ute richter et al- overexpression of tropinone reductases alters alkaloid composition in atropa...

8/3/2019 Ute Richter et al- Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cult

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RESEARCH PAPER

Overexpression of tropinone reductases alters alkaloid

composition in Atropa belladonna root cultures

Ute Richter, Grit Rothe, Anne-Katrin Fabian, Bettina Rahfeld and Birgit Drager*

Institute of Pharmaceutical Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, D-06120Halle/Saale, Germany

Received 22 June 2004; Accepted 28 October 2004

Abstract

The medicinally applied tropane alkaloids hyoscy-

amine and scopolamine are produced in Atropa bella-

donna L. and in a small number of other Solanaceae.

Calystegines are nortropane alkaloids that derive from

a branching point in the tropane alkaloid biosynthetic

pathway. In A. belladonna root cultures, calystegine

molar concentration is 2-fold higher than that of hyo-

scyamine and scopolamine. In this study, two tropinone

reductases forming a branching point in the tropane

alkaloid biosynthesis were overexpressed in A. bella-

donna. Root culture lines with strong overexpression of

the transcripts contained more enzyme activity of the

respective reductase and enhanced enzyme products,

tropine or pseudotropine. High pseudotropine led to an

increased accumulation of calystegines in the roots.

Strong expression of the tropine-forming reductase was

accompanied by 3-fold more hyoscyamine and 5-fold

more scopolamine compared with control roots, and

calystegine levels were decreased by 3090%of control.

In some of the transformed root cultures, an increase of

total tropane alkaloids was observed. Thus, transform-

ation with cDNA of tropinone reductases successfully

altered the ratio of tropine-derived alkaloids versus

pseudotropine-derived alkaloids.

Key words: Atropa belladonna, calystegine, gene transform-

ation, hyoscyamine, overexpression, scopolamine, tropane

alkaloids, tropinone reductase.

Introduction

The manipulation of metabolite flux in secondary meta-

bolism in order to enhance or decrease individual products

is a major goal of plant biotechnology. Tropane alkaloids of

medicinal application, such as hyoscyamine and scopol-

amine are found in a limited number of solanaceous plants.

They are obtained for industrial use predominantly frommembers of the genera Atropa, Datura, Hyoscyamus, and

Duboisia. These plants contain additional tropane alka-

loids, calystegines, which are characterized by the loss of

the methyl group on the bridge nitrogen and by three to five

hydroxyl groups on the tropane skeleton (Fig. 1). In con-

trast to hyoscyamine and scopolamine, calystegines are not

esterified. The biosynthesis of calystegines requires the first

enzymatic steps of the general tropane alkaloid pathway

(Draeger, 2004). Reduction of tropinone to the stereo-

isomeric alcohols tropine and pseudotropine constitutes

the diversion of calystegine and tropine ester alkaloid

formation.Roots are the major organs of tropane alkaloid bio-

synthesis. Root cultures of Atropa belladonna L. form the

tropine-derived alkaloids hyoscyamine (9 lmol g1 drymass) and scopolamine (1 lmol g1 dry mass). Calyste-gines accumulate in these root cultures at two to three times

the molar concentration of the ester alkaloids (Rothe et al.,

2003). Two tropinone reductases responsible for tropane

alcohol formation have been isolated and characterized

from root cultures of several species, Datura stramonium L.(Nakajima et al., 1993b, 1998; Portsteffen et al., 1994,

1992) , Hyoscyamus nigerL. (Draegeret al., 1988; Hashimoto

et al., 1992), and Atropa belladonna (Draeger and Schaal,

1994). Each reductase was found to be strictly stereospe-cific. cDNAs coding for both tropinone reductases were

cloned from D. stramonium (Nakajima et al., 1993b)

and from H. niger (Nakajima et al., 1993a; Nakajima

and Hashimoto, 1999). In wild-type roots, pseudotropine

as a product of tropinone reduction undergoes a rapid

* To whom correspondence should be addressed. Fax: +49 345 55 27021. E-mail: [email protected]: H6H, hyoscyamine-6b-hydroxylase; PMT, putrescine N-methyltransferase; TRI, tropine-forming tropinone reductase; TRII, pseudotropine-forming tropinone reductase.

