Journal of Experimental Botany, Vol. 54, No. 382,
Regulation of Carbon Metabolism Special Issue, pp. 477±488, January 2003
DOI: 10.1093/jxb/erg040
Decreased sucrose content triggers starch breakdown and
respiration in stored potato tubers (Solanum tuberosum)
Mohammad-Reza Hajirezaei1,5, Frederik BoÈrnke1, Martin Peisker1, Yasuhiro Takahata2, Jens Lerchl3,
Ara Kirakosyan4 and Uwe Sonnewald1
1
Institut fuÈr P¯anzengenetik und Kulturp¯anzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany
Abstract
To change the hexose-to-sucrose ratio within phloem
cells,
yeast-derived
cytosolic
invertase
was
expressed in transgenic potato (Solanum tuberosum
cv. DesireÂe) plants under control of the rolC promoter. Vascular tissue speci®c expression of the
transgene was veri®ed by histochemical detection of
invertase activity in tuber cross-sections. Vegetative
growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the
sprout growth of stored tubers was much delayed,
indicating impaired phloem-transport of sucrose
towards the developing bud. Biochemical analysis of
growing tubers revealed that, in contrast to sucrose
levels, which rapidly declined in growing invertaseexpressing tubers, hexose and starch levels
remained unchanged as compared to wild-type controls. During storage, sucrose and starch content
declined in wild-type tubers, whereas glucose and
fructose levels remained unchanged. A similar
response was found in transgenic tubers with the
exception that starch degradation was accelerated
and fructose levels increased slightly. Furthermore,
changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration.
Considering that sucrose breakdown was restricted
to phloem cells it is concluded that, in response to
phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether
elevated hexose and/or hexose-phosphates or
decreased sucrose levels are responsible for the
metabolic changes observed, sucrose content was
decreased by tuber-speci®c expression of a bacterial
sucrose isomerase. Sucrose isomerase catalyses the
reversible conversion of sucrose into palatinose,
which is not further metabolizable by plant cells.
Tubers harvested from these plants were found to
accumulate high levels of palatinose at the expense
of sucrose. In addition, starch content decreased
slightly, while hexose levels remained unaltered,
compared with the wild-type controls. Similar to low
sucrose-containing invertase tubers, respiration and
starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers.
In contrast to invertase transgenics, however, no
accumulation of phosphorylated intermediates was
observed. Therefore, it is concluded that sucrose
depletion rather than increased hexose metabolism
triggers reserve mobilization and respiration in
stored potato tubers.
Key words: Invertase, potato tuber, respiration, starch
breakdown, sucrose isomerase, sucrose sensing.
Introduction
During potato tuber development four different developmental stages can be distinguished: (i) tuber induction, (ii)
storage phase, (iii) dormancy, and (iv) sprouting.
Molecular, biochemical and cell biological changes during
tuberization have been studied in great detail. During the
stolon-to-tuber transition, sucrose utilization changes from
2
Present address: Kyushu National Agricultural Experiment Station, Nishigoshi, Kumamoto, 861-1192, Japan.
Present address: Plant Science Sweden AB, Herman Ehles VaÈg 4, 26831 SvaloÈv, Sweden.
Present address: Faculty of Biology, Yerevan State University, Alex Manoogian St. 1, 375025 Yerevan, Armenia.
5
To whom correspondence should be addressed. Fax: +49 39482 5515. e-mail: [email protected]
3
4
Journal of Experimental Botany, Vol. 54, No. 382, ã Society for Experimental Biology 2003; all rights reserved
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 26, 2014
Received 8 April 2002; Accepted 6 September 2002
478 Hajirezaei et al.
net synthesis of reserve compounds to net degradation.
During this process, starch and protein breakdown
outweighs their synthesis leading to the formation of
soluble sugars and amino acids. As the transport sugar,
sucrose is formed in parenchyma cells and transported
via the phloem system towards the developing sprout.
Within the developing sprout sucrose is hydrolysed and
utilized to support growth and development. Since
visible sprout growth precedes detectable starch degradation, the initial growth phase is most likely initiated by
preformed sucrose. This assumption has recently been
supported by increasing the sucrose content of tubers by
tuber-speci®c expression of a cytosolic pyrophosphatase,
which leads to accelerated sprouting (Farre et al., 2001;
discussed in Sonnewald, 2001). Due to increased
sucrose demand in developing sprouts, soluble sugar
levels decrease in storage parenchyma cells. Falling
sugar levels might serve as a signal to trigger starch
breakdown to provide suf®cient assimilates for sprout
growth. Based on this hypothesis, it is tempting to
speculate that sucrose levels may act as sink signals to
adapt reserve mobilization in storage parenchyma cells
according to sink demand. To test this hypothesis,
transgenic potato plants with altered sucrose levels were
created. To inhibit the phloem transport of sucrose, a
cytosolic yeast-derived invertase was expressed in
phloem cells of transgenic potato plants. Tubers
harvested from these transgenic plants were analysed
for their sprouting behaviour, carbohydrate content and
rate of respiration. sucrose levels dramatically decreased
in tuber tissue due to invertase activity. In agreement
with the hypothesis that low sucrose levels would
trigger starch breakdown, accelerated starch turnover in
stored potato tubers was found. Irrespective of the
observed reserve mobilization, sprout growth of transgenic tubers was strongly delayed. Since sucrose
hydrolysis leads to an accelerated hexose metabolism,
analysis of these plants did not allow differences
between sucrose and hexose effects to be distinguished.
