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The Influence of Organic and Inorganic Chemical Factors on Cell Growth and Anthraquinone Formation in Suspension Cultures of Galium vernum
Biochem. Physiol. Pllanzen 186, 117 - 124 ( 1990) VEB Gustav Fischer Verlag lena
The Influence of Organic and Inorganic Chemical Factors on Cell Growth and Anthraquinone Formation in Suspension Cultures of Galium vernum J. STROBEL, M. HIEKE, E. GEBAUER, E. WIND!), and D. GROGER Institut flir Biochemie der Pflanzen, Akademie der Wissenschaften der DDR, Halle (SaaIe), G.D.R. I) VEB Chemiekombinat Bitterfeld Key Term Index: suspension culture, optimization, growth, anthraquinones; Galium vernum
Summary Cell suspension cultures of Galium vernum are able to produce anthraquinones. The effects of medium components and supplements such as sucrose, phosphate, growth regulators and biotic or abiotic elicitors on growth and anthraquinone production were investigated. Highest cell growth and anthraquinone formation were obtained by increased sucrose and phosphate concentrations.
Introduction Due to their anthraquinone content many plants of the Rubiaceae have been used for the preparation of natural dyes or starting material for semisynthetic compounds. A large number of Rubiaceae species are also able to accumulate a variety of anthraquinones when cultured in vitro, e.g. Morinda spp. (LEISTNER 1975; ZENK et al. 1975, 1984; INOUE et al. 1981; IGBAVBOA et al. 1985; YAMAMOTO et al. 1987), Rubia spp. (SUZUKI et al. 1982, 1984, 1985), Cinchona spp. (MULDER-KRIEGER et al. 1982, 1984; WUNSMA et al. 1984, 1986a and b; KHOURI et al. 1986) and Galium spp. (BAUCH and LEISTNER 1978a and b; WILSON and MARRON 1978; INOUE et al. 1984). In contrast to most secondary metabolites anthraquinone concentrations in plant cell cultures are among the highest known at present. Many Rubiaceae species produce more anthraquinones on a dry weight basis in cell culture than the whole plant (SCHULTE et al. 1984). Recently, the production of anthraquinones by plant cell cultures has been reviewed by LEISTNER (1985) and KOBLITZ (1988). Their formation in cultured cells was reported to be regulated by nutritional and hormonal factors (ZENK et al. 1975; BAUCH and LEISTNER 1978a; SCHULTE et al. 1984; HARKES et al. 1985; WIJNSMA et al. 1985, 1986b; KHOURI et al. 1986), however, no generalization could be made in regard to the optimal production of these metabolites. In a first screening we initiated cell cultures from some Galium species in order to obtain anthraquinone producing in vitro systems which are suitable for optimization and scale up. Among these species Galium vernum ScoP. (Cruciata glabra (L.) EHRENDF.) was found to be suitable, well growing and producing anthraquinones. It was our aim to optimize this cell culture of G. vernum with regard to quantitative and qualitative (changes in the anthraquinone Abbreviations: NAA, I-naphthaleneacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; 4-Me, 4acid; 4-CI, 4-chlorophenoxyacetic acid; 2-1, 2-iodophenoxyacetic acid;, 4-1, 4iodophenoxyacetic acid; HPLC , high performance liquid chromatography; TLC, thin layer chromatography; ElMS, electron impact mass spectra; dw, dry weight
methylphenoxya<-:~tic
BPP 186 (1990) 2
117
spectrum) anthraquinone production. In cultured cells growth regulators of the auxin type, as well as sucrose concentration and elicitors have been established as stimulants of secondary metabolite productivity (ZENK et al. 1975, 1984; WOLTERS and EILERT 1983; SCHULTE et al. 1984; DICOSMO and MISAWA 1985; WIJNSMA et al. 1985, 1986b). Materials and Methods Culture conditions Hypocotyl segments of Galium vernum seedlings were induced to form callus on solid MS medium (MURASHIGE and SKOOG 1962) containing2mgl- 1 NAA, 0.2 mgl- I kinetin, 30 gl-I sucrose and 7 gl-I agar (MS standard medium). The callus culture has been maintained in our laboratory since 1985 and was transferred onto fresh medium every 4 weeks. After a few months suspension cultures were established by transferring the callus material in a liquid MS standard medium. The suspensions were cultivated in 200 ml Erlenmeyer flasks containing 30 ml fresh medium and 10 ml inoculum on gyrotary shakers (80-100 rpm) at 26± 1°C in the dark and subcultured every 7 days. Several other Murashige/Skoog media with 2,4-D instead of NAA in different amounts (1- 5 mg I-I) and with other vitamin supplements; e.g. Ca-pantothenate or vitamin solution according to KOBLITZ and HAGEN (1962), as well as some modifications ofB5 medium (GAMBORG et al. 1968) which contained 2.0 mg I-I 2,4-D and/or 1-2 mg I-I NAA were investigated during 3 subcultures (stability investigations). The MS standard medium with special supplements or increased sucrose and/or phosphate levels was used to study the optimization of anthraquinone productivity (Table 1). For these purposes the cells were harvested after 12-14 days. Phenoxyacetic acids were synthesized from chloroacetic acid and correspondingly substituted phenols according to KOELSCH (1931).
Extraction and determination of the anthraquinones The anthraquinones were extracted from freeze-dried cells in a Soxhlet apparatus with boiling 80 % aqueous ethanol until the tissue was colorless. The nutritional medium was free of pigments. The anthraquinone concentrations were estimated according to ZENK et al. (1975) with alizarin as standard compound. Values given in Table I are averages of 2-4 analyses.
Characterization of anthraquinone aglycones The ethanolic extracts were evaporated in vacuo at 30°C to an aqueous residue containing the free and glycosidically bound anthraquinones. The aqueous residue was hydrolyzed with 4 N H 2S04 for 15 min at 80°C and extracted with chloroform. The major anthraquinones were characterized by co-chromatography with authentic samples on TLC plates (silicia gel 60 PF254 , Merck), HPLC, UV-VIS and ElMS data. The TLC solvent system applied for the aglycones was: toluene-methanol (9: 1). HPLC of the free anthraquinones was performed on a HP 1090 from Hewlett Packard using a Lichrosorb RP 18 column (4.6·250 mm). The anthraquinones were eluted with methanol/O.l % H3P04 at a flow rate of 1 ml min -I and detected at 280 or 430 nm. UV-VIS spectra were recorded on the Shimadzu double beam spectrophotometer UV 210 A. The ElMS were obtained using a mass spectrograph of the research institute "M. v. ARDENNE" (DRESDEN,
G.D.R).