Journal of Experimental Botany, Vol. 56, No. 412, Society for Experimental Biology 2005; all rights reserved

Journal of Experimental Botany, Vol. 56, No. 412, pp. 645652, February 2005

doi:10.1093/jxb/eri067 Advance Access publication 10 January, 2005


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conversion to calystegines, while the other product tropineis esterified to yield hyoscyamine and scopolamine (Rothe

et al., 2001). Therefore it appears possible that individual

enhancement of the tropane alcohol levels would enhancethe levels of the respective end-products.

It is shown here that overexpression of TRI or TRII

resulted in higher enzyme activity of either type of re-

ductase and also in an increase in the formation of the

respective enzyme products tropine or pseudotropine.

Overexpression of the tropinone reductase I or II shifted

the ratio of tropine-derived products versus pseudotropine-

derived products.

Materials and methods

Transformation and root cultures

The cDNA of tropinone reductases I or II from D. stramonium

(EMBL accession numbers L20473, L20474) were cloned into thebinary plasmid pBI121 (Jefferson et al., 1987) carrying a kanamycinresistance gene, a b-glucuronidase (GUS) reporter gene, and theCaMV 35S-promotor. Insertion of cDNA for tropinone reductases Ior II using the restriction sites BamHI and SacI removes the GUS

gene. The plasmids were transformed into competent cells of Agro-bacterium rhizogenes strain 15834 as described (Hoefgen andWillmitzer, 1988). Different leaves from different plants of thewild-type A. belladonna were shaken three times for 5 min ina suspension of A. rhizogenes, which contained the TRI- or TRII-plasmid for transformation. The inoculated leaves were cultured onsolid MS-medium (Murashige and Skoog, 1962) containing 1.5%agar, Gamborgs B5 vitamins (Gamborg et al., 1968), 50 mg l

1

kanamycin, 500 mg l1

carbenicillin, and 0.1 mg l1

1-naphthylacetic acid at 23 8C. Roots developed on the leaves after 12 months.They were separated and cultured in Gamborgs B5 liquid mediumcontaining 20 mg l

1kanamycin and 250 mg l

1carbenicillin to

remove agrobacteria. Vector control root cultures carried the pBI 121plasmid with intact GUS gene transferred by A. tumefaciens. Rootcultures of wild-type and of vector control were taken from sterile

plants. Initially 20 trI-transformed and 29 trII-transformed rootculture lines were obtained from independent transformation events.Successful transformation was inferred by the growth on kanamycinand was confirmed by PCR and by dot blot analysis. The root culturesgrew with different morphology. Some lines grew as short, thick, andslightly translucent roots; most other lines had typical hairy rootsappearance with thin long roots and many side branches. Root culturelines were cultivated in 300 ml conical flasks in 70 ml Gamborgs B5medium with 40 mg l

1ampicillin on a gyratory shaker (100 rpm,

22 8C) in the dark and routinely subcultured after 28 d. For eachmeasurement three flasks were harvested. Roots were blotted dry forthe determination of fresh mass and dried to constant mass before drymass measurement.

PCR for plasmid insertion

Genomic DNA was isolated from 14-d-old root cultures. Plasmidinsertion was tested by PCR with two different primer pairs for eachkind of transformation. The following primers were used to proveTRI transformation: CaMV 35S forward 59-AAA CCT CCT CGGATT CCA-39; TRI reverse (1) 59-TTC CTT ATG TAT CAC CACCC-39 and TRI forward 59-ATG GAA GAA TCA AAA GTG TCC-39; TRI reverse (2) 59-TTA AAA CCC ACC ATT AGC TGT-39.DNA of TRII transformants was amplified with these four primers:CaMV 35S forward 59-AAA CCT CCT CGG ATT CCA-39; TRIIreverse (1) 59-TTT GAA CCC CTT ACT TCT CC-39 and TRIIforward 59-ATG GCT GAA GGT GAA T-39; TRII reverse (2) 59-ACA ATT AGC CAT A AG TCC ACC-39. PCR amplification wasperformed with 40 cycles (1 min 95 8C; 1 min primer annealingtemperature, 1 min 72 8C). The amplified fragments were separated ina 1.5% agarose gel, controlled for the expected size and confirmed to