To circumvent enhanced hexose metabolism, the sucrose
content was decreased by tuber-speci®c expression of an
apoplastic sucrose isomerase from Erwinia rhapontici
(BoÈrnke et al., 2002b). The enzyme catalyses the
reversible conversion of sucrose into palatinose which
is not metabolizable by plant cells. As a consequence of
sucrose isomerase activity, a nearly quantitative conversion of sucrose into palatinose was observed. By
contrast with invertase-expressing plants, hexose metabolism was not stimulated. The investigation of starch
levels during potato tuber storage revealed accelerated
starch breakdown. This observation strongly suggests
that sucrose levels are responsible for the regulation of
metabolic processes during the sink-to-source transition
of potato tubers.
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hydrolytic to sucrolytic breakdown (Appeldoorn et al.,
1997; Hajirezaei et al., 2000). This process is associated
with a switch from apoplastic to symplastic phloem
unloading (Viola et al., 2001). Despite numerous studies
on the effect of growth regulators on tuberization,
endogenous tuberization factors have not been identi®ed.
Recent reports, however, suggest that sugars play a crucial
role in the regulation of cellular functions (for a review see
Pego et al., 2000). During seed development of Vicia faba,
for instance, high glucose levels correlate with high
metabolic activity whereas high sucrose levels correlate
with starch accumulation (Borisjuk et al., 1998). In potato
tubers, a similar situation has been hypothesized. By
ectopic expression of a yeast-derived cytosolic invertase
the hexose-to-sucrose ratio could be signi®cantly increased
(Sonnewald et al., 1997). As a consequence, the metabolic
activity of growing potato tubers was shifted from starch
synthesis towards glycolysis. This result suggested that
either low sucrose or elevated cytosolic glucose levels
would be responsible for the metabolic changes observed.
To distinguish between both situations, Trethewey et al.
(2001) expressed a bacterial sucrose phosphorylase in
transgenic potato plants. Due to the phosphorolytic
cleavage of sucrose, the formation of cytosolic glucose
could be circumvented. Interestingly, glycolysis and
respiration were induced in sucrose phosphorylase
expressing potato tubers. On the basis of these results,
the authors speculate that induction of glycolysis occurs
via a glucose-independent mechanism. Although interesting, the data do not unequivocally prove that low levels of
sucrose are sensed. Accelerated hexose metabolism and/or
fructose signalling could similarly explain the results
observed.
In contrast to numerous studies concerning metabolic
regulation in growing potato tubers, much less is known
about the regulatory processes during potato tuber
dormancy. The onset of dormancy starts during tuber
initiation and ends with the growth of a visible sprout.
This period is de®ned as the time during which sprout
growth will not occur even under otherwise favourable
conditions. The length of the dormancy period is
dependent on both the genetic background and the
environmental conditions during tuber development.
Post-harvest environmental conditions have only limited
impact on the sprouting behaviour. Therefore, the period
is classi®ed as endodormancy (Lang et al., 1987). It is
hypothesized that dormancy is regulated by the relative
concentrations of growth promoters and inhibitors
(Hemberg, 1985). Gibberellins and cytokinins are generally considered as growth promoters, whereas abscisic
acid and ethylene are believed to inhibit sprout growth.
Despite intensive studies, no unequivocal evidence for
the phytohormonal control hypothesis could be obtained.
During the sink (growing) to source (sprouting) transition of potato tubers, cellular metabolism shifts from a
Sucrose signalling in potato tubers 479
Materials and methods
Plants, bacterial strains and media
Potato plants (Solanum tuberosum) were obtained through
Vereinigte Saatzuchten eG (cv. Desiree) or Bioplant (cv. Solara),
Ebstorf, Germany. Plants in tissue culture were grown under a 16/8 h
light/dark period on Murashige and Skoog medium (Murashige and
Skoog, 1962) containing 2% sucrose. Plants used for biochemical
analysis were grown in individual pots (diameter 20 cm, depth 13
cm) in the greenhouse. E. coli strain XL1-blue (Stratagene, La Jolla)
was cultivated using standard techniques (Sambrook et al., 1989).
