Journal of Experimental Botany, Vol. 58, No. 7, pp. 1603–1615, 2007
doi:10.1093/jxb/erl295 Advance Access publication 12 April, 2007
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Calystegines in potatoes with genetically engineered
carbohydrate metabolism
Ute Richter1, Uwe Sonnewald2 and Birgit Dräger1,*
1
Department of Pharmacy, Faculty of Science I Biosciences, Martin Luther University Halle-Wittenberg,
Hoher Weg 8, D-06120 Halle, Germany
2
Department of Biochemistry, Faculty of Science II, Friedrich-Alexander-University Erlangen-Nuremberg,
Staudtstr. 5, D-91058 Erlangen, Germany
Received 14 August 2006; Revised 29 November 2006; Accepted 4 December 2006
Abstract
Calystegines are hydroxylated nortropane alkaloids
derived from the tropane alkaloid biosynthetic pathway. They are strong glycosidase inhibitors and occur
in vegetables such as potatoes, tomatoes, and cabbage.
Calystegine accumulation in root cultures was described
to increase with carbohydrate availability. Whether this
is indicative for the in planta situation is as yet unknown. Potatoes are model plants for the study of
carbohydrate metabolism. Numerous transgenic potato lines with altered carbohydrate metabolism are
available, but rarely were examined for alterations in
secondary metabolism. In this study, calystegine accumulation and expression of biosynthetic enzymes were
related to genetic modifications in carbohydrate metabolism in potato tubers. Tubers contained more soluble sugars due to overexpression of yeast invertase
in the apoplast or in the cytosol, or due to antisense
suppression of sucrose synthase. It is shown that the
major part of calystegines in tubers originated from
biosynthesis in plant roots. Yet, tuber calystegine
levels responded to genetic alterations of carbohydrate metabolism in tubers. The strongest increase in
calystegines was found in tubers with suppressed sucrose synthase activity. Transcripts and enzyme activities involved in calystegine biosynthesis largely
concurred with product accumulation. Whole plant
organs were examined similarly and displayed higher
calystegines and corresponding enzyme activities in
roots and stolons of plants with enhanced soluble
sugars. Increases in calystegines appear to be linked
to sucrose availability.
Key words: Antisense, calystegine, carbohydrate metabolism,
invertase, overexpression, potato, sucrose synthase, tropane
alkaloid biosynthesis.
Introduction
Due to novel drugs based on natural alkaloids (Cordell
et al., 2001; Newman et al., 2003; Ortholand and
Ganesan, 2004) and in particular on the tropane skeleton
(Gross, 2004), the importance of tropane alkaloids has increased during the last decade. Tropane alkaloids for medicinal applications such as hyoscyamine and scopolamine
occur in a limited number of Solanaceae; economically
utilized species are Atropa belladonna, Datura stramonium, Hyoscyamus niger, and Duboisia species. Calystegines are nortropane alkaloids derived from the tropane
biosynthetic pathway (Fig. 1) with a more widespread occurrence within angiosperm plants (Dräger, 2004). Calystegines were found in Solanaceae, in Convolvulaceae, in
Moraceae, in Erythroxylaceae (Brock et al., 2005), and
in Brassicaceae (Brock et al., 2006). Practical interest in
calystegines focuses mainly on two areas: they are strong
glycosidase inhibitors with therapeutical potential (Asano
et al., 2000); and they are food and feed constituents with
possible toxic effects (Molyneux et al., 1995; Haraguchi
et al., 2003; Ikeda et al., 2003).
The highest concentrations of calystegines (1.6% dry
mass) were recorded in small sprouts of potato tubers
(Keiner and Dräger, 2000). In root cultures, where
calystegines are formed, their biosynthesis can be enhanced by soluble sugars, sucrose being most effective
(Rothe et al., 2001; Rothe and Dräger, 2002). Not only is
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: PMT, putrescine N-methyltransferase; TR, tropinone reductase.
ª 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1604 Richter et al.
Fig. 1. Calystegines derive from the tropane alkaloid biosynthetic
pathway and share the first enzymatic steps.
sucrose the most important transport form of carbohydrates in plants, but sucrose-specific induction of plant secondary metabolism has also been repeatedly described.
Anthocyanins in a cell culture of Vitis vinifera were enhanced by sucrose; the effect was mediated by calcium,
calmodulin, and protein kinases (Vitrac et al., 2000).
Arabidopsis thaliana mutants that accumulate high sucrose
levels show strong anthocyanin accumulation, irrespective
of whether the mutation affects a sucrose transporter (Lloyd
and Zakhleniuk, 2004) or plastidial phosphoglucomutase
(Solfanelli et al., 2006). Whether sugar availability influences calystegine biosynthesis in planta has not been
studied so far.
