Plant Physiol. (1988) 86, 0161-0165 0032-0889/88/86/016 1/05/$0 1.00/0 Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of bracteatum' Received for publicaton April 1, 1987 and in revised form September 21, 1987

STEVEN D. CLINE AND CARMINE J. COSCIA* Downloaded from https://academic.oup.com/plphys/article/86/1/161/6083039 by guest on 30 September 2021 E. A. Doisy Department ofBiochemistry, St. Louis University School ofMedicine, St. Louis, MO 63104

ABSTRACT lated biosynthesis ofsecondary metabolites in inoculated tissues. These compounds, which have been referred to as phytoalexins, Fungal elicitor preparations from either homogenized mycelia of Den- frequently function as low mol wt antimicrobial compounds and dryphionpenicilatum (Cda.) Fr., a specific pathogen ofPapa er species, are a proposed defense strategy ofthe host to pathogen aggression or conidia of Verticillium dakliae Kleb., a general pathogen, were added (3-5 and references cited therein). to 14-day-old suspension cultures of Papa'er bracteatum. tissue Here we report the effects of two fungal elicitor preparations, cultures were grown either in the presence or absence of 0.1 milligram of one produced from a specific pathogen ofPapaver, Dendryphion 2,4-dichlorophenoxyacetic acid per liter and 0.5 millm of6-benzylam- penicillatum, and one obtained from a general pathogen, inopurine per liter. Dendryphion extracts elicited an accumulation of the Verticillium dahliae, on the accumulation of alkaloids in cell benzophenanthridine allaloid, sanguinarie, which was not greatly influ- suspension cultures of Papaver bracteatum. We focused on the enced by hormone deprivation. Millimolar concentrations of dopamine benzophenanthridine alkaloid, sanguinarine, and the morphinan were detected under all conditions. was found when celis were alkaloid, thebaine, and their precursor, dopamine. cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor- dose-dependent manner only under conditions of hormonal deprivation, MATERIALS AND METHODS resulting in an elevation of sangirine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in Plant Cell Cultures. Papaver bracteatum Arya II Lindl. cell the medium (23 milligrams per liter), with 85% of the alkaloid associated culture was initiated and maintained for 2 years on Murashige with a lOOg sedimenting fraction that, upon light microscopic inspection, and Skoog's revised tobacco medium (MSRT)2 as previously proved to be devoid ofcells. In bioassays, sninarine showed significant described (18, 19). biological activity at concentrations as low as 5 to 10 micrograms per Culture Conditions. Papaver cell suspension cultures were gen- milliliter against three general plant pathogens, Verticillium dahliae, erated from 14- to 21-d-old static cultures grown at 21 to 24°C Botrytiscinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion in a 12-h fluorescent light cycle on MSRT containing 0.1 mg/L was less affected sanguinarine addition and an to 2,4-D and 0.5 mg/L BA (18, 19). Approximately 30 to 40 g of by displayed ability beige callus was transferred to 250 ml of liquid MSRT media metabolize the alkaloid as evidenced by its loss from the media, subse- with or without hormone supplement and incubated 7 d on a quent accumulation in the mycelia, and ultimate disappearance over a 48- rotary shaker at 21 to 24°C in a 12-h fluorescent light cycle. Ten- hour period. By comparison, dopamine and thebaine were less toxic to ml aliquots ofthe cell suspension were added to flasks containing the general plant pathogens. 