Agrobacterium tumefaciens strain C58C1 containing pGV2260
(Debleare et al., 1985) was cultivated in YEB medium (Verveliet
et al., 1975).
Plasmid construction and potato transformation
To obtain phloem-speci®c cytosolic invertase expression the truncated suc2 gene from Saccharomyces cerevisiae encoding the
mature invertase was placed under transcriptional control of the rolC
promoter from Agrobacterium rhizogenes as described in Lerchl
et al. (1995). Construction of the chimeric sucrose isomerase gene
has been described in BoÈrnke et al. (2002a). Direct transformation of
Agrobacterium tumefaciens strain C58C1:pGV2260 was performed
as described by HoÈfgen and Willmitzer (1988). Potato transformation using Agrobacterium-mediated gene transfer was performed as
described by Rocha-Sosa et al. (1989).
Preparation and analysis of samples for enzyme activities
To measure enzyme activities, 100±200 mg potato tuber slices were
homogenized in 0.5 ml 100 mM 4-(2-hydroxyethyl)-1-piperazine
ethanesulphonic acid (Hepes)-KOH, pH 7.5, 2 mM MgCl2, 1 mM
EDTA, 1 mM EGTA, 5 mM mercaptoethanol, 15% glycerine, and
0.1 mM Pefabloc phosphatase inhibitor. After centrifugation for 10
min, 13 000 rpm at 4 °C, the supernatant was frozen immediately for
further analysis. Invertase and Sucrose synthase activities were
determined as described in Hajirezaei et al. (1994). Amylase
activities were measured using the megazyme kit (Megazyme
Ireland). The procedure is based on the cleavage of a bond within the
blocked p-nitrophenyl maltosaccharide substrate. The non-blocked
reaction product containing p-nitrophenyl substituent is instantly
cleaved to glucose and free p-nitrophenyl by the excess quantities of
glucoamylase and a-glucosidase. The phenolate colour is developed
on the addition of Trizma base. For the measurement, 15 ml of extract
was added to a buffer containing 100 mM MES-KOH, pH 6.2, 1 mM
EDTA, and 1% mercaptoethanol. The reaction was started by adding
15 ml of substrate and the mixture was incubated at 37 °C for 30±60
min. After incubation, the reaction was stopped by adding 250 ml 1%
Tris solution (w/v). The developed colour was detected at 405 nm
using an Elisa Reader (Tecan Deutschland GmbH). Starch
phosphorylase was determined as described in Steup (1990). An
aliquot of extract was added to a buffer containing 25 mM imidazolHCl, pH 6.9, 5 mM MgCl2, 1 mM EDTA, 5 mM sodium molybdate,
0.6 mM NAD, 2.5 mM glucose-1,6-bisphosphate, 0.4 U phosphoglucomutase, 0.4 U glucose-6-P dehydrogenase, and 0.1% soluble
starch. The reaction was started by adding of 2 mM sodium
dihydrogen phosphate, pH 7.0 and the increase of NAD was detected
at 340 nm.
Histochemical staining of invertase
Staining of endogenous and yeast invertase was performed using an
enzyme-coupled assay. The procedure is based on the cleavage of
sucrose by invertase to glucose and fructose. In the presence of
oxygen, the glucose produced is converted to gluconolactone and
hydrogen peroxide by glucose oxidase. The oxidized coloured
diaminobenzidine is visualized by adding diaminobenzidine as
substrate and peroxidase. For the assay tuber discs (thickness: 2 mm,
diameter 2±3 cm) were washed three times with bi-distilled water
and incubated for 2 h at 37 °C in a buffer containing 0.38 M sodium
phosphate, pH 6.0, 0.1 mM DTT, 0.3 mg ml±1 diaminobenzidine
(DAB, Sigma, Germany), 0.25 mg ml±1 horseradish peroxidase
(Sigma, Germany), 100 mM purest sucrose and 0.02 mg ml±1 glucose
oxidase. After incubation the buffer was replaced by the same buffer
without DTT and a brown colour developed after 1±3 h. As the
control, discs were preincubated at 80 °C for 30 min (data not shown).
Metabolite determination
Metabolites were extracted essentially as described in Jelitto et al.
(1992). 50±300 mg tissue material was frozen immediately in liquid
nitrogen. After homogenizing, the frozen material was ground to a
®ne powder, 1.5 ml of 16% (w/v) trichloroacetic acid (TCA) in
diethylether (4 °C) was added and the tissue further homogenized.
After incubating the extract on dry ice for 15 min, 0.8 ml of 16%
TCA (w/v) in water containing 5 mM EGTA (4 °C) was added to the
homogenate, which was then left for an additional 3 h at 4 °C.
Following centrifugation for 5 min at 15 000 rpm, the water phase
was washed 3±4-fold with 600 ml water-saturated ether each time
and thereafter neutralized with 5 M KOH/1 M triethanolamine.