Potato is a model plant for the investigation of carbohydrate metabolism. The number of plants with geneti-
cally engineered carbohydrate metabolism is steadily
increasing (Geigenberger et al., 2004), but the impact of
carbohydrate alteration on plant secondary metabolism has
only scarcely been considered. For potatoes overexpressing genes of fructan formation, the glycoalkaloids achaconine and a-solanine were determined to be in the
same concentration range as in the non-transformed parent
lines (Zywicki et al., 2005). To study the impact of altered
carbohydrate availability on calystegine biosynthesis,
transgenic potato lines overexpressing yeast invertase in
the cytosol or in the apoplast (Sonnewald et al., 1997) or
plants with suppressed sucrose synthase activity (Zrenner
et al., 1995) were selected. Expression of the transgenes
was limited to tubers by the use of the tuber-specific
patatin promoter for the invertase gene (Rocha-Sosa et al.,
1989) or by choosing an isoform of sucrose synthase
that is expressed predominantly in tubers (Fu and Park,
1995). Alteration of primary metabolism in tuber lines
had been investigated before: tubers overexpressing invertase in the apoplast were characterized by elevated
glucose and decreased sucrose contents. Plants produced
larger tubers and an increased total yield (Sonnewald
et al., 1997). Levels of several primary metabolites, for example, amino acids, organic acids, disaccharides such as
maltose, isomaltose, and trehalose, and sugar phosphates
were affected differentially in those tubers (Roessner et al.,
2001). Plants overexpressing cytosolic invertase in the
tuber produced a higher number of tubers, but with
reduced starch content per fresh mass and with increased
soluble sugars (Sonnewald et al., 1997), sugar phosphates,
and amino acids (Trethewey et al., 1998). Unexpectedly,
higher cytosolic invertase did not increase sink strength
and, in consequence, starch content. Instead, sucrose breakdown and resynthesis were activated (Trethewey et al.,
1999) and entailed, together with higher glycolytic
activity (Trethewey et al., 1999; Hajirezaei et al., 2000),
increased carbon dioxide release (Trethewey et al., 1998)
and reduced oxygen levels in those tubers (Bologa et al.,
2003). Sucrose synthase is the predominant enzyme for
sucrose breakdown in starch-accumulating and growing
potato tubers (Fernie and Willmitzer, 2001). Sucrose
synthase suppression in tubers leads to reduced tuber
yield in number and dry mass. The tubers contain less dry
mass, less starch, higher sucrose, fructose, and glucose
contents, and, as a reaction to the metabolic alterations,
higher invertase activities (Zrenner et al., 1995).
Early grafting experiments demonstrated that tropane
alkaloids are synthesized in plant roots from where they
are transported to aerial parts of the plant (James and
James, 1954). Enzymes of tropane alkaloid biosynthesis
were localized in roots (Hashimoto and Yamada, 2003).
Accordingly, altered carbohydrate metabolism in tubers
would possibly not influence tropane alkaloid biosynthesis
in roots. However, cloning of cDNAs coding for the
first enzyme specific for tropane alkaloids, putrescine
Calystegines in potatoes with altered carbohydrates 1605
N-methyltransferase (PMT) (Stenzel et al., 2006), and
coding for pseudotropine-forming tropinone reductase
(TRII) (Keiner et al., 2002) from potato tuber sprouts
suggested that calystegines in sprouts are derived from de
novo synthesis in this tissue. Potato tubers are stem tissues, thus calystegine biosynthesis in potato appears not to
be limited to roots. Immunolocalization of TRII in tuber
sprouts, in stolons, and in roots confirmed the view that
calystegine biosynthesis takes place in different potato
tissues (Kaiser et al., 2006).
In this study, the biosynthetic capacity for tropane
alkaloids in potato was monitored, and tubers and plant tissues were identified as organs with calystegine biosynthesis and storage. Further, the genetic transformation of
carbohydrate metabolism in tubers was related to accumulation of calystegines in the whole plant. Biosynthetic
activity was probed by analysis of calystegines, their
metabolites, and of TRII and PMT at the mRNA and
protein levels. The tropine-forming tropinone reductase
(TRI) was included in the measurements in order to gain
more insight into the significance of this enigmatic enzyme in potato. It had been cloned and localized in potato
tissues that accumulate calystegines. The catalytic characteristics differ somewhat from TRIs found in, for example,
Datura stramonium or Hyoscyamus niger, yet with a range
of ketone substrates, the highest activity of potato TRI
was found with tropinone. The products tropine or
esterified tropine, however, are not detected in the potato
tissues (Kaiser et al., 2006).
Materials and methods
Plant material
Solanum tuberosum L. cv. Désirée, wild-type tubers (WT2) were
supplied by Saatzucht Lange AG (Bad Schwartau, Germany).
Transgenic plants were produced as described in Sonnewald et al.
(1997) for invertase expression and in Zrenner et al. (1995) for
suppressed sucrose synthase activity. Tubers were harvested from
plants, which were cultivated as hydroponics for 3 months in the
greenhouse. All together, 14 independent transformed potato lines
were examined (Table 1) together with wild-type tubers of the batch
that was initially used for transformation (WT1) and independent
Désirée wild-type tubers (WT2). Hydroponically grown tubers were
harvested and stored as indicated in Fig. 2. They were kept for
3 months at a cool temperature of 12–15 C, transferred to 4 C for
a short period (2 d), and subsequently to room temperature. This
treatment was considered optimal for the induction of sprouting and
was considered as dormancy of freshly harvested tubers (Sonnewald, 2001). The treatment was successful in wild-type and U-IN1
tubers; sprouts developed 3–4 weeks after transfer to room temperature. Sprouting was delayed in tubers of line RSSa; sprouts
appeared only 2 months after transfer to room temperature. All
tubers were observed up to 6 months after transfer to room temperature (288 d after harvest). Tubers of the line U-IN2 instead of
sprouting turned black and rotted. Consequently, no sprouts and
plants were obtained from the line U-IN2. Tubers of lines U-IN1,
RSSa, and wild type (WT1) were planted in pots with garden soil
and kept in a greenhouse with 16 h light, 8 h darkness at 23 C and
Table 1. Transgenic potato lines
Carbohydrate contents and activity of overexpressed or suppressed enzymes in tubers after harvesting (Zrenner et al., 1995; Sonnewald et al., 1997).
