Plant Physiol. (1 995) 108: 1553-1 560

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of

Hyoscyam us m u ticus’

Fabricio Medina-Bolivar and Hector E. Flores*

Graduate Program in Plant Physiology (F.M.-B.) and Department of Plant Pathology/Biotechnology lnstitute (H.E.F.), The Pennsylvania State University, 31 5 Wartik Laboratory, University Park, Pennsylvania 16802

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. I n contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspen- sions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog pfluorophenylalanine (PFP). Cells that could grow in media containing 400 PM PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyo- scyamine. Hairy root cultures were regenerated from both PFP- sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxy- acetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine path- way upon the regeneration of root cultures.

.

The use of root cultures for physiological and biochem- ical studies, in particular hairy roots obtained by transfor- mation with Agrobacterium rhizogenes (Chilton et al., 1982; Willmitzer et al., 19821, has increased steadily in recent years (Signs and Flores, 1990). Hairy roots can express root-specific pathways and have shown stable production of alkaloids, polyacetylenes, sesquiterpenes, naphthoqui- nones, and other secondary metabolites (Signs and Flores, 1990). They have been used as a model system for the study of photosynthesis and photoautotrophy in roots (Flores et al., 1993). Hairy roots also respond to funga1 elicitors by de novo expression of phytoalexins and can transform xeno- biotics into bioactive metabolites (Flores and Curtis, 1992; Flores et al., 1994). In addition to producing low-molecular- weight secondary metabolites, hairy roots have been used

’ Supported by a grant from the National Science Foundation

* Corresponding author; e-mail hector-flores@agcs.psu.edu; fax (BCS-09110288).

1-814-863-1357.

to study the biosynthesis of root-specific defense-related proteins (Savary and Flores, 1994).

Many hairy root cultures of solanaceous species have been established to date and have been shown to produce tropane alkaloids (Flores and Filner, 1985; Knopp et al., 1988). In some cases, alkaloid levels are higher in these root cultures than in the entire plant (Mano et al., 1986; Maldo- nado-Mendoza et al., 1993). We have reported that Hyoscy- amus muticus hairy roots constitutively produce hyoscya- mine (Flores and Filner, 1985), an anticholinergic agent used in the treatment of Parkinson’s disease and organo- phosphate poisoning, that causes smooth muscle relaxation (Cordell, 1981). Hyoscyamine is biosynthetically derived from putrescine and Phe (Fig. 1). Putrescine is the precur- sor for the tropine ring (Leete, 1979), and Phe is used in the biosynthesis of the aromatic moiety of the alkaloid. It has been previously accepted that hyoscyamine derives from the esterification of tropine with tropic acid. However, increasing evidence suggests that phenyllactic acid, but not tropic acid, is the intermediate in the biosynthesis of hyo- scyamine (Robins et al., 1994).

The production of hyoscyamine is very tightly linked to the degree of morphological organization of the culture (Flores, 1987; Lindsey and Yeoman, 1983; Robins et al., 1991a). Hyoscyamine has been shown to accumulate only in organized systems, such as hairy roots, but is present only in trace amounts or is completely absent in undiffer- entiated systems such as callus cultures (Flores and Filner, 1985). Root cultures, however, have the disadvantage of not being readily amenable to selection pressures, such as can be applied to disperse cell-suspension cultures. We have shown that Hyoscyamus hairy roots can be converted into cell suspensions by growth in 2,4-D-containing me- dium. Conversely, these root-derived cell suspensions can readily be converted back to root cultures by withdrawal of the growth regulator (Flores, 1987). The ability to intercon- vert these two morphologically (and biochemically) dis- tinct phenotypes allowed for screening at the cell leve1 of genetic mutants or somaclonal variants, which may harbor a desired trait. The selected characteristic could then be expressed in root cultures regenerated from cell suspen-

Abbreviations: MS, Murashige and Skoog salts; MSD, MS me- dium supplemented with l mg/L 2,4-D; PFP, p-fluorophenylala- nine.

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1554 Medina-Bolivar and Flores Plant Physiol. Vol. 108, 1995

?

I

H,N-(CH&-NHz

Putrescine . .

