TY - JOUR
AU - Dräger, Birgit
AB - Abstract The medicinally applied tropane alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna 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 hyoscyamine and scopolamine. In this study, two tropinone reductases forming a branching point in the tropane alkaloid biosynthesis were overexpressed in A. belladonna. 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 30–90% of control. In some of the transformed root cultures, an increase of total tropane alkaloids was observed. Thus, transformation with cDNA of tropinone reductases successfully altered the ratio of tropine-derived alkaloids versus pseudotropine-derived alkaloids. Atropa belladonna, calystegine, gene transformation, hyoscyamine, overexpression, scopolamine, tropane alkaloids, tropinone reductase Introduction The manipulation of metabolite flux in secondary metabolism in order to enhance or decrease individual products is a major goal of plant biotechnology. Tropane alkaloids of medicinal application, such as hyoscyamine and scopolamine are found in a limited number of solanaceous plants. They are obtained for industrial use predominantly from members of the genera Atropa, Datura, Hyoscyamus, and Duboisia. These plants contain additional tropane alkaloids, 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 contrast 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. Fig. 1. View largeDownload slide Reduction of tropinone as branch point in the formation of tropane alkaloids. PMT, putrescine N-methyltransferase; TRI, tropine-forming tropinone reductase; TRII, pseudotropine-forming tropinone reductase; H6H, hyoscyamine 6β-hydroxylase. Fig. 1. View largeDownload slide Reduction of tropinone as branch point in the formation of tropane alkaloids. PMT, putrescine N-methyltransferase; TRI, tropine-forming tropinone reductase; TRII, pseudotropine-forming tropinone reductase; H6H, hyoscyamine 6β-hydroxylase. Roots are the major organs of tropane alkaloid biosynthesis. Root cultures of Atropa belladonna L. form the tropine-derived alkaloids hyoscyamine (9 μmol g−1 dry mass) and scopolamine (1 μmol g−1 dry mass). Calystegines 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 niger L. (Draeger et al., 1988; Hashimoto et al., 1992), and Atropa belladonna (Draeger and Schaal, 1994). Each reductase was found to be strictly stereospecific. 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 conversion to calystegines, while the other product tropine is esterified to yield hyoscyamine and scopolamine (Rothe et al., 2001). Therefore it appears possible that individual enhancement of the tropane alcohol levels would enhance the 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 reductase 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 the binary plasmid pBI121 (Jefferson et al., 1987) carrying a kanamycin resistance gene, a β-glucuronidase (GUS) reporter gene, and the CaMV 35S-promotor. Insertion of cDNA for tropinone reductases I or II using the restriction sites BamHI and SacI removes the GUS gene. The plasmids were transformed into competent cells of Agrobacterium rhizogenes strain 15834 as described (Hoefgen and Willmitzer, 1988). Different leaves from different plants of the wild-type A. belladonna were shaken three times for 5 min in a suspension of A. rhizogenes, which contained the TRI- or TRII-plasmid for transformation. The inoculated leaves were cultured on solid MS-medium (Murashige and Skoog, 1962) containing 1.5% agar, Gamborg's B5 vitamins (Gamborg et al., 1968), 50 mg l−1 kanamycin, 500 mg l−1 carbenicillin, and 0.1 mg l−1 1-naphthyl acetic acid at 23 °C. Roots developed on the leaves after 1–2 months. They were separated and cultured in Gamborg's B5 liquid medium containing 20 mg l−1 kanamycin and 250 mg l−1 carbenicillin to remove agrobacteria. Vector control root cultures carried the pBI 121 plasmid with intact GUS gene transferred by A. tumefaciens. Root cultures of wild-type and of vector control were taken from sterile plants. Initially 20 trI-transformed and 29 trII-transformed root culture lines were obtained from independent transformation events. Successful transformation was inferred by the growth on kanamycin and was confirmed by PCR and by dot blot analysis. The root cultures grew with different morphology. Some lines grew as short, thick, and slightly translucent roots; most other lines had typical hairy roots appearance with thin long roots and many side branches. Root culture lines were cultivated in 300 ml conical flasks in 70 ml Gamborg's B5 medium with 40 mg l−1 ampicillin on a gyratory shaker (100 rpm, 22 °C) in the dark and