% Dry mass
Invertase apoplastic
WT
U-IN1-3
U-IN1-33
U-IN1-41
Invertase cytosolic
WT
U-IN2-17
U-IN2-30
U-IN2-34
U-IN2-33
U-IN2-42
Sucrose synthase antisense
WT
RSSa-99
RSSa-108
RSSa-112
RSSa-114
RSSa-129
RSSa-130
a
Bologa et al. (2003).
Enzyme activity (nmol min
Sucrose
Glucose
Fructose
Invertase
2.0
0.6
0.2
0.4
0.1
3.2
4.1
4.0
0.1
0.1
0.1
0.1
9
82
598
479
2.0
0.2
0.2
0.4
Not known, phenotype
weaker than U-IN2-30a
Not known, phenotype
weaker than U-IN2-33
0.2
5.8
5.5
3.5
0.1
0.2
0.1
0.1
24
452
358
346
1.6
1.3
1.0
2.6
2.6
2.9
Not known, phenotype
weaker than RSSa-99
0.2
0.3
0.4
7.1
0.3
2.8
0.1
0.2
0.5
2.2
0.4
0.7
1
mg
1
protein)
Sucrose synthase
36 500
20 900
10 200
1400
16 500
3100
1606 Richter et al.
twice for 20–60 min at 65 C, 0.25 M NaH2PO4, 2% SDS, 1 mM
EDTA; and twice for 20 min at 65 C, 0.05 M NaH2PO4, 1% SDS,
1 mM EDTA. Northern blots were rehybridized with an 18S rRNA
probe from Lycopersicon esculentum as loading control for RNA
(Dobrowolski et al., 1989).
Fig. 2. Storage temperature and sampling dates of potato tubers.
Samples were taken 10 d (sample 1), 92 d (sample 2), 99 d (sample 3),
106 d (sample 4), 120 d (sample 5), 155 d (sample 6), and 288 d
(sample 7) after harvest. The filled arrow indicates sprouting of wildtype and U-IN1 tubers. The open arrow indicates sprouting of RSSa
tubers.
50% relative humidity. Two months after transfer to soil, plants
began to flower.
Sampling and statistical treatment
Large and small tubers were analysed separately throughout the
sampling but, for clarity of the graphs, data are shown for large
tubers only. After cold treatment and transfer to room temperature,
tuber eyes, together with peel, were analysed at all sampling dates.
Pith was analysed for calystegines at the first sampling date only,
and sprouts were analysed when they were available. Peel was cut
from the tubers by a scalpel and was found to contain cork; it was
not thicker than 2 mm. Pith was taken as cubes (5 mm edge length)
from the middle of the tubers. Tuber vascular bundle was cut from
the vascular ring (;3 mm) that is visible as a translucent zone in
tuber transverse slices (;5 mm thick). Other plant tissues were cut
as vertical sections (stolon, tuber sprout, and root, 1–4 cm length)
or complete leaves and used for calystegine analysis or tropinone
application directly after harvest. Samples size for calystegine
analysis and tropine/pseudotropine analysis was 0.1 g dry mass.
Dry mass was determined after lyophilization. Sample numbers are
indicated for each individual graph. Significant differences between
data were evaluated by t-tests using the algorithm embedded in
Microsoft Excel (Microsoft Corporation, Seattle, WA, USA).
P-value limits are given with each graph.
Northern blot
Tissues from S. tuberosum were immediately frozen in liquid
nitrogen. Total RNA was isolated using TRIzol Reagent (Gibco
BRL and Invitrogen) or using the phenol–chloroform method
(Reinbothe et al., 1992). Extraction buffer [10 mM TRIS-HCl
pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% SDS (w/v)] and precipitation buffer (4 M LiCl in 20 mM sodium acetate pH 5.2) were
adjusted. A 20 lg aliquot of total RNA was separated in a 1.2%
formaldehyde–agarose gel and blotted onto a nylon membrane
Hybond N+, (Amersham Biosciences). Northern blots were
hybridized for 16 h at 42 C in 0.25 M NaH2PO4, pH 7.2, 0.25 M
NaCl, 7% SDS, 1 mM EDTA, 50% formamide, 10% (w/v) PEG
(Mr 6000 Da), and 100 lg ml 1 herring sperm DNA (Amasino,
1986). The following [a-32P]dATP-labelled probes were used for
hybridization: trI, 400 bp fragment of the 3#-terminus; trII,
a 400 bp fragment of the 3#-terminus (Keiner et al., 2002), and
pmt, a 200 bp fragment of the 5#-terminus (Stenzel et al., 2006).
