Phytochemicals in Plant Cell Cultures

Morgan

PART I

Phenylpropanoids, Naphthoquinones, and Anthraquinones

CHAPTER 1

Coumarins and Furanocoumarins

Ulrich Matern, Heiner Strasser, Hilke Wendorff and Daria Hamerski,     Department of Plant Biochemistry, Biological Institute II, University of Freiburg, Freiburg, Federal Republic of Germany

Publisher Summary

This chapter discusses the induced accumulation of coumarins, the regulation of their biosynthesis, and their potential physiological function. Cultured plant cells do not accumulate secondary metabolites as readily as their parent plants. However, as coumarins are produced in large quantities in various plants belonging to families Ructacae, Umbelliferae, and Solanaceae, cell cultures derived from these plants either lack coumarins, or their coumarin content is low. Moreover, the amount of coumarin produced in cell cultures may vary from one transfer of cells to another. These differences cannot be attributed to genetic incompetence of the cultured cells, but result rather from abnormal gene expression. Coumarins isolated from cell cultures grown on standard growth media are referred as constitutive coumarins. The level of secondary metabolities in cultured cells may further be increased by several means.

I. Introduction

II. Coumarins in Crown Gall Tumors

III. Constitutive Coumarins

IV. Induced Coumarins

V. Biosynthesis

VI. Regulation of Biosynthesis

VII. Physiological Significance

References

I INTRODUCTION

Cultured plant cells do not, as a rule, accumulate secondary metabolites as readily as their parent plants (Barz and Ellis, 1981; Dougall, 1981; Berlin, 1983; Ellis, 1984; Heinstein, 1985a,b). For example, whereas coumarins are produced in large quantities in various plants belonging to the families Rutaceae, Umbelliferae, and Solanaceae, cell cultures derived from these plants either lack coumarins, or their coumarin content is comparatively low (Murray et al., 1982). Moreover, the amount of coumarin produced in cell cultures may vary from one transfer of cells to another. These differences cannot be attributed to genetic incompetence of the cultured cells, but result rather from abnormal gene expression (Muhitch and Fletcher, 1985). Growth media are adjusted primarily to sustain rapid growth of cells, and it has been suggested that selective genome expression corresponds closely with cell maturation (Yeoman et al., 1980). For simplicity, coumarins isolated from cell cultures grown on standard growth media are referred to here as constitutive coumarins. This term need not imply, however, that their synthesis is independent of the growth-medium composition.

The level of secondary metabolites in cultured cells in general may be increased by several means. Individual high-producing cells can be selected for propagation from a heterogeneous cell population. Although this approach has been successful in some instances (Radwan and Kokate, 1980; Yamamoto et al., 1982; Ohta and Yatazawa, 1982), Ellis (1984) showed that, under otherwise constant growth conditions, cell clones selected for high secondary metabolite contents eventually segregate into a heterogeneous population of low average yield. Basing their approach on the observation that polyamines extend the life span of mature nondividing cells, Muhitch and Fletcher (1985) investigated the effect of culture age and polyamine addition on the production of phenolics in Paul’s scarlet rose suspension cultures. Addition of polyamines indeed induced changes in the type of phenolics in the culture as well as in their quantity. Neither one of these approaches, however, has been followed for production of coumarins. Production media have also been developed, based mainly on increased sucrose and reduced inorganic phosphate levels, as well as on a modified hormone regime (Phillips and Henshaw, 1977; Knobloch and Berlin, 1980; Sugano et al., 1975). The effect of growth-medium composition on the production of coumarins has been extensively studied in tobacco suspension cultures (Okazaki et al., 1982a,b).

Fungal-cell-wall-derived glucan fractions (elicitors) have been used to induce the accumulation of secondary metabolites with potential antimycotic activity (phytoalexins) in various cultured cells (Ebel et al., 1976; Tietjen and Matern, 1984; Ellis, 1984; Heinstein, 1985a; see also Chapter 9, Volume 4, this treatise). Coumarins also accumulate in various diseased plants (for review, see Murray et al., 1982), and this protocol has been successfully employed to induce coumarin synthesis in parsley (Tietjen et al., 1983).