60 to 70 ml of MSRT with or without hormone and allowed to incubate for 7 d. Preparation of Fungal Elicitors and Elicitation Protocols. A homogenized, sterile fungal extract from the poppy pathogen, Dendryphion penicillatum, was prepared from vegetative myce- lium grown for 4 to 5 d in MSRT liquid media with 1 g/L casein Attempts to stimulate alkoid production in cell cultures have at 21 to 24C. The mycelium was filtered, washed with deionized frequently entailed conditions which could be classified as stress- water, frozen at 1 5°C for 24 to 48 h, and 1 g fresh weight mycelia related. Interest in studying the accumulation of alkaloids in (8.7% dry weight) homogenized in 13 volumes of 0.1 M phos- Papaver species has followed the premise that these secondary phate buffer (pH 7.0) and filter sterilized (0.2 um) prior to metabolites may be stress compounds formed in response to addition to cell suspensions. Between 0 and 3.0 ml of fungal extremes in culture conditions. Some success in stimulating elicitor preparation was then added to cell suspensions and aLkaloid production has been achieved by altering hormone levels incubated for an additional 6 d. (17-19), adding protein synthesis inhibitors (14), or lowering Verticillium elicitor was prepared from an aqueous suspension growth temperatures (21). Recently, enhancement of alkaloid of conidia washed from 14-d-old cultures grown at 21 to 24°C production by cell suspension cultures has been achieved through on potato dextrose agar plates and adjusted to 1.5 to-2.0 x 106 the use ofbiotic elicitor preparations from fungal plant pathogens conidia/ml. The suspension was autoclaved for 10 min and 0 to (7, 9, 10, 12). The basis for elicitor induction ofalkaloids follows 10 ml added to 60 to 70 ml of the 14-d-old P. bracteatum the concept that host-pathogen interactions often lead to stimu- suspension cultures grown in the presence or absence of 2,4-D and BA. In hormone deprivation studies, cell suspensions were ' This work was supported by National Science Foundation grant PCM 83-14368 and National Institutes of Health training grant HL- 2Abbreviations: MSRT, Murashige-Skoog revised tobacco medium: 07050. BA, 6-benzylaminopurine; EDso, 50% effective dose. 161 162 CLINE AND COSCIA Plant Physiol. Vol. 86, 1988 maintained for 1, 5, 10, 15, or 20 d in hormone-free MSRT prior to adding the conidial elicitor preparation. Cells were harvested by filtration through miracloth 4 d after elicitor addition, and levels of alkaloid and dopamine were determined. The filtered a medium was also used for alkaloid determinations. In later experiments the filtered medium was centrifuged at lOOg to yield L ai 500 an alkaloid-rich pellet. c Alkaloid and Dopamine Analysis. Alkaloids and dopamine were quantitied as previously described (19, 23, 24). Briefly, total 0 alkaloids were separated on extrelut columns and the sanguinar- IU ine and thebaine contents were determined by HPLC with UV detection. A catecholamine fraction was isolated by alumina chromatography, and dopamine was measured by HPLC with O0 electrochemical detection. The amount of tissue extracted for Downloaded from https://academic.oup.com/plphys/article/86/1/161/6083039 by guest on 30 September 2021 alkaloid analysis ranged from 0.2 to 1.0 g, while for dopamine it 0 5 10 15 20 ranged from 50 to 200 mg. The alkaloid content in media was Sanguinarine (mg/ml) determined by removing a 1.0-ml aliquot of medium from each FIG. 1. Effect of sanguinarine on growth of fungal plant pathogens in flask at cell harvest, loading it directly onto an extrelut column suspension culture. D. penicillatum (*), R. solani (A), V. dahliae (U), with 0.5 ml methanol and following the same procedure used and B. cinerea (0) were inoculated into suspension culture containing 0 for cell extracts. to 20 sg/ml sanguinarine. Dry weight of each fungus was determined Bioassay of Fungal Pathogens. Four fungal plant pathogens after 4 to 5 d of growth at room temperature. Each value represents the were screened for growth response in the presence of varying mean of three experiments (* P < 0.05). concentrations of sanguinarine, dopamine (Sigma) or thebaine (Mallinckrodt). Sanguinarine was purified to homogeneity from a practical grade (K and K Labs) and characterized by mass a C, spectrometry (19). Three broad-host-ranged genera: Botrytis ci- L 'a 4i c .a 0 nerea Pers. ex Fr., Rhizoctonia solani Kuehn. and Verticillium L) bt c11 dahliaeKleb. plus a specific pathogen ofPapaver, D. penicillatum L (Cda.) Fr., were used in this experiment. Dendryphion was iso- c41 c10 L 3 a lated from infested seeds ofP. bracteatum (16), whereas the other c10 c 3 ul10 fungal species were collected from various non-Papaver hosts. co c