After hybridization, blots were washed with these buffers: 5 min at
room temperature, 23 SSC (0.3 M NaCl, 0.03 M sodium citrate);
Protein extraction and quantification
Plant and tuber tissues were frozen in liquid nitrogen and ground in
a mortar. Proteins were extracted using TRIS-HCl-saturated phenol
(Meyer et al., 1988). After 30 min incubation, the phenol phase was
separated and proteins were precipitated for at least 4 h with 0.1 M
methanolic ammonium acetate solution. After centrifugation, the
protein pellet was dissolved in buffer containing 68 mM TRIS-HCl
pH 6.8, 10% glycerol (v/v), 2.3% SDS (w/v), and 5% mercaptoethanol. For quantification, 5 ll solutions were spotted on Whatman
paper. After staining for 20 min in ESEN solution (0.2% Coomassie
Brilliant Blue R 250, 25% isopropanol, 10% acetic acid) and destaining in water, the protein–dye complex was eluted with 0.5%
SDS and the absorbance of the complex was measured at 578 nm
(Esen, 1978). Total proteins (5–50 lg) were separated by 12.5%
SDS-PAGE (Laemmli, 1970) and transferred to a Hybond P
membrane (Amersham-Biosciences) using tank blotting. Membranes were blocked (45 min) in milk protein buffer [10 mM
TRIS-HCl pH 7.4, 150 mM NaCl, 0.2% (w/v) Tween-20, 5% (w/
v) defatted milk powder]. After this, membranes were incubated (at
least 4 h) with primary antibodies against TRI or TRII (Kaiser
et al., 2006) in milk protein buffer at a 1:100 dilution. After three
wash steps (10 min) in buffer containing 10 mM TRIS-HCl pH 7.4,
150 mM NaCl, and 0.2% (w/v) Tween-20, membranes were treated
with the secondary antibody (anti-rabbit IgG alkaline phosphatase
conjugate) in milk protein buffer at a 1:4000 dilution. Spots of
secondary antibody binding were visible after incubation with
0.15 mM nitro blue tetrazolium chloride (NBT) and 0.15 mM 5bromo-4-chloro-3-indolyl phosphate in buffer containing 100 mM
TRIS-HCl pH 9.7, 100 mM NaCl, 50 mM MgCl2 for 10–15 min.
The reaction was stopped with 50 mM TRIS-HCl pH 7.5, 150 mM
NaCl, 0.02% (w/v) sodium azide, and 20 mM EDTA.
Calystegine analysis
Fresh mass and dry mass (by lyophilization) were determined for all
tissues. Samples were taken from at least three different tubers and
treated separately for each data point. Calystegines were extracted
with methanol:water (1:1 v/v), purified with a strong acid cation
exchange column (Merck Darmstadt), and measured by gas
chromatography as described by Richter et al. (2005). Individual
calystegines A3, B2, and, in some sprout samples, B4 were
identified, calculated, and added to give total calystegines. Data
were averaged and standard deviations were calculated. Measurements with >50% deviation from average (without the critical
measurement) were considered as outliers.
Tropinone, tropine, and pseudotropine analysis
Tuber tissues were cut with a sterile blade into peel sections
(>1 mm thick), vascular bundle (;3 mm thick), and cubes of pith
tissue (;5 mm). Other plant tissues were cut as vertical sections
(stolon, tuber sprout, and root, 1–4 cm length) or complete leaves
and flowers with three independent samples per tissue and treatment. Plant sections were incubated with 2 mM tropinone solution
(48 h) at 23 C, 100 rpm, 16 h light, and 8 h darkness. Control
samples were incubated in distilled water. The samples were
extracted with methanol:water (1:1 v/v) followed by a purification
step with Extrelut columns (Merck, Darmstadt). Samples were analysed by gas chromatography as described by Richter et al. (2005)
and gas chromatography–mass spectrometry as described by Brock
Calystegines in potatoes with altered carbohydrates 1607
et al. (2005) with some modifications: column, DB-5 (30 m30.32
mm id30.25 lm film thickness); injection volume, 5 ll. Limits of
detection were 0.1 lmol g 1 dry mass for all three metabolites.
Results
Calystegine accumulation
Calystegine concentrations were followed in potato tuber
tissues over a period of 288 d after harvest. Tubers of
transgenic and control lines were not uniform in size, but
appeared roughly as two size groups, small tubers of
2–4 cm diameter and larger tubers mostly >4 cm in
diameter; examples have been illustrated (Tauberger
et al., 1999). Large and small potato tubers were shown
to differ in composition of carbohydrates (De Wilde et al.,
2006); therefore, the two tuber size groups were analysed
separately. Calystegine concentrations of tubers and of all
other tissues were related to dry mass, and tuber dry mass
percentage of all lines was followed during the 288 d of
storage (Fig. 3). No significant differences in dry mass per
fresh mass were found between large and small tubers
(not shown), but dry mass of tubers of the transformed
plant lines differed markedly. Potatoes transformed with
yeast invertase in the cytosol and with antisense sucrose
synthase were shown before to have a decreased tuber dry
mass ratio per fresh mass directly after harvest (Zrenner
et al., 1995; Sonnewald et al., 1997) due to lower starch
formation per fresh mass. During storage, dry mass percentage stayed approximately constant for wild-type
tubers; the same was the case for tuber lines with apoplastic invertase and most lines with suppressed sucrose synthase, although at lower levels than for wild-type tubers.