The concentrations of metabolites and ATP/ADP were determined
photometrically as in Stitt et al. (1989) using a dual wavelength
spectral photometer (Sigma-ZWS II, Biochem). The recovery of
small, representative amounts of each metabolite through the
extraction has been documented (Hajirezaei et al., 1994).
Determination of soluble sugars and starch
Soluble sugars and starch were quanti®ed in tuber samples extracted
with 80% ethanol, 20 mM Hepes-KOH, pH 7.5 as described in
Hajirezaei et al. (2000). Palatinose content was measured using
Dionex HPLC system (Sunnyvale, CA, USA) as described in BoÈrnke
et al. (2002a). The quanti®cation of palatinose content was carried
out on a peak area basis.
Measurement of respiration rate in whole potato tubers
Gas exchange measurements were carried out using an infrared gas
analysis in an open system (Compact minicuvette System CMS-400,
Walz GmbH, Effeltrich, Germany). Whole tubers were enclosed
in a standard chamber MK-022/A and the release of CO2 was
monitored continuously. Chamber temperature and dew point
temperature of the air entering the chamber were adjusted to 20 °C
and 13 °C, respectively. Measurements were done at a gas ¯ow rate
of 1500 cm3 s±1 and ambient CO2 concentration of about 100 mmol
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Enzymes and reagents
Enzymes and biochemicals were purchased from Boehringer
(Mannheim, FRG) or Sigma (Deisenhofen, FRG).
Preparation and determination of invertase activity and sugar
contents in vascular tissues and parenchyma cells
To verify phloem-speci®c expression of invertase, vascular tissue
and parenchyma from the central pith were collected and used for the
measurement of soluble invertase activity and sugar contents. To
harvest different tissues, tuber cross-sections (2 mm thickness) were
placed on a light box and vascular tissue was cut as 1 mm strips
along the slices. For parenchyma preparation, samples were
collected from the middle part of the same cross-sectioned tuber.
All samples were frozen immediately for further analysis. Soluble
invertase activity and sugar measurement were done as described in
Hajirezaei et al. (1994).
480 Hajirezaei et al.
mol±1. CO2 evolution rate referred to tuber fresh weight is given as
nmol CO2 g±1 s±1.
Results and discussion
Phloem-speci®c expression of cytosolic yeast-derived
invertase in transgenic potato plants
A chimeric gene consisting of the rolC promoter, the
truncated suc2 gene from yeast encoding the mature
invertase protein, and the octopine synthase polyadenylation signal (Lerchl et al., 1995) was used to obtain phloemspeci®c expression of invertase in transgenic potato plants.
After Agrobacterium tumefaciens-mediated gene transfer
95 regenerated plants (subsequently named DIN8) were
screened for enzymatic activity in roots using an invertase
activity gel assay (von Schaewen et al., 1990; data not
shown). Thirty preselected plants were multiplied in tissue
Fig. 2. Measurement of soluble sugar contents in different parts of
tuber slices containing a yeast invertase in phloem cells. Sugar
contents were determined in vascular tissue-enriched samples
indicated as blank bars and in parenchyma cells shown as ®lled bars.
Untransformed controls are displayed as C and transformed tuber as
87. Results are given as mmol g±1 FW and are means of ®ve
independent replicates 6SE.
culture and duplicates transferred to the greenhouse. No
detrimental effects on plant growth or development were
observed for greenhouse-grown DIN8 plants (data not
shown). DIN8 plants had a similar size, growth rate,
branching pattern of the shoot, leaf appearance, timing of
¯owering, and tuber yield as compared to untransformed
control plants. For a detailed biochemical analysis, three
independent DIN8 lines (numbers 30, 87 and 90) were
selected and ampli®ed in tissue culture.
Histochemical detection of invertase activity in potato
tuber cross-sections veri®es phloem-restricted
expression of the chimeric gene
To verify the cell-speci®c invertase expression, a histochemical invertase assay was performed. Cross-sections of
potato tubers were collected and washed three times with
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Fig. 1. Histochemical staining of invertase and measurement of
soluble acid invertase. For invertase staining, cross-sectioned tuber
slices were washed with water, incubated in a buffer and the resulting
brown colour was visualized (upper part). Acid invertase activity was
measured in vascular tissue-enriched samples (V) and parenchyma (P)
samples. (A) Cross-section of untransformed tuber, (B) cross-section
of the transgenic line 87, (C) the activity of the soluble acid invertase
in untransformed controls displayed as C and transformed tubers 87,
90 and 30. Results of invertase activity are given as nmol min±1 g±1
FW and are means of ®ve independent replicates 6SE.
Sucrose signalling in potato tubers 481
bi-distilled water to remove endogenous soluble sugars.
Subsequently, slices were incubated in a sucrose-containing buffer and invertase activity was visualized by the
formation of a brown colour (see materials and methods).