Results and Discussion Growth of the Cell Culture
Galium vernum yellow-orange to red-brown colored cells grow as a homogeneous suspension. Fig. 1 shows the changes with time in growth and anthraquinone production of cells cultured in the MS standard medium. The cell growth reached its maximum after approximately 9 d and the anthraquinone production after 11 d indicating a constitutively formation of the anthraquinones. 118
BPP 186 (1990) 2
0
o
I
o
0
I
:::::
Cl
E
...c 100 ... CIJ C
0
U
CIJ C
0
.!:
:J C"
~ .c 50 c
...
5
«
a
5
10
15
Days
Fig. 1. Changes in cell growth (dry weight) and total anthraquinone production of Galium vernum suspension cultures in the MS standard culture medium.
Characterization of the Anthraquinone Aglycones Acid hydrolysis of the anthraquinone glycosides and TLC and/or HPLC revealed the presence of 5 major and 8 minor compounds. The anthraquinone spectrum was identical in all tested nutritional media variants. As major constituents could be detected: - Alizarin UV-VIS (Amax, MeOH): 248, 264, 277, 330 (sh.), 434 nm ElMS (mlz): 240 (M+!100), 212, 184, 138 RF value: 0.28 - Purpurin UV-VIS (Amax. MeOH): 218, 260, 280 (sh), 515 nm ElMS (mlz): 256 (M+!lOO), 228, 208, 182 RF value: 0.21 - Lucidin UV-VIS (Amax, MeOH): 242, 246, 280, 330,415 nm ElMS (mlz): 270 (M+), 252 (100),224, 196, 168, 139 RF value: 0.33 - Rubiadin UV-VIS (Amax, MeOH): 242 (sh.), 243, 260, 279, 308, 410 nm ElMS (mlz): 254 (M+!100), 226, 225,197,169, 152,141,115,105 RF value: 0.38 EPP 186 (1990) 2
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- Alizarin-l-methylether UV-VIS ("max, MeOH): 242 (sh.), 249, 270, 284, 385 nm ElMS (mlz): 254 (M+), 237, 236, 225, 211, 209, 208 (100), 183, 180, 152 RF value: 0.45 The physical data obtained are in accordance with literature values (WIJNSMA and VERPOORTE 1986).
Effect of Nutritional and Hormonal Factors Efforts to raise the anthraquinone levels produced by plant cell cultures by varying nutritional and hormonal conditions have been performed with remarkable success (BAUCH and LEISTNER 1978a; SCHULTE et al. 1984; ZENK et al. 1984; KHOURI et al. 1986). The first approach adopted here was to optimize the suspension culture of G. vernum by changing the basal media MS and B5 to study the stability of cell growth and anthraquinone production. Three subcultures of G. vernum cells in 7 different MS and 5 different B5 media were investigated. The applied variants of MS media were also used for other plant cell cultures in our laboratory. Considering the results of BAUCH and LEISTNER (1978a) with Galium mollugo suspension cultures modified B5 media have been developed. The best results concerning growth of the cells and anthraquinone productivity were obtained using our MS standard medium (13.5 g dw 1-' and 105 mg anthraquinones 1-'). Therefore, we used this medium for further modifications. By subculturing in the other MS and B5 media cell growth and anthraquinone forming capacity were already reduced after the first subculture. It has been found in Morinda, Rubia and Galium cell culture systems that anthraquinone synthesis is remarkably influenced by different growth regulators (ZENK et al. 1975, 1984; SCHULTE et al. 1984). Furthermore, the optimal sucrose and phosphate concentrations were determined (Table 1). In these experiments we changed either the concentration (1 - 10 mg 1-1) and substitution pattern of growth regulators or the phosphate concentration (170- 370 mg 1-1) under the conditions of normal (30 g 1-1) and increased (60-80 g 1-1) sucrose levels. In contrast to reported results from other Galium species producing increased amounts of anthraquinones after application of high concentrations of different growth regulators (SCHULTE et al. 1984), the cell culture of G. vernum did not respond in the same manner. Comparative investigations should be performed with other media. An increased cell growth was obtained by higher (60- 80 g 1-') sucrose levels. The yield of biomass was enhanced (up to 23 g dw 1-') by doubling the sucrose concentration from 30-60 g 1-1. An increase of the sucrose level to 80 g 1-1 did not further improve cell growth. These results correspond with the results of WIJNSMA et al. (1986b). A maximum of anthraquinone production (200 mg 1-') in the suspension culture of G. vernum was only reached by a combined increase of sucrose and phosphate concentrations. However, these results are in contradiction to the findings of KNOBLOCH and BERLIN (1983) who demonstrated that phosphate often influences negatively the formation of secondary metabolites.