Dry mass decreased during storage in lines with cytosolic
overexpression of invertase (U-IN2), confirming that high
Fig. 3. Dry mass percentage of potato tuber pith tissue fresh mass of
transformed (U-IN-1, U-IN-2, RSSa, RSSa-112) and control (WT)
potato lines. Data were taken from six tubers of various size derived
from every potato line, i.e. every data point in the graph contains 6–30
measurements. Standard deviation was <30% for every data point.
glycolytic activity and respiration entailed loss of carbon
as CO2 (Trethewey et al., 1998, 1999; Junker et al., 2004).
Among tubers with antisense sucrose synthase expression,
line 112 differed from the others by a gradual loss of dry
mass. Line 112 shows the strongest transformation effect
expressed as diminished sucrose synthase activity and
enhanced sucrose accumulation (Zrenner et al., 1995).
Calystegine distribution within potato tubers was compared 10 d after harvest. The predominant calystegines in
all tissues were calystegine A3 and B2 with a largely 1:1
molar ratio, confirming previous data (Keiner and Dräger,
2000). In all tuber lines, total calystegines were significantly higher in tuber peel than in the pith (Fig. 4). Higher
calystegine concentrations in peel than in tuber pith had
been noted before for wild-type tubers of various cultivars
(Keiner and Dräger, 2000; Friedman et al., 2003).
Most tubers overexpressing invertase (lines U-IN1 and UIN2) did not differ significantly from wild-type tubers in
calystegine content. Among tubers silenced for sucrose
synthase activity, line RSSa-112, the line with the
strongest transformation effect, contained more calystegines than any other line. No consistent difference in
calystegine concentration was detected between large and
small tubers.
Calystegine contents were followed in all tuber lines
during storage. As the tuber peel constantly contained the
majority of calystegines, pith analysis was omitted for the
following sampling dates. Tubers of the two sizes at later
sampling dates, like the tubers 10 d after harvest, did not
differ markedly in calystegine content. For the subsequent
samples, calystegines are given as average concentrations
in large tubers. A continuous decrease in calystegine content of tuber peel is evident for all lines (Fig. 5a–f). The
concomitant increase in eyes and sprouts indicates transfer
of calystegines from peel to sprouts. Those tuber eyes that
were analysed after 120 d (Fig. 5g) still appeared dormant, but large deviations of samples of these seemingly
dormant eyes suggest that eyes begin to accumulate
calystegines before sprouting becomes visible. Tuber peel
and sprouts of line RSSa-112 contained the highest
calystegine concentrations over all storage stages; sprouts
accumulated approximately 1% calystegines in the dry
mass (Fig. 5h).
Plants were cultivated from those tubers that sprouted
(lines U-IN1, RSSa, and wild type); they were analysed
for calystegines at the flowering stage. Stolons and roots
were the organs with the highest calystegines, with stolons
revealing about half of the calystegine content of roots
(Fig. 6a, b). In roots of two lines overexpressing apoplastic invertase (lines 33 and 41) and in most sucrose
synthase antisense lines, calystegines were markedly elevated compared with the wild type (Fig. 6b). Although
genetic alterations of carbohydrate metabolism in all transgenic lines were predominantly expressed in tubers, effects
on calystegine accumulation are recognized in roots.
1608 Richter et al.
Fig. 4. Total calystegines in potato tubers of 4–6 cm diameter (size 1) and 2–4 cm diameter (size 2) in pith and peel. Data represent the average of
three samples; vertical lines above the bars indicate the standard deviation. *Significantly different from wild type 1 (P <0.01).
Calystegines in young leaves appeared higher in some of
the U-IN1 and RSSa lines (Fig. 6d), however, with considerable variation, and the total concentration was much
lower than in roots and stolons. A similar pattern was
found for flowers (Fig. 6c), and old leaves also contained
very low concentrations of total calystegines irrespective
of carbohydrate alteration in tubers (Fig. 6e). Wild-type
potato cultivars had been analysed before, with a similar
calystegine distribution in plant roots and leaves as shown
for wild-type tissues here (Keiner and Dräger, 2000).
Calystegine biosynthesis
Calystegine accumulation in all potato lines was related to
the expression of enzymes involved in their biosynthesis.
RNA gel blot analysis was performed for transcripts of
both TRI and TRII, and for PMT. As the metabolic role of
S. tuberosum TRI is not clear, but distinction from TRII
was important, both enzymes were always monitored together. Protein levels for TRI and TRII were estimated by
western blotting, and enzyme activities were determined
by quantifying the tropinone reduction products tropine
and pseudotropine.
Within tuber tissues, transcripts encoding TRII responsible for calystegine formation were found mainly in the
sprouts of those potato lines whose tubers sprouted (Fig.
7A). Weak signals of trII were detected in some peel
samples (Table 2), although the calystegine concentration
was high in tuber peel. For trI, in comparison, weak transcript signs were seen in a few sprout samples (not shown),
but trI transcripts were detected in other tuber tissues of
all storage stages, in peels, and sometimes in the pith.