Most of this chapter is dedicated to a discussion of the induced accumulation of coumarins, the regulation of their biosynthesis, and their potential physiological function. Phenylcoumarins and isocoumarins, which originate from isoflavonoid (Brown, 1981) and polyketide intermediates (Stoessl and Stothers, 1978), respectively, are not considered, nor are angular furanocoumarins, which have never been isolated from cultured cells.

II COUMARINS IN CROWN GALL TUMORS

Brown and Tenniswood (1974) reported that normal tobacco callus tissue contains bergapten (for chemical structures see Table I, 27) and bound umbelliferone (2), esculetin (8), and scopoletin (10), whereas the corresponding crown gall tumor tissue cultures induced with Agrobacterium tumefaciens lack umbelliferone and bergapten. Instead, higher levels of esculetin and scopoletin were found, suggesting that, in tumors, the biosynthetic flow is diverted from the umbelliferone–furanocoumarin pathway in favor of esculetin and scopoletin (Fig. 1). Similarly, Reichling et al. (1979) reported that crown gall tumor tissues induced in Matricaria chamomilla produced neither the flavonoids nor the coumarins herniarin (4) and umbelliferone (2), typical for this plant.

Table I

Chemical Structures of Coumarins

Fig. 1 Patterns in coumarin biosynthesis. Coumarin numbers refer to Table I.

III CONSTITUTIVE COUMARINS

Most of the information on coumarins produced in cultured cells has been summarized by Murray et al. (1982). Cultures of the rutaceous species Ruta graveolens, R. graveolens ssp. hortensis, and Thamnosma montana are particularly rich sources of coumarins (Table II). Rutacultin (12), which was originally thought to be produced only by cultured cells, was later also isolated from Ruta roots (Novak et al., 1973). Umbelliprenin (6), on the other hand, which is one of the major coumarins in young Thamnosma seedlings, was not accumulated by the corresponding cell cultures (Kutney et al., 1973). One may speculate that Citrus cultures at least should also produce coumarins under appropriate growth conditions, because the scopoletin (10) content of Citrus leaves, which had been originally proposed as a diagnostic marker for young tree decline, was shown to be closely related to leaf age (Wheaton and Feldman, 1979).

Table II

Constitutive Coumarins from Cultured Plant Cells

Suspension cultures of Hydrangea macrophylla (Saxifragales) (Table II) accumulated appreciable amounts of umbelliferone (∼1 mg per 5 g fresh weight of cells) and of the corresponding β-glucoside skimmin (∼1 mg per 15 g fresh weight of cells) as well as two isocoumarinic derivatives and daphnetin 8-monomethyl ether (14). Callus cultures of Swertia japonica (Gentianaceae) (Table II) were shown to accumulate scopoletin and scopolin (11) at approximately 14 and 140 μg per gram dry weight of cells, respectively.

IV INDUCED COUMARINS

The induced accumulation of scopoletin in cultured cells and its excretion from the cells has been investigated most thoroughly in several members of the Solanaceae. Early experiments employing Atropa belladonna root tissue culture (Mothes and Kala, 1955) revealed that, after feeding with l-phenylalanine, scopoletin was mostly excreted by the cells, whereas newly formed umbelliferone accumulated within the tissue. Later, A. belladonna callus culture was shown to contain scopoletin (Vakkari, 1980), and treatment of the culture with 0.05 mM l-methionine increased its amount from 0.7 to 1.1 mg per gram fresh weight of tissue.

Tobacco plants accumulate scopoletin after infection with various pathogens (for review, see Murray et al., 1982). Fritig and Hirth (1971) reported that healthy tobacco tissue cultures already contain large quantities of scopoletin and that virus infection does not significantly change this amount. Okazaki et al. (1982a) later showed that accumulation of scopolin and scopoletin in tobacco tissue cultures strongly depends on the sucrose and phosphate levels as well as on the amino acid composition of the growth medium. Furthermore, scopoletin was mostly recovered from the culture filtrate while scopolin accumulated within the cells. Addition of several plant hormones increased the amount of both scopoletin and scopolin (Okazaki et al., 1982b). Surprisingly, addition of 2,4-dichlorophenoxyacetic acid (2,4-D) enhanced the uptake of exogenously supplied scopoletin by the cells and its glucosylation to scopolin (11), whereas kinetin, indolyl-3-acetic acid (IAA), and naphthaleneacetic acid (NAA) adversely affected the quantity of scopoletin in the culture filtrate. In other cultured cells, an inhibitory effect of 2,4-D on the biosynthesis of phenolic acids had been observed (Sugano et al., 1975).