120
BPP 186 (1990) 2
Table I. The effect of II/odified MS media Ivith different amounts of growth regulators, sucrose and phosphate on cell growth and anthraquinone production in cell su~pension cultures of Galium vernum. A: Other growth regulators instead of NAA and increased sucrose concentrations: Growth regulator [1-10 mg I-I]
Sucrose [g I-I]
Cell growth [g dw I-I]
Anthraquinone yield [mgl- I]
a 4-Me 4-CI ) d 2-I 4-I
30 30 30 30 30
13.8 12.9-13.4 12.5-13.9 12.3-13.2 13.3-13.9
108 107-114 100-114 98-117 104-111
b 4-Me} 4-CI e
60 60 60
22.9 20.5-21.5 19.6-20.6
127 135-139 128-144
c
80 80 80
19.7 17.8-19.9 18.2-19.3
121 114-126 124-127
4-Me} 4-CI e
B: MS standard medium with increased phosphate and sucrose concentrations:
a b c d e
=
KH 2 P04 [mg I-I]
Sucrose [g I-I]
Cell growth 19 dw I-I]
Anthraquinone yield [mg I-I]
170 a 170 170 370 370
30 60 80 30 60
13.4 22.9 19.7 10.6 23.0
102 127 121 67 200
MS standard medium containing 2 mg I-I NAA and 30 g I-I sucrose (control)
= MS standard medium containing 2 mg I-I NAA and 60 g I-I sucrose (control)
= MS standard medium containing 2 mg I-I NAA and 80 g I-I sucrose (control) = modified MS media containing 1, 2, 4 or 5 mg I-I of the growth regulators = modified MS media containing 3 or 10 mg I-I of the growth regulators
Effect of Biotic and Abiotic Elicitors It is widely accepted that elicitors can induce or stimulate the synthesis of secondary metabolites in plant cell cultures (WOLTERS and EILERT 1983; DICOSMO and MISAWA 1985). Biotic elicitors comprise for example microbial cell-wall material, glucan polymers and glycoproteins. Abiotic elicitors include e.g. ultraviolet irradiation and heavy metals. The most extensively studied substances are microbial elicitors and heavy metals. Testing the influence of elicitors on G. vernum cells we used a yeast elicitor, prepared from Saccharomyces according to SCHUMACHER et al. (1987), as well as vanadium (VOS0 4 . 5 H20) and copper (CUS04' 5 H20) salts. It was found that the effect of the yeast elicitor (O.I-J.Og I-I medium) and of heavy metals (l0-50 mg 1-1 medium) was low (max. BPP 186 (1990) 2
121
anthraquinone yield 134 mg 1- I). In spite of high elicitor concentrations the cell culture of G. vernum did not respond with substantially higher anthraquinone accumulation. That means that the applied yeast elicitor was not effective in this system. Surprisingly, the cell culture of G. vernum was able to tolerate relatively high concentrations of heavy metal salts. Conclusions
In accordance with results from investigations with other plant cell cultures the here described experiments demonstrate that optimal culture conditions have to be found for each plant species. Several medium components and supplements such as growth regulators and elicitors known to be stimulators of secondary metabolism did not considerable increase anthraquinone formation and did not change anthraquinone spectrum. Cell suspension cultures of other Galium species were established to produce high concentrations of anthraquinones (up to 1.5 g 1-1, SCHULTE et al. 1984). But further experiments are necessary to optimize the anthraquinone production by cell culture of G. vernum. Confirming our observations FIDGEON and WILSON (1987) discussed a NAA requirement for growth and cell division of G. mollugo suspension cultures recently. Acknowledgements The authors are very greatful to Dr. J. SCHMIDT (IBP Halle) for ElMS and Dr. G. SCHNEIDER (IBP Halle) for HPLC.
References BAUCH, H.-J, and LEISTNER, E.: Aromatic metabolites in cell suspension cultures of Galium mollugo L. Planta Med. 33, 105-123 (1978a). BAUCH, H.-J., and LEISTNER, E.: Attemps to demonstrate incorporation of labelled precursors into aromatic metabolites of Galium mollugo L. Planta Med. 33, 124-127 (1978b). DICOSMO, F., and MISAw A, M. : Eliciting secondary metabolism in plant cell cultures. Trends Biotechnol. 3, 318-322 (1985). FIDGEON, c., and WILSON, G.: Growth regulation of Galium mollugo L. cell suspensions by 1naphthalene acetic acid. J. Exp. Bot. 38, 1491-1500 (1987). GAMBORG, O. L., MILLER, R. A., and OJIMA, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). HARKES, P. A. A., KRUBOLDER, L., LIBBENGA, K. R., WUNSMA, R., NSENGIYAREMGE, T., and VERPOORTE, R.: Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein. Plant Cell Tissue Organ Cult. 4, 199-214 (1985). IGBAVBOA, D., SIEWECKE, H.-J., LEISTNER, E., ROEWER, 1., HUESEMANN, W., and BARz, W.: Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida BENTH. Planta 166, 537-544 (1985). INOUE, K., NAYESHIRO, H., INOUYE, H., and ZENK, M. H.: Anthraquinones in cell suspension cultures of Morinda citrifolia. Phytochem. 20, 1693-1700 (1981). INOUE, K., SHIOBARA, Y., NAYESHIRO, H., INOUYE, H., WILSON, G., and ZENK, M. H.: Biosynthesis of anthraquinones and related compounds in Galium mollugo cell suspension cultures. Phytochem. 23,307-311 (1984). KHOURI, H. E., IBRAHIM, R. K., and RIDEAU, M.: Effects of nutritional and hQrmonalfactors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra. Plant Cell Rep. 5, 423-426 (1986).