Some of the transgenic lines, i.e. lines U-IN1-41, U-IN217, U-IN2-33, RSSa-108, and RSSa-112, showed stronger
trI transcripts in tuber peel than the equivalent wild-type
potatoes, indicating that altered tuber carbohydrate metab-
olism may influence trI expression (Fig. 7B). However, signal variation between several samples was considerable in
strength, and large and small tubers sometimes showed
contrasting trI transcript levels. The concordant observations are the clear distinction of trI and trII signals and the
presence of trI transcripts in all tuber samples with
varying intensity.
Whole plants contained transcripts for both TRs in
almost every tissue to varying degrees (Table 2). Stolon
tissue contained trII transcript levels in all transformed
lines (Fig. 7C). Transcripts of trI were also visible in most
stolon tissues, mostly at low levels. The strongest signals
for trII transcripts were observed in roots of all lines (Fig.
7D). In addition, young leaves repeatedly revealed trII
signals, in particular, in the lines with the strongest transformation effects, U-IN1-33 and RSSa-112 (not shown).
Young leaves, however, contain only small amounts of
calystegines (Fig. 6d). Transcripts coding for PMT were
less abundant throughout all plant lines. They were detected in roots only (Fig. 7D); all other tissue samples
from other plant organs were negative (not shown). PMT
occurrence confined to roots was found before in tobacco
(Hibi et al., 1994) and in Atropa belladonna (Suzuki
et al., 1999). In tubers or tuber sprouts, pmt transcripts
were not detected on any northern blot in this study;
however, S. tuberosum pmt cDNA had initially been
cloned from sprouting tuber eyes (Stenzel et al., 2006).
Northern blots of potato tuber sprouting eyes and sprouts
of up to 6 mm length displayed pmt transcripts; in longer
and more developed sprouts, the pmt transcript was not
detected (Stenzel et al., 2006). Due to the limited number
of transgenic tubers available, sprouting eyes could not be
included for transcript analysis in this study.
Protein accumulation of both TRs was monitored by
western blots of protein extracts from tuber tissues of all
Calystegines in potatoes with altered carbohydrates 1609
Fig. 5. Calystegine levels in potato tuber peel (a–f), dormant tuber eyes (g), and sprouts (h) during storage. Days after harvest are indicated in each
panel. Bars signify the standard deviation. Each column comprises 3–6 independent samples. WT sprouts (WT1) were divided into <1 cm and >1 cm
length. *Significantly different from WT1 (P <0.05).
potato lines, and from lines that produced plants in young
and old leaves, flowers, stolons, and roots. TRII protein
was detected in roots and stolons of all potato lines. TRI
protein appeared to be more abundant; it was detected in
all tuber tissues and in flowers and stolon (data not
shown). A difference in TR protein levels between the
wild type and transformed potato lines was not observed
by western blotting. In a previous study, TRII was localized by immunostaining in roots, stolons, and in phloem
companion cells of tuber sprouts, but the overall TRII protein level was low as judged by western blotting (Kaiser
et al., 2006). As the purified antibody preparations against
TRI and TRII do not cross-react and possess approximately the same detection limits [65–100 lg l 1 (Kaiser
et al., 2006)], TRII expression seems to be limited to
certain tissues and developmental stages. For S. tuberosum PMT, no antibodies are available.
In order to compare TR transcripts and enzyme protein
levels due to reductase activity in situ, tropinone in aqueous solution was applied to tuber and plant tissues. Product
analysis after 2 d of incubation showed a clear separation
of tuber and other plant tissues. In tuber peel, pith, and
vascular tissue, tropine was the only product of tropinone
reduction; pseudotropine was not detected (Fig. 8a–c),
1610 Richter et al.
Fig. 6. Calystegine levels in potato plant tissues. Bars signify the standard deviation. Each column comprises three independent samples. n.a., Not
analysed. *Significantly different from wild type (P <0.05).
Table 2. Results of northern blotting for trI and trII, summarized for potato tuber tissues and for other plant parts
+ strong signal; – weak signal.
TR transcripts in tissues
Wild type
U-IN-1
U-IN-2
RSSa
Tuber peel, 10 d
after harvest
Tuber peel
(storage time)
Tuber pith
Tuber sprouts
Young leaves
Old leaves
Flowers
Stolons
Roots
trI; trII not detected
trI; trII not detected
trI; trII not detected
trI; trII not detected
trI (92–120 d); trII
(92 d, 99 d)
trI –; trII not detected
trI –; trII
trI –; trII
trI –; trII
trI; trII –
trI –; trII
trI –; trII +
trI (92–120 d); trII – (99 d)
trI (92–120 d);
trII not detected
tr –; trII not detected
–
–
–
–
–
–
trI (92–99 d); trII
not detected
trI –; trII not detected
trI; trII line 129 +
trI –; trII line 112 +
trI –; trII
trI; trII not detected
trI –; trII
trI –; trII line 129 +
trI –; trII not detected
trI; trII line 33 +
trI –; trII line 33 +
trI –; trII
trI; trII
trI –; trII +
trI –; trII +
except for minor amounts (<1 lmol g 1 dry mass) in a
few peel samples (not shown). In contrast, stolon, leaves,
flowers, roots, and tuber sprouts (Fig. 8c–i) formed
pseudotropine only. Reduction product accumulation may
be related to enzyme activity in situ with some provisos:
(i) compartmentation and transport of tropinone within the
tissue may delay reduction; and (ii) reduction products
may undergo further metabolism. However, as small tis-
sue sections are easily perfused by water-soluble compounds, and as tropinone is known to be taken up by plant
tissues (Keiner and Dräger, 2000; Scholl et al., 2001), the
fast accumulation of tropine (up to 30 lmol g 1 dry
matter in tuber peel) and pseudotropine (up to 180 lmol g 1
dry matter in roots) indicates that under these conditions,
when tropinone is not limiting, both TRs are highly active
in their respective tissues. This corresponds to all these
Calystegines in potatoes with altered carbohydrates 1611
(2 mM tropinone in the incubation solution) is present in
the tissue. When a higher tropinone concentration (5 mM)
was applied to potato tissues, tropine was also detected in
roots, stolon, and tuber peel, in addition to large amounts
of pseudotropine (Kaiser et al., 2006).