be correct by nucleotide sequencing (ABI Prism 377 DNA Se-quencer, Perkin Elmer).

Northern blot and dot blot

For northern blot, 14-d-old root cultures were harvested and imme-diately frozen in liquid nitrogen. Total RNA was isolated withTRIzol Reagent (Gibco BRL at Invitrogen). RNA (20 lg) wasseparated in a 1.2% formaldehyde-agarose gel and blotted ontoa Hybond N

+membrane (Amersham).

For dot blot, genomic DNA of each root line (0.5, 1, 2, and 5 lg)was directly applied onto a nylon membrane (Hybond N

+,

NCH3

O

TRIITRI

PMT

putrescine

NH2NH2

N

CH3

OH

NCH3

OH

N - methylputrescine

tropinone

tropine pseudotropine

NH2NH

CH3

N

CH3

O tropic acidhyoscyamine calystegine A3

HN

OH

HOOH

O

N

CH3

O tropic acid

scopolamine

HN

HOOH

OH

calystegine A5

HN

OH

HOOH

OH

calystegine B2

H6H

Fig. 1. Reduction of tropinone as branch point in the formation oftropane alkaloids. PMT, putrescine N-methyltransferase; TRI, tropine-forming tropinone reductase; TRII, pseudotropine-forming tropinonereductase; H6H, hyoscyamine 6b-hydroxylase.

646 Richteret al.


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Amersham). DNA was fused to the membrane by UV cross-linking.The membrane was incubated for 5 min each in denaturation buffer(1.5 M NaCl, 0.5 M NaOH), neutralization buffer (1.5 M NaCl,0.5 M TrisHCl pH 7.0), and 23 SSC (0.3 M NaCl, 0.03 M Na-citratepH 7.0).

Dot blot and northern blot membranes were hybridized for 16 h at42 8C in a buffer containing 50% formamide, 53 SSC, 53 Denhardtssolution, 1% SDS, 10% dextran sulphate, and 50 lg ml1 herringsperm DNA. Dot blots were hybridized with a [a-32P]dATP-labelled

DNA probe of the CaMV 35S promoter. Northern blots werehybridized with the [a-32P]dATP labelled double-stranded cDNA ofTRI or TRII fromD. stramonium. Blots werewashed four times underthe following conditions: 5 min, room temperature, 23 SSC, 0.1%SDS; 30 min, 58 8C, 13 SSC, 0.1% SDS; 30 min, 58 8C, 0.23 SSC,0.1% SDS; 15 min, 60 8C, 0.13 SSC, 0.1% SDS. Equal loading ofRNA was shown by rehybridization with a 18S rRNA probe from

Lycopersicon esculentum (Dobrowolski et al., 1989).

Alkaloid analysis

For alkaloid analysis 31-d-old root cultures were harvested anddivided into three portions for individual determination of tropaneester alkaloids, calystegines, and tropane alcohols. Fresh mass anddry mass were determined. Hyoscyamine, scopolamine, and calyste-gines were analysed as described by Rothe and Draeger (2002) usingthe GC-column described below. Tropinone, tropine, and pseudo-tropine were extracted twice with 10 ml g

1fresh mass MeOH/H2O

(1:1 v:v). Extracts were evaporated and adjusted to 1 ml with water.After the addition of 50 ll of 26% ammonia, extracts were applied toan Extrelut

column (Merck). After 20 min incubation on the

column, alkaloids were eluted with 234 ml CHCl3 and 4 mlCHCl3/MeOH (9:1 v:v). The eluates were concentrated and dissolvedin a total volume of 150 ll ethyl acetate. Samples were analysed bygas chromatography: HP5 column (30 m3320 lm30.25 lm),stationary phase: methylsiloxan 95%, phenylsiloxan 5%; mobilephase: helium, injection: pulsed splitless. Temperature programme:start: 65 8C, 10 8C min

1up to 120 8C, hold 2 min, 10 8C min

1up to

240 8C, hold 1 min.

