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Plant Physiol. (1999) 121: 123-134
Antisense Repression of Hexokinase 1 Leads to an Overaccumulation
of Starch in Leaves of Transgenic Potato Plants But Not to Significant
Changes in Tuber
Carbohydrate Metabolism1
Jon Veramendi2, *,
Ute Roessner,
Andreas Renz,
Lothar Willmitzer, and
Richard N. Trethewey3
Max Planck Institut für Molekulare Pflanzenphysiologie, Karl
Liebknecht Strasse 25, 14476 Golm, Germany
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ABSTRACT |
Potato
(Solanum tuberosum L.) plants transformed with sense and
antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the
wild-type activity in antisense transformants to 485% activity in
sense transformants) and a 7-fold variation in developing tubers (from
32% of the wild-type activity in antisense transformants to 222%
activity in sense transformants). Despite the wide range of hexokinase
activities, no change was found in the fresh weight yield, starch,
sugar, or metabolite levels of transgenic tubers. However, there was a
3-fold increase in the starch content of leaves from the antisense
transformants after the dark period. Starch accumulation at the end of
the night period was correlated with a 2-fold increase of glucose and a
decrease of sucrose content. These results provide strong support for
the hypothesis that glucose is a primary product of transitory starch
degradation and is the sugar that is exported to the cytosol at night
to support sucrose biosynthesis.
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INTRODUCTION |
The route by which carbon is exported from the chloroplast to the
cytosol to support Suc biosynthesis remains under critical discussion
(Trethewey and Smith, 1999 ). The presence of a triose-P translocator in
the envelope of chloroplasts and the role of this translocator in the
support of Suc biosynthesis during the day has long been established
(Stitt, 1990). However, there is increasing evidence that Glc, and not
triose-P, is the predominant form in which carbon is exported from the
chloroplast at night.
First, the presence of a Glc transporter in the chloroplast envelope
has been demonstrated in spinach (Schäfer et al., 1977), Arabidopsis (Trethewey and ap Rees, 1994a, 1994b), and tobacco (Häusler et al., 1998). Second, a mutant line of Arabidopsis defective in chloroplast Glc transport is characterized by an excess-starch phenotype; the amount of transient starch in leaves at
the end of the light period was around five times that found in
wild-type leaves (TC265; Caspar et al., 1991; Trethewey and ap
Rees, 1994b). It is conceivable that an inability to export Glc leads
to a feedback inhibition of the pathway of starch degradation and the
observed excess-starch phenotype. This hypothesis was supported by
experiments with wild-type Arabidopsis leaves indicating that there is
a net glycolytic flux at night, which would be an impossibility if the
primary export of carbon was at the triose-P level. Third, Schleucher
et al. (1998) have recently reported the use of NMR to distinguish
between Glc synthesized from hexose export and that derived from
triose-P; starch degradation was allowed to occur in the presence of
2H-enriched water, and the ratio of labeling in
Glc at the different carbon atoms was used to determine the relative
contribution of the two routes of export. These authors concluded that
in tomato and bean leaves, more than 75% of the carbon exported from
chloroplasts at night is in the form of hexose.
If the predominant route of carbon export from the chloroplast at night
is at the level of Glc, then a cytosolic hexokinase (HK) (EC 2.7.1.1)
is required to phosphorylate Glc to Glc-6-P, thus activating the hexose
unit for Suc biosynthesis. In plants there have been several different
reports about glucokinase (GLK) (Glc phosphorylating) or HK activities.
The difference between GLK and HK is a functional classification: the
former phosphorylates strictly Glc, while the latter is capable of
phosphorylating a range of hexoses (e.g. Fru and Man).
Glc-phosphorylating activities have thus far been reported in the
following plant organs: tomato fruit, GLK (Martinez-Barajas and
Randall, 1998); maize endosperm, GLK (Doehlert, 1989); maize
roots, HK (Galina et al., 1995); maize leaves, HK (Schnarrenberger,
1990); castor bean endosperm, HK (Miernyk and Dennis, 1983); pea seeds,
HK and GLK (Turner et al., 1977; Turner and Copeland, 1981); spinach
leaves, HK (Baldus et al., 1981; Schnarrenberger, 1990); Arabidopsis
leaves, HK (Jang et al., 1997); pea leaves, HK (Schnarrenberger, 1990);
tobacco leaves, HK (Sindelar et al., 1998); soybean nodules, HK
(Copeland and Morell, 1985); and avocado, HK (Copeland and Tanner,
1988). Glc-phosphorylating activities have been reported to be
associated with the mitochondrial envelope in avocado (Copeland and
Tanner, 1988), spinach (Schnarrenberger, 1990), and maize roots (Galina et al., 1995), and to be soluble in the cytosol of soybean nodules (Copeland and Morell, 1985) and spinach (Schnarrenberger, 1990). There has been no convincing report of HK/GLK activity located within
the stroma of plastids.