Discussion
Fig. 7. Transcript levels for enzymes of the tropane alkaloid pathway
(trII, pmt) compared with trI in tubers and plant tissues observed after
northern blotting. Tuber sprouts (A) of the lines U-IN1 and RSSA that
sprouted, and peels (B) of all transgenic tuber lines and of both wild
types (WT1, WT2) were examined. For plant stolons (C) and roots (D)
of the regenerated lines, only WT1 were used for comparison. 18S
rRNA was monitored as loading control.
tissues containing low levels of tropinone only, if the metabolite is not applied externally (data not shown).
There is concordance of enzyme product measurements
with transcript accumulation, as tissues with strong trII
transcripts such as root and stolons show the highest
pseudotropine formation after tropinone application. Tropine formation in tuber tissue also largely concurs with
the transcript accumulation, but remains enigmatic considering the metabolic significance. In non-treated tuber tissue, no tropine or tropine esters are found, although in all
potato tissues low concentrations of tropinone were detected. It is assumed that the potato TRI must have another metabolic significance in situ. Tropinone is reduced
to tropine only if an unnaturally high concentration
Calystegine biosynthesis in potato
From the data on accumulation of calystegines, their
metabolites, transcripts, and enzyme activities, a model of
calystegine biosynthesis and transport in potato plants can
be proposed (Fig. 9). PMT as the key enzyme for tropane
alkaloid formation appears to restrict the overall metabolic
activity in the pathway; transcripts were found in roots
(Fig. 7D) and in sprouting tuber eyes only (Stenzel et al.,
2006). The metabolite tropinone, present in low concentrations throughout the whole potato plant, is distributed
from the roots into the aerial parts of the plant and into the
stolon. TRs are active in all plant organs including the
stolon, TRII activity exceeding that of TRI except in tuber
tissues (Fig. 8). Strong TRII activity does not allow
tropinone to accumulate to high concentrations in tissues,
indicating that tropinone supply contributes to the overall
flux limitation. TRII activity, in contrast, does not appear
as rate-limiting for calystegine biosynthesis. Tropinone entering the aerial parts and stolon is readily reduced to
pseudotropine, which is then metabolized to calystegines.
In tubers, where no PMT and only low TRII activities
were found, the major part of calystegine accumulation
appears to be the result of tropinone reduction in the stolon and subsequent metabolism to calystegines. When
tuber eyes sprout, a sudden decrease of calystegines in
tuber peel is observed (Fig. 5b, c). Calystegines are obviously transferred from the tuber peel to the eyes and thereafter to sprouts (Fig. 5g, h). In growing tuber sprouts,
PMT and TRII activity provide additional metabolites for
calystegine formation. Roots that contain high TRII activity and high calystegine levels may also export calystegines to aerial parts of the plants and to tubers.
This model of calystegine accumulation involves many
metabolite transport steps. Transport processes as an essential part of the regulatory instruments for many alkaloids
and other secondary compounds in biosynthesis are just
being detected (Kutchan, 2005; Yazaki, 2005). In contrast
to alkaloids, for which transport was attributed to ATP
transporters (Shitan et al., 2003; Otani et al., 2005) or pHdependent efflux carriers (Li et al., 2002), calystegines are
hydrophilic sugar-mimic compounds. A plant sucrose transporter was identified that transports phenol glucosides
(Chandran et al., 2003), indicating that this type of transporter should receive attention when looking for calystegine transport. For further elucidation of overall
biosynthetic regulation, cloning of transport proteins and
1612 Richter et al.
Fig. 8. Formation of tropine (black columns) or pseudotropine (grey columns) in potato tissues supplied with 2 mM tropinone solution for 48 h. Bars
signify the standard deviation. Each column comprises 3–4 independent samples (the exception is h, U-IN1-41, two samples). n.a., Not analysed;
n.d., not detected; o, detected, but below 0.1 lmol g 1 dry matter. *Significantly different from wild type (P <0.05).
also of further enzymes involved in calystegine biosynthesis will be mandatory, for example, enzymes that demethylate and hydroxylate the tropane alkaloid skeleton.