Enzyme activity

Fourteen-day-old root cultures were harvested, frozen in liquidnitrogen, ground in a mortar with a suspension of 40% insolublepolyvinylpyrrolidone and extracted with a buffer containing 0.1 Mpotassium phosphate pH 7.8, 0.25 M sucrose, 3.0 mM DTT, 1.0 mMEDTA, and 10 mg g

1fresh mass ascorbic acid. Tropinone

reductases were precipitated in 4075% saturation of ammoniumsulphate. The protein pellet was dissolved in buffer (0.02 Mpotassium phosphate pH 7.0, 0.25 M sucrose, 1.0 mM DTT, and25% glycerol). Total tropinone reductase activity was measuredspectrophotometrically with 5 mM tropinone as substrate and 0.2 mMNADPH as co-substrate in 0.1 M potassium phosphate buffer pH 6.2.The decrease of NADPH absorption was determined at 340 nm at30 8C. The blank assay contained the same mixture without tropinone.Specific TRI activity was determined with 5 mM 3-quinuclidinone.Protein content was measured in the 75% ammonium sulphate pellet(Bradford, 1976) with bovine serum albumin as the standard protein.

Enzyme product determination

Enzyme reduction products of 5 mM tropinone were accumulatedwith root protein extracts (see above) and a NADPH regeneratingsystem using 1 mM glucose-6-phosphate, 0.5 mM NADP, and 2 unitsglucose-6-phosphate dehydrogenase. The reaction was stopped with26% ammonia after 2 h incubation at 30 8C. The mixture was appliedto an Extrelut

column (Merck). Tropinone, tropine, and pseudo-

tropine were analysed by gas chromatography as described above.

Results

Selection of transformed root lines

All root cultures, 20 trI-transformed lines, 29 trII-transformed

lines, vector controls, and wild type, were examined for

their alkaloid production. A broad range of increased and

decreased levels of individual alkaloids was observed. In

the trI-transformants, accumulation of tropine was seen aswell as enhanced levels of hyoscyamine and scopolamine.

In many of the trII- transformants, calystegine levels were

enhanced, and in some of the cultures the metabolite

pseudotropine accumulated (data not shown). Based on

the alkaloid content obtained for all the transformed root

cultures, six root culture lines containing the trI insert and

representing the range of transformants, as well as eight

lines representing the trII transformation results, together

with a non-transformed and an empty vector-transformed

control culture line were chosen for detailed examination.

All selected cultures displayed typical hairy root growth

characteristics.

Transcript levels of trI and trII

All root lines that were selected had incorporated the cDNA

of TRI or TRII, however, the transcription of the transgene

varied. Strong expression of trIwas repeatedly observed in

four root lines, 1-2, 1-3, 1-5 as well as 1-6, and the lines 2-6,

2-7, and 2-8 showed the strongesttrIItranscription (Fig. 2).

Root cultures of Datura stramonium, known for their high

constitutive expression of tropinone reductase I (Portsteffen

et al., 1994), were taken as a positive control and gave

a weak signal with the trI probe, while wild-type and the

empty vector control of A. belladonna gave no visible

signal with either probe. The control roots contain active

tropinone reductases (see below), but transcript con-centrations were obviously too low to be detected. In

transgenic root cultures with no visible transcript level, 1-1,

1-4, 2-1, and 2-2, it cannot be decided whether additional

mRNA is present. Clearly, the culture lines with a distinct

signal on the northern blot can be considered as strongly

overexpressing the respective transgene.