The most thorough characterization of the kinetic properties of HK
isoforms has been undertaken in potato (Solanum tuberosum L.) (Renz and Stitt, 1993; Renz et al., 1993). These authors reported the presence of three isoforms in potato tubers, all of which had a
high affinity for Glc and Man but a significantly lower affinity for
Fru. All three HK activities had affinities for ATP that were around
10-fold higher than for other nucleoside triphosphates. One of the HK
activities (denoted HK1) was inhibited by Glc-6-P with a
physiologically relevant Ki of 4.1 mM; the inhibition was non-competitive with
respect to Glc. The balance of the three activities was subject to
change according to the developmental status of the tubers.
Glc-phosphorylating activity was very low in growing tubers but
increased during storage and sprouting, with HK1 activity becoming much
more dominant in relation to the activity of HK2; HK3 was always
negligible (Renz et al., 1993). No information was provided about the
compartmentation of these three isoforms or their relative activities
within leaf tissue.
HK has recently been implicated as a sensor in the sugar-dependent
regulation of gene expression in plants (Koch, 1996; Jang and Sheen,
1997; Jang et al., 1997; Zhou et al., 1998; Smeekens, 1998). The
phenomenon of carbon catabolite repression has been well described
in microorganisms, especially in Saccharomyces cerevisiae, in which a large number of genes are regulated by HK in accordance with the Glc levels in the cell (Ma and Botstein, 1986; Rose et al., 1991; Ronne, 1995). In mammals, GLK plays a key role
in Glc sensing by the insulin-secreting pancreatic  -cells. This has
been shown by analysis of transgenic mice with altered GLK activity
(Grupe et al., 1995) and by a high correlation between a form of type
II diabetes and the occurrence of GLK mutations in humans (Bell et al.,
1996).
In higher plants it is generally accepted that sugars regulate the
expression of genes involved in many different processes, although
whether mechanisms are at work that are similar to those found in
microorganisms and mammals is subject to much debate (Koch, 1996;
Smeekens, 1998). Jang et al. (1997) have cloned two HKs from
Arabidopsis and performed different bioassays with transgenic Arabidopsis lines that overexpressed or contained reduced amounts of HK
activity following antisense inhibition. Wild-type seedlings grown on
6% (w/v) Glc plates showed a phenotype characterized by
suppression of hypocotyl and root elongation and by the greening and
expansion of cotyledons. Transgenics that overexpressed HK showed
hypersensitivity to 6% (w/v) Glc (stunted hypocotyls,
cotyledons, and roots), while antisense transgenic lines were
relatively hyposensitive to Glc. These authors concluded that the HK
protein acts as a sensor for Glc levels and that the absence or
increased abundance of this sensor led to the observed dramatic
phenotypical changes through alterations in gene expression. However,
no data were presented on the biochemical characterization of the
Arabidopsis lines, leaving open the question of whether the
phenotypical changes were an indirect consequence of an altered
metabolic system.
In this paper we present a biochemical and physiological analysis of
transgenic potato lines in which the activity of an isoform of HK has
been modulated using either overexpression or antisense approaches. We
chose to work with potato because it is a well-characterized system
with a strong sink organ. In particular, we evaluated whether inhibition of HK in leaves would provide data to support the hypothesis that Glc is the primary form in which carbon is exported from the
chloroplast at night.
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MATERIALS AND METHODS |
Plant Material
Potato (Solanum tuberosum L. cv Desirée) plants
were supplied by Saatzucht Lange (Bad Schwartau, Germany). Plants were
grown in the greenhouse at 15°C to 22°C under a 16-h light/8-h dark photoperiod (natural daylight was supplemented to give a minimum photon
flux of 250 µmol m 2
s1). Developing tubers (10-25 g fresh weight)
were harvested from healthy, 2- to 3-month-old plants and used for the
determination of enzyme activities, metabolic intermediates, starch,
and sugars. Mature tubers were harvested from senescent plants and used
for yield analysis. The analysis of carbohydrates in leaves was
performed on 8-week old plants grown in the greenhouse from May through June (12 plants/line). For each measurement, three source leaf discs (1 cm in diameter) were harvested from individual plants at the following
times: 4:30 AM, 10 AM, 2:30
PM, and 8 PM, immediately frozen in liquid nitrogen, and stored at  20°C prior to analysis.
mRNA Extraction and Northern Analysis
Total RNA was isolated from 2 g fresh weight of tuber tissue,
as described by Logemann et al. (1987). mRNA was extracted from 300 µg of total RNA using a mRNA purification kit (Dynabeads, Dynal,
Germany), and separated on agarose-formaldehyde gels (approximately 4 µg per sample). Standard conditions were used for the transfer of RNA
to membranes and for the subsequent hybridization (Sambrook et al.,
1989). A potato ubiquitin cDNA probe was used as a control to
standardize loading in each lane. Densitometric analysis was performed
using a phosphor imager (BAS-1500, Fuji).