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KNOBLOCH, K.-H., and BERLIN, 1.: Influence of phosphate on the formation of indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus. Plant Cell Tissue Organ Cult. 2, 333-340 (1983). KOBLITZ, H.: Anthraquinones. In: Cell Culture and Somatic Cell Genetics of Plants (Eds. F. CONSTABEL and 1. K. VASIL), Vol. 5, pp. 113-139, Academic Press, New York and London 1988. KOBLITZ, H., und HAGEN, J.: Vergleichende Untersuchungen tiber das Wachstum isolierter Karottengewebe auf halbsynthetischen Substraten und auf einem neuen vollsynthetischen Medium. Flora 152, 447-457 (1962). KOELSCH, C. F.: The identification of phenols. J. Amer. Chern. Soc. 53, 304-305 (1931). LEISTNER, E.: Isolierung, Identifizierung und Biosynthese von Anthrachinonen in Zellsuspensionskulturen von Morinda citri/olia. Planta Med., Suppl. 214-224 (1975). LEISTNER, E.: Biosynthesis of chorismate-derived quinones in plant cell cultures. In: Primary and Secondary Metabolism of Plant Cell Cultures (Eds. K. H. NEUMANN, W. BARZ, and E. REINHARD), pp. 215-224, Springer-Verlag, Berlin-New York 1985. MULDER-KRIEGER, T., VERPOORTE, R., DE WATER, A., VAN GESSEL, M., VAN OEVEREN, B. C. J. A., and BAERHEIM SVENDSEN, A.: Identification of the alkaloids and anthraquinones in Cinchona ledgeriana callus cultures. Planta Med. 46, 19-24 (1982). MULDER-KRIEGER, T., VERPOORTE, R., VAN DER KREEK, M., and BAERHEIM SVENDSEN, A.: Identification of alkaloids and anthraquinones in Cinchona pubescens callus cultures; the effect of plant growth regulators and light on the alkaloid content. Planta Med. 50, 17-21 (1984). MURASHlGE, T., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497 (1962). ROBINS, R.I., PAYNE, 1., and RHODES, M. 1. c.: The production of anthraquinones by cell suspension cultures of Cinchona ledgeriana. Phytochem. 25, 2327-2334 (1986). SCHULTE, U., EL-SHAGI, H., and ZENK, M. H.: Optimization of 19 Rubiaceae species in cell culture for the production of anthraquinones. Plant Cell Rep. 3, 51-54 (1984). SCHUMACHER, H.-M., GUNDLACH, H., and ZENK, M. H.: Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures. Plant Cell Rep. 6, 410-413 (1987). SUZUKI, H., MATSUMOTO, T., and OBI, Y.: Anthraquinones in cell suspension cultures of Rubia cordifolia. In: Plant Tissue Culture (A. FUJIWARA, ed.), pp. 285-286, Maruzen-Tokyo 1982. SUZUKI, H., MATSUMOTO, T., and MIKAMI, Y.: Effects of nutritional factors on the formation of anthraquinones by Rubia cordifolia plant cells in suspension culture. Agric. BioI. Chern. 48, 603-610 (1984). SUZUKI, H., MATSUMOTO, T., and MIKAMI, Y.: Effects of physical factors and surface active agents on the formation of anthraquinones by Rubia cordifolia cells in suspension culture. Agric. BioI. Chern. 49, 519-520 (1985). WIJNSMA, R., VERPOORTE, R., MULDER-KRIEGER, T., and BAERHEIM SVENDSEN, A.: Anthraquinones in callus cultures of Cinchona ledgeriana. Phytochem. 23, 2307-2311 (1984). WIJNSMA, R., Go, 1. T. K. A., VAN WEERDEN, 1. N., HARKES, P. A. A., VERPOORTE, R., and BAERHEIM SVENDSEN, A.: Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spec. Plant Cell Rep. 4, 241-244 (1985). WIJNSMA, R., Go, J. T. K. A., HARKES, P. A. A., VERPOORTE, R., and BAERHEIM SVENDSEN, A.: Anthraquinones in callus cultures of Cinchona pubescens. Phytochem. 25, 1123-1126 (1986a). WIJNSMA, R., VERPOORTE, R., HARKES, P. A. A., VAN VLIET, T. B., TEN HOOPEN, H. J. G., and BAERHEIM SVENDSEN, A.: The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkaloids and anthraquinones. Plant Cell Tissue Organ Cult. 7, 21-29 (1986b). WUNSMA, R., and VERPOORTE, R.: Anthraquinones in the Rubiaceae. In: Progress in the Chemistry of Organic Natural Products (Eds. W. HERZ, H. GRIESEBACH, G. W. KIRBY and C. TAMM), Vol. 49, pp. 79-149, Springer-Verlag, Wien-New York 1986. WILSON, G., and MARRON, P.: Growth and anthraquinone biosynthesis by Galium mollugo L. cells in batch and chemostat culture. 1. Exp. Bot. 29, 837-851 (1978). BPP 186 (1990) 2
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WOLTERS, B., und EILERT, U.: Elicitoren - Ausli:iser der Akkumulation von Pflanzenstoffen. Dtsch. Apotheker Ztg. 123, 659-667 (1983). YAMAMOTO, H., TABATA, M., and LEISTNER, E.: Cytological changes associated with induction of anthraquinone synthesis in photoautotrophic cell suspension cultures of Morinda lucida. Plant Cell Rep. 6,187-190 (1987). ZENK, M. H., EL-SHAGI, H., and SCHULTE, U.: Anthraquinone production by cell suspension cultures of Morinda citrifolia. Planta Med., Suppl. 79-101 (1975). ZENK, M. H., SCHULTE, U., and EL-SHAGI, H.: Regulation of anthraquinone formation by phenoxyacetic acids in Morinda cell cultures. Naturwiss. 71, 266 (1984). Received September 7, 1989; revised form accepted October 17, 1989 Authors' address: Dr. JORGEN STROBEL, Dipl.-Biol. MARGIT HIEKE, Ing. ELKE GEBAUER, Prof. Dr. DETLEF GROGER, Institute of Plant Biochemistry, Academy of Sciences of the G.D.R., Weinberg 3, Halle (Saale), DDR-4050.
Biochem. Physiol. Pflanzen 186, 124 (1990) VEB Gustav Fischer Verlag Jena
B uchbesprechung BIACS, P. A., GRUIZ, K., and KREMMER, T. (Eds): Biological Role of Plant Lipids. Proceedings ofthe 8th International Symposium on the Biological Role of Plant Lipids, held at Budapest, Hungary, July 25-28,1988.626 S., zahlr. Abb. und Tab., Akademiai Kiad6, Budapest 1989, Preis: Ft 785. It has become a well received tradition of scientists involved in plant lipid research to present the latest progress biannually at an international forum and to contribute in a highly focused manner to a proceedings volume of the conference. The state of art is presented here by 138 papers, most of them 4 pages, that are neatly grouped into 8 chapters. This arrangement allows to scan special fields and gauge new developments rather fast. The first 4 chapters (Lipid metabolism; Structural and functional organization of lipids; Biosynthesis and function of prenyllipids; Carrier proteins, genetics of plant lipids) are devoted to basic plant lipid research from the biochemical, biophysical, genetic and molecular biology point of view. These chapters are heavily supplement by the applied side of plant lipid research in chapters 5 to 7 (Biocides, interaction with plant lipids; Biotechnology of lipids; Nutritional aspects; Development, environment, stress). A tremendous increase in our understanding of plant lipid metabolism was observed over the last years, e.g., 'prokaryotic' and 'eukaryotic' lipid paths in higher plants, acyl exchange and characteristic fatty acid patterns in oilseed triglycerides, molecular biology of fatty acid synthase. Nevertheless, unresolved questions are addressed in many papers of this volume, such as plastidal and nonplastidal desaturation, medium-chain fatty acid biosynthesis, molecular biology of lipids, phosphatidylinositolcycle, to name only a few. Budapest, being a much sought-after meeting place for scientists from East and West, has thus provided a truly international forum which is well reflected in this book. Although produced from camera-ready manuscripts, uniform headings and an excellent layout facilitate the reading. The editors added author, subject, and taxa indices, a special service that is highly appreciated. F. SPENER, Munster
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The Influence of Organic and Inorganic Chemical Factors on Cell Growth and Anthraquinone Formation in Suspension Cultures of Galium vernum J. STROBEL, M. HIEKE, E. GEBAUER, E. WIND!), and D. GROGER Institut flir Biochemie der Pflanzen, Akademie der Wissenschaften der DDR, Halle (SaaIe), G.D.R. I) VEB Chemiekombinat Bitterfeld Key Term Index: suspension culture, optimization, growth, anthraquinones; Galium vernum
Summary Cell suspension cultures of Galium vernum are able to produce anthraquinones. The effects of medium components and supplements such as sucrose, phosphate, growth regulators and biotic or abiotic elicitors on growth and anthraquinone production were investigated. Highest cell growth and anthraquinone formation were obtained by increased sucrose and phosphate concentrations.