Tropinone reductase activity

In many, but not all, transformed root cultures, biomass was

enhanced compared with non-transformed wild-type roots.

Agrobacterium rhizogenes line 15834 contains the Ri-

plasmid that confers higher auxin formation and enhanced

auxin sensitivity and led to faster growth in some of thetransformed root cultures (Fig. 3). Wild-type roots for

comparison were cultured without auxin addition.

For individual measurement of TRI and TRII activity,

purification and chromatographic separation of TRI and

TRII from A. belladonna root tissue is possible. Enzyme

activity after chromatographic separation, however, does

not reflect the level of activity in the tissue because of the

instability of the TRI enzyme. Genuine TRI activity may be

Overexpression of tropinone reductases 647


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distinguished from total TR activity in protein extracts aftera fast partial purification using 3-quinuclidinone as sub-

strate. 3-Quinuclidinone is reduced by TRI with similar

velocity as tropinone (Boswell et al., 1999; Hashimoto

et al., 1992; Portsteffen et al., 1994), but not by A.

belladonna TRII (Draeger and Schaal, 1994) or by any

other TRII tested. The specific TRI activity calculated from

3-quinuclidinone reduction is given in Fig. 4 together with

the total TR activity measured with tropinone as substrate.

TRII activity can be determined as the difference between

total TR activity and specific TRI activity. The content of

extractable protein per gram biomass in trI-transformants

was higher in general (0.40.75 mg g1 fresh mass) than in

trII- and in empty vector-transformed roots (0.10.25 mgg1 fresh mass); no explanation can be given for this

observation yet. Irrespectively, whether tropinone reduc-

tase activity was based on root biomass or extractable

protein mass, it was significantly enhanced in four trI-

transformants 1-2, 1-3, 1-5, and 1-6 corresponding to

higher trI transcript seen by northern blotting. Only one

root line, 2-6, of the trII-transformants had higher tropinone

reductase activity than the control cultures. In the other trII-

transformants, total tropinone reductase activity was de-

creased to less than half of the control level, in spite of

considerable mRNA levels in some lines seen on the

northern blot. For the root lines, where enzyme activity

was decreased and no tr transcript was observed, cosup-pression would appear as a possible explanation, provided

that more sensitive mRNA detection will allow transcript

monitoring in wild-type roots for comparison. It is evidentthat in the trI-transformed lines 1-2, 1-3, and 1-6, the

majority of the elevated tropinone reductase activity can be

attributed to TRI. The transgene is obviously expressed and

translated into active protein in these cultures. Control

cultures and trII-transformants show no 3-quinuclidinone

reduction, but must be assumed to contain TRI activity, as

they form tropine and hyoscyamine. The instability of the

TRI enzyme after extraction is the most probable reason for

the loss of small amounts of activity. Directly after

extraction soluble protein was incubated with tropinoneand a NADPH regenerating system (Fig. 5). More than 85%

of total tropinone was reduced to tropine by protein

preparations from root line 1-2. The root lines 1-3, 1-5,

and 1-6 also showed considerable tropine formation, while

in all other root lines pseudotropine was the major product,

and total product formation consisted of less than 5% of

total tropinone. Root line 2-6 formed slightly more pseudo-

tropine than the control root extracts corresponding to the

Fig. 2. Accumulation oftrI- and trII-transcripts in root culture lines of A. belladonna. Probes were labelled with cDNA of tropinone reductase I and oftropinone reductase II, respectively. Rehybridization with 18S rRNA of Lycopersicon esculentum served as loading control. WT, wild-type; VC, vectorcontrol; Ds, Datura stramonium RNA as positive control; lines 1-1 to 1-6 transformed with trI; lines 2-1 to 2-8 transformed with trII.