Yeast Complementation Assays
A full-length cDNA encoding a HK from potato (StHK1; accession no.
X94302) was isolated as an EST in the laboratory of Dr. Udo Schmitz
(Institut für Genbiologische Forschung, Berlin) and kindly made
available for this project. The full-size StHK1 was subcloned as a
1.7-kb EcoRI fragment into the p195XE (YEplac195 derivative)
yeast expression vector. Two strains of Saccharomyces cerevisiae were used for complementation: DBY2219
(hxk1, hxk2; Ma and Botstein, 1986) and YSH7.4-3C
(glk1, hxk1, hxk2; De Winde et al.,
1996). The medium for the selection of the transformed colonies
contained 0.67% (w/v) yeast nitrogen base (Difco, Detroit, MI) with
amino acids and
(NH4)2SO4,
2% (w/v) Bacto agar (Difco), and 2% (w/v) Fru (DBY2219 strain) or 2%
(w/v) Glc (YSH7.4-3C strain). Crude extracts of the transformed
YSH7.4-3C cultures were prepared at 4°C as follows: 100 mL of a fresh
culture (A595 between 0.7 and 0.9)
were centrifuged at 4,000g for 10 min, washed with 10 mL of
cold water, centrifuged at 4,000g for 10 min, and
resuspended in 3.5 mL of extraction buffer (Trethewey et al., 1998,
without BSA). Following lysis with a French press, the lysate was
centrifuged at 12,000g for 10 min and the supernatant was
desalted using PD-10 columns (Pharmacia). Aliquots of 100 µL were
immediately frozen in liquid nitrogen and stored at 80°C until
analysis. HK1 activity was assayed using the pyruvate kinase/lactate
dehydrogenase test detailed by Renz and Stitt (1993), and the
phosphorylation coefficient was also calculated as described by these
authors.
Preparation and Selection of Transgenic Lines
The full-length 1.7-kb StHK1 EST was initially subcloned into the
plasmid pSK. For the overexpression construct a
BamHI/SalI fragment was introduced in the sense
orientation into the vector pBinAR-Kan (Liu et al., 1990) between the
CaMV 35S promoter and the ocs terminator. For the antisense
construction, a 1.3-kb/HindIII fragment of the StHK1 cDNA
was subcloned into pSK in the reverse orientation. An
Asp718/SalI fragment was then introduced into the
vector pBinAR-Kan between the CaMV 35S promoter and the ocs terminator (Liu et al., 1990). Both constructs were introduced into
potato by Agrobacterium tumefaciens-mediated transformation (Rocha-Sosa et al., 1989), and transgenic plantlets were selected on
kanamycin-containing medium (Dietze et al., 1995). Initial screening of
around 100 lines was performed by determining HK activity in the leaves
of plants grown in 6-cm pots in a phytotron. A second activity screen
was then performed with six plants per line for seven preselected lines
grown in the greenhouse.
HK Purification from Potato Tubers
The purification procedure was based on that described by Renz et
al. (1993). Developing tubers were harvested from non-senescent plants
grown in the greenhouse during the spring season and stored at 4°C
prior to use. Peeled and sliced potato tuber tissue (approximately 7 g) was homogenized in a blender (Waring) for 1 min in 30 mL of
extraction buffer (50 mM HEPES-KOH, pH 8.0, 5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM benzamidine, 1 mM
 -aminocaproic acid, 0.5 mM PMSF, and 2% [w/v]
polyvinylpolypirrolidone). The homogenate was filtered, centrifuged,
refiltered, and then applied to a DE-52 cellulose column (18 cm long,
1.6 cm in diameter). The column was washed with buffer (50 mM HEPES-KOH, pH 8.0, 5 mM
MgCl2, and 1 mM DTT) and eluted
in a linear gradient of 0 to 0.4 M KCl at a flow rate of
1.5 mL min1, and 60 1-mL fractions were
collected. Twenty-five microliters of each fraction was checked for HK
activity as described below. Two different groups of fractions,
corresponding to HK1 and HK2 activities, were further purified by
affinity chromatography. HK1 fractions were desalted by passage through
PD-10 columns (Pharmacia) prior to loading onto an affinity
chromatography column (Matrex Blue A, Amicon, Beverly, MA). HK2
was collected from the flow-through of the chromatography column and
concentrated with a Centricon 10 membrane (Amicon). Purified samples of
HK1 and HK2 were used to determine the dependence of activity on pH.