routinely subcultured after 28 d. For each measurement three flasks were harvested. Roots were blotted dry for the determination of fresh mass and dried to constant mass before dry mass measurement. PCR for plasmid insertion Genomic DNA was isolated from 14-d-old root cultures. Plasmid insertion was tested by PCR with two different primer pairs for each kind of transformation. The following primers were used to prove TRI transformation: CaMV 35S forward 5′-AAA CCT CCT CGG ATT CCA-3′; TRI reverse (1) 5′-TTC CTT ATG TAT CAC CAC CC-3′ and TRI forward 5′-ATG GAA GAA TCA AAA GTG TCC-3′; TRI reverse (2) 5′-TTA AAA CCC ACC ATT AGC TGT-3′. DNA of TRII transformants was amplified with these four primers: CaMV 35S forward 5′-AAA CCT CCT CGG ATT CCA-3′; TRII reverse (1) 5′-TTT GAA CCC CTT ACT TCT CC-3′ and TRII forward 5′-ATG GCT GAA GGT GAA T-3′; TRII reverse (2) 5′-ACA ATT AGC CAT A AG TCC ACC-3′. PCR amplification was performed with 40 cycles (1 min 95 °C; 1 min primer annealing temperature, 1 min 72 °C). The amplified fragments were separated in a 1.5% agarose gel, controlled for the expected size and confirmed to be correct by nucleotide sequencing (ABI Prism 377 DNA Sequencer, Perkin Elmer). Northern blot and dot blot For northern blot, 14-d-old root cultures were harvested and immediately frozen in liquid nitrogen. Total RNA was isolated with TRIzol® Reagent (Gibco BRL at Invitrogen). RNA (20 μg) was separated in a 1.2% formaldehyde-agarose gel and blotted onto a Hybond™ N+ membrane (Amersham). For dot blot, genomic DNA of each root line (0.5, 1, 2, and 5 μg) was directly applied onto a nylon membrane (Hybond™ N+, 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 Tris–HCl pH 7.0), and 2× SSC (0.3 M NaCl, 0.03 M Na-citrate pH 7.0). Dot blot and northern blot membranes were hybridized for 16 h at 42 °C in a buffer containing 50% formamide, 5× SSC, 5× Denhardt's solution, 1% SDS, 10% dextran sulphate, and 50 μg ml−1 herring sperm DNA. Dot blots were hybridized with a [α-32P]dATP-labelled DNA probe of the CaMV 35S promoter. Northern blots were hybridized with the [α-32P]dATP labelled double-stranded cDNA of TRI or TRII from D. stramonium. Blots were washed four times under the following conditions: 5 min, room temperature, 2× SSC, 0.1% SDS; 30 min, 58 °C, 1× SSC, 0.1% SDS; 30 min, 58 °C, 0.2× SSC, 0.1% SDS; 15 min, 60 °C, 0.1× SSC, 0.1% SDS. Equal loading of RNA 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 and divided into three portions for individual determination of tropane ester alkaloids, calystegines, and tropane alcohols. Fresh mass and dry mass were determined. Hyoscyamine, scopolamine, and calystegines were analysed as described by Rothe and Draeger (2002) using the GC-column described below. Tropinone, tropine, and pseudotropine were extracted twice with 10 ml g−1 fresh mass MeOH/H2O (1:1 v:v). Extracts were evaporated and adjusted to 1 ml with water. After the addition of 50 μl of 26% ammonia, extracts were applied to an Extrelut® column (Merck). After 20 min incubation on the column, alkaloids were eluted with 2×4 ml CHCl3 and 4 ml CHCl3/MeOH (9:1 v:v). The eluates were concentrated and dissolved in a total volume of 150 μl ethyl acetate. Samples were analysed by gas chromatography: HP5 column (30 m×320 μm×0.25 μm), stationary phase: methylsiloxan 95%, phenylsiloxan 5%; mobile phase: helium, injection: pulsed splitless. Temperature programme: start: 65 °C, 10 °C min−1 up to 120 °C, hold 2 min, 10 °C min−1 up to 240 °C, hold 1 min. Enzyme activity Fourteen-day-old root cultures were harvested, frozen in liquid nitrogen, ground in a mortar with a suspension of 40% insoluble polyvinylpyrrolidone and extracted with a buffer containing 0.1 M potassium phosphate pH 7.8, 0.25 M sucrose, 3.0 mM DTT, 1.0 mM EDTA, and 10 mg g−1 fresh mass ascorbic acid. Tropinone reductases were precipitated in 40–75% saturation of ammonium sulphate. The protein pellet was dissolved in buffer (0.02 M potassium phosphate pH 7.0, 0.25 M sucrose, 1.0 mM DTT, and 25% glycerol). Total tropinone reductase activity was measured spectrophotometrically with 5 mM tropinone as substrate and 0.2 mM NADPH as co-substrate in 0.1 M potassium phosphate buffer pH 6.2. The decrease of NADPH absorption was determined at 340 nm at 30 °C. 