A slight staining of the endogenous invertase was detected
throughout control tubers (Fig. 1A). As shown in Fig. 1B,
invertase activity of transgenic plants was strongly
enhanced in vascular tissues, as compared to untransformed controls (Fig. 1A). This result was con®rmed by
measuring invertase activity in tissue samples enriched for
vascular tissue or containing mainly parenchyma cells. As
shown in Fig. 1C acid invertase activity of parenchyma
cells was only slightly elevated (lines 87 and 90) or
unaltered (line 30) in transgenic tuber samples, as compared to untransformed controls. In vascular tissueenriched samples, however, invertase activity of transgenic
tubers was more than 10-fold higher than in control
extracts (Fig. 1C). Tissue-speci®c activity of invertase was
further supported by measuring carbohydrate levels in
vascular tissue-enriched and parenchyma samples. Cellspeci®c expression of invertase was expected to result in
an increased hexose-to-sucrose ratio in phloem cells. As
shown in Fig. 2, hexose content of vascular tissue-enriched
samples from transgenic tubers was strongly increased (up
to 10-fold) at the expense of sucrose which strongly
decreased as compared to untransformed tubers. In
parenchyma cells, the contents of hexoses remained
unchanged while sucrose content decreased only slightly
in transformed tubers compared to untransformed tubers
(Fig. 2). The slightly reduced sucrose content in parenchyma cells may be explained by phloem-speci®c invertase
activity acting as a strong sucrose sink, rather than
unspeci®c invertase expression. Comparing these results
with results obtained from transgenic potato plants
expressing cytosolic invertase under the control of a
tuber-speci®c promoter (Sonnewald et al., 1997) strongly
supports the phloem-speci®c expression of the yeastderived invertase. Tuber-speci®c invertase expression
leads to a massive increase of glucose (10±20-fold) and a
dramatic decrease (down to 4%) of sucrose in parenchyma
cells (Sonnewald et al., 1997).
Phloem-speci®c expression of cytosolic invertase
results in non-sprouting potato tubers
Recently, it was shown that removal of cytosolic pyrophosphate changed the sprouting behaviour of transgenic
potato tubers dramatically (Hajirezaei and Sonnewald,
1999). It was postulated that the inhibition of sprouting
was most likely due to reduced sucrose export and its
subsequent utilization (Hajirezaei and Sonnewald 1999).
This assumption is supported by the ®nding that removal
of PPi in phloem cells of transgenic tobacco plants resulted
in photoassimilate accumulation in leaves and reduced
growth of tobacco plants (Lerchl et al., 1995;
Geigenberger et al., 1996). It was concluded that inorganic
pyrophosphate was essential for long-distance sucrose
transport. Based on these observations it was expected that
phloem restricted sucrose hydrolysis would result in a
disconnection between storage parenchyma cells and the
developing bud. Thereby, sprout growth should be
inhibited. As expected a strong delay in sprouting was
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Fig. 3. In¯uence of the expression of a yeast invertase in phloem cells on potato tuber sprouting. Tubers were lifted and stored for 5 months at
room temperature. (A) untransformed control, (B) line 87, (C) line 30, and (D) line 90.
482 Hajirezaei et al.
observed in the case of DIN8 tubers (Fig. 3B±D). After 5
months of storage, control tubers showed well-developed
sprouts (Fig. 3A) whereas DIN8 tubers did not develop
sprouts exceeding the 1±2 mm stage. Even after
prolonged storage (8 months) transgenic tubers did not
develop sprouts larger than 3 mm (data not shown). The
observation suggests that the initiation of sprouting
(indicative for the end of the dormancy period) is
independent of phloem-bound sucrose transport, whereas
sprout growth is highly dependent on phloem-mediated
sucrose supply.
Fig. 4. Effect of a phloem targeted yeast invertase on carbohydrate
metabolism during storage. Slices of untransformed and transformed
tubers were cut and frozen immediately for the measurement of
soluble and insoluble sugar contents. Samples were taken from freshly
harvested tubers and tubers stored at room temperature for 10, 16 and
28 weeks. FW/DW ratio was determined from the same tuber slices
used for sugar measurements. Untransformed controls are shown as C
and transformed tubers as 87, 30 and 90. Results are given as mmol
g±1 FW and are means of ®ve (soluble sugars) and eight (starch)
independent replicates 6SE.
Fig. 5. Enzyme activities in potato tubers expressing a yeast invertase
in the phloem cells. Tubers were stored for 150 d at room
temperature. The untransformed control is indicated as the ®lled bars
and transgenic lines as blank bars. 87, 90 and 30 represent the
independent transgenic lines. Tuber slices were harvested and
homogenized in a buffer as described in Materials and methods.
Measurements are means of ®ve replicates 6SE. Results are given as
nmol g±1 FW min±1.