Introduction Due to their anthraquinone content many plants of the Rubiaceae have been used for the preparation of natural dyes or starting material for semisynthetic compounds. A large number of Rubiaceae species are also able to accumulate a variety of anthraquinones when cultured in vitro, e.g. Morinda spp. (LEISTNER 1975; ZENK et al. 1975, 1984; INOUE et al. 1981; IGBAVBOA et al. 1985; YAMAMOTO et al. 1987), Rubia spp. (SUZUKI et al. 1982, 1984, 1985), Cinchona spp. (MULDER-KRIEGER et al. 1982, 1984; WUNSMA et al. 1984, 1986a and b; KHOURI et al. 1986) and Galium spp. (BAUCH and LEISTNER 1978a and b; WILSON and MARRON 1978; INOUE et al. 1984). In contrast to most secondary metabolites anthraquinone concentrations in plant cell cultures are among the highest known at present. Many Rubiaceae species produce more anthraquinones on a dry weight basis in cell culture than the whole plant (SCHULTE et al. 1984). Recently, the production of anthraquinones by plant cell cultures has been reviewed by LEISTNER (1985) and KOBLITZ (1988). Their formation in cultured cells was reported to be regulated by nutritional and hormonal factors (ZENK et al. 1975; BAUCH and LEISTNER 1978a; SCHULTE et al. 1984; HARKES et al. 1985; WIJNSMA et al. 1985, 1986b; KHOURI et al. 1986), however, no generalization could be made in regard to the optimal production of these metabolites. In a first screening we initiated cell cultures from some Galium species in order to obtain anthraquinone producing in vitro systems which are suitable for optimization and scale up. Among these species Galium vernum ScoP. (Cruciata glabra (L.) EHRENDF.) was found to be suitable, well growing and producing anthraquinones. It was our aim to optimize this cell culture of G. vernum with regard to quantitative and qualitative (changes in the anthraquinone Abbreviations: NAA, I-naphthaleneacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; 4-Me, 4acid; 4-CI, 4-chlorophenoxyacetic acid; 2-1, 2-iodophenoxyacetic acid;, 4-1, 4iodophenoxyacetic acid; HPLC , high performance liquid chromatography; TLC, thin layer chromatography; ElMS, electron impact mass spectra; dw, dry weight
methylphenoxya<-:~tic
BPP 186 (1990) 2
117
spectrum) anthraquinone production. In cultured cells growth regulators of the auxin type, as well as sucrose concentration and elicitors have been established as stimulants of secondary metabolite productivity (ZENK et al. 1975, 1984; WOLTERS and EILERT 1983; SCHULTE et al. 1984; DICOSMO and MISAWA 1985; WIJNSMA et al. 1985, 1986b). Materials and Methods Culture conditions Hypocotyl segments of Galium vernum seedlings were induced to form callus on solid MS medium (MURASHIGE and SKOOG 1962) containing2mgl- 1 NAA, 0.2 mgl- I kinetin, 30 gl-I sucrose and 7 gl-I agar (MS standard medium). The callus culture has been maintained in our laboratory since 1985 and was transferred onto fresh medium every 4 weeks. After a few months suspension cultures were established by transferring the callus material in a liquid MS standard medium. The suspensions were cultivated in 200 ml Erlenmeyer flasks containing 30 ml fresh medium and 10 ml inoculum on gyrotary shakers (80-100 rpm) at 26± 1°C in the dark and subcultured every 7 days. Several other Murashige/Skoog media with 2,4-D instead of NAA in different amounts (1- 5 mg I-I) and with other vitamin supplements; e.g. Ca-pantothenate or vitamin solution according to KOBLITZ and HAGEN (1962), as well as some modifications ofB5 medium (GAMBORG et al. 1968) which contained 2.0 mg I-I 2,4-D and/or 1-2 mg I-I NAA were investigated during 3 subcultures (stability investigations). The MS standard medium with special supplements or increased sucrose and/or phosphate levels was used to study the optimization of anthraquinone productivity (Table 1). For these purposes the cells were harvested after 12-14 days. Phenoxyacetic acids were synthesized from chloroacetic acid and correspondingly substituted phenols according to KOELSCH (1931).
Extraction and determination of the anthraquinones The anthraquinones were extracted from freeze-dried cells in a Soxhlet apparatus with boiling 80 % aqueous ethanol until the tissue was colorless. The nutritional medium was free of pigments. The anthraquinone concentrations were estimated according to ZENK et al. (1975) with alizarin as standard compound. Values given in Table I are averages of 2-4 analyses.
Characterization of anthraquinone aglycones The ethanolic extracts were evaporated in vacuo at 30°C to an aqueous residue containing the free and glycosidically bound anthraquinones. The aqueous residue was hydrolyzed with 4 N H 2S04 for 15 min at 80°C and extracted with chloroform. The major anthraquinones were characterized by co-chromatography with authentic samples on TLC plates (silicia gel 60 PF254 , Merck), HPLC, UV-VIS and ElMS data. The TLC solvent system applied for the aglycones was: toluene-methanol (9: 1). HPLC of the free anthraquinones was performed on a HP 1090 from Hewlett Packard using a Lichrosorb RP 18 column (4.6·250 mm). The anthraquinones were eluted with methanol/O.l % H3P04 at a flow rate of 1 ml min -I and detected at 280 or 430 nm. UV-VIS spectra were recorded on the Shimadzu double beam spectrophotometer UV 210 A. The ElMS were obtained using a mass spectrograph of the research institute "M. v. ARDENNE" (DRESDEN,
G.D.R).