Scopoletin accumulation on infection has been demonstrated in Helianthus annuus (Cohen and Ibrahim, 1975; Tal and Robeson, 1986) as well as in elicitor-treated Gossypium arboreum (Zeringue, 1984). This coumarin was, however, not reported from cotton suspension cultures induced with fungal conidia (Heinstein, 1985b).

Dark-grown parsley suspension cultures, lacking coumarins, accumulate fairly large quantities of isopimpinellin (33), bergapten (27), xanthotoxin (31), psoralen (25), and graveolone (24) in response to elicitor treatment (Tietjen et al., 1983). Whereas an elicitor isolated from Phytophthora megasperma f.sp. glycinea predominantly induced the accumulation of psoralen and graveolone, bergapten and xanthotoxin were most abundant in cultures treated with an elicitor from Alternaria carthami. In both cases, most of the induced coumarins were recovered from the culture filtrate (Tietjen et al., 1983). Similarly, dark-grown cell cultures of Ammi majus L. rapidly accumulate the coumarins ammirin (23), isopimpinellin (33), bergapten (27), marmesin (16), umbelliferone (2), and a compound tentatively identified as an isomer of marmesin in response to elicitor treatment (Hamerski et al., 1987). The stereochemistry of ammirin, however, has not been confirmed. As in parsley, the bulk of these coumarins were recovered from the culture filtrate. For biosynthetic reasons (see Section V), induced Ammi majus cultures may represent an ideal system to study the flow of label from l-[¹⁴C]phenylalanine into the coumarins, and possibly also to provide a clue to the conversion of 4-coumaric acid to umbelliferone (Fig. 1). Elicitor-induced coumarin accumulation has also been observed in dark-grown cultures of Conium maculatum, Anethum graveolens (D. Hamerski and U. Matern, unpublished) and Arracacia esculenta (K. Harter and U. Matern, unpublished). Unusually, induced Arracacia cultures accumulated demethylsuberosin (5) in their culture fluid besides umbelliferone, an umbelliferone ether, marmesin, bergapten and isopimpinellin. Within the Umbelliferae, carrot cultures appear to be exceptional. Despite contradictory reports on the presence of coumarins in healthy garden carrot (Ivie et al., 1982; Ceska et al., 1986), esculetin (8) (Khandobina et al., 1982) and scopoletin (10) as well as various isocoumarins were isolated from diseased garden or mauve-coloured carrot (Coxon et al., 1973; N. Saleh, National Research Center, Cairo, Egypt, personal communication). Nevertheless, accumulation of scopoletin has not been reported from induced carrot cultures (Kurosaki and Nishi, 1983). It appears possible that the 2,4-D concentration that sustained growth of cells in these experiments may have hindered the formation of scopoletin.

The rapid induction of coumarin accumulation in cultured cells leads to the question of the factors involved in the induction process. Because a direct interaction of elicitor with either enzyme proteins or nucleic acids appears unlikely, one must propose an intracellular signaling system. Although no conclusive information is available, a quick drop in cytoplasmic inorganic phosphate level concomitant with an inhibition of phosphate uptake occurs in parsley cells on addition of elicitor (Strasser et al., 1983). Furthermore, expression of the full effect requires the presence of the elicitor for at least 20 min (Strasser and Matern, 1986). An involvement of polyphosphoinositides in signal transduction has been assumed. However, a careful analysis revealed no significant elicitorinduced changes in the relative labeling of phosphoinositides by either [2-³H]inositol, [2-³H]glycerol or [³²P]orthophosphate within 20 min following addition of the elicitor (Strasser et al., 1986).