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Irrespective of decreased sprout growth, starch
breakdown and dry matter loss is highly accelerated in
low sucrose-containing invertase-expressing tubers
during storage
Based on the results discussed, it was speculated that, due
to the cell-speci®c manipulation of sucrose hydrolysis,
reserve mobilization of tuber parenchyma cells would be
delayed as compared to wild-type tubers. To test this
assumption, potato tubers from transgenic and wild-type
plants were harvested from 60-d-old plants before the onset
Sucrose signalling in potato tubers 483
tubers. The three hexose phosphates, glc6P, fru6P and
glc1P showed similar behaviour in growing and stored
tubers of DIN8 lines. Transgenic tubers contained between
2.2±5-fold higher levels of hexose phosphates compared
with control tubers (Table 1). Similar responses were
found for the concentrations of the glycolytic intermediates 3-phosphoglycerate (3PGA) and phosphoenolpyruvate (PEP). While in growing tubers of DIN8 lines, the
level of 3PGA increased 2.1±2.6-fold, stored DIN8 tubers
contained 1.5±2.0 times higher levels. The concentration
of PEP increased up to 3.0-fold in growing DIN8 tubers
and up to 1.8-fold in stored Din8 tubers compared to
control tubers. Pyruvate content of growing DIN8 tubers
was indistinguishable from wild-type controls, but increased 2.0±3.2-fold in stored DIN8 tubers. The levels of
ATP and ADP did not differ signi®cantly between DIN8
and wild-type tubers.
The rate of respiration is strongly enhanced in DIN8
tubers
To investigate whether the accelerated starch breakdown
of DIN8 tubers would correlate with changes in enzyme
activities, enzymes involved in sucrose and starch
mobilization were measured in DIN8 and wild-type
tubers stored for 5 months at room temperature. As
shown in Fig. 5 sucrose synthase activity increased 1.4±
1.7-fold in DIN8 tubers, as compared to control tubers.
A comparison of starch-degrading enzymes showed a
slight decrease of starch phosphorylase, unaltered aamylase and moderately increased b-amylase activities.
Although, slight changes in enzyme activities could be
detected, it is unlikely that these changes are responsible
for the major changes in starch turnover.
According to these results, starch degradation of DIN8
tubers is highly accelerated which leads to a substantial
loss of dry matter. In parallel, sprout growth was strongly
inhibited. This con¯icting observation indicates that
excess carbohydrates are most likely channelled towards
alternative pathways. The increased content of glycolytic
intermediates suggests that the ¯ux through glycolysis and
possibly respiration may be enhanced. To test this
assumption, the respiration rate of intact growing and
stored tubers was measured. As shown in Fig. 6 the rate of
respiration markedly decreases from growing to stored
tubers, irrespective of the genetic background.
Nevertheless, DIN8 tubers are characterized by an elevated
respiration rate, compared with controls. In growing
tubers, the rate of respiration of DIN8 tubers was found
to be only moderately increased. This difference became
more pronounced during storage. During storage, respiration rates of DIN8 tubers were found to be 2±4-fold
higher, compared with controls (Fig. 6). This suggests that,
in the absence of sprout growth, excess carbohydrates are
utilized to fuel the respiratory chain.
Hexose-phosphates and glycolytic intermediates
accumulate in stored DIN8 tubers
Isomerization of sucrose as an alternative tool to
decrease sucrose levels in potato tubers
In order to investigate the consequences of phloemspeci®c yeast invertase expression on pathways involved
in sucrose degradation, starch synthesis and glycolysis,
concentrations of different intermediates in growing tubers
and tubers stored for 5 months at room temperature were
measured. As shown in Table 1, in growing DIN8 tubers a
reduction of uridinediphosphate glucose (UDPGlc) was
found which, along with fructose, is the product of the
cleavage of sucrose by sucrose synthase. In contrast to
growing tubers, the UDPGlc level did not change signi®cantly in stored tubers of DIN8 lines compared to control
The results discussed so far indicate that (i) phloemmediated sucrose transport is required for sprout growth
and (ii) that low sucrose may act as a signal to communicate sink demand and to regulate reserve mobilization in
source tubers. On the basis of the results a working
hypothesis has been developed which is summarized in
Fig. 7. According to the model, sucrose is transported via
the phloem system from the storage parenchym to the
developing sprout. As a consequence, the sucrose content
decreases in parenchyma cells which may act as a `sink
signal' to stimulate reserve mobilization. Phloem-speci®c
Accelerated starch breakdown of DIN8 tubers is not
paralleled by signi®cant changes in the activity of
starch-degrading enzymes
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of ¯owering. Subsequently, samples for carbohydrate
analysis were taken from freshly harvested tubers (growing) and tubers stored for 10, 16, and 28 weeks at room
temperature. As shown in Fig. 4, glucose, fructose and
starch levels did not signi®cantly differ between growing
DIN8 and wild-type tubers. However, sucrose levels were
strongly decreased in growing DIN8 tubers (Fig. 4C).