Biochemical Analysis
Metabolites, starch, and sugars were determined as described by
Trethewey et al. (1998). The recoveries of metabolites in the TCA
extracts were found to be: Glc-6-P, 108% ± 10%; Fru-6-P, 117% ± 10%; Glc-1-P, 119% ± 11%; and 3-phosphoglycerate, 95% ± 7%
(mean ± SE; n = 6). HK activity was
assayed as described by Renz et al. (1993) with the following
modifications: NAD was used instead of NADP, the Glc-6-P dehydrogenase
was from Leuconostoc mesenteroides, the increase in
absorbance was measured at 340 nm, and the final volume of the reaction
was 300 µL.
Statistical Analysis
The word "significant" is used in the text only when the
change in question has been confirmed to be statistically significant (P < 0.05) with the t test incorporated
into Microsoft Excel 7.0.
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RESULTS |
Molecular Characterization of StHK1
A full-length cDNA encoding StHK1 was isolated as an EST in the
group of Dr. Udo Schmitz and kindly made available for this project
(accession no. X94302). The cDNA encodes a protein of some 498 amino
acids; no evidence was found of an N-terminal targeting peptide. The
sequence of StHK1 shows a high similarity to the sequences already
reported from Arabidopsis (Jang et al., 1997): 71% nucleotide identity
to AtHXK1 and 70% identity to AtHXK2. At the amino acid level, the
identity with AtHXK1 and AtHXK2 was 69% and 67%, respectively, while
the computed similarities were 81% and 79% using the BESTFIT
algorithm incorporated into the Genetics Computer Group package,
version 8.0. Comparison with HKs from yeast revealed 35% identity and
55% similarity with both HXK1 and HXK2. Conserved binding domains for
sugar and ATP (two regions for phosphate, one for adenosine) could be
identified in the amino acid sequence of StHK1 (Bork et al., 1993).
Analysis of mRNA northern blots using the StHK1 cDNA as a probe
revealed a band of around 1.7 kb. The transcript was present in young
and mature leaves, stems, roots, stolons, and developing and mature tubers (data not shown).
StHK1 Can Complement Two Yeast Deficient Mutants and Phosphorylate
Both Glc and Fru
To determine whether the protein product of the potato StHK1 cDNA
is enzymatically active, we performed a functional complementation of
two different yeast strains: DBY2219 (lacking HK activity) and
YSH7.4-3C (lacking HK and GLK activity). Transformed yeast cells were
able to grow on a selective medium containing Fru (DBY2219) or Glc
(YSH7.4-3C) as the only carbon source. The vector p195XE alone was not
able to complement the mutants (Fig. 1).
The kinetic properties of HK1 were analyzed in extracts of complemented
YSH7.4-3C cultures (Table I). HK1 showed
a high affinity for Glc and Man (Km
values were 33 and 29 µM, respectively). The
Km for Fru was 50 times higher than
that for Glc and Man. The Vmax was
found to be higher for Glc and Fru than for Man. The estimated values for the phosphorylation coefficient showed a high selectivity of HK1
for Glc and Man compared with Fru. The
Km and
Vmax of HK1 with respect to ATP was
detected to be 103 µM and 360 nmol
min1 mg1 protein,
respectively. No HK activity was found in the original yeast strain
YSH7.4-3C or following expression of the empty p195XE vector.
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| Figure 1.
Complementation of the yeast YSH7.4-3C
hxk1/hxk2/glk1 triple mutant. Potato HK1 cDNA provided
catalytic activity to support the growth of the yeast mutant on the Glc
plate. A, Two independent transformants; B, culture transformed with
the empty plasmid p195XE.
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Table I.
Kinetic constants of the StHK1 (expressed in the
YSH7.4-3C yeast strain) for Glc, Fru, and Man
The Km and Vmax values
were calculated from Eadie-Hofstee plots. Each value is the mean of
three independent assays (SE was 2%-16% of the mean).
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Overexpression and Antisense Transgenic Potato Lines Showed a Broad
Range of HK Activities
Antisense ( HK1) and overexpression (PHK1) transgenic potato
plants were selected following screening at the level of HK activity. Three lines of HK1 were found (8, 13, and 82), all of which showed a
reduction in activity compared with wild type of up to 78% in source
leaves and 76% in developing tubers (Table
II). Four lines of PHK1 were selected (5, 70, 89, and 95), all of which demonstrated up to a 4-fold increase in
HK activity in leaves and up to a 2-fold increase in tubers (Table II).