V BIOSYNTHESIS

Coumarin (1) and umbelliferone originate from l-phenylalanine, most likely via formation of 2- and 2,4-dihydroxycinnamic acid, respectively (Fig. 1) (Murray et al., 1982). Umbelliferone may be further converted by additional oxidation to, for example, esculetin (8) (Brown, 1985). Alternatively, umbelliferone may be prenylated, with subsequent formation either of a fused pyrone ring to form, for example, graveolone (24), or of a fused furan ring and loss of a C3 fragment to yield the various furanocoumarins (Fig. 1) (Murray et al., 1982).

The formation of scopoletin is an exception to the scheme just outlined, because ferulic acid has been described as its immediate precursor in tobacco (Fig. 1) (Murray et al., 1982). Nevertheless, enzymes isolated from tobacco tissue cultures methylate esculetin to scopoletin and isoscopoletin (Tsang and Ibrahim, 1979; Blume, 1982), although not with exclusive substrate specificity. A probably nonphysiological hydroxylation of coumarin to umbelliferone was accomplished with Conium maculatum and Catharanthus roseus but not with Apocynum cannabinum cell cultures (Carew and Bainbridge, 1976). On the other hand, labeled coumarin administered to Russet Burbank potato leaves was transported basipetally and recovered unchanged from roots and tubers (Gawronska et al., 1982), even though potato naturally contains 7-oxygenated coumarins. Glycosylation, which has been observed in various cell cultures, is probably not required in biosynthesis (Fritig et al., 1970), serving rather to facilitate vacuolar storage (Rataboul et al., 1985; Werner and Matile, 1985). Ibrahim and Boulay (1980) partially purified a glucosyltransferase from tobacco cultures, which specifically glucosylates the 7-hydroxyl group in esculetin (8) and daphnetin (13), and to a lesser extent, that of umbelliferone (2), scopoletin (10), and hydrangetin (14). Tabata et al. (1984) fed esculetin to suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides, and Nicotiana tabacum. All three cultures formed the 6-O-β-glucoside esculin (9), but only Gardenia additionally synthesized some 7-O-β-glucoside. Unexpectedly, no scopolin (11) was reported from the experiments employing tobacco cultures, although such cultures reportedly contain esculetin 6-O-methyltransferase and scopoletin 7-O-glucosyltransferase activities (Blume, 1982). Esculin accumulated exclusively within the cells, and 2,4-D stimulated its formation from exogeneously supplied esculetin.

Cinnamic acid 4-hydroxylase, a microsomal enzyme, has been studied from parsley cultures (Scheel and Sandermann, 1975). This enzyme activity is induced on elicitor treatment and serves routinely for control of induction efficiency in our current research (see below). A crucial step in the biosynthesis of coumarins is the ortho-hydroxylation postulated to precede lactonization of either cinnamic acid (Gestetner and Conn, 1974; Ranjeva et al., 1977) or 4-coumaric acid (Kindl, 1971), thus linking general phenylpropanoid metabolism with the coumarin-specific pathway. In all three reports, the hydroxylating activity was ascribed to chloroplast fractions. Despite continued efforts, however, ortho-hydroxylation of either cinnamic acid, 4-coumaric acid, 4-coumaroyl-CoA, or 5-coumaroyl shikimic acid ester could not be confirmed in extracts from various induced cell cultures (H. Wendorff and U. Matern, unpublished). No attempts were made in these experiments to isolate plastids, because the parsley cells that accumulate coumarins efficiently on induction have been subcultured continuously in the dark for approximately 20 years and most likely lack normal plastids.

The prenylation of umbelliferone to yield demethylsuberosin (5) (Fig. 2) was accomplished in vitro by an enzyme isolated from Ruta graveolens cell cultures (Dhillon and Brown, 1976). The reaction is dependent on manganese and requires dimethylallyl diphosphate as cosubstrate. The enzyme was solubilized from isolated chloroplasts and partially purified. This observation has so far favored plastids as the sole site of coumarin synthesis. However, it is known that HMGCoA-reductase—an enzyme responsible for the biosynthesis of dimethylallyl diphosphate—is active in both plastids and microsomal preparations assigned to the endoplasmic reticulum (Suzuki and Uritani 1976). Only the microsomal enzyme activity appears to be induced upon elicitor treatment of potato (Oba et al., 1985) and cultured parsley (Tietjen and Matern, 1983) or Ammi majus cells (D. Hamerski and U. Matern, unpublished). Moreover, furanocoumarin specific O-methyltransferases (see below) are not associated with chloroplasts (Brown, 1985).

Fig. 2 Coumarin-specific enzyme reactions associated with the membranes of the endoplasmic reticulum in elicitor-induced Ammi majus cells. DMAPP = dimethylallyl diphosphate.

The enzymatic cyclization of demethylsuberosin (5) to (+)marmesin (16) (Fig. 2) was demonstrated using microsomes from elicitor-induced Ammi majus cells in the presence of NADPH and oxygen (Hamerski and Matern, 1988). Inhibition studies showed this reaction to be catalyzed by a cytochrome P450-monooxygenase, thus implying an oxidative cyclization via the epoxide. However, no intermediate could be detected under any incubation condition. Although these results appear not to support the mechanism postulated for the formation of marmesin by Brown et al. (1970), a short-lived intermediate epoxide can not be ruled out completely due to the fact that enzymes attacking oxiranes are generally known to possess very high catalytic activities (Wistuba and Schurig, 1986). Marmesin synthase activity has also been found in microsomes from induced parsley (Wendorff, 1987) and Arracacia cells (K. Harter and U. Matern, unpublished) and is, in all cases, associated with the endoplasmic reticulum.

Recently, we could also demonstrate the NADPH-dependent conversion of synthetic (±)[3-¹⁴C]marmesin into psoralen (25) (Fig. 2) by microsomes prepared from elicitor-induced parsley cells (Wendorff and Matern, 1986) (Fig. 2). Again, inhibition studies suggested a cytochrome P450-dependent mechanism for the psoralen synthase reaction. Our results are in accord with the reaction sequence postulated previously for the biosynthesis of psoralen (Murray et al., 1982). Microsomes derived from cells induced with Phytophthora elicitor, but not those from Alternaria elicitor-induced cells, catalyzed the NADPH-dependent formation of yet another product from the racemic marmesin mixture. Preliminary experiments suggest that this compound may be converted further to graveolone (24) by microsomes. Extensive dilution experiments employing either authentic (+)marmesin or authentic (—)marmesin (=nodakenetin) (22) revealed that only the former is converted to both psoralen and the product tentatively identified as a graveoloneintermediate. The mechanism of both of these reactions is unknown at present. However, assuming initial 3′-hydroxylation of (+)marmesin in both the cis- and trans-orientation, a subsequent break of the trans-vicinal bond must formally release water and acetone from the cis-hydroxylated substrate to yield psoralen (Fig. 3). Likewise, a 1-oxo-3-hydroxy-isopentyl-substituted umbelliferone anion would be the initial product from relocation of charge in trans-hydroxylated marmesin. This would then cyclize to graveolone (Fig. 3). The postulated trans-hydroxylated substrate for the latter reaction had been isolated from Xanthoxylum arnottianum Maxim. (Ishii et al., 1973) and named xanthoarnol. This reaction scheme is still a hypothesis. Nevertheless, it is of great interest to see the previously reported differential induction of cells by the two elicitors (Tietjen et al., 1983) reflected in the catalytic properties of isolated microsomes.

Fig. 3 Hypothetic sequence of reactions as suggested for psoralen and graveolone synthesis catalyzed by microsomal enzyme activities from elicitor-induced parsley cells. The letter B represents an enzyme base.

The formation of bergapten (27) has been suggested as proceeding from either 5-hydroxylated marmesin or via psoralen and bergaptol (26). The endoplasmic membrane fractions from elicitor-induced Ammi majus cells catalyzed only the latter reaction (Fig. 2) (D. Hamerski and U. Matern, unpublished). Bergaptol synthase was also identified as a cytochrome P450-dependent monooxygenase. The close spatial association of all the enzymes which sequentially catalyze the formation of bergaptol from umbelliferone (Fig. 2) and the fact that exogeneously supplied marmesin—in contrast to umbelliferone—is not readily incorporated into the psoralens make it likely that furanocoumarin synthesis occurs in the lumen of the endoplasmic reticulum and not as previously suggested in the plastids.