During storage, sucrose levels of DIN8 and wild-type
tubers gradually decreased. Irrespective of the timedependent decrease of sucrose, DIN8 sucrose levels always
remained lower as compared to the wild type. During
storage, hexose levels of DIN8 tubers remained comparable to the wild type controls. These moderate changes of
soluble sugars would have been expected because of the
cell-speci®c expression of invertase. Analysing for starch
accumulation, however, revealed a different picture. In
growing potato tubers, no obvious difference in the amount
of starch accumulation between transgenic and wild-type
tubers was detectable, however, this changed drastically
during storage. While starch breakdown was hardly
signi®cant in the case of wild-type tubers, DIN8 tubers
were found to degrade large amounts of starch (Fig. 4D).
This accelerated starch breakdown was paralleled by a
signi®cant loss of dry matter (Fig. 4E).
484 Hajirezaei et al.
Table 1. In¯uence of a phloem-targeted yeast invertase on metabolite concentrations during dormancy
Samples were harvested from tubers of growing plants and tubers stored for 150 d at room temperature. Results are mean 6SE
(n=8 (control) and n=4 (transgenic lines)) independently harvested tubers.
Growing tubers
Control
Din-87
Din-90
Din-30
UDPGlc
Glc-6-P
Fru-6-P
Glc-1-P
3PGA
PEP
Pyruvate
ATP
ADP
81.162.4
128612
36.262.3
10.061.0
65.067.8
22.362.5
5.160.7
38.063.3
17.462.3
64.465.5
542636
167610
34.261.9
14264.9
40.362.3
7.062.1
26.061.9
13.060.8
49.764.2
513612
15565.9
32.161.3
13768.2
55.465.5
7.161.0
33.063.5
14.262.0
49.262.5
590630
17364.7
33.262.0
17265.2
64.362.2
6.562.4
35.163.8
16.061.0
27.465.2
260625
82.064.0
16.262.5
19166.0
53.863.5
4.360.9
19.861.4
12.061.7
29.9611
303614
83.063.0
18.363.2
15166.5
48.662.2
3.860.5
23.660.5
11.961.3
33.166.3
313620
98.067.0
19.262.1
200614
60.363.2
6.160.6
29.565.6
15.361.2
Tubers stored for150 d at RT
UDPGlc
Glc-6-P
Fru-6-P
Glc-1-P
3PGA
PEP
Pyruvate
ATP
ADP
41.763.0
11766.0
32.861.9
7.560.7
10363.5
33.562.4
1.960.3
24.761.8
14.261.0
Fig. 6. In¯uence of the expression of a yeast invertase in phloem cells
on respiration rate. Whole growing tubers (shown as G) and whole
tubers stored for 3, 6, 10 and 18 weeks at room temperature from
control (®lled bars) and transgenic lines (blank bars) were used to
measure the respiration rate. 87, 90 and 30 represent the independent
transgenic lines. Data represent mean values and standard errors
(n=11) of CO2 evolution rates from growing and stored tubers.
Results are given as nmol g±1 s±1.
expression of invertase leads to four major changes as
compared with untransformed controls: (i) due to sucrose
hydrolysis, sucrose levels decline, (ii) nutrient supply of
the developing sprout is blocked, (iii) the rate of respiration is stimulated, and (iv) starch breakdown is accelerated. Since invertase expression leads to severe changes in
sprout development, it cannot be ruled out that unknown
`sink signals' exist, regulating starch breakdown and
respiration in storage parenchyma cells. In order to
substantiate the results obtained with the phloem-speci®c
invertase-expressing potato lines, it was decided to use a
second set of experiments employing an additional transgenic approach. the generation of transgenic potato plants
expressing a chimeric sucrose isomerase gene from
Erwinia rhapontici under the control of the tuber-speci®c
patatin class I B33 promoter (BoÈrnke et al., 2002b) has
recently been described. The gene product was targeted to
the apoplasm where it catalyses the conversion of sucrose
into the non-metabolizable sucrose isomer palatinose.
Biochemical analyses revealed a nearly quantitative conversion of sucrose into palatinose within these transgenic
tubers. Palatinose production only slightly affected starch
accumulation, resulting in an only marginally decreased
starch content (BoÈrnke et al., 2002b). The common feature
of both invertase-expressing and sucrose isomeraseexpressing potato tubers is the drastically reduced sucrose
content. However, since palatinose is not further metabo-
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 26, 2014
Intermediates
(nmol g±1 FW)
Sucrose signalling in potato tubers 485
lized its accumulation does not provide precursors for any
further enzymatic reactions which is in contrast to the more
global metabolic impact of sucrose cleavage by invertase
and the subsequent release of hexoses which preferentially
feed into the glycolytic pathway. The comparison of both
transgenic genotypes would allow for a clear dissection of
metabolic effects mediated by a reduced sucrose content
per se and of effects exerted by some downstream
metabolism. In order to test this hypothesis, three previously described sucrose isomerase-expressing potato lines
(BoÈrnke et al., 2002b) were subjected to a thorough
biochemical analysis.