In the wild-type plants, HK activity was always higher in tubers than
in leaves; in the PHK1 lines this relationship was inverted. Northern
analysis of mRNA preparations from the selected lines corroborated the
enzyme activity data (Fig. 2).
Densitometric analysis of the blots indicated that there was up to a
10-fold reduction in transcript levels in the HK1 lines and up to a
15-fold increase in the PHK1 lines.
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Table II.
Hexokinase activity in antisense (HK1) and
overexpression (PHK1) transgenic lines
Plants were grown in the greenhouse in the autumn/winter season in
3.5-L pots. Leaf samples were taken from 8-week-old plants, tuber
samples from 12-week-old plants. Data are presented as the means ± SE; n = 6.
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| Figure 2.
Northern-blot analysis of transgenic plants with
altered expression of StHK1. mRNA was extracted from developing tubers
of greenhouse-grown plants. The filter was hybridized with a
1.3-kb/HindIII cDNA fragment derived from the StHK1
cDNA. The histogram shows the ratio of StHK1 mRNA to potato ubiquitin
mRNA (used as a control) in each transformed line (antisense lines 8, 13, and 82 and sense lines 5, 70, 89, and 95). WT, Wild type.
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StHK1 cDNA Corresponds to the Partially Purified HK1 Described
Previously
To determine whether the StHK1 cDNA corresponded to any of the
three HK activities previously described by Renz et al. (1993), we
chose an antisense (HK1-13) and an overexpression line (PHK1-70) for
a partial purification experiment using the same method described by
Renz et al. (1993). Separation of wild-type tuber extracts after
elution from the DE-52 cellulose column showed a pattern similar to
that described by Renz et al. (1993). Two different activities (named
HK1 and HK2 by Renz et al., 1993) could be separated (Fig.
3). The PHK1-70 sample showed a large
increase in the first peak; in this line both HK peaks were of similar
size (Fig. 3B). In the antisense line HK1-13, the first peak almost
disappeared (Fig. 3C).
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| Figure 3.
Separation of HK activities from developing potato
tubers of the wild type (A), and the PHK1-70 (B) and HK1-13 (C)
lines after elution from a cellulose column with a KCl gradient.  ,
HK activity; dashed lines, KCl concentration.
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One of the differences between HK1 and HK2 is the dependence of their
activities on pH (Renz and Stitt, 1993). Therefore, we performed a pH
experiment with completely separated HK1 and HK2 preparations from the
wild type and from both transgenics. We used an affinity chromatography
column to obtain full separation of the two isoforms, and HK1 bound to
the column, while HK2 passed through. This step also served as a
control to ensure that the HK1 and HK2 enzyme activities assigned here
correspond to the HK1 and HK2 previously named by Renz et al. (1993).
HK2 had a broad pH response, with only a 20% decrease in activity at
pH 9.6 in relation to pH 7.5. HK1 activity, however, decreased quite strongly in the same pH range (up to 60%) (Fig.
4A). Similar patterns were observed with
extracts derived from wild-type and transgenic plants. Our results were
in full agreement with the previous observations from Renz and Stitt
(1993).
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| Figure 4.
Dependence on pH of HK1 and HK2 activities
purified from tubers of the HK1-70 line (A) and HK1 activity following
expression of the StHK1 cDNA in the YSH7.4-3C yeast strain (B). The
assays were carried out with 50 mM MES-KOH (pH 5.0-6.5),
50 mM imidazole (pH 6.0-7.4), or 50 mM
Tris-HCl (pH 7.0-9.5).  , HK1;  , HK2; , HK1 (yeast).
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In addition, we performed a partial purification of the HK activity
produced following expression of the StHK1 cDNA in a yeast strain
deficient in Glc-phosphorylating activities (YSH7.4-3C). The crude
extract was desalted and loaded onto an affinity chromatography column
as previously described. As expected, the HK activity bound to the
column and enzyme activity was exclusively present in the eluate, but
was not found in the flow-through (data not shown). However, we were
surprised to observe that the dependence of this HK activity on pH was
strikingly different from that of HK1 from plant extracts. The HK1
activity extracted from yeast showed a broader range of maximal
activity and relatively high activity at acidic pH (Fig. 4B). These
differences could be explained by posttranslational processing of HK1
in yeast that is different from that which occurs in potato tubers.
Tuber Yield from the Transgenic Lines
We determined the yield of tubers from the antisense and
overexpression lines (Fig. 5A). Because
yield trials are subjected to considerable variation between
individuals for each line, we grew 15 plants in large 20-cm pots, and
all plants were transferred simultaneously from tissue culture to the
greenhouse. No significant differences were found in the tuber fresh
weight per plant of the overexpression lines. In the antisense lines,
only line 13 showed a significant yield reduction (21%) relative to
the wild type. There were no significant changes in the mean tuber
number per plant in the antisense lines, whereas three of the four
sense lines showed a reduction (Fig. 5B).