0

2

4

6

8

10

12

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC WT

fresh mass in gram per flask

Fig. 3. Root biomass of culture lines of Atropa belladonna after 28 d.Lines 1-1 to 1-6 transformed with tropinone reductase I. Lines 2-1 to 2-8transformed with tropinone reductase II. VC, root line transformed withempty vector; WT, wild-type, non-transformed root line; error bars:standard deviation, n=3.

648 Richteret al.


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higher TRII activity in the enzyme assay. In summary, root

lines 1-2, 1-3, 1-5, and 1-6 clearly express more TRI

activity, while within the trII-transformants only root line

2-6 appears to be enhanced in reductase activity.

Alkaloids and alkaloid precursors

The levels of tropinone, tropine, and pseudotropine are usually

low in root cultures of A. belladonna (Rothe et al., 2001).

Tropinone hardly accumulates to concentrations higher than

0.2lmol g1 dry mass; tropine and pseudotropine are directly

incorporated into esterified alkaloids and calystegines. Thetropinone content of 1.2 lmol g1 dry mass in root line 2-6 isextraordinary and may be seen in relation to the strikingly high

tropine content of the root line (Fig. 6A). The trI-transformed

root lines 1-2, 1-3, 1-5, and 1-6 displayed higher tropine

content and decreased pseudotropine. Root lines 1-1 and 1-4,

by contrast, contained decreased tropane alcohols correspond-

ing to the decreased tropinone reductase activity (Figs 4, 5).

Pseudotropine was 4.5 times higher than control levels in trII-

transformed root line 2-6. The accumulation of alkaloid end-

products in the transformed root lines reflects the expression

levels andenzyme activity of thetwo tropinone reductases(Fig.

6B). Significantly more hyoscyamine was accumulated in the

trI-transformed root lines 1-2, 1-3, 1-5, and 1-6. Root line 1-4also contained slightly more hyoscyamine, although trItran-

script levels and in vitro enzyme activities were low. Scopol-

amine also increased up to 5-fold in the trI-transformed root

lines, indicating either that the activity of hyoscyamine 6b-hydroxylase was enhanced or that more hyoscyamine was

available for the enzyme. In addition, these root lines contained

12 lmol 6-hydroxyhyoscyamine g1 dry mass (not shown).

The hyoscyamine 6b-hydroxylase protein comprises two

enzymatic activities, hydroxyl transfer to hyoscyamine and

dehydration to scopolamine. The hydroxylase activity may be

2050-fold stronger than the epoxidase activity (Yun et al.,

1992), leading to release of 6-hydroxyhyoscyamine in tissues,

where high hyoscyamine concentrations are available or added

from outside (Rochaetal., 2002). The major calystegines inA.

belladonna are calystegine A3, A5, and B2. The usual ratio in

root cultures is 5:1:1 (A3:A5:B2). This ratio remained unaltered

in the trI-transformed root lines, irrespective of the total

calystegine concentration. In the trII-transformed root lines,the ratio was shifted in favour of calystegine B2(A3:A5:B2=5.4:1:2). Root line 2-6 contained an extraordinary

high calystegine concentration, indicating thattrIIoverexpres-

sion led to enhanced pseudotropine, which was converted into

calystegines. Hyoscyamine appeared unaltered, although the

tropine levels in this root line were very high as well (Fig. 6).

Discussion

Products of the tropine-forming tropinone reductase I,

hyoscyamine and scopolamine, are important pharmaceuti-

cal and source compounds for derivative synthesis. It has

been shown here for the first time that an augmentation ofthe TRI-products in Atropa belladonna is possible by

directing the metabolite tropinone into the tropine-branch

of the pathway. Transformation with cDNA of TRI suc-

cessfully altered the ratio of tropine-derived alkaloids versus

pseudotropine-derived alkaloids (Fig. 6C). While control

and empty vector-transformed roots contain twice as much

TRII-products as TRI-products, there is an obvious pre-

ponderance of TRI-products in trI-transgenic root cultures.