Fungal isolates were grown on MSRT agar plates supplemented cn'o a 0 1 u with g/L casein at 21 to 24°C in the dark. Flasks containing 75 10 Go I ml of MSRT liquid medium were inoculated with three (3-mm x diameter) agar plugs obtained from the advancing mycelial edge. After inoculation, sanguinarine, dopamine, or thebaine was added to cultures, and the flasks were placed onto a shake incubator at 21 to 24°C in a 12-h fluorescent light cycle and Incubation Period (h) allowed to incubate for 4 to 5 d. The vegetative mycelium was FIG. 2. Changes in sanguinarine concentration in medium and my- filtered and tissue weights were determined. celium of D. penicillatum in suspension culture. Mycelial agar plugs of Dendryphion were inoculated into 75 ml MSRT + 1 g/L casein and RESULTS incubated on a rotary shaker for 48 h. Sanguinarine was then added to a final concentration of 10 gg/ml. At 12- to 24-h intervals, flask contents Bioassay Studies with Fungal Pathogens. The pathogens were were filtered, providing medium and mycelial fractions and subsequently grown in the presence of sanguinarine, dopamine, or thebaine. analyzed for their sanguinarine content. Mycelia sanguinarine values are The greatest effect on growth of all fungi tested was found when expressed as percent controls in which an equal concentration of sangui- sanguinarine was added to the media (Fig. 1). For Botrytis and narine was added to medium devoid of Dendryphion (* P < 0.05). Rhizoctonia, EDso values were estimated to be less than 5 ug/ ml, while for Verticillium, they were about 15 ug/ml. Dendry- despite the fact that dopamine additions were two orders of phion, the poppy pathogen, was least affected by sanguinarine magnitude greater than the maximum amount of sanguinarine concentrations of up to 20 ,g/ml. Initially, at 5 Ag/ml a 50% added (data not shown). Thebaine had little or no effect on the reduction in growth was found. However, as the concentration growth of the general pathogens, Botrytis and Rhizoctonia, even of sanguinarine was increased, a positive growth response fol- at concentrations as high as 500 isg/ml. An estimated EDso of lowed. Sanguinarine amended medium assumes a bright orange 200 ,ug/ml was observed for the poppy pathogen, Dendryphion. color and in bioassays with the general pathogens, the color of Treatment of Papaver Cell Suspension Cultures with Dendry- the medium remained unchanged. During growth studies with phion Elicitor Preparation. Initially, the time dependency of Dendryphion, a complete loss of coloration was observed over a alkaloid accumulation after addition of Dendryphion elicitor 4-d incubation period. Concentrations of sanguinarine in the preparation, was examined with P. bracteatum suspension cul- medium were monitored during a period of 48 h in the presence tures grown in the presence of 2,4-D and BA. These tests, of Dendryphion and found to progressively decrease (Fig. 2). performed on 14-d-old cell suspension cultures, revealed a max- Concurrently, sanguinarine content in the mycelium increased, imum cellular accumulation of sanguinarine that was 2 orders peaking at 24 h of incubation and decreasing thereafter. The of magnitude greater than nonelicited controls. The response total quantity detected in both medium and tissue by HPLC-UV occurred between 2 and 5 d after adding the elicitor extract (Fig. at 48 h accounted for only about 5% of the original amount of 3). Dopamine concentrations fluctuated but remained in the sanguinarine added to medium. These results suggest that the mmolar range over the incubation period. Subsequent experi- alkaloid was metabolized. ments examining the relationship between elicitor dosage and In contrast, dopamine did not affect the growth of Verticillium, the alkaloid cellular response were performed using a 6-d elicitor Rhizoctonia, or Dendryphion in shake culture bioassay tests, exposure period. STIMULATION OF SANGUINARINE PRODUCTION 163

600 3.0 in the absence of 2.4-D and BA produced sanguinarine in con- centrations comparable to cells grown in the presence of phyto- A hormones. However, one reaction to the elicitor, a change in color of the medium to brownish-orange and a darkening of the

L 400 cells, occurred 24 to 48 h sooner in the absence of hormone. '°L Under these conditions, sanguinarine was produced in a dose- 3l -1C dependent manner, while dopamine concentrations decreased c4 (data not shown). Cells grown without hormone accumulated L.a 2001 ca thebaine ranging between 1.5 and 2.0 fresh weight, consist- .o-. ytg/g C,O ent with earlier findings (19), although production was not in'a0 elicitor-dependent. Treatment of Papaver Cell Suspension Cultures with Verticil-

0 0 lium Elicitor Preparation. Initially, Verticillium elicitor-treated 0 24 48 72 96 120 144 168 cell suspensions of P. bracteatum were evaluated to compare the Downloaded from https://academic.oup.com/plphys/article/86/1/161/6083039 by guest on 30 September 2021 Incubation Period (h) effect of hormonal deprivation on elicitor activation of alkaloid FIG. 3. Time course of the effect of Dendryphion elicitor on sangui- and dopamine production. Elicited 14-d-old cell suspensions, narine and dopamine production in P. bracteatum cell suspension cul- grown in the presence of 2,4-D and BA generated very little tures. One-ml of a standard elicitor preparation (80 mg) was added to sanguinarine, while cell suspensions grown without hormone 14-d-old suspension cultures grown in 70 ml of MSRT with hormone. produced larger quantities upon addition of elicitor preparation Cells were harvested by filtration at 24- to 48-h intervals through 7 d and to each flask (Table I). The natural predisposition for sanguinar- cellular dopamine and sanguinarine concentrations determined. Each ine production was apparent under hormone-free conditions value represents the mean of four experiments (* P < 0.05). illustrated by the nonelicited controls, which accumulated about 7-fold more sanguinarine than nonelicited controls grown in the

800 presence of hormone. The effect of preincubation in hormone- 3.0 free media on the ability to elicit sanguinarine production was 3 further examined when 14-d-old static cultures were transferred

E 2.0 600 to hormone-free media and subsequently elicited at 4- to 5-d v intervals through 20 d. Elicited suspensions were compared to i2.0 non-elicited controls over this time course. A difference in cel- lular sanguinarine levels between Verticillium-elicited cells and c0 400 o E controls was seen on d 1 after transfer to hormone-free media 1.0 and then not until approximately 15 d later (Table II). The most 1. 0 E significant change in the elicitor response was in the concentra- Ul L3 200 a tions of sanguinarine in the medium which were elevated over controls for the entire time period and showed a 20-fold increase on d 20. Dopamine concentrations did not change as a result of 0 0o elicitation but decreased 40 to 60% over the 20-d period in both 0.0 0.5 1.0 1.5 2.0 2.5 controls and elicited cultures. Elicitor (ml) Verticillium conidial elicitation of sanguinarine was dose-de- FIG. 4. Effect of Dendryphion elicitor on alkaloid and dopamine pendent for both cells and media (Fig. 5), as observed for the content in P. bracteatum cell suspension cultures grown with 2,4-D and Dendryphion-derived elicitor. The highest accumulation of san- BA. Dendryphion elicitor was added to 14-d-old suspension cultures guinarine occurred when 1.5 x 106 conidia were added per flask. grown in 70 ml ofMSRT with hormone. Cells were harvested by filtration Addition of larger amounts to optimize alkaloid production has 6 d following elicitor addition (80 mg fr wt/ml) and dopamine and not been undertaken. Consistent with previous tests, dopamine sanguinarine concentrations determined. A l-ml aliquot of medium was concentrations fluctuated but remained in the mmolar range. used for sanguinarine analysis. Each value represents the mean of four Cell growth of elicited suspensions did not differ significantly experiments (* P < 0.05). from the controls in these experiments. As the elicitor dose was increased, the proportion of total medium to total cellular san- When Dendryphion elicitor preparation was added to P. brac- guinarine also rose. The ratio of total sanguinarine