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Plant Physiol. (1998) 117: 85-90
Tomato Fructokinases Exhibit Differential Expression and
Substrate Regulation1
Yoshinori Kanayama2,
David Granot,
Nir Dai,
Marina Petreikov,
Arthur Schaffer,
Ann Powell, and
Alan B. Bennett*
Mann Laboratory, Department of Vegetable Crops, University of
California, Davis, California 95616 (Y.K., A.P., A.B.B.); and Institute
of Field and Garden Crops, The Volcani Center, Bet Dagan 50250, Israel
(D.G., N.D., M.P., A.S.)
 |
ABSTRACT |
Two
divergent genes encoding fructokinase, Frk1 and
Frk2, have been previously shown to be expressed in
tomato (Lycopersicon esculentum L.) and have now been
further characterized with regard to their spatial expression and the
enzymic properties of the encoded proteins. Frk1 and
Frk2 mRNA levels were coordinately induced by exogenous
sugar, indicating that both belong to the growing class of
sugar-regulated genes. However, in situ hybridization indicated that
Frk1 and Frk2 were expressed in a
spatially distinct manner, with Frk2 mRNA primarily
localized in cells of the fruit pericarp, which store starch, and
Frk1 mRNA distributed ubiquitously in pericarp tissue.
To evaluate the biochemical characteristics of the products of the
Frk1 and Frk2 genes, each cDNA was
expressed in a mutant yeast (Saccharomyces cerevisiae)
line defective in hexose phosphorylation and unable to grow on glucose
or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast
conferred the ability to grow on Fru and exhibited fructokinase
activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a
substrate, only Frk2 activity was inhibited at high Fru concentrations.
These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in
plant cells specialized for starch storage.
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INTRODUCTION |
Suc translocated from leaves to sink tissue may be stored directly
or metabolized by Suc synthase and/or invertase to provide hexose and
hexose phosphate for storage or metabolism. In both Suc synthase- and
invertase-mediated metabolic pathways, Fru is formed as a metabolic
product and must be phosphorylated for further metabolism. Two enzymes,
hexokinase (EC 2.7.1.1) and fructokinase (EC 2.7.1.4), are able to
phosphorylate Fru in plants. Hexokinase can effectively utilize several
hexoses, including Fru and Glc, whereas fructokinase specifically
phosphorylates Fru. Fructokinase is likely to be of primary importance
in phosphorylation of Fru in plants because the affinity of
fructokinase for Fru is much higher than that of hexokinase (Renz and
Stitt, 1993 ).
Sink tissues such as potato (Solanum tuberosum L.) tubers
and tomato (Lycopersicon esculentum L.) fruit have been
useful to study the mechanism of Suc import and metabolism. During the
early stages of tomato fruit development, it has been proposed that Suc
is imported symplastically and metabolized primarily by Suc synthase
(Wang et al., 1994; Ruan and Patrick, 1995). Suc synthase activity is
correlated, both temporally and spatially, with starch synthesis in
developing tomato fruit (Wang et al., 1994). Suc synthase mRNA is
localized in vascular tissue and in the tissue surrounding seeds,
suggesting that this enzyme plays a role in sugar import and in
providing carbohydrates to developing seeds in young tomato fruit. In
potato two isoforms of Suc synthase have been shown to be expressed
differentially, and one of them is associated with sink function (Fu
and Park, 1995). In the Suc-synthase-mediated pathway of Suc
assimilation, effective phosphorylation of Fru appears to be necessary
to maintain C flow to starch synthesis and respiration, since
Suc-synthase activity is inhibited by free Fru (Wolosiuk and Pontis,
1974; Schaffer and Petreikov, 1997b). Thus, the relationship between
Suc synthase and fructokinase may be of critical importance in the sink
metabolism of Suc.