After multiplication in tissue culture the plants were
transferred to the greenhouse. As observed before (BoÈrnke
et al., 2002b) sucrose isomerase-expressing plants were
indistinguishable from control plants with respect to size,
growth rate, overall appearance, and tuber yield. The
plants were allowed to set tubers and after harvest a portion
was directly used for biochemical analyses, whereas the
remaining tubers were stored at room temperature before
analysis. As described earlier (BoÈrnke et al., 2002b)
expression of the Erwinia rhapontici sucrose isomerase
within the apoplasm of transgenic tubers evoked a nearly
quantitative shift of sucrose into palatinose (Fig. 8). Starch
content was only slightly decreased in growing transgenic
tubers. The picture changed distinctly when tubers stored
for 80 d were used for the analyses. Hexose content was
now elevated compared with control tubers whereas
sucrose levels remained lower in the transgenics. The
difference in starch content between control and transgenic
tubers was now much more pronounced with starch levels
being 66% in line 5, 56% in line 12 and 60% in line 26,
respectively, lower.
Palatinose production has no impact on metabolite
contents in transgenic potato tubers
In order to investigate the impact of sucrose conversion
into palatinose on sucrose degradation and glycolysis, the
contents of different intermediates of these pathways was
determined. In contrast to the phloem-speci®c invertaseexpression, sucrose isomerase-expression had no impact
on UDPGlc levels in freshly harvested tubers, whereas
stored tubers showed a reduction of this metabolite up to
42% compared with the control tubers (Fig. 9A). Hexose
phosphate levels increased in freshly harvested transgenic
tubers, while, in stored transgenic tubers, the levels
decreased up to 45% compared with the control. 3-PGA
content remained unchanged in both growing and stored
tubers.
Stored potato tubers expressing sucrose isomerase
differ in respiration rate from the untransformed control
Although the levels of glycolytic intermediates in
palatinose-accumulating potato tubers remain largely
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Fig. 7. Schematic presentation of the events occuring in tubers which contain a yeast invertase in phloem cells.
486 Hajirezaei et al.
unaltered, starch breakdown is heavily accelerated during
storage. To ®nd out whether this would affect the
respiration rate a similar experiment as described for the
invertase-expressing lines was conducted. Respiration
rates of the untransformed control and that of sucrose
isomerase-expressing lines did not differ during growth
and dramatically decreased during storage. However, after
80 d of storage the respiration rates of transgenic tubers
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 26, 2014
Fig. 8. Impact of a chimeric PalI gene, sucrose isomerase, on
carbohydrate metabolism in potato tubers. Samples were taken from
growing tubers (left side of the ®gure) and tubers stored for 80 d at
room temperature (right side of the ®gure). Contents of (A) palatinose,
(B) glucose, (C) fructose, (D) sucrose, and (E) starch were determined
as described in Materials and methods. Values of untransformed and
transformed tubers (®lled bars) represent the mean of ®ve (A, B, C, D)
and eight (E) independent measurements 6SE. 5, 12 and 26 represent
the independent transgenic lines. Results are given as mmol g±1 FW.
Fig. 9. In¯uence of of a chimeric PalI gene, sucrose isomerase, on
intermediates in growing tubers (left) and tubers stored for 80 d at
room temperature (right). (A) UDPGlucose, (B) the sum of glucose-6phosphate, fructose-6-phosphate and glucose-1-phosphate and (C) 3phosphoglycerate. 5, 12 and 26 represent the independent transgenic
lines. Data are presented as the mean 6SE of measurements on ®ve
independent tubers. Results are given as nmol g±1 FW.
Sucrose signalling in potato tubers 487
were signi®cantly higher than those of control tubers
(Fig. 10).
Conclusion
Fig. 10. Effect of a chimeric PalI gene, sucrose isomerase, on the
respiration rate of intact tubers. Data represent mean values and
standard errors of CO2 evolution rates from growing tubers (n=5),
tubers stored for 30 d (n=5) and tubers stored for 80 d (n=9).
Untransformed control is displayed as C and transformed tubers as 5,
12 and 26. Results are given as nmol g±1 s±1.
Acknowledgements
We wish to thank Andrea Knospe and Sybille Freist for doing plant
transformation, Enk Geyer and the greenhouse personnel for taking
care of the greenhouse plants and H Ernst for photographic work.
Furthermore, we are grateful to Hannelore Apel for the measurement
of respiration rate and to Ulrike Schlereth for the excellent technical
assistance.
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