Results and Discussion Growth of the Cell Culture
Galium vernum yellow-orange to red-brown colored cells grow as a homogeneous suspension. Fig. 1 shows the changes with time in growth and anthraquinone production of cells cultured in the MS standard medium. The cell growth reached its maximum after approximately 9 d and the anthraquinone production after 11 d indicating a constitutively formation of the anthraquinones. 118
BPP 186 (1990) 2
0
o
I
o
0
I
:::::
Cl
E
...c 100 ... CIJ C
0
U
CIJ C
0
.!:
:J C"
~ .c 50 c
...
5
«
a
5
10
15
Days
Fig. 1. Changes in cell growth (dry weight) and total anthraquinone production of Galium vernum suspension cultures in the MS standard culture medium.
Characterization of the Anthraquinone Aglycones Acid hydrolysis of the anthraquinone glycosides and TLC and/or HPLC revealed the presence of 5 major and 8 minor compounds. The anthraquinone spectrum was identical in all tested nutritional media variants. As major constituents could be detected: - Alizarin UV-VIS (Amax, MeOH): 248, 264, 277, 330 (sh.), 434 nm ElMS (mlz): 240 (M+!100), 212, 184, 138 RF value: 0.28 - Purpurin UV-VIS (Amax. MeOH): 218, 260, 280 (sh), 515 nm ElMS (mlz): 256 (M+!lOO), 228, 208, 182 RF value: 0.21 - Lucidin UV-VIS (Amax, MeOH): 242, 246, 280, 330,415 nm ElMS (mlz): 270 (M+), 252 (100),224, 196, 168, 139 RF value: 0.33 - Rubiadin UV-VIS (Amax, MeOH): 242 (sh.), 243, 260, 279, 308, 410 nm ElMS (mlz): 254 (M+!100), 226, 225,197,169, 152,141,115,105 RF value: 0.38 EPP 186 (1990) 2
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- Alizarin-l-methylether UV-VIS ("max, MeOH): 242 (sh.), 249, 270, 284, 385 nm ElMS (mlz): 254 (M+), 237, 236, 225, 211, 209, 208 (100), 183, 180, 152 RF value: 0.45 The physical data obtained are in accordance with literature values (WIJNSMA and VERPOORTE 1986).
Effect of Nutritional and Hormonal Factors Efforts to raise the anthraquinone levels produced by plant cell cultures by varying nutritional and hormonal conditions have been performed with remarkable success (BAUCH and LEISTNER 1978a; SCHULTE et al. 1984; ZENK et al. 1984; KHOURI et al. 1986). The first approach adopted here was to optimize the suspension culture of G. vernum by changing the basal media MS and B5 to study the stability of cell growth and anthraquinone production. Three subcultures of G. vernum cells in 7 different MS and 5 different B5 media were investigated. The applied variants of MS media were also used for other plant cell cultures in our laboratory. Considering the results of BAUCH and LEISTNER (1978a) with Galium mollugo suspension cultures modified B5 media have been developed. The best results concerning growth of the cells and anthraquinone productivity were obtained using our MS standard medium (13.5 g dw 1-' and 105 mg anthraquinones 1-'). Therefore, we used this medium for further modifications. By subculturing in the other MS and B5 media cell growth and anthraquinone forming capacity were already reduced after the first subculture. It has been found in Morinda, Rubia and Galium cell culture systems that anthraquinone synthesis is remarkably influenced by different growth regulators (ZENK et al. 1975, 1984; SCHULTE et al. 1984). Furthermore, the optimal sucrose and phosphate concentrations were determined (Table 1). In these experiments we changed either the concentration (1 - 10 mg 1-1) and substitution pattern of growth regulators or the phosphate concentration (170- 370 mg 1-1) under the conditions of normal (30 g 1-1) and increased (60-80 g 1-1) sucrose levels. In contrast to reported results from other Galium species producing increased amounts of anthraquinones after application of high concentrations of different growth regulators (SCHULTE et al. 1984), the cell culture of G. vernum did not respond in the same manner. Comparative investigations should be performed with other media. An increased cell growth was obtained by higher (60- 80 g 1-') sucrose levels. The yield of biomass was enhanced (up to 23 g dw 1-') by doubling the sucrose concentration from 30-60 g 1-1. An increase of the sucrose level to 80 g 1-1 did not further improve cell growth. These results correspond with the results of WIJNSMA et al. (1986b). A maximum of anthraquinone production (200 mg 1-') in the suspension culture of G. vernum was only reached by a combined increase of sucrose and phosphate concentrations. However, these results are in contradiction to the findings of KNOBLOCH and BERLIN (1983) who demonstrated that phosphate often influences negatively the formation of secondary metabolites.