,/ Phenylalanine

Caffeoyl putrescine

Tropine Tropic acid

-0-c-c- I

Hyoscyamine

Figure 1. Biosynthetic pathways of hyoscyamine and caffeoyl putrescine.

sions. In this report we demonstrate that the above ap- proach can be used to develop root clones with improved yields of hyoscyamine. This was done by selection and subsequent regeneration of hairy root-derived cells resis- tant to PFP, a toxic analog of Phe (Fig. 2). The changes in putrescine- and Phe-derived compounds present in the parental and resistant cell and root clones are discussed.

MATERIALS AND METHODS

Seed Germination and Establishment of Hairy Root Cultures

Seeds of Hyoscyumus muticus L. obtained from the Insti- tut fiir Pflanzengenetik und Kulturpflanzenforschung (Gatersleben, Germany) were surface sterilized with 10% (v/v) household bleach for 15 min, washed five times with sterile water, and germinated on MS medium (Murashige and Skoog, 1962) supplemented with 0.4 mg/L MgSO, 7H,O, 3% SUC, 0.35% Gelrite (Scott Laboratories, West War- rack, RI), and 0.5 mg/L GA,. Hairy roots were established as described previously (Signs and Flores, 1990). Briefly, 3-week-old seedlings and isolated leaves were inoculated in the stem or the petiole with a 2-day-old culture of

Agrobucterium rhizogenes strain 15834 (American Type Cul- ture Collection, Rockville, MD). Hairy roots that developed at the infection site were transferred to MS medium sup- plemented with 500 mg/L cefotaxime (Claforan; Hoescht- Roussel Pharmaceuticals, Somerville, NJ) and subcultured at least once on this medium before transfer to antibiotic- free medium. Root tips were then subcultured in 125-mL Erlenmeyer flasks containing 50 mL of MS medium and maintained on a shaker (90 rpm) at 25°C in the dark.

Establishment of Callus and Cell-Suspension Cultures

Hairy root segments (1 cm) were transferred to solid MSD and cultured under continuous light. After 4 weeks, a friable green callus was obtained and subcultured onto fresh medium every 3 weeks. For initiation of suspension cultures, approximately 0.1 g of callí was transferred to 50-mL Erlenmeyer flasks containing 20 mL of liquid MSD. Vigorously growing cell suspensions were maintained by subculturing them (1 /10 dilution) into fresh medium every 2 weeks. AI1 cultures were maintained on a shaker (90 rpm) at 25°C under continuous light.

Selection and Cloning of PFP-Resistant Cell Clones

Cell clones resistant to PFP were obtained by sequential subculture of cells in MSD with increasing concentrations of filter-sterilized PFP (10-1000 p ~ ) . Cultures shnwing in-

Hairy Root

I ------- I

callus induction I I Cell Suspension I

I

PFP-sensitive

selection for PFP-resistance 1 regeneration I

Hairy Root )______

Figure 2. Scheme for selection of root-derived cell clones resistant to amino acid analogs and regeneration of roots from resistant cell clones. PFP-sensitive cells were derived from the parental hairy root HyBsl O. PFP-resistant cells were selected from PFP-sensitive cells and induced to differentiate into PFP-resistant hairy roots. PFP-sen- sitive hairy roots were regenerated from PFP-sensitive cells and were used as a control.

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Manipulation of Tropane Alkaloid Biosynthesis 1555

dications of resistance to the amino acid analog were sin- gle-cell cloned using a double filter paper technique adapted from Horsch and Jones (1980). Briefly, 2-week-old cell suspensions were filtered sequentially through nylon sieves with mesh sizes of 210,149,105, and 74 pm; the last filtrate consisted of single cells. After we determined the cell density with a hemocytometer, the cell suspension was diluted to 1000 cells/mL with MSD and 0.5 mL was placed on a filter paper disc (transfer disc, Whatman No. 3, 5.5 cm). The transfer disc containing single cells was placed on top of a second filter paper disc (guard disc, VWR grade 415, 9 cm). A feeder layer (1 mL) of cell aggregates (>74 pm) from the parental cell clone was used for a11 cultures and placed under the guard disc. The entire plate was fed by solid MSD containing the same concentration of PFP used in the selection procedure. After 4 to 5 weeks, colonies developing on the transfer disc were subcultured into fresh solid medium containing the same concentration of PFP. Putative PFP-resistant calli were transferred into fresh me- dium every 3 weeks. These cultures were later established in liquid medium and subcultured as described above. The stability of a11 putative PFP-resistant cell clones was con- firmed by at least four transfers into medium lacking PFP before returning to PFP-containing medium. Only cell clones maintaining the resistant trait were maintained and used in further experiments.

