very electron dense (Fig. 1 c). Isolated lipid droplets showed a very similar picture compared with those which are located in whole cells. After Os04 -fixation and conventional embedding isolated lipid droplets had no contrast. They arc surrounded by a small border of cytoplasm, but not by a membrane (Fig. 1 d). In preparation procedures without dehydration in acetone or ethanol, e.g. glutaraldehyde/urea embedding, lipid droplets show after fixation with Os0 4 a very strong contrast (Fig.1e). Os0 4 react with the lipids, but the reaction products, which are soluble in acetone or ethanol, are lost during dehydration. Light and electron microscopical investigations revealed that the fraction, used for the alkaloid determination, consists of lipid droplets partially surrounded by small border of cytoplasm.
506
D.
NEuMANN
et
al.
Fig. 1. Eleclro11 1J,icruscopy of alkaloid producing Claviceps cells and of isolated lipid dropletes. a) Cells of the outer layer of a sclerotium of Cla1:iceps purpurea. Note the large area occupied by lipid droplets (l). Fixation: Glutaraldehydej0s0 4 • 8,000 x. b) Cells of a submerged alkaloid producing mltnre of Clariceps purpurcn. Besides several nuclei (n) and well contrasted n·toplasm a lot of lipid droplets (I) e;m be seen. Fix;:tion: Glutaraldehyde; Os0 4 • 4,000 x. c) Ultrathin cryoseetiun of a sderotial cell of Claviceps ]JUrpurea. Note the strong contn1st of the lipid droplets (I). Fixation: Glutaraldehyde/Os0 4 • 8,000 x.
Localization of Alkaloids in Sclerotia
507
The lipid droplets of the sclerotia contain after isolation in buffer of pH 12 90 flg alkaloidjml, compared with 8 flgfml in the lower phase of the gradient. In lipid droplets of submerged cultures we found 22 flgfml alkaloid and 10 flgfml in the lower phase of the gradient. Using an extraction buffer with pH 7 we found reversed results, this means the main part of alkaloids was located in the lower phase of the gradient. These experiments indicate that the ergot alkaloids in sclerotia and submerged cultures are apparently localized in the lipid droplets. We can not exclude that during homogenization and centrifugation a part of the alkaloid fraction especially ergometrine and clavines will be dissolved in the buffer phase. This assumption is supported also by the reversed distribution after isolation of lipid droplets with buffer of pH 7. Lipid droplets represent a metabolically inactive storage compartment. Alkaloids can be accumulated in this compartment without any influence on the metabolism. Storage of secondary plant products means not that the compounds remains for ever in the storage compartment. Lipid droplets of the sclerotia! cells are phagocytized by the developing vacuoles during germination and the lipids are metabolized inside the vacuoles (STREIBLOVA et al. 1978). The fate of the alkaloids is unknown, but one can speculate, that alkaloids are also metabolized by hydrolytic enzymes inside the vacuoles. This assumption is also supported by the results of GROGER (1958). In submerged cultures a similar pattern is observed. After transfer to a new medium the cells grow and develope vacuoles, which take up lipid droplets and metabolize their lipids (unpublished). Most of the microorganisms cultivated under submerged conditions are able to excrete overproduced metabolites into the surrounding medium. Some strains of Claviceps purpurea can store ergot alkaloids within the mycelium in submerged cultures. But there are also other strains known which excrete about 90% of the produced alkaloid, despite of having lipid droplets for the intracellular storage of the ergolines. Apparently an additional mechanism for the transport across the cytoplasm has been developed. Ultrastructural evidence for a compartmentated transport is still lacking. Another possibility could be the translocation in the endoplasmic reticulum similar to the alkaloid transport in some higher plants (NEUMANN 1975). References CLAUTRIAU, G.: Localisation et signification des alcaloides dans quelques graines. Ann. Soc. belg. Microscopie 18, 37 (1894). CROMWELL, B. T.: Experiments on the origin and function of berberine in Berberis darwinii. Biochem. z. 27, 860 (1933). GRi:iGER, D.: Weitere Untersuchungen iiber den Alkaloid-Stoffwechsel des Mutterkorns. Die Kulturpflanze 6, 243-257 (1958).
d) Isolated lipid droplets of sclerotia of Claviceps purpurea after embedding in Durcupan ACM. The lipid droplets (1) show no surrounding membrane, they are covered by a thin layer of cytoplasm. Fixation: Os0 4 • 40,000 x. e) Isolated lipid droplets after embedding in glutaraldehyde/urea. The lipid droplets show a strong contrast. Fixation: Os0 4 • 24,000 x . 33 Biochem. Physiol. Pflanzen, Bd. 17 4
508
D. NEUM\NN eta!., Localization of Alkaloids in Sclerotia
KLEINIG, H., STEINI{U, C., KoPP, C., and ZAAR, K.: Oleosomes (spherosomes) from Daucus carota suspension culture cells. Planta 140, 233-237 (1978). LuFT, J. H.: Permanganate- a new fixative for electron microscopy. J. biophys. biochem. Cytol. 2, 799 (1956). MoLLE, PH.: La localisation des alcaloides dans solanacees. Ann. Soc. belg. Microscopie 21, 8-20 (1895). MOTHES, K., and SILBER, A.: Die Konstanz des Alkaloidgehaltes bei verschiedenen Rassen von Mutterkorn. Forschungen und Fortschritt 28, 46-61 (1954). MuNSCHE, D.: Zytologische Untersuchungen zur Biosynthese des Damascenins in Nigella damascena. Flora 154, 317-324 (1964). NEUMANN, D.: Beitriige zur Physiologic der Alkaloide. VI. Ultrastruktur der Parenchymzellen und Speicherung von Alkaloid in Blattstecklingen von N1:cotiana rustica L. Biochem. Physiol. Pflanzen 168, 511-518 (1975). - Interrelationship between morphology of alkaloid storage cells and the possible mechanism of alkaloid storage. Nova Acta Leopoldina, Suppl. 7, 77-81 (1976). - and MuLLER, E.: Intrazelluliirer Nachweis von Alkaloiden in Pflanzenzellen im Iicht- und elektronenmikroskopischen MaBstab. Flora lii8, 479-492 (1967). - - Beitriige zur Physiologie der Alkaloide. III. Chelidonium majus und Sanguinaria canadensis. Ultrastruktur der Alkaloidbehiilter, Alkaloidaufnahme und -verteilung. Biochem. Physiol. Pflanzen 163, 375-391 (1972). PEASE, D. C., and PETERSON, R. G.: Polymerisable glutaraldehyde- urea mixtures as polar, watercontaining embedding media. J. Ultrastr. Res. 41, 133-139 (1972). STREIBLOVA, E., Rue, llf., and KYBAL, J.: Fine structure of imbibed sclerotia! cells of Calviceps purpurea (Fr.) Tul. revealed by freeze-etching. Zeitschr. Allgem. Mikrobiol. 18, 123-134 (1978). Received February 20, 1979.
Author's address: Herrn Dr. DIETER NEUMANN, Institut fiir Biochemic der Pflanzen der Ad W der DDR, DDR- 402 Halle (Saale), Weinbergweg.