120
BPP 186 (1990) 2
Table I. The effect of II/odified MS media Ivith different amounts of growth regulators, sucrose and phosphate on cell growth and anthraquinone production in cell su~pension cultures of Galium vernum. A: Other growth regulators instead of NAA and increased sucrose concentrations: Growth regulator [1-10 mg I-I]
Sucrose [g I-I]
Cell growth [g dw I-I]
Anthraquinone yield [mgl- I]
a 4-Me 4-CI ) d 2-I 4-I
30 30 30 30 30
13.8 12.9-13.4 12.5-13.9 12.3-13.2 13.3-13.9
108 107-114 100-114 98-117 104-111
b 4-Me} 4-CI e
60 60 60
22.9 20.5-21.5 19.6-20.6
127 135-139 128-144
c
80 80 80
19.7 17.8-19.9 18.2-19.3
121 114-126 124-127
4-Me} 4-CI e
B: MS standard medium with increased phosphate and sucrose concentrations:
a b c d e
=
KH 2 P04 [mg I-I]
Sucrose [g I-I]
Cell growth 19 dw I-I]
Anthraquinone yield [mg I-I]
170 a 170 170 370 370
30 60 80 30 60
13.4 22.9 19.7 10.6 23.0
102 127 121 67 200
MS standard medium containing 2 mg I-I NAA and 30 g I-I sucrose (control)
= MS standard medium containing 2 mg I-I NAA and 60 g I-I sucrose (control)
= MS standard medium containing 2 mg I-I NAA and 80 g I-I sucrose (control) = modified MS media containing 1, 2, 4 or 5 mg I-I of the growth regulators = modified MS media containing 3 or 10 mg I-I of the growth regulators
Effect of Biotic and Abiotic Elicitors It is widely accepted that elicitors can induce or stimulate the synthesis of secondary metabolites in plant cell cultures (WOLTERS and EILERT 1983; DICOSMO and MISAWA 1985). Biotic elicitors comprise for example microbial cell-wall material, glucan polymers and glycoproteins. Abiotic elicitors include e.g. ultraviolet irradiation and heavy metals. The most extensively studied substances are microbial elicitors and heavy metals. Testing the influence of elicitors on G. vernum cells we used a yeast elicitor, prepared from Saccharomyces according to SCHUMACHER et al. (1987), as well as vanadium (VOS0 4 . 5 H20) and copper (CUS04' 5 H20) salts. It was found that the effect of the yeast elicitor (O.I-J.Og I-I medium) and of heavy metals (l0-50 mg 1-1 medium) was low (max. BPP 186 (1990) 2
121
anthraquinone yield 134 mg 1- I). In spite of high elicitor concentrations the cell culture of G. vernum did not respond with substantially higher anthraquinone accumulation. That means that the applied yeast elicitor was not effective in this system. Surprisingly, the cell culture of G. vernum was able to tolerate relatively high concentrations of heavy metal salts. Conclusions
In accordance with results from investigations with other plant cell cultures the here described experiments demonstrate that optimal culture conditions have to be found for each plant species. Several medium components and supplements such as growth regulators and elicitors known to be stimulators of secondary metabolism did not considerable increase anthraquinone formation and did not change anthraquinone spectrum. Cell suspension cultures of other Galium species were established to produce high concentrations of anthraquinones (up to 1.5 g 1-1, SCHULTE et al. 1984). But further experiments are necessary to optimize the anthraquinone production by cell culture of G. vernum. Confirming our observations FIDGEON and WILSON (1987) discussed a NAA requirement for growth and cell division of G. mollugo suspension cultures recently. Acknowledgements The authors are very greatful to Dr. J. SCHMIDT (IBP Halle) for ElMS and Dr. G. SCHNEIDER (IBP Halle) for HPLC.
References BAUCH, H.-J, and LEISTNER, E.: Aromatic metabolites in cell suspension cultures of Galium mollugo L. Planta Med. 33, 105-123 (1978a). BAUCH, H.-J., and LEISTNER, E.: Attemps to demonstrate incorporation of labelled precursors into aromatic metabolites of Galium mollugo L. Planta Med. 33, 124-127 (1978b). DICOSMO, F., and MISAw A, M. : Eliciting secondary metabolism in plant cell cultures. Trends Biotechnol. 3, 318-322 (1985). FIDGEON, c., and WILSON, G.: Growth regulation of Galium mollugo L. cell suspensions by 1naphthalene acetic acid. J. Exp. Bot. 38, 1491-1500 (1987). GAMBORG, O. L., MILLER, R. A., and OJIMA, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). HARKES, P. A. A., KRUBOLDER, L., LIBBENGA, K. R., WUNSMA, R., NSENGIYAREMGE, T., and VERPOORTE, R.: Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein. Plant Cell Tissue Organ Cult. 4, 199-214 (1985). IGBAVBOA, D., SIEWECKE, H.-J., LEISTNER, E., ROEWER, 1., HUESEMANN, W., and BARz, W.: Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida BENTH. Planta 166, 537-544 (1985). INOUE, K., NAYESHIRO, H., INOUYE, H., and ZENK, M. H.: Anthraquinones in cell suspension cultures of Morinda citrifolia. Phytochem. 20, 1693-1700 (1981). INOUE, K., SHIOBARA, Y., NAYESHIRO, H., INOUYE, H., WILSON, G., and ZENK, M. H.: Biosynthesis of anthraquinones and related compounds in Galium mollugo cell suspension cultures. Phytochem. 23,307-311 (1984). KHOURI, H. E., IBRAHIM, R. K., and RIDEAU, M.: Effects of nutritional and hQrmonalfactors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra. Plant Cell Rep. 5, 423-426 (1986).
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KNOBLOCH, K.-H., and BERLIN, 1.: Influence of phosphate on the formation of indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus. Plant Cell Tissue Organ Cult. 2, 333-340 (1983). KOBLITZ, H.: Anthraquinones. In: Cell Culture and Somatic Cell Genetics of Plants (Eds. F. CONSTABEL and 1. K. VASIL), Vol. 5, pp. 113-139, Academic Press, New York and London 1988. KOBLITZ, H., und HAGEN, J.: Vergleichende Untersuchungen tiber das Wachstum isolierter Karottengewebe auf halbsynthetischen Substraten und auf einem neuen vollsynthetischen Medium. Flora 152, 447-457 (1962). KOELSCH, C. F.: The identification of phenols. J. Amer. Chern. Soc. 53, 304-305 (1931). LEISTNER, E.: Isolierung, Identifizierung und Biosynthese von Anthrachinonen in Zellsuspensionskulturen von Morinda citri/olia. Planta Med., Suppl. 