Hairy Root Regeneration

PFP-resistant cells were transferred to solid MSD. After 1 month, the resulting calli were transferred onto a hormone- free medium. Hairy roots developed after 4 to 5 weeks and were subcultured on solid MS medium. This transfer se- quence allowed the regeneration of hairy roots with the same morphological and biochemical phenotype as the parental root clone (see "Results"). Selection pressure was maintained by including 100 PM PFP in the medium throughout the regeneration procedure. Resistance to PFP was evaluated by incubating the regenerated roots in liq- uid MS medium with increasing concentrations of PFP (0-1000 p ~ ) . As a control, hairy roots were also regener- ated from nonselected hairy root-derived cells and evalu- ated under the same conditions.

Analysis of Cinnamoyl Putrescines

Cinnamoyl putrescines were extracted by a method adapted from Berlin et al. (1982). Lyophilized cells (50 mg) or roots (50 mg) were homogenized in 3 mL of methanol: CHCl,:H,O (12:5:3, v/v/v) and centrifuged for 10 min. The supernatant was partitioned into two phases by addition of 1 mL of chloroform and 1 mL of water. The methanol-water phase was evaporated to dryness and the resulting residue was dissolved in 50% methanol. Extracts were separated by TLC on silica gel 60 CF,,, plates (Merck, No. 13144) with butano1:acetic acid:water (4:1:1, v/v/v) as solvent. Cin- namoyl putrescines were detected under UV light, identi- fied by comparison to R,s of authentic standards (kindly provided by Prof. C. Martin, Institut National de la Recher- che Agronomique, Dijon, France). Quantification was done

by scraping bands from the plates and eluting with 50% methanol. of the compounds was determined in a Beckman (DU-65) spectrophotometer. Cinnamoyl putre- scines were also visualized and quantified by spraying the TLC plates with ninhydrin (Wagner et al., 1984) and scan- ning with a Pharmacia LKB Ultroscan laser densitometer.

Analysis of Hyoscyamine

Hyoscyamine was analyzed by the method of Kamada et al. (1986). Briefly, lyophilized cells (100 mg) or roots (100 mg) were extracted with CHCl,:methanol:NH,OH (15:5:1, v/v/v). After homogenization and incubation for 1 h at 25"C, the extract was filtered and washed twice with 1 mL of chloroform. The filtrate was evaporated to dryness un- der air and then 5 mL of chloroform and 2 mL of 0.5 M

sulfuric acid were added to the residue. The aqueous phase was adjusted to pH 10 with 28% ammonium hydroxide, and alkaloids were extracted twice with 1 mL of chloro- form. This mixture was then evaporated to dryness and dissolved in methanol. Extracts were separated by TLC on silica gel 60 CF,,, plates using to1uene:ethylacetate:dieth- ylamine (721, v/v/v) as solvent. Hyoscyamine was de- tected by spraying twice with Dragendorff's reagent and 5% sodium nitrite (Wagner et al., 1984) and identified by reference to authentic standards (hyoscyamine hydrochlo- ride, Sigma). Quantification of hyoscyamine was per- formed on a Pharmacia LKB UltroScan XL laser densitom- eter at a fixed wavelength (A = 633 nm).

Identity of the alkaloids was also independently con- firmed by a HPLC method modified from that of Fliniaux et al. (1993). The alkaloid extracts dissolved in methanol were filtered through a 0.2-mm Nalgene (Nalgo Co., Roch- ester, NY) cellulose acetate syringe filter. Extracts were analyzed using a Waters 600E system equipped with a U6K injector and 990 photodiode array detector. Samples were separated isocratically on a Nova-Pak C,, (Waters) steel column (3.9 X 150 mm) using a mobile phase of 12.5% (v/v) acetonitrile and 87.5% (v/v) aqueous phosphoric acid (0.3%, v/v) adjusted to pH 2.2 with triethylamine, at a flow rate of 0.8 mL/min. Identification and quantification of hyoscyamine in samples was done by reference to the retention time of authentic hyoscyamine standards.