Calystegines and carbohydrate metabolism
Calystegines in potato tubers and plants respond to genetic
alterations in carbohydrate metabolism by different levels
of accumulation. Tubers expressing cytosolic invertase
(U-IN2) contain a reduced dry mass, indicating reduced
starch (Fig. 3) due to enhanced glycolysis and respiration;
they did not sprout during the period of observation.
Reduced or delayed tuber sprouting was reported before
for tubers overexpressing cytosolic invertase in companion cells (Hajirezaei et al., 2003). In those tubers,
calystegines decreased after harvest faster than in wildtype tubers (Fig. 5a–d). Calystegine levels in tubers overexpressing apoplastic invertase (lines U-IN1) were not
significantly different from those in the wild type (Figs 4,
5), but were higher in roots of the respective plants (Fig.
6), roots of line U-IN1-33 containing the highest calystegine levels, followed by U-IN1-41 and U-IN1-3. The
results matched the transformed phenotype measured as
invertase activity and carbohydrate alteration, where
U-IN1-33 was strongest (Sonnewald et al., 1997).
Overexpression of invertase, however, was controlled by
the B33 patatin promoter that is active predominantly in
tubers (Rocha-Sosa et al., 1989). The effect of invertase
overexpression in tubers on calystegines in roots may be
explained either by low B33-directed transgene expression
also in roots and stems (Rocha-Sosa et al., 1989) being
sufficient for calystegine increase or by pleiotropic metabolism in roots caused by altered tuber carbon metabolism.
Individual potato lines differed in the extent of sucrose
synthase suppression, line RSSa-112 showing the strongest transgene effect. These tubers contained markedly less
dry mass, i.e. less starch. Sucrose synthase antisense
tubers delayed sprouting for 4–6 weeks and generated
intact plants. Tuber peel and sprouts of line RSSa-112
contained the highest calystegine concentrations registered
in this study, with a maximal content of 1% dry mass in
sprouts (Figs 4, 5). Roots of this line also contained more
calystegines than wild-type tissues (Fig. 6a, b). Although
trII transcript was not visibly enhanced (Fig. 7D),
tropinone-reducing activity was stronger than in the wild
type (Fig. 8f). Antisensing of sucrose synthase was
focused on tubers by targeting the sucrose synthase isoform 4 that is predominantly expressed in tuber tissue
Calystegines in potatoes with altered carbohydrates 1613
In summary, calystegines in potato tubers increase with
increasing sucrose levels, as observed in sucrose synthase
antisense tubers. Tubers expressing invertase in the
apoplast or in the cytosol contain high glucose levels, low
sucrose, and unchanged or decreased calystegines (Rothe
et al., 2001; Rothe and Dräger, 2002). Roots of U-IN1
plants, however, contain elevated calystegines. They await
examination of their carbohydrate levels.
Sucrose had been identified as an inducer of alkaloid
formation in root cultures of A. belladonna; it could not
equally be replaced by glucose, fructose, or other metabolizable sugars (Rothe et al., 2001; Rothe and Dräger,
2002). In order to pinpoint links of sucrose or of sucrose
combined with subsequent metabolites to calystegine biosynthesis and accumulation, metabolic profiling appears
the method of choice for future investigations. Correlation
of carbohydrates and other metabolites with calystegines
will highlight metabolic patterns that are combined with
calystegine formation in an unbiased manner (Bino et al.,
2004; Weckwerth et al., 2004). The monosaccharidemimic structure of calystegines suggests not only that they
are transported like sugars but that they may directly
interfere with carbohydrate metabolism. Inhibition of plant
invertase by calystegine B2 was demonstrated; interestingly, invertase of invading fungi was not inhibited (Höke
and Dräger, 2004). Plant invertases are important regulators of growth, secondary metabolism, and defence
(Roitsch et al., 2003; Roitsch and González, 2004). It is
tempting to speculate on the role of calystegines participating in a control loop of carbohydrate metabolism by
their glycosidase inhibitory activity; however, data are not
yet sufficient to elaborate on such considerations.
Acknowledgements
Fig. 9. Model of tropane alkaloid biosynthesis and transport in potato.
The first specific enzyme PMT is restricted to root and sprouting tuber
eyes. Tropinone is transported from roots and sprouting eyes to aerial
plant tissues and tuber sprouts, respectively, and metabolized without
accumulation. Calystegines are also translocated from roots to aerial
parts and from tuber peel to sprouting eyes.
(Fu and Park, 1995): either suppression of sucrose synthase 4 in roots, although low (Fu and Park, 1995), is
sufficient to induce metabolic alteration in the tissues, or
calystegine accumulation in roots indirectly reacts to
altered carbohydrate metabolism in tubers. Slight but
significant changes in soluble sugars, sugar phosphates,
glycolytic intermediates, and the ATP:ADP ratio were
demonstrated in roots of sucrose synthase 4 antisense
plants (Biemelt et al., 1999). Glucose, glucose phosphates,
fructose, fructose-6-phosphate, and starch were decreased
compared with wild-type roots, sucrose was not affected,
and the ATP/ADP ratio was diminished.
The excellent technical help of Ursula Ködel, Beate Schöne, and
Anja Wodak, and helpful comments on the manuscript by AnnaCarolin Meier at the Pharmaceutical Institute in Halle are gratefully
acknowledged. The Deutsche Forschungsgemeinschaft financially
supported the group of BD.
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