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 accumulated with root protein extracts (see above) and a NADPH regenerating system using 1 mM glucose-6-phosphate, 0.5 mM NADP, and 2 units glucose-6-phosphate dehydrogenase. The reaction was stopped with 26% ammonia after 2 h incubation at 30 °C. The mixture was applied to an Extrelut® column (Merck). Tropinone, tropine, and pseudotropine 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 as well 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 trI was 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 strongest trII transcription (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 concentrations 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. Fig. 2. View largeDownload slide Accumulation of trI- and trII-transcripts in root culture lines of A. belladonna. Probes were labelled with cDNA of tropinone reductase I and of tropinone reductase II, respectively. Rehybridization with 18S rRNA of Lycopersicon esculentum served as loading control. WT, wild-type; VC, vector control; 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. Fig. 2. View largeDownload slide Accumulation of trI- and trII-transcripts in root culture lines of A. belladonna. Probes were labelled with cDNA of tropinone reductase I and of tropinone reductase II, respectively. Rehybridization with 18S rRNA of Lycopersicon esculentum served as loading control. WT, wild-type; VC, vector control; 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. 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 the transformed root cultures (Fig. 3). Wild-type roots for comparison were cultured without auxin addition. Fig. 3. View largeDownload slide 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 2-8 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. Fig. 3. View largeDownload slide 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 2-8 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. 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 distinguished from total TR activity in protein extracts after a fast partial purification using 3-quinuclidinone as substrate. 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 (0.4–0.75 mg g−1 fresh mass) than in trII- and in empty vector-transformed roots (0.1–0.25 mg g−1 fresh mass); no explanation can be given for this observation yet. Irrespectively, whether tropinone reductase 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 decreased 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, cosuppression would appear as a possible explanation, provided that more sensitive mRNA detection will allow transcript monitoring in wild-type roots for comparison. It is evident that 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 tropinone and 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 pseudotropine than the control root extracts corresponding to the 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. Fig. 4. View largeDownload slide Tropinone reductase activity in root culture lines of Atropa belladonna. Total tropinone reductase activity was measured photometrically by NADPH consumption with tropinone as substrate. Specific TRI activity was measured with 3-quinuclidinone as substrate. 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-transformed root line; error bars: standard deviation, n=3. Fig. 4. View largeDownload slide Tropinone reductase activity in root culture lines of Atropa belladonna. Total tropinone reductase activity was measured photometrically by NADPH consumption with tropinone as substrate. Specific TRI activity was measured with 3-quinuclidinone as substrate. 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-transformed root line; error bars: standard deviation, n=3. Fig. 5. View largeDownload slide Products of tropinone reduction in protein extracts from root culture lines of Atropa belladonna. 5 mM tropinone were incubated with protein extracts and a NADPH regenerating system. Reduction products, measured by gas chromatography, are expressed as a percentage of tropinone 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-transformed root line. Fig. 5. View largeDownload slide Products of tropinone reduction in protein extracts from root culture lines of Atropa belladonna. 5 mM tropinone were incubated with protein extracts and a NADPH regenerating system. Reduction products, measured by gas chromatography, are expressed as a percentage of tropinone 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-transformed root line. 