in cells and teatum cell suspension cultures grown in the presence of 2,4-D medium was 1:2 in the absence of elicitor, increasing to 1:3 at and BA, sanguinarine accumulation in cells was found to be the highest concentration of elicitor preparation. After filtration elicitor-dose-dependent (Fig. 4). Cellular concentrations of do- through Miracloth to remove whole cells, the filtrate was sepa- pamine, a precursor of sanguinarine, decreased over the 6 d of rated into lOOg supernatant and pelleted fractions. Extrelut elicitor exposure. The magnitude of sanguinarine accumulation prefractionation of 1 ml aliquots ofthe supernatant and noncen- peaked at an elicitor concentration of 120 mg fresh weight/flask trifuged culture medium determined that between 76 to 91% of (1.5 ml elicitor) where over 460 gg of sanguinarine per g fresh the extractable sanguinarine in the medium was located in the weight was found to accumulate in the cell tissue. At the highest IOOg pellet fraction. elicitor concentration, a decrease in the sanguinarine and dopa- mine content was observed. This was accompanied by extensive DISCUSSION browning of tissue and cessation of growth indicative of a toxic response. Growth of the cell suspensions treated with up to 120 Among the various secondary metabolites which have been mg fresh weight of elicitor preparation per flask was not signifi- elicited in cell cultures, plant alkaloids have only recently re- cantly different from controls (1 18%). A reduction in cell growth ceived attention. Eilert et al. have been successful in stimulating was observed after treatment with additional elicitor (65%). indole alkaloid production in Catharanthus roseus (7), acridone Concomitantly, sanguinarine concentration in the medium in- epoxides in Ruta graveolens (8), and sanguinarine in Papaver creased proportional to elicitor addition (Fig. 4). Thebaine was somniferum cell cultures (6, 9) following treatment with general not detected under these growth conditions. fungal elicitors. Heinstein (12) reported on the production of Dendryphion-elicited cell suspensions of P. bracteatum grown morphinan alkaloids in P. somniferum and harringtonia alka- 164 CLINE AND COSCIA Plant Physiol. Vol. 86, 1988 Table I. Combined Effect of Verticillium Elicitor and Hormone Supplement on Cellular Sanguinarine and Dopamine Production in P. bracteatum From an elicitor preparation of Verticillium (1.5 x 106 conidia/ml) 0, 1 or 3 ml were added to 14-d-old suspension cultures grown in 70 ml ofMSRT with 2,4-D and BA or hormone-free. Elicitor values are expressed as the final concentration ofconidia in the cell suspension medium. Cells were harvested by filtration 4 d after elicitation, and cellular dopamine and sanguinarine levels were determined. Each value represents the mean ± SEM of three experiments. Statistical comparisons were made between hormone-supplemented and hormone- free conditions (* P < 0.05). Media Elicitor 2,4-D + BA supplemented Hormone-free Sanguinarine Dopamine Sanguinarine Dopamine conidia/ml ug/gfresh wt mg/gfresh wt lAg/gfresh wt mg/gfresh wt Downloaded from https://academic.oup.com/plphys/article/86/1/161/6083039 by guest on 30 September 2021 0 9.5 ± 3.8 1.74 ± 0.25 69.4 11.0* 1.37 ± 0.27 2 x 104 7.8 ± 5.6 1.77 ± 0.40 556.4 ± 102.1* 1.42 ± 0.27 6 x 104 6.0 ± 3.5 1.76 ± 0.06 4758.8 ± 653.7* 1.64 ± 0.32

Table II. Effect ofPreincubation in Hormone-Free Media on Verticillium-Elicited Sanguinarine and Dopamine Production in Cell Suspension Cultures ofP. bracteatum Static cultures were transferred to 60 ml hormone-free MSRT and incubated for 1, 5, 10, 15, or 20 d prior to adding 5 ml/flask of a Verticillium conidial elicitor preparation (2.0 x 106/ml). Cells were harvested after 4 d and cellular dopamine, sanguinarine, and media sanguinarine concentrations determined. Each value represents the mean ofthree experiments. Statistical comparison were made between elicited and non-elicited paired observations at each preincubation period (* P < 0.05). Preincubation Cellular Sanguinarine Media Sanguinarine Cellular Dopamine Period Nonelicited Elicited Nonelicited Elicited Nonelicited Elicited d Ag/gfresh wt ;Lg/ml mg/gfresh wt 1 42.1 ± 4.1 143.7 ± 13.5* 0.34 ± 0.05 0.61 ± 0.04* 1.80 ± 0.05 2.00 ± 0.23 5 148.1 ± 26.9 172.4 ± 40.0 0.32 ± 0.06 0.47 ± 0.08* 1.54 ± 0.31 1.66 ± 0.63 10 93.6± 11.2 109.0± 11.0 0.15 ±0.01 0.37±0.07* 1.25±0.04 0.92±0.15 15 73.9 ± 8.1 194.5 ± 13.5* 0.33 ± 0.25 2.27 ± 0.35* 1.49 ± 0.30 1.21 ± 0.12 20 147.5 ± 19.1 649.4 ± 304.5* 0.12 ± 0.01 2.59 ± 1.76* 1.08 ± 0.05 0.73 ± 0.15 elicitor preparation from conidia ofthe general pathogen, Verti- 50S cillium, coupled to hormone deprivation, gave higher yields of sanguinarine than those previously reported (6, 9). 