Precursor Feeding Experiments

Twelve root tips (1 cm long) were used to inoculate 50-mL Erlenmeyer flasks containing 20 mL of MS medium. These cultures were grown as previously described. Radio- active precursors of hyoscyamine were added to both PFP- sensitive and PFP-resistant hairy root cultures at three times. On d 6, 12, and 18 of culture, 10 pL of labeled precursor [1,4-'4C]putrescine (37.7 kBq, 3.87 GBq/mmol; NEN) or [U-'4C]Phe (37.7 kBq, 14.99 GBq/mmol; ICN) were added to the flasks. After incubation for 24 h, the roots were harvested, pooled, and washed with NaCl (0.061 M) and immediately frozen at -80°C. Cinnamoyl putrescines and tropane alkaloids were extracted as de- scribed above from the lyophilized roots and separated by TLC. Incorporation of radioactivity into the various metab-

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1556 Medina-Bolivar and Flores Plant Physiol. Vol. 108, 1995

PFP (nM)

Figure 3. Effect of PFP on growth of PFP-sensitive and PFP-resistantcells and hairy roots of H. muticus. Fresh weight was determinedafter 21 d of culture and is indicated as a percentage of control flasks(no PFP). A, PFP-sensitive (HyBslO) and PFP-resistant (HyBs10.R1and HyBslO.R2) cell clones. B, PFP-sensitive (HyBslO andHyBs10-1) and PFP-resistant (HyBs10.R1-3 and HyBslO.R2-4) hairyroot clones.

olites was measured directly on the TLC plate with aBioscan System 200 Imaging Scanner (Bioscan Inc., Wash-ington, DC). Radioactive hyoscyamine and caffeoyl pu-trescine were identified by comparison to the RF values ofauthentic standards after visualization under UV light orwith the reagents described previously. Radioactivity inthese bands was further determined by scraping the bandsinto a scintillation vial containing 4 mL of Ecoscint A(National Diagnostics, Manville, NJ) and measuring themin a Beckman 5000 TA liquid scintillation spectrometer.Total recoveries of radioactivity ranged from 85 to 90%.

RESULTS

Formation of Hairy Roots and Cell-Suspension Cultures

Twelve hairy root clones of H. muticus were establishedby transformation with A. rhizogenes strain 15834. Fastgrowing, friable calli were obtained from all clones byculturing hairy roots in MSD. These cultures were thenused to initiate the cell-suspension cultures. Of all theroot-derived cell cultures tested, HyBslO was the first inwhich putative resistance to PFP was detected. The resis-tant trait was maintained in this clone after at least foursubcultures in PFP-free medium. Therefore, this clone wasused for root regeneration, subsequent characterization ofthe PFP-resistant cell and root cultures, and comparisonwith the parental root clone.

Selection for PFP-Resistant Cells

Two PFP-resistant cell clones (HyBslO.Rl andHyBslO.R2) were obtained by sequential subculture of the

parental root clone HyBslO in increasing concentrations ofPFP (up to 400 U.M). The resistant cells were able to grow atnearly normal rates in the presence of 400 /HM PFP, whereasgrowth of the parental cell clone was completely inhibitedunder these conditions (Fig. 3A). Both sensitive and resis-tant clones exhibited a friable phenotype and showed sta-ble growth for at least 15 months. The stability of thePFP-resistant trait was evaluated by subculturing the resis-tant clones for at least four passages in PFP-free mediumand then subculturing back into medium with 400 JU.M PFP.Cell clones that maintained the resistant characteristic werethen cloned from single cells (see "Materials and Meth-ods"). Single cell-derived colonies were then used in theroot-regeneration experiments. The cloning method hasalso allowed for screening of cells with higher resistance toPFP (F. Medina-Bolivar and H.E. Flores, unpublishedresults).

Regeneration of Hairy Roots and Their Sensitivity to PFP

Hairy roots were regenerated from both PFP-sensitiveand PFP-resistant cell clones. The typical hairy root phe-notype was recovered when the cells were first subculturedin medium containing 1 mg/L 2,4-D for at least 1 month,followed by subculture to hormone-free medium (Fig. 4).This specific transfer sequence was required for regenera-tion of "normal" hairy roots. When cell cultures were trans-ferred directly to MS, abnormal root tips resulted that didnot exhibit the original morphological phenotype. All re-generation media were supplemented with PFP to main-tain the selection pressure on the resistant phenotype dur-ing the differentiation process. Growth of the parentalhairy root clone (HyBslO) was completely inhibited by 100fjiM PFP (Fig. 3B). No differences in PFP resistance wereobserved between this clone and the one regenerated fromPFP-sensitive cells (HyBslO-1). Growth of root clones re-generated from PFP-resistant cells was reduced by approx-imately 50% of control at 100 /U.M PFP, indicating thatresistance to PFP was somewhat lower in hairy roots thanin the cells from which they were regenerated. However,