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 0.2 μmol g−1 dry mass; tropine and pseudotropine are directly incorporated into esterified alkaloids and calystegines. The tropinone content of 1.2 μmol g−1 dry mass in root line 2-6 is extraordinary 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 corresponding 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 and enzyme activity of the two 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 1-4 also contained slightly more hyoscyamine, although trI transcript levels and in vitro enzyme activities were low. Scopolamine also increased up to 5-fold in the trI-transformed root lines, indicating either that the activity of hyoscyamine 6β-hydroxylase was enhanced or that more hyoscyamine was available for the enzyme. In addition, these root lines contained 1–2 μmol 6-hydroxyhyoscyamine g−1 dry mass (not shown). The hyoscyamine 6β-hydroxylase protein comprises two enzymatic activities, hydroxyl transfer to hyoscyamine and dehydration to scopolamine. The hydroxylase activity may be 20–50-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 (Rocha et al., 2002). The major calystegines in A. 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 that trII overexpression 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). Fig. 6. View largeDownload slide Alkaloids and their precursors in 31-d-old root culture lines of Atropa belladonna. All compounds were measured by gas chromatography and expressed as μmol g−1 dry mass. 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-transformed root line. (A) Precursors of tropane alkaloids, error bars: standard deviation, n=3. (B) Alkaloids, error bars: standard deviation, n=3. (C) Ratio between TRI-derived alkaloids versus TRII-derived alkaloids. Fig. 6. View largeDownload slide Alkaloids and their precursors in 31-d-old root culture lines of Atropa belladonna. All compounds were measured by gas chromatography and expressed as μmol g−1 dry mass. 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-transformed root line. (A) Precursors of tropane alkaloids, error bars: standard deviation, n=3. (B) Alkaloids, error bars: standard deviation, n=3. (C) Ratio between TRI-derived alkaloids versus TRII-derived alkaloids. Discussion Products of the tropine-forming tropinone reductase I, hyoscyamine and scopolamine, are important pharmaceutical and source compounds for derivative synthesis. It has been shown here for the first time that an augmentation of the TRI-products in Atropa belladonna is possible by directing the metabolite tropinone into the tropine-branch of the pathway. Transformation with cDNA of TRI successfully 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 preponderance of TRI-products in trI-transgenic root cultures. Preceding experiments for altering the tropane alkaloid pathway 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 (Nakajima et 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 a Duboisia hybrid (Moyano et al., 2002). Increase in total alkaloid production was 2-fold in H. muticus, but not observed 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 the plants 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, 3–4-fold higher total leaf alkaloid level was striking (Yun et al., 1992). In these studies, only the products derived from TRI were monitored; calystegines 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 calystegine levels, pseudotropine as a metabolite does not necessarily accumulate. Enhanced tropine formation also caused higher hyoscyamine and scopolamine levels, but only 2–3-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 hyoscyamine accumulation was shown as a result of methyl jasmonate 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 are restricted to the pericycle of a short region of young roots of A. belladonna (Suzuki et al., 1999a, b), tropinone reductases in the roots of H. 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 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 accumulation. This implies that with increased TRI-products a decrease in calystegines occurred simultaneously, for example, in line 1-3. In the root line 2-6, however, total alkaloids were 233 μmol g−1 dry mass, by far the highest content of all root cultures. The root line clearly overexpressed 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 only precursors 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 with PMT 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 tropane alkaloid 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. Abbreviations: H6H, hyoscyamine-6β-hydroxylase; PMT, putrescine N-methyltransferase; TRI, tropine-forming tropinone reductase; TRII, pseudotropine-forming tropinone reductase. 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TI - Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures
JO - Journal of Experimental Botany
DO - 10.1093/jxb/eri067
DA - 2005-01-10
UR - https://www.deepdyve.com/lp/oxford-university-press/overexpression-of-tropinone-reductases-alters-alkaloid-composition-in-BL2uVmKhB7
SP - 645
EP - 652
VL - 56
IS - 412
DP - DeepDyve
ER -