A 5-O-methyltransferase and an 8-O-methyltransferase accepting linear furanocoumarins (26, 30, and 32) as substrates were isolated from Ruta graveolens cell cultures and purified to homogeneity (Thompson et al., 1978, Sharma et al., 1979). Both enzymes exhibited the same molecular mass of between 85 and 110 kDa. The former enzyme specifically methylated the hydroxyl group in the position ortho to the side chain in furanocoumarins, whereas the latter enzyme appeared to be less specific accepting 8-hydroxylated simple coumarins like daphnetin (13), also. Two methyltransferases with similar substrate specificities were recently described from elicitor-induced parsley cells (Hauffe et al., 1986). 5-Hydroxyxanthotoxin (32) was a better substrate than bergaptol (26) for the 5-O-methyltransferase, which is in accord with the proposed biosynthesis of isopimpinellin (33) via xanthotoxin (31) (Murray et al., 1982). The parsley methyltransferases possess molecular masses of 67 and 73 kDa, respectively, and possibly consist of two subunits.

VI REGULATION OF BIOSYNTHESIS

Inducible cell cultures appear to be well suited for regulatory studies. Elicitor induction, however, usually triggers several rather than one particular pathway, including among others lignin biosynthesis. It thus remains difficult to evaluate the relative significance of, for example, changes in the general phenylpropanoid metabolism for coumarin synthesis, as long as no isoforms of individual enzymes can be exclusively assigned to coumarin biosynthesis.

The coordinated induction of phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate:CoA ligase, and dimethylallyl diphosphate:umbelliferone dimethylallyltransferase in parsley cells in response to elicitor treatment has been reported (Hahlbrock et al., 1981; Tietjen and Matern, 1983). The latter enzyme activity, specifically involved in the biosynthesis of coumarins, reached its maximum several hours later than that of the other enzymes and was assigned to a separate regulatory group of enzymes (Tietjen and Matern, 1983). Yet another transferase activity (dimethylallyl diphosphate:umbelliferone O-dimethylallyltransferase) found in the endoplasmic reticulum of elicitor-induced Ammi majus cells also showed such a delay in activation (Hamerski and Matern, 1988). A similar induction time course in response to elicitor was reported for the two coumarin-specific O-methyltransferase activities in cultured parsley cells (Hauffe et al., 1986). In the cases of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, de novo synthesis has been shown upon induction. This appears to be due to transient regulation of the respective translation and transcription rates (Kuhn et al., 1984; Chappell and Hahlbrock, 1984; Schmelzer et al., 1985).

VII PHYSIOLOGICAL SIGNIFICANCE

The significance of coumarins is considered here only as it relates to infection. In differentiated plants, furanocoumarins are frequently excreted into schizolysigenous containers (Andon and Denisova, 1974) or into the waxy surface (Städler and Buser, 1984), exposing them to possible invaders. Similarly, cell cultures rapidly excrete most of the coumarins synthesized in response to elicitor treatment into the culture fluid (Tietjen et al., 1983; Hamerski et al., 1981).

Furanocoumarins are toxic to various organisms, and their potential use as phytoalexins has been reviewed (Murray et al., 1982; Brown, 1981). On the other hand, a role for simple coumarins like scopoletin (10) in limiting spread of a pathogen is more difficult to define. Stoessl and Hohl (1981) argued that the direct antimycotic activity of scopoletin is negligible. However, it may contribute indirectly to the general defense reaction. Scopoletin activates the plant’s hexose monophosphate pathway under stress (Hoover et al., 1977). Furthermore, an inhibitory effect on the pectinolytic enzymes of pathogens has been reported (Ravise and Kirkiacharian, 1976). Scopoletin may also be oxidized by particular isoperoxidases (Reigh et al., 1973), thus mediating the action of peroxidases (Wheatley and Schwabe, 1985) in a way similar to that postulated for the flavone apigenin (Yamauchi and Minamide, 1985).

ACKNOWLEDGMENTS

Part of the work described in this chapter was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. Valuable discussions with S. A. Brown, Peterborough, Ontario, Canada; R. C. Beier, College Station, Texas; and W. Heller, Munich, are gratefully acknowledged. We thank C. Beggs for critical reading of the manuscript.

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