Figure 4. Hairy roots regenerated from PFP-resistant callus of H.muticus (clone HyBslO.R2). Hairy root-derived cell suspensionswere transferred to MSD followed by subculture on hormone-freemedium to induce root regeneration. www.plantphysiol.orgon April 4, 2019 - Published by Downloaded from

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Manipulation of Tropane Alkaloid Biosynthesis 1557

these regenerated roots were still clearly resistant to PFP as compared to the parental root clone.

Production of Caffeoyl Putrescine and Hyoscyamine in PFP-Sensitive and PFP-Resistant Cell Clones

Caffeoyl putrescine was the major cinnamoyl putrescine present in both PFP-sensitive and PFP-resistant cell clones. The levels of this compound were quantified during a time-course experiment in one PFP-sensitive cell clone (HyBslO) and one PFP-resistant cell clone (HyBslO.R2). The PFP-resistant cells accumulated approximately 2 times more caffeoyl putrescine than the PFP-sensitive cells (Fig. 5). The concentration of these putrescine conjugates tends to increase with age of the cell culture. Hyoscyamine was not detected in either PFP-sensitive or PFP-resistant cell clones, in agreement with previous results (Flores, 1987). Overproduction of caffeoyl putrescine has also been re- ported in tobacco cell cultures resistant to PFP (Berlin et al., 1982).

Production of Caffeoyl Putrescine and Hyoscyamine in PFP-Sensitive and PFP-Resistant Hairy Roots

The time course of caffeoyl putrescine and hyoscyamine levels in regenerated roots is shown in Figure 6. PFP- resistant hairy roots (clone HyBslO.R2-4) accumulated al- most twice as much caffeoyl putrescine than the PFP-sen- sitive clone during the first few days of culture (Fig. 6). The concentration of caffeoyl putrescine then decreased ap- proximately 45% from d 3 to 12 and stabilized after d 15. This pattern differs markedly from that found in the pa- rental PFP-sensitive and PFP-resistant cells, in which the concentration of caffeoyl putrescine increased with time (Fig. 5). The levels of caffeoyl putrescine found in the regenerated roots were, in general, lower than those found in the root-derived cell cultures.

The production of hyoscyamine showed the same gen- eral pattern in both PFP-sensitive and PFP-resistant hairy roots, namely, it remained low during the early stages of

HyBslOR2

PFP-resistant

HyBslO & PFP-sensitive

o ! O 10 2 0 30

Days

Figure 5. Time course of caffeoyl putrescine production in PFP- sensitive and PFP-resistant cell clones. Each point represents the average yield of three flasks. DW, Dry weight.

7

I A I ,

1

7 - 0

O O 1 0 2 0 30

Days

Figure 6. Time course of caffeoyl putrescine and hyoscyamine pro- duction in PFP-sensitive and PFP-resistant hairy roots. Each point represents the average yield of three flasks. A, PFP-sensitive hairy root clone HyBsl O-1 regenerated from PFP-sensitive cell clone HyBsl O. B, PFP-resistant hairy roots (clone HyBslO.R2-4) regener- ated from PFP-resistant cells (clone HyBslO.R2). DW, Dry weight.

growth and increased greatly as the root cultures reached a stationary phase (Fig. 6). However, PFP-resistant hairy root clones accumulated higher levels (nearly 2-fold) of hyoscy- amine than the PFP-sensitive clone (Figs. 6 and 7). No differences in hyoscyamine production were observed be- tween the parental root and the hairy roots regenerated from the PFP-sensitive cell clone.

Precursor Feeding Experiments

The results presented above suggest that the root cul- tures regenerated from the PFP-resistant cell clone have a greater ability to synthesize hyoscyamine. During the ex- ponential growth phase, these root cultures also show a clear inverse correlation between cinnamoyl putrescine and tropane alkaloid levels (Fig. 6). Therefore, we followed the course of label incorporation from ['4C]putrescine or [14C]Phe into these compounds in PFP-sensitive and PFP- resistant root cultures. Incorporation of [14CIputrescine into caffeoyl putrescine was higher than incorporation into hyoscyamine in both PFP-sensitive and PFP-resistant root cultures. The highest incorporation of putrescine into caf- feoyl putrescine was found in the 6-d-old cultures. In con- trast, the highest incorporation into hyoscyamine was ob-

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I 558 Medina-Bolivar and Flores

.Bl" 0 HyBslO

3

PFP-sensitive

F n p 2 - PFP-resistanl

Y E"

5 1 -

C a , - - 6 E .