0

200

400

600

800

1000

1200

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC WT

total reductase activity

specific TRI activity

nkat / g protein

Fig. 4. Tropinone reductase activity in root culture lines of Atropabelladonna. Total tropinone reductase activity was measured photomet-rically by NADPH consumption with tropinone as substrate. Specific TRIactivity was measured with 3-quinuclidinone as substrate.Lines 1-1 to 1-6transformed with tropinone reductase I. Lines 2-1 to 2-8 transformed with

tropinone reductase II. VC, root line transformed with empty vector; WT,wild-type, non-transformed root line; error bars: standard deviation, n=3.

0

5

10

15

20

25

30

35

40

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC

% tropine

% pseudotropine

85,3%

WT

Fig. 5. Products of tropinone reduction in protein extracts from rootculture lines ofAtropa belladonna. 5 mM tropinone were incubated withprotein extracts and a NADPH regenerating system. Reduction products,measured by gas chromatography, are expressed as a percentage oftropinone in the assay. Lines 1-1 to 1-6 transformed with tropinone

reductase I. Lines 2-1 to 2-8 transformed with tropinone reductase II. VC,root line transformed with empty vector; WT, wild-type, non-transformedroot line.



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Preceding experiments for altering the tropane alkaloidpathway were performed with different enzymes. Until

today, four enzymes of the pathway have been cloned in

total, putrescine N-methyltransferase PMT (Suzuki et al.,

1999a), hyoscyamine-6-hydroxylase H6H (Matsuda et al.,

1991), and the two tropinone reductases examined here

(Nakajimaet al., 1993b). Overexpression in tropane alkaloid-

producing plant species has been attempted before for PMT

and H6H. PMT overexpression was studied in root cultures

and intact plants of A. belladonna (Rothe et al., 2003;

Sato et al., 2001). Although the PMT enzyme product N-

methylputrescine was augmented in root cultures and intact

plants, the levels of hyoscyamine and scopolamine were not

increased. PMT was also overexpressed in root cultures of

Datura metel, Hyoscyamus muticus (Moyano et al., 2003)

and in aDuboisia hybrid (Moyano et al., 2002). Increase in

total alkaloid production was 2-fold in H. muticus, but notobserved for D. metel or the Duboisia hybrid. It was

concluded that PMT expression alone is not sufficient to

exert a major influence on alkaloid production, and that

subsequent steps in the alkaloid biosynthesis are regulatory.

Overexpression results for H6H differed, largely depending

on the plant chosen for transformation. A. belladonna-

regenerated plants transformed with H6H contained more

than 80% scopolamine in the leaf alkaloids, some of theplants in addition formed more total alkaloids (Yun et al.,

1992). H6H overexpression in root cultures of H. muticus

cultures resulted in a maximal scopolamine content of c.

10% of that of hyoscyamine (Jouhikainen et al., 1999). A

Duboisia hybrid wild-type plant that contained about 80%scopolamine and 20% hyoscyamine retained an almost

similar alkaloid content after H6H transformation and plant

regeneration (Palazon et al., 2003). Increased formation of

total tropane alkaloids upon transformation was observed in

some of the transformants, when H6H was introduced into

H. muticus-cultured roots (Jouhikainen et al., 1999). In A.

belladonna intact plants, 34-fold higher total leaf alkaloid

level was striking (Yun et al., 1992). In these studies, only

the products derived from TRI were monitored; calyste-

gines were not included in the alkaloids measurements.

For the A. belladonna root culture lines, TRI and TRII

product quantities are given. Enhanced pseudotropine

formation is directly connected with enhanced calysteginelevels, pseudotropine as a metabolite does not necessarily

accumulate. Enhanced tropine formation also caused higher

hyoscyamine and scopolamine levels, but only 23-fold

higher than in control roots, and tropine accumulated

additionally. Presumably, esterification and formation of

the tropic acid moiety is a limiting factor for the total

hyoscyamine formation as well as tropine availability.