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| Figure 5.
Yield (A), average tuber number (B), and average
tuber size (C) of transgenic lines altered in HK activity (antisense
lines 82, 8, and 13 and sense lines 89, 5, 70, and 95) and the wild
type (WT). For each line, 15 plants were grown in 3.5-L pots in the
greenhouse during the spring season. Mature tubers were harvested from
senescent plants. Data are presented as the means ± SE.
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Transgenic Tubers Did Not Show Changes in Starch and Sugar Contents
The sugar and starch contents of developing tubers are shown in
Tables III and IV. The analysis was performed on material from the same
set of plants for which the HK activities have been presented in Table
II. No significant differences were found in the antisense lines,
except for the increase in Glc observed in line 13 (Table III). However, given the large variation
in all of the Glc samples measured (a general phenomenon in tuber
metabolism) no particular significance was attached to this. The
overexpression lines also showed no clear differences; one of the lines
(PHK1-89) contained elevated Suc, and there was a tendency for a
reduction in the Glc levels, although high variability was again
observed (Table IV).
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Table III.
Carbohydrate contents of antisense transgenic
tubers (HK1)
The same set of antisense plants described in Table II were also used
for starch and sugar measurements.
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Table IV.
Carbohydrate contents of overexpression transgenic
tubers (PHK1)
The same set of overexpression plants described in Table II were also
used for starch and sugar measurements.
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Glycolytic Metabolites in the Transgenic Tubers Were Essentially
Unchanged
The measurements of glycolytic intermediates presented in Tables
V and VI
were confirmed by recovery experiments and low SEs. The
wild-type values were comparable to those previously reported for
potato tubers (Burrell et al., 1994; Geigenberger et al., 1998;
Trethewey et al., 1998). In the antisense lines there was a tendential
decrease of Glc-6-P and Glc-1-P, but these differences were not
significant. Hexose-P levels were unchanged in the overexpression
lines. In 3-phosphoglycerate there were no significant changes except
in line PHK1-70; the tendency was for a lower 3-phosphoglycerate
content in the overexpression lines. The PPi content of the antisense
lines was also not significantly changed compared with the wild type.
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Table V.
Metabolite contents of tubers from selected
antisense lines (HK1)
Metabolites were determined in the same samples used for carbohydrate
analysis presented in Table III. The data are presented as the
means ± SE of measurements on six individual plants
per line.
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Table VI.
Metabolite contents of tubers from selected
overexpression lines (PHK1)
Metabolites were determined in the same samples used for carbohydrate
analysis presented in Table IV. The data are presented as the
means ± SE of measurements on six individual plants
per line.
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HK1 Plants Accumulated Transitory Starch in Leaves after the
Dark Period
In preliminary experiments, we observed a significant increase of
starch in leaves of the antisense plants, but no changes in the
overexpression plants. We therefore performed a time-course experiment
with three antisense lines (12 plants per line) in the greenhouse: we
harvested samples from source leaves at four time points during the
course of the day (Fig. 6). Plants from the different lines and the controls were placed randomly in the greenhouse to neutralize the consequences of any position effects. At
the first time point (at dawn), the antisense plants showed a 2.1- to
3.2-fold increase in starch relative to the wild type (Fig. 6A). The
rate of starch accumulation during the morning in wild-type and
transgenic plants was similar: The difference between the first and
second time points was 73 µmol g1 fresh
weight for the wild type and between 57 and 90 73 µmol g1 fresh weight in the HK1 transgenics. The
situation changed in the evening (between the third and fourth time
points), with an increase of 78 µmol g1 fresh
weight in the wild type, but a significantly reduced accumulation in
the HK1 transgenics (between 16 and 49 µmol
g1 fresh weight). At the end of the light
period, the amount of starch present in the source leaves of the HK1
transgenics was only moderately higher (around 25%) than in the wild
type.
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| Figure 6.
Diurnal changes in leaf starch (A), Glc (B), and
Suc (C) in wild-type () and in the HK1 lines 8 (), 13 (),
and 82 (×). At each time point, samples were taken from
mature source leaves and the data presented represent the means ± SE of measurements on 12 plants per line.