.- PFP-sensilive

PFP-resistant v)

I

Root and cell clones

Figure 7. Hyoscyamine contents in the parenta1 hairy root clone (HyBsl O), root-derived PFP-sensitive and PFP-resistant cells, and regenerated PFP-sensitive and PFP-resistant hairy roots. Al l cultures were analyzed after 21 d of growth. DW, Dry weight.

served in the 18-d-old cultures. Putrescine incorporation into hyoscyamine and caffeoyl putrescine was significantly higher in the PFP-resistant roots than in the PFP-sensitive roots (Fig. 8). Incorporation of Phe followed a similar pat- tern to that of putrescine. No significant differences in label incorporation of [14C]Phe into hyoscyamine and caffeoyl putrescine were observed between the PFP-sensitive and PFP-resistant roots in any of the cultures (Fig. 9).

a, C o v)

.- FA PFP-resistant 2

a L 8 o

f 6 a 4

2

n

.I-

l- Y

6 12 18

Days

Figure 8. lncorporation of [ I ,4-'4C]putrescine into hyoscyamine (A) and caffeoyl putrescine (B) in PFP-sensitive (HyBsl O-1) and PFP- resistant (HyBsl0.R-2) hairy roots. The 6-, 12-, and 18-d-old cultured roots were incubated with the labeled putrescine and harvested after 24 h for hyoscyamine and caffeoyl putrescine determinations.

1.21 A

Plant Physiol. Vol. 108, 1995

1 - - 0.8 & 2 0.4

n

C

8 0 C .- L

PFP-sensitive B .- C 4 3.Q

tZd PFP-resistant

6 12 18 Days

Figure 9. lncorporation of [U-l4C1 Phe into hyoscyamine (A)and caf- feoyl putrescine (6) in PFP-sensitive (HyBs10-1) and PFP-resistant (HyBs1O.R-2) hairy roots. The 6-, 12-, and 18-d-old cultured roots were incubated with labeled Phe and analyzed after 24 h for hyo- scyamine and caffeoyl putrescine determinations.

DlSCUSSlON

We have described a method for enhancing hyoscyamine production in hairy root cultures based on selection for PFP resistance in hairy root-derived cell suspensions and sub- sequent regeneration of PFP-resistant cells into roots. Re- sistance to PFP in cells and roots was associated with an increased yield of cinnamoyl putrescines, particularly caf- feoyl putrescine. These results are consistent with those observed in PFP-resistant carrot and tobacco cells (Palmer and Widholm, 1975; Berlin et al., 1981). In carrot, resistance to PFP was associated with increased levels of free Phe, which possibly arose from an alteration in the Phe biosyn- thetic pathway (Berlin et al., 1981). However, in tobacco cells PFP resistance was correlated with higher activities of Arg decarboxylase, Orn decarboxylase, and Phe ammonia lyase (Berlin et al., 1982). Because these three enzymes are involved in the formation of cinnamoyl putrescines, their results suggest the coordinate regulation of putrescine and Phe pools to meet the demand for secondary metabolite synthesis. This could also apply to the biosynthesis of hyoscyamine in root cultures, which occurs from the same precursors.

The levels of caffeoyl putrescine showed distinct changes in cell and root cultures of H. muticus during time-course experiments. Concentrations of caffeoyl putrescine in- creased with the age of the cell culture. In contrast, in root cultures the compound showed an early peak and later decreased as the roots became older. The feeding experi- ments confirmed that labeled putrescine and Phe were mainly directed into the formation of caffeoyl putrescine in the young root cultures. Our results are consistent with the

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Manipulation of Tropane Alkaloid Biosynthesis 1559

report of Parr (1992) that showed that incorporation of putrescine into putrescine conjugates was highest at the initial stage of growth in root cultures of Datuva stvamonium.

Two main functions have been suggested for cinnamoyl putrescines in plants. Their presence seems to be correlated with flower development and male fertility (Cabanne et al., 1977). It has also been suggested that these compounds are involved in the hypersensitive response conferring resis- tance to pathogens (Sun et al., 1991). Di- or polyamines conjugated to cinnamic acids or their derivatives can form alkaloids with complex structures, some of which may be part of a defense mechanism or connected with ion trans- port (Meyer and Abdallah, 1980; Smith et al., 1983). It has been suggested that a putrescine-utilizing mutant cell clone of tobacco may metabolize putrescine to y-aminobutyric acid via a cinnamoyl putrescine intermediate (Flores and Filner, 1985). Similarly, our results suggest that cinnamoyl putrescines may be turned over in H. muticus hairy roots and possibly be used as a precursor for the biosynthesis of hyoscyamine. We are currently studying the incorporation of labeled caffeoyl putrescine into hyoscyamine to further explore the relation between the hyoscyamine and caffeoyl putrescine pathways.

The diversion of putrescine and Phe into both the cin- namoyl putrescine and tropane alkaloid pathways may represent a key regulatory point for the production of hyoscyamine. Previously, Parr (1992) indicated that two sites are important for the biosynthesis of tropane alka- loids: an early one at the partitioning of putrescine into primary versus secondary metabolites and another at the esterification of tropine. Since hyoscyamine and caffeoyl putrescine share biosynthetic precursors and the hyoscya- mine pathway is not expressed in cell suspensions, the precursors (putrescine and Phe) are likely diverted into the synthesis of caffeoyl putrescine. In roots, however, the same precursors seem to be partly diverted into hyoscya- mine. Our feeding experiments with PFP-sensitive and PFP-resistant hairy roots showed that putrescine incorpo- ration into hyoscyamine and caffeoyl putrescine was higher in the PFP-resistant clone. This suggests that pu- trescine pools may be limiting to hyoscyamine biosynthesis in H. muticus roots.

The ability to interconvert roots and cells in vitro has facilitated the selection for H . muficus metabolic variants discussed in this report. We propose that the same ap- proach could be applied to other root and cell cultures that express a diversity of metabolic pathways (Dhoot and Hen- shaw, 1977; Kurz and Constabel, 1985; Oksman-Caldentey and Strauss, 1986; Robins et al., 1991b; Sierra et al., 1991). For example, the pools of the amino acid Trp available for the synthesis of indole alkaloids could be increased by selecting for resistance to 5-methyltryptophan. Diversion of Trp into the alkaloid pathway could occur upon regen- eration of roots from the cell cultures. Severa1 indole alka- loids are known to occur in roots, such as the Cathavanthus alkaloids and camptothecin. In the Solanaceae, this ap- proach could also be directed toward enhancing the pro- duction of scopolamine. This tropane alkaloid is derived

from hyoscyamine via two oxidative reactions catalyzed by hyoscyamine-6P-hydroxylase (Yun et al., 1993). Scopol- amine production has been reported in root cultures of Brugmansia candida (Giulietti et al., 1993) and Datuva spp. (Kamada et al., 1986; Jaziri et al., 1988; Knopp et al., 1988). We have recently established hairy root cultures of B. candida and developed a root-cell-root interconversion pro- toco1 for Datuva spp. (F. Medina-Bolivar and H.E. Flores, unpublished observations). The application of the above selection protocol to these root cultures is underway.

In addition to tropane alkaloids, which are mostly con- stitutive intracellular secondary metabolites, hairy root cul- tures of H. muticus have been shown to produce and release sesquiterpenes (lubimin and solavetivone) upon elicitation with a funga1 extract of Rhizoctonia solani (Flores and Cur- tis, 1992; Reddy et al., 1993). Sesquiterpenes and tropane alkaloids share a common precursor, acetoacetate, which is used in the formation of both the tropine ring of hyoscya- mine (Leete, 1979), and in the synthesis of the isoprenoid unit of the sesquiterpenes (Cane, 1981). Our system can thus be used to study the regulation of precursor flow in both constitutive and inducible pathways.

ACKNOWLEDGMENTS

We wish to acknowledge Paula Michaels for her technical as- sistance. We also thank Laura Butterfield and Brett Savary for their helpful comments during the preparation of this manuscript.

Received February 3, 1995; accepted April24, 1995. Copyright Clearance Center: 0032-0889 /95 / 108/ 1553 / 08.

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