Enhanced tropine formation without equivalent hyoscya-

mine accumulation was shown as a result of methyl jas-

monate elicitation in root cultures of D. stramomium(Zabetakis et al., 1999). Different localization of enzymes

and precursors of tropane alkaloid formation in the root

tissues has been demonstrated. While PMT and H6H arerestricted to the pericycle of a short region of young roots of

A. belladonna (Suzuki et al., 1999a, b), tropinone reduc-

tases in the roots ofH. niger are located in the endodermis

and outer cortex (TRI) or in the pericycle, endodermis, and

inner cortex (TRII) (Nakajima and Hashimoto, 1999). The

tissues of tropinone formation and of esterification on the

way to hyoscyamine are not known, but the necessity for

metabolite transport in alkaloid biosynthesis becomes

0

20

40

60hyoscyamine

scopolamine

calystegines

122+/-18B

0

20

40

60

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC WT

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC WT

tropinone

tropine

pseudotropine

mol / dm

mol / dm

77.7+/-12A

0

1

2

3

4

5ratio TRI: TRII derived alkaloidsC

1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 VC WT

Fig. 6. Alkaloids and their precursors in 31-d-old root culture lines of Atropa belladonna. All compounds were measured by gas chroma-tography and expressed as lmol g

1 dry mass. Lines 1-1 to 1-6transformed with tropinone reductase I. Lines 2-1 to 2-8 transformed withtropinone reductase II. VC, root line transformed with empty vector; WT,wild-type, non-transformed root line. (A) Precursors of tropane alkaloids,error bars: standard deviation, n=3. (B) Alkaloids, error bars: standarddeviation, n=3. (C) Ratio between TRI-derived alkaloids versus TRII-derived alkaloids.

650 Richteret al.


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evident. Thus, local precursor availability can differentially

restrict the end-product formation, and also if more tropane

alcohol metabolites are formed by overexpression of the

tropinone reductases. In root culture lines transformed with

TRI, a strong increase in TRI-products was observed, but

only a moderate increase of total tropane alkaloid accumu-

lation. This implies that with increased TRI-products a de-

crease in calystegines occurred simultaneously, forexample, in line 1-3. In the root line 2-6, however, total

alkaloids were 233 lmol g1 dry mass, by far the highestcontent of all root cultures. The root line clearly overex-

pressed TRII, but at the same time accumulated tropine; the

ratio of alkaloids remained unchanged. It is assumed that

enforcement of tropinone formation led to the general

increase in alkaloids.

These observations enforce the concept that not onlyprecursors or the first specific metabolites regulate the flux

through the pathway, but also that the enhanced activity of

the later metabolic enzymes may cause an augmentation of

total end-products. Strong total alkaloid augmentation after

simultaneous transformation of H. niger-root cultures withPMT and H6H is impressive (Zhang et al., 2004). Such

experiments help to fathom the overall capacity of plant

tissues for alkaloid biosynthesis, transport, and storage.

Obviously, a multitude of interactive regulatory parameters

determines the total alkaloid formation, of which the

expression level of each individual enzyme is only one of

the multiple influences.

The impact of calystegine accumulation in A. belladonna

and other Solanaceae is still unknown. It will be of interest

to examine more TRII-transformants to see whether TRII-

products and calystegines may be increased specifically and

to what extent. Regeneration of intact plants must be

attempted as the next step in the investigation of tropanealkaloid pathway regulation and for an insight into the

metabolic role of calystegines in the plant. If successful TRI

overexpression is accompanied by a loss of calystegines,

the consequences for the whole plant performance must be

carefully examined.

Acknowledgements

We thank Professor Takashi Hashimoto, Nara Institute of Scienceand Technology, for the cDNA of Datura stramonium TRs and formany useful hints and comments at the beginning of this work. Theexcellent technical assistance of Anja Wodak, Ursula Kodel, andBeate Schone is gratefully acknowledged. The authors thank Dr LoreWestphal, Institute of Plant Biochemistry, Halle, for criticallyreading the manuscript. The work was financially supported by theDeutsche Forschungsgemeinschaft (grant to BD).

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