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The higher starch content in the transgenics at dawn was correlated
with a significant increase of 1.6- to 2.1-fold in Glc in the HK1
transgenic lines relative to the wild type (Fig. 6B). Glc levels
equilibrated during the course of the day, and no significant differences between wild type and transgenics were observed at the end
of the light period. Moreover, the increased starch level at dawn in
the transgenics correlated with reduced Suc levels at the start of the
day in two of the three HK1 transgenics (Fig. 6C). Interestingly, at
the time points 10 AM and 2:30 PM, source leaves of the transgenics had tendentially higher Suc content than the
wild type (significant for line 13 at 2:30 PM). There were
no differences in Fru levels between the wild type and the transgenics
(data not shown).
| |
DISCUSSION |
Antisense inhibition of HK in potato leaves leads to an
excess-starch phenotype. We report a 30% increase in leaf starch at the end of the light period and a 3-fold increase in starch
accumulation at the end of the night period. This is exactly what would
be expected if Glc were the predominant route by which carbon is exported from chloroplasts at night. The phenotype of the antisense potato plants was comparable to that of the TC265 Arabidopsis mutant
line. TC265 is inhibited in the export of Glc from the chloroplasts,
and this deficiency leads to a 5-fold accumulation of starch in leaves
(Trethewey and ap Rees, 1994a, 1994b). These two mutant/transgenic
lines are defective at adjacent steps in the proposed pathway of Suc
biosynthesis at night and together provide convincing evidence that
export of Glc is the predominant route by which carbon leaves the
chloroplast at night. The fact that both lines have an excess-starch
phenotype at the end of the night strongly indicates that there are
feedback mechanisms that restrict the degradation of transitory starch.
However, the data also indicate that in both the potato antisense lines
and the Arabidopsis mutant, significant turnover of starch still
occurs. In both cases, it is impossible at the level of experimental
observation to determine if the amount of turnover is identical to the
wild type or if there is a modest reduction. We favor the latter
proposal, because a small reduction in turnover of starch could lead to
the observed large accumulation in leaf starch over the lifetime of the
plant. Significant turnover of starch may still be possible in the
antisense lines because inhibition of the HK activity is incomplete;
the most severe lines had an 78% reduction in activity in the leaves.
The occurrence of degradation in the mutant Arabidopsis line indicates
that there is some export of carbon via the triose-P translocator at
night (Trethewey and ap Rees, 1994a, 1994b).
The uncertainties in the measurement of starch are of the same order of
magnitude as the measured levels of soluble sugars in leaves;
therefore, the results of the Suc and Glc determinations must be
evaluated independently of whether starch turnover is altered in the
transgenic potato lines. Measurements of sugar levels in the leaves of
transgenic potato plants indicated that Glc is significantly elevated
in all three lines at the end of the night period, and in two of the
three lines for one-half of the day period. This is a clear indication
that there is a buildup of Glc following starch degradation in the
leaves. We propose that the residual HK activity is not strong enough
to bring all of this Glc rapidly into metabolism. The accumulation of
Glc is a further indication of the involvement of HK in the pathway of interconversion of starch to Suc at night.
The argument that the HK1 potato transgenics are a phenocopy of the
Arabidopsis TC265 lines requires that HK be located in the cytosol. We
found no evidence of a chloroplast-targeting peptide in the sequence of
the StHK1 cDNA, and there was no lengthy N-terminal extension found in
similarity comparisons with the HKs from yeast. We also performed
chloroplast import experiments with the in vitro translation product of
the StHK1 cDNA but found no evidence of any processing of the
translation product (controls were performed; data not shown). We
therefore conclude that HK1 is located outside of the chloroplast.
Several experiments with transgenic lines have demonstrated a
considerable flexibility in the overall day/night rhythms of carbohydrate allocation and export in potato. For example, in potato
plants in which the triose-P translocator was inhibited through
antisense repression, a 3-fold increase in starch in the leaves was
observed during the light period (Riesmeier et al., 1993) and there was
a subsequent elevated rate of export of carbohydrate from the leaves
during the night period (Heineke et al., 1994; Häusler et al.,
1998). Conversely, leaf-specific inhibition of ADP-Glc
pyrophosphorylase in potato led to a 60% reduction in the transitory
starch content of leaves (Leidreiter et al., 1995); there was a
compensating higher mobilization of photoassimilates from the leaf
during the day. Thus, there is a considerable potential for flexibility
in the timing and partitioning of carbohydrate allocation. Based upon
the previous results with other mutants and transgenics, it may be
expected that the disturbance in transitory starch metabolism in the
HK1 potato lines would lead to an altered partitioning of
carbohydrate within leaves and between sink and source organs. This
could be indicated by the tendentially higher Suc levels in the leaves
(significant in line 13 at the 2:30 PM time point),
however, more experimentation is required to adequately resolve this
question.