Fructokinase has been purified and characterized from several plants,
and most studies suggest the presence of at least two fructokinase
isoforms. For example, fructokinase isoforms can be separated by
ion-exchange chromatography from potato (Gardner et al., 1992; Renz and
Stitt, 1993), spinach (Schnarrenberger, 1990), barley (Baysdorfer et
al., 1989), avocado (Copeland and Tanner, 1988), and maize (Doehlert,
1989), and in some cases the isoforms have been shown to differ in
inhibition by Fru and/or in their specificity for nucleotide
triphosphates. Recently, two fructokinases were purified from young,
green tomato fruit. In this case, the isoforms separated by
ion-exchange chromatography exhibited almost identical kinetic
characteristics, and it was not clear whether the isoforms represented
the products of distinct fructokinase genes (Martinez-Barajas and
Randall, 1996). The cDNAs encoding two divergent fructokinases (Frk1
and Frk2) in tomato have been isolated and shown to be differentially
regulated (Kanayama et al., 1997; Martinez-Barajas et al., 1997).
A number of genes encoding enzymes involved in carbohydrate metabolism,
including invertase, Suc synthase, Suc-P synthase, amylase, starch
phosphorylase, adenosine 5 -diphospho Glc pyrophosphatase, and starch
synthase, have been shown to be induced by sugar levels, and it has
been proposed that their expression in sink tissues may be modulated by
carbohydrate status (Muller-Rober et al., 1990; Visser et al., 1991;
Koch et al., 1992; Quick and Schaffer, 1996). In maize Xu et al. (1996)
showed differential patterns of expression of Suc synthase and
invertase genes in the presence of Glc, and classified genes as sugar
enhanced, sugar repressed, or starvation tolerant. Because fructokinase
may function cooperatively with enzymes involved in starch
biosynthesis, it is possible that fructokinase gene expression may also
be responsive to carbohydrate status (Schaffer and Petreikov, 1997a).
Fructokinase has been the only gene encoding an enzyme required for
Suc-to-starch conversion that had not been demonstrated to be sugar
responsive (Quick and Schaffer, 1996).
Here we describe experiments that assess the sugar regulation of
fructokinase gene expression in tomato and provide enzymic characterization of two divergent fructokinase gene products from tomato. The results suggest that the two divergent tomato fructokinases may play distinct roles in sink metabolism.
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MATERIALS AND METHODS |
Yeast Transformation
Yeast (Saccharomyces cerevisiae) transformation
and culture were as described in Dai et al. (1997). The yeast strain
used was DFY632-MATa, ura3-52, hxk1::LEU2,
hxk2::LEU2, glk1::LEU2, lys1-1, leu2-1 (Walsh et
al., 1991). Yeast cells were grown on yeast
extract-peptone-galactose medium, consisting of 1% yeast extract
(Difco, Detroit, MI), 2% Bacto Peptone (Difco), and 110 mm
(2%) Gal (Sherman et al., 1986). Selective media for URA auxotrophic growth ( URA, +sugar) contained 0.5%
(NH4)2SO4,
0.17% yeast N2 base without amino acids (Difco),
0.2% casamino acids (Difco), 0.004% adenine (Sigma), 0.008% Trp
(Sigma), and 110 mm of either Gal, Fru, or Glc.
The yeast shuttle vector pFL61, containing the URA3 gene as a selective
marker and the constitutive phosphoglycerate kinase promoter and
terminator (Minet et al., 1992), was used for transformation. A
full-length Frk1 or Frk2 cDNA was cloned
downstream of the phosphoglylcerate kinase promoter in pFL61 to yield
pFL61-Frk1 and pFL61-Frk2. Yeast transformations were carried out by
growing DFY632 cells in yeast extract-peptone-galactose liquid medium
to mid-logarithmic phase, treating the cells with lithium acetate
according to the method of Ito et al. (1983), and selecting for
transformants on URA, +Gal plates.
Protein Extraction and Fructokinase Activity
Protein extraction from yeast were carried out as described in Dai
et al. (1997). DFY632 yeast cells transformed with either pFL61,
pFL61-Frk2, or pFL61-Frk1 were grown in 40 mL of URA, +Gal liquid
medium for 72 h to approximately 5 × 107 cells mL 1. Cells were
centrifuged for 5 min at 6,000 rpm, washed twice with water, and
resuspended in 0.5 mL of water. Two-hundred-fifty milliliters of the
cells was extracted twice with 500 mL of extraction buffer (50 mm Hepes pH 7.5, 1 mm EDTA, and 1 mm PMSF) by vortexing with 250 mL of glass beads. Following
vortexing for 90 s, the mixture was centrifuged for 5 min at
12,000g at 4°C, and the supernatant was brought to 80%
(NH4)2SO4
saturation. After centrifugation at 12,000g at 4°C, the
pellet was resuspended in 0.5 mL of washing buffer (50 mm
Hepes, pH 7.5, 1 mm EDTA, and 1 mm DTT),
desalted on a G-25 Sephadex column, and used as the crude enzyme
extract for subsequent enzymatic analysis.