214-224 (1975). LEISTNER, E.: Biosynthesis of chorismate-derived quinones in plant cell cultures. In: Primary and Secondary Metabolism of Plant Cell Cultures (Eds. K. H. NEUMANN, W. BARZ, and E. REINHARD), pp. 215-224, Springer-Verlag, Berlin-New York 1985. MULDER-KRIEGER, T., VERPOORTE, R., DE WATER, A., VAN GESSEL, M., VAN OEVEREN, B. C. J. A., and BAERHEIM SVENDSEN, A.: Identification of the alkaloids and anthraquinones in Cinchona ledgeriana callus cultures. Planta Med. 46, 19-24 (1982). MULDER-KRIEGER, T., VERPOORTE, R., VAN DER KREEK, M., and BAERHEIM SVENDSEN, A.: Identification of alkaloids and anthraquinones in Cinchona pubescens callus cultures; the effect of plant growth regulators and light on the alkaloid content. Planta Med. 50, 17-21 (1984). MURASHlGE, T., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497 (1962). ROBINS, R.I., PAYNE, 1., and RHODES, M. 1. c.: The production of anthraquinones by cell suspension cultures of Cinchona ledgeriana. Phytochem. 25, 2327-2334 (1986). SCHULTE, U., EL-SHAGI, H., and ZENK, M. H.: Optimization of 19 Rubiaceae species in cell culture for the production of anthraquinones. Plant Cell Rep. 3, 51-54 (1984). SCHUMACHER, H.-M., GUNDLACH, H., and ZENK, M. H.: Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures. Plant Cell Rep. 6, 410-413 (1987). SUZUKI, H., MATSUMOTO, T., and OBI, Y.: Anthraquinones in cell suspension cultures of Rubia cordifolia. In: Plant Tissue Culture (A. FUJIWARA, ed.), pp. 285-286, Maruzen-Tokyo 1982. SUZUKI, H., MATSUMOTO, T., and MIKAMI, Y.: Effects of nutritional factors on the formation of anthraquinones by Rubia cordifolia plant cells in suspension culture. Agric. BioI. Chern. 48, 603-610 (1984). SUZUKI, H., MATSUMOTO, T., and MIKAMI, Y.: Effects of physical factors and surface active agents on the formation of anthraquinones by Rubia cordifolia cells in suspension culture. Agric. BioI. Chern. 49, 519-520 (1985). WIJNSMA, R., VERPOORTE, R., MULDER-KRIEGER, T., and BAERHEIM SVENDSEN, A.: Anthraquinones in callus cultures of Cinchona ledgeriana. Phytochem. 23, 2307-2311 (1984). WIJNSMA, R., Go, 1. T. K. A., VAN WEERDEN, 1. N., HARKES, P. A. A., VERPOORTE, R., and BAERHEIM SVENDSEN, A.: Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spec. Plant Cell Rep. 4, 241-244 (1985). WIJNSMA, R., Go, J. T. K. A., HARKES, P. A. A., VERPOORTE, R., and BAERHEIM SVENDSEN, A.: Anthraquinones in callus cultures of Cinchona pubescens. Phytochem. 25, 1123-1126 (1986a). WIJNSMA, R., VERPOORTE, R., HARKES, P. A. A., VAN VLIET, T. B., TEN HOOPEN, H. J. G., and BAERHEIM SVENDSEN, A.: The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkaloids and anthraquinones. Plant Cell Tissue Organ Cult. 7, 21-29 (1986b). WUNSMA, R., and VERPOORTE, R.: Anthraquinones in the Rubiaceae. In: Progress in the Chemistry of Organic Natural Products (Eds. W. HERZ, H. GRIESEBACH, G. W. KIRBY and C. TAMM), Vol. 49, pp. 79-149, Springer-Verlag, Wien-New York 1986. WILSON, G., and MARRON, P.: Growth and anthraquinone biosynthesis by Galium mollugo L. cells in batch and chemostat culture. 1. Exp. Bot. 29, 837-851 (1978). BPP 186 (1990) 2
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WOLTERS, B., und EILERT, U.: Elicitoren - Ausli:iser der Akkumulation von Pflanzenstoffen. Dtsch. Apotheker Ztg. 123, 659-667 (1983). YAMAMOTO, H., TABATA, M., and LEISTNER, E.: Cytological changes associated with induction of anthraquinone synthesis in photoautotrophic cell suspension cultures of Morinda lucida. Plant Cell Rep. 6,187-190 (1987). ZENK, M. H., EL-SHAGI, H., and SCHULTE, U.: Anthraquinone production by cell suspension cultures of Morinda citrifolia. Planta Med., Suppl. 79-101 (1975). ZENK, M. H., SCHULTE, U., and EL-SHAGI, H.: Regulation of anthraquinone formation by phenoxyacetic acids in Morinda cell cultures. Naturwiss. 71, 266 (1984). Received September 7, 1989; revised form accepted October 17, 1989 Authors' address: Dr. JORGEN STROBEL, Dipl.-Biol. MARGIT HIEKE, Ing. ELKE GEBAUER, Prof. Dr. DETLEF GROGER, Institute of Plant Biochemistry, Academy of Sciences of the G.D.R., Weinberg 3, Halle (Saale), DDR-4050.
Biochem. Physiol. Pflanzen 186, 124 (1990) VEB Gustav Fischer Verlag Jena
B uchbesprechung BIACS, P. A., GRUIZ, K., and KREMMER, T. (Eds): Biological Role of Plant Lipids. Proceedings ofthe 8th International Symposium on the Biological Role of Plant Lipids, held at Budapest, Hungary, July 25-28,1988.626 S., zahlr. Abb. und Tab., Akademiai Kiad6, Budapest 1989, Preis: Ft 785. It has become a well received tradition of scientists involved in plant lipid research to present the latest progress biannually at an international forum and to contribute in a highly focused manner to a proceedings volume of the conference. The state of art is presented here by 138 papers, most of them 4 pages, that are neatly grouped into 8 chapters. This arrangement allows to scan special fields and gauge new developments rather fast. The first 4 chapters (Lipid metabolism; Structural and functional organization of lipids; Biosynthesis and function of prenyllipids; Carrier proteins, genetics of plant lipids) are devoted to basic plant lipid research from the biochemical, biophysical, genetic and molecular biology point of view. These chapters are heavily supplement by the applied side of plant lipid research in chapters 5 to 7 (Biocides, interaction with plant lipids; Biotechnology of lipids; Nutritional aspects; Development, environment, stress). A tremendous increase in our understanding of plant lipid metabolism was observed over the last years, e.g., 'prokaryotic' and 'eukaryotic' lipid paths in higher plants, acyl exchange and characteristic fatty acid patterns in oilseed triglycerides, molecular biology of fatty acid synthase. Nevertheless, unresolved questions are addressed in many papers of this volume, such as plastidal and nonplastidal desaturation, medium-chain fatty acid biosynthesis, molecular biology of lipids, phosphatidylinositolcycle, to name only a few. Budapest, being a much sought-after meeting place for scientists from East and West, has thus provided a truly international forum which is well reflected in this book. Although produced from camera-ready manuscripts, uniform headings and an excellent layout facilitate the reading. The editors added author, subject, and taxa indices, a special service that is highly appreciated. F. SPENER, Munster
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