40 Sanguinarine was first isolated from Papaver callus by Furuya 3 et al. (11). Subsequently, other investigators (15, 18-20) have c) confirmed it to be a major alkaloidal product of Papaver cell L 301 LD c1 L. .001- cultures. In contrast, this compound appears to be a trace con- 0 0 coa 20f stituent (<10 ug/g fresh weight) in seedlings (24) and mature E (2). to L1 a4 It Our data as well as others show that sanguinarine C] (12, 13, 22) 10 has significant biological activity. The compound reduced growth in 3 of 4 fungal plant pathogens tested at 10' M. The exception 0 to this was with the Papaver pathogen, D. penicillatum, which 4.0 6.0 displayed minimum susceptibility to sanguinarine. The lack of Elicitor (ml) response to Dendryphion to sanguinarine may be due to its ability FIG. 5. Effect of Verticillium elicitor on alkaloid and dopamine con- to metabolize the compound as evidenced by the loss of the tent in P. bracteatum cell suspension cultures grown without hormone. aLkaloid from the medium, followed by subsequent accumulation An autoclaved, Verticillium conidial suspension (1.5 x 106 conidia/ml) and apparent disappearance from the mycelium. This suggests was added to 14-d-old cell suspensions ofP. bracteatum grown in 60 ml that the success of this pathogen, may be related to the detoxifi- MSRT with 2,4-D and BA. Cells were harvested by filtration 4 d following cation of sanguinarine, should it be an elicited product in whole elicitor addition and dopamine and sanguinarine concentrations deter- plant tissue as in cell culture. Detoxification of induced com- mined. A l-ml aliquot of medium was used for sanguinarine analysis. pounds such as phytoalexins as a criterion for establishing path- Each value represents the mean of four experiments (* P < 0.05). ogenicity has been supported by others (3). Sanguinarine appears to be a candidate as a phytoalexin in light of its relative absence loids in Cephalotaxus harringtonia after exposure to a fungal in whole plant tissue and its induction at least in tissue culture. elicitor. Moreover, this alkaloid possesses significant biological potency In the present study sanguinarine but not the morphinan against incompatible pathogens. Furthermore, its role as a phy- alkaloid, thebaine, was generated in P. bracteatum cell cultures toalexin is supported by the interesting relationship between the treated with elicitor preparations from a specific poppy pathogen, apparent ability of Dendryphion to metabolize sanguinarine in Dendryphion. In addition, exposure of P. bracteatum to an vitro and its specific pathogenicity. Of course, extensive in vivo STIMULATION OF SANGUINARINE PRODUCTION 165 studies would be necessary to prove that sanguinarine is a phy- sanguinarine was represented as a solubilized portion in the toalexin. medium. Failure to include both fractions in the analysis would Verticillium elicitor preparations proved to be the more potent greatly underestimate the contribution of medium sanguinarine of the two elicitors utilized here. In one experiment, cellular to total production. sanguinarine alone accumulated in tissue to approximately 10% Although no attempts were made to optimize yields, the release ofthe dry weight (Table I). Maximum production in subsequent of considerable proportions of the accumulated alkaloid suggests tests was estimated at 9% when total cellular and medium the potential of this approach for the generation of secondary fractions were combined (Fig. 5). The highest level of sanguinar- metabolites and the study of the regulation of their synthesis. ine reported by Eilert et al. (9) was 3% dry weight found in P. Acknowledgments-We thank Michael D. Rush for assistance in this work and somniferum cell cultures treated with a Botrytis elicitor prepara- Dr. Eugene Himelick for supplying cultures of Verticillium dahliae. tion. Elicitor preparations from Verticillium have been reported to stimulate morphinan alkaloid production in P. somniferum LITERATURE CITED

(12). 1. BERLIN J, E FORCHE, V WRAY, J HAMMER, W HOSEL 1983 Formation of Downloaded from https://academic.oup.com/plphys/article/86/1/161/6083039 by guest on 30 September 2021 Sanguinarine accumulation has been shown previously in P. benzophenanthridine alkaloids by suspension cultures of Eschscholtzia cal- bracteatum (19), P. somniferum (25) and Eschschohzia califor- ifornica. Z Naturforsch 38: 346-352 2. BOHM, H 1981 Papaverbracteatum Lindl-results and problems ofthe research nica (1) cell suspensions grown under hormone-free conditions. on a potential medicinal plant. Pharmazie 36: 660-667 Hormonal deprivation alone has been found to increase cellular 3. DARVILL AG, P ALBERSHEIM 1984 Phytoalexins and their elicitors-a defense sanguinarine 4-fold despite an extensive growth period ofseveral against microbial infection in plants. Annu Rev Plant Physiol 35: 243-275 months (25). In the present experiments with Verticillium, non- 4. 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KAMIMURA S, M AKUTSU, M NISHIKAWA 1976 Formation of thebaine in the a decrease in its levels as alkaloids are suspension culture of Papaver bracteatum. Agr Biol Chem 40: 913-919 synthesis preventing 18. KUTCHAN TM, S AYABE, CJ COSCIA 1985 Cytodifferentiation and Papaver produced. However, we cannot exclude the possibility that do- alkaloid accumulation. In JD Phillipson, MF Roberts, M Zenk, eds, The pamine synthesis is constant, but its conversion to sanguinarine Chemistry and Biology of Isoquinoline Alkaloids. Springer-Verlag, Berlin, occurs at the expense of the synthesis of other alkaloids in this pp 28 1-294 labeled and 19. KUTCHAN TM, S AYABE, RJ KRUEGER, EM COSCIA, CJ COSCIA 1983 Cytodif- highly bifurcated pathway. By introducing tyrosine ferentiation and alkaloid accumulation in cultured cells of Papayer bractea- phenylalanine to media of cell suspension cultures of P. somni- tum. Plant Cell Rep 2: 281-284 ferum, evidence for the elicited induction of sanguinarine syn- 20. LOCKWOOD GB, 1981 Orientalidine and isothebaine from cell cultures of thesis was obtained (6). However, the mechanism of this induc- Papaver bracteatum. Phytochemistry 20: 1463-1464 21. LOCKWOOD GB, 1984 Alkaloids of cell suspensions derived from four Papaver tion has not been clarified. spp and the effect of temperature stress. Z Pflanzenphysiol 1 14: 361-363 During the elicitation process, substantial amounts of sangui- 22. MITCHER LA, YH PARK, D CLARK, GW CLARK III 1978 Antimicrobial agents narine accumulated in medium. The ratio of cell to medium from higher plants. An investigation of Hunnemannia fumariaefolia pseu- sanguinarine elicited by Verticillium and hormone deprivation doalcoholates of sanguinarine and chelerythrine. J Nat Prod 41: 145-150 23. ROBERTS MF, D MCCARTHY, TM KUTCHAN, CJ COSCIA 1983 Localization of ranged from 1:2 to 1:3. These proportions contrast with those enzymes and alkaloidal metabolites in Papaver latex. Arch Biochem Biophys previously reported for P. somniferum elicited cell suspensions 222: 599-609 where the cell to medium ratio was either 2:1 or 1:1 (6). In this 24. RUSH MD, TM KUTCHAN, CJ COSCIA 1985 Correlation of the appearance of study, concentrations ofsanguinarine in the medium were largely morphinan alkaloids and laticifer cells in germinating Papaver bracteatum seedlings. Plant Cell Rep 4: 237-240 attributed to a IOOg pelleted, membrane-associated, cell-free 25. SCHUCHMANN R, E WELLMANN 1983 Somatic embryogenesis oftissue cultures fraction which yielded significant amounts of the alkaloid after ofPapaver somniferum and and its relationship to alkaloid methanol extraction. Only about 15% of the total medium and lipid metabolism. Plant Cell Rept 2: 88-91