Fructokinase activity was measured by an enzyme-linked assay, according
to a modification of Huber and Akazawa (1985). Assays contained, in a
total volume of 1 mL, 30 mm Hepes-NaOH (pH 7.5), 1 mm MgCl2, 0.6 mm EDTA, 9 mm KCl, 1 mm NAD, 1 mm ATP, 2 units of NAD-dependent Glc-6-P dehydrogenase, and 2 units of
phosphoglucoisomerase. The reaction was initiated with Fru at
concentrations ranging from 0.1 to 10 mm for Frk2 and to 50 mm for Frk1. Reactions were carried out at 37°C and
A340 was monitored continuously.
Sugar Induction of Fructokinase mRNA Accumulation
Fully expanded cotyledons of tomato (Lycopersicon
esculentum) seedlings were incubated in various sugar solutions
(100 mm), and RNA was extracted from them as described by
Mito et al. (1996) for gel-blot analysis. RNA gel-blot analysis was
carried out as described by Kanayama et al. (1997) using total RNA
obtained.
In Situ RNA Hybridization
The in situ hybridization was performed essentially as described
by Van de Wiel et al. (1990). Tissue of tomato fruit of approximately 10 mm in diameter, harvested from greenhouse-grown plants, was fixed
with 4% (w/v) paraformaldehyde and 0.25% (v/v) glutaraldehyde in 0.01 m sodium phosphate buffer, pH 7.2, at room temperature, dehydrated, and embedded in paraplast. Sections, 8 µm, attached to
poly-1-Lys-coated slides were deparaffinized with xylene
and rehydrated. They were subsequently pretreated with 1 µg
mL1 proteinase K in 100 mm
Tris-HCl, pH 7.5, containing 50 mm EDTA at 37°C for 30 min, and with 0.1 m triethanolamine, pH 8.0, at room
temperature for 10 min, following the addition of acetic anhydride to a
final concentration of 0.25% (v/v). They were then dehydrated and
dried under a vacuum until hybridization. The Frk1 35S-labeled sense and antisense RNA probes were
synthesized with T7 and SP6 RNA polymerase from a SpeI- or
EcoRV-linearized pCRII vector (Invitrogen, Carlsbad, CA)
containing a partial Frk1 cDNA, as described in Kanayama et
al. (1997). The Frk2 sense and antisense RNA probes were
synthesized with T3 and T7 RNA polymerase from the XhoI- or
SmaI-linearized pBluescript SK vector containing 911 nucleotides of the 5 end of Frk2 cDNA, as described in
Kanayama et al. (1997). The probes were partially degraded to 150 nucleotides by heating at 60°C in 0.2 m
Na2CO3/0.2 m
NaHCO3.
Sections were hybridized with RNA probes in 50% (v/v) formamide, 300 mm NaCl, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.02% (w/v) Ficoll, 0.02% (w/v) PVP, 0.02%
(w/v) BSA, 10% (w/v) dextran sulfate, 60 mm DTT, and 0.15 mg mL1 yeast tRNA at 42°C for 16 h.
After washing three times in 4× SSC and 5 mm DTT at room
temperature, slides were treated with 50 µg
mL1 RNase A in 500 mm NaCl, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA at 37°C for 30 min, and washed four times in the same buffer with 5 mm DTT
at 37°C for 20 min. After a low-stringency wash in 2× SSC with 1 mm DTT at room temperature, the final wash consisted of
0.1× SSC with 1 mm DTT at 37°C. Slides were dehydrated
in graded ethanol (each with 300 mm ammonium acetate) and
100% ethanol. After vacuum drying, slides were coated with NTB2
nuclear emulsion (Kodak), diluted 1:1 with 600 mm ammonium
acetate, and exposed for 21 d for Frk1 or for 10 d
for Frk2 at 4°C. They were developed in D19 developer
(Kodak) for 5 min at 15°C and fixed (Kodak). Sections were stained
with 2 g of KI and 1 g of I2 in 300 mL
of water for 5 min and then in 0.02% (w/v) toluidine blue for 5 min.