Our choice of potato for this study provided the opportunity to examine
the role of HK in a heterotrophic tissue. We found no significant
differences in the metabolism of the potato tubers from either the
antisense or the overexpression lines. This, coupled with the
observation that there was no great change in the yield of tubers per
plant, indicates that HK1 does not play a significant role in the
normal function of tuber induction and development. We were able to
assign the StHK1 cDNA to the HK1 activity previously described in
tubers by Renz et al. (1993) following analysis of the antisense and
overexpression transgenic lines. In these studies we found that the HK1
activity peak was enhanced or reduced in the overexpression and
antisense lines, respectively, and that the HK1 activity when
overexpressed in either potato or yeast demonstrated the same behavior
on an affinity chromatography column as that previously described for
HK1 by Renz et al. (1993). However, we were concerned to note that the
pH curve for the HK1 activity purified from potato was different from
that obtained following expression of the StHK1 cDNA in yeast. This
indicates that some posttranslational modifications may occur in one or
more of the systems, and demonstrates that caution is required in
interpreting and comparing kinetic data from heterologous systems.
Our observations that the yield of tubers, a strong sink tissue, was
largely unchanged in the antisense and overexpression lines indicates
that the overall physiology of the transgenic plants was not greatly
affected. More significant changes might have been expected if HK were
an important molecular sensor providing a direct link between carbon
status, gene expression, and photosynthesis. However, our demonstration
that inhibition of HK leads to significant effects on the biochemistry
of leaves indicates the need for caution in the assignment of a
function for this enzyme as a molecular signal of Glc status in plant
leaves (Jang et al., 1997). There are a myriad of regulatory factors
that can link metabolic status to gene expression, enzyme activity, and
flux in leaf tissue, and once the metabolic network is disrupted it
becomes extremely difficult to deconvolute cause and effect. Much more
detailed work will be required before the mechanisms behind the effects described by Jang et al. (1997) are truly understood. At this stage,
however, we do not identify HK as a target in the regulatory networks
of plant metabolism for the manipulation of sink strength in crop
plants.
Previous analysis of lines overexpressing a yeast invertase in the
cytosol of tubers (Sonnewald et al., 1997) and in combination with a
GLK (Trethewey et al., 1998) indicated that there was a switch in
partitioning in tubers of these lines away from starch biosynthesis and
toward glycolysis. Investigation of several other transgenic lines
(antisense against ADP-Glc pyrophosphorylase alone and in combination
with yeast invertase in the cytosol or apoplast) indicated that this
change of partitioning is most closely associated with an increased
cytosolic cleavage of Suc (Trethewey et al., 1999). In lines that show
enhanced glycolysis, this is mediated in part by an induction in the
activity of some key enzymes of glycolysis (e.g. phosphofructokinase,
pyruvate kinase, triose-P isomerase, and glyceraldehyde 3-P
dehydrogenase).
The question of the nature of the signal that connects the altered
metabolism at the level of Suc cleavage to an induction in the activity
of the glycolytic pathway is a fascinating one. The signal might
conceivably be low Suc, high Glc, or an elevated flux from Suc to
Glc-6-P. This last possibility is an interesting one in view of the
evidence from yeast systems that HK might be capable of sensing the
flux that it is catalyzing. However, the results of this study do not
provide any support for the hypothesis that HK1 is a central regulatory
element in potato tubers. We now believe that it is more likely that a
signal related to the cytosolic Suc levels is the decisive factor
controlling carbohydrate partitioning in potato tubers.
In conclusion, the studies presented in this paper provide convincing
evidence that HK1 is involved in the pathway of carbon export from
chloroplasts at night. However, we have not found any evidence that HK1
is involved in Glc sensing in potato, nor do we have any indication
that this enzyme catalyzes is a decisive step in the metabolism of
potato tubers.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Alexander von Humboldt Foundation to J.V.
2
Present address: Departamento de
Producción Agraria, Universidad Pública de Navarra. Campus
Arrosadía, 31006 Pamplona, Spain.
3
Present address: Metanomics GmbH & Co. KGaA,
Tegeler Weg 33, 10589 Berlin, Germany.
*
Corresponding author; e-mail jon{at}upna.es; fax
34-948-232191.
Received March 17, 1999;
accepted May 22, 1999.
| |
ACKNOWLEDGMENTS |
We are indebted to Dr. David Botstein for the DBY2219
yeast strain, to Dr. Joris Winderickx for the YSH7.4-3C yeast strain, and to Dr. José J. Sánchez-Serrano for the ubiquitin cDNA
from potato. We are also grateful to Romy Ackermann for performing the
potato transformation and to Bruno Marty, Olaf Woiwode, and Frank Huhn
for taking good care of the plants in the greenhouse. The comments of
Dr. Alisdair Fernie were most valuable in the preparation of the
manuscript.
| |
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Abstract
Introduction