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RESULTS |
Induction of Fructokinase mRNA Accumulation by Sugar
To assess the regulation of expression of the Frk1 and
Frk2 genes, mRNA corresponding to each gene was assayed in
cotyledons incubated in buffer or in the presence of various sugars.
Both Frk1 and Frk2 mRNA abundance increased by
the exogenous application of Glc, Fru, or Suc, although the abundance
of Frk1 mRNA was consistently lower than that of
Frk2 (Fig. 1). Frk1
mRNA levels were essentially undetectable in the absence of exogenous
sugar treatment, whereas basal levels of Frk2 mRNA were
readily detected (Fig. 1). Treatment with mannitol or
3-o-methylglucose did not influence the abundance of
Frk1 and Frk2 mRNA, indicating that the sugar
regulation of fructokinase gene expression is not due to the osmotic
effects of sugar treatment, but is most likely related to cellular C
metabolite status.
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| Figure 1.
Total RNA was isolated following the incubation
in water or in 100 mm mannitol (Man),
3-o-methylglucose (OMG), Glc, Fru, or Suc for 15 h.
After quantifying abundance of mRNA with a phosphor imager, the x-ray
film was exposed for 12 and 2 d, respectively, for Frk1 and
Frk2.
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The time dependence of Frk1 and Frk2 mRNA
accumulation in response to exogenous sugars was similar for both genes
(Fig. 2). Frk1 and
Frk2 mRNA increased markedly 1.7 h after the addition of 100 mm Glc, and then increased only slightly over the
next 5 to 6 h. Starch accumulation in tissues exposed to Glc was
not measured. Similarly, the concentration dependence of Frk1 and Frk2
mRNA accumulation was similar, with the expression of both genes
significantly enhanced by 4 mm Glc and not repressed by up
to 100 mm Glc, a concentration that suppressed maize
invertase and Suc synthase levels (Koch et al., 1992; Xu et al., 1996). Based on these results, we conclude that both Frk1 and
Frk2 gene expression is sugar regulated in cotyledon tissue
and that this regulation appears to be coordinate for both genes.
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| Figure 2.
Total RNA was isolated from tomato cotyledons
incubated with 100 mm Glc for varying periods or with
varying concentrations of Glc for 15 h. The x-ray film was exposed
for 12 and 2 d, respectively, for Frk1 and Frk2.
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In Situ Localization of Frk1 and Frk2
Transcripts in Fruit Tissue
Because Frk1 and Frk2 appeared to be
coordinately regulated by sugar, we also examined whether they showed
the same spatial pattern of expression in developing fruit tissue.
Young, green fruit of approximately 10 mm in diameter were harvested and used for in situ hybridization with both Frk1 and
Frk2 hybridization probes. The localization of starch and
mRNA for Suc synthase has already been reported at this stage of tomato
fruit development (Wang et al., 1994). Because Frk2 mRNA is
approximately 20-fold more abundant than Frk1 mRNA, sections
to be hybridized with the Frk2 probe were exposed for
10 d and the sections hybridized with the Frk1 probes
were exposed for 21 d. Starch granules at this stage of fruit
development were also localized by staining with KI/I2 and were found to be localized in the inner
cell layers of the pericarp wall (Fig. 3,
A and D). It is interesting that Frk1 mRNA appeared to be
distributed ubiquitously in all cells of the pericarp tissue (Fig. 3B),
whereas Frk2 mRNA was most abundant in the inner cell layers
of the pericarp wall (Fig. 3E). The spatial pattern of Frk2
mRNA accumulation was very similar to the spatial pattern of starch
deposition. We previously showed that the developmental pattern of
Frk2 mRNA accumulation closely paralleled the temporal pattern of starch accumulation in tomato fruit (Kanayama et al., 1997).
Together, these results suggest that Frk2 gene expression may be both temporally and spatially coupled with starch accumulation, although the results do not rule out the possibility that the less-abundant Frk1 expression may contribute to the total
fructokinase activity and to starch accumulation in these tissues.
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| Figure 3.
Cross-sections of fruit approximately 10 mm in
diameter were hybridized with the Frk1 probe in B and C, and
with the Frk2 probe in E and F. Antisense probes were used
in B and E, and sense (control) probes were used in C and F. Hybridization signals are visible as white dots in dark-field
microscopy of the sections. The arrows in E indicate the concentrated
signals of Frk2 transcripts. Bright-field microscopy of the
sections hybridized with the antisense or sense probe are shown in A
and D. The dyes used in staining these sections were toluidine blue and
KI/I2. Bars = 200 µm; PE, pericarp; E, epidermis; V,
vascular tissue; and ST, starch granule.
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Characterization of Frk1 and Frk2 Products Expressed in Yeast Cells
To assess the biochemical characteristics of the Frk1 and Frk2
isoforms of fructokinase, each cDNA was cloned into a yeast expression
vector, pFL61, and expressed in DFY632, a yeast triple mutant that is
unable to phosphorylate either Glc or Fru. We previously demonstrated
that DFY632 cells expressing the tomato Frk1 cDNA were able
to grow on Fru but not on Glc, indicating that Frk1 encodes
a genuine fructokinase with a high specificity for Fru (data in top
panel of Fig. 4 republished from Kanayama
et al. [1997]). DFY632 cells expressing the tomato
Frk2 cDNA (pFL-Frk2) are also able to grow on Fru, but not
Glc, confirming that tomato Frk2, similar to
Frk1, also encodes a genuine fructokinase (Fig. 4).
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| Figure 4.
The Frk1 and Frk2 cDNAs were subcloned into pFL61
and expressed in yeast cells. Yeast cells transformed with pFL61 were
used as a control. Fru, Glc, or Gal (110 mm each) were
added to selective media (URA) for URA auxothrophic strains. The top
panel illustrating the ability of pFL-Frk-1 to complement the yeast
mutant was previously published (Kanayama et al., 1997) and is shown
here for direct comparison with similar results using pFL-Frk2.
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Fructokinase activity in crude protein extracts from both yeast
lines expressing Frk1 and Frk2 cDNAs was measured
at various concentrations of Fru (Fig.
5). The activity of both Frk1 and Frk2
obeyed Michaelis-Menten kinetics at substrate concentrations below 0.2 mm. However, Frk2 activity was inhibited by concentrations of Fru higher than 0.5 mm, whereas Frk1 activity was not
inhibited by concentrations of Fru up to 50 mm. In
addition, the affinity of Frk2 for Fru was much higher than that for
Frk1, with the respective Km values for Fru
of 1.3 mm (Frk1) and 0.054 mm (Frk2) calculated from Lineweaver-Burke plots.
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| Figure 5.
Relative activity was based on the maximal
activity observed for each enzyme (166 nmol mg1 protein
min1 for Frk1 at 50 mm Fru and 15 nmol
mg1 protein min1 for Frk2 at 0.2 mm Fru). Inset shows an expanded axis for Frk2 activity
over the low concentration range. Mutant yeast cells transformed with
the plasmid vector alone did not show any Fru phosphorylation
activity.
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DISCUSSION |
Isoforms of fructokinase previously have been separated by
ion-exchange chromatography and characterized from a number of plant
tissues, including tomato fruit (Martinez-Barajas and Randall, 1996).
It is likely that the FKI and FKII described by Martinez-Barajas and
Randall (1996) are both products of the Frk2 gene because both FKI and FKII were shown to be inhibited by Fru, similar to Frk2.
The calculated molecular mass of Frk2 is 34.8 kD (Kanayama et al.,
1997), which is close to the size of FKI and FKII (35 kD)
(Martinez-Barajas and Randall, 1996, 1997). The differences in pI
reported for FKI and FKII could be due to posttranslational modification of the Frk2 polypeptide, because it appears that Frk2 is
encoded by a single gene in tomato (Kanayama et al., 1997; Martinez-Barajas and Randall, 1997).
Potato fructokinase can be separated into three isoforms by
anion-exchange chromatography (Renz et al., 1993). The first two peaks
of activity (FK1 and FK2) are high in sink tissues (tubers) but very
low in leaves. In contrast, the third peak of activity (FK3) is similar
in sink (tuber) and source (leaf) tissues and FK3 is not inhibited by
Fru, as are the other isoforms (Gardner et al., 1992). The tomato
Frk1 gene is similar to potato FK3 in that it is expressed
in all organs (Kanayama et al., 1997) and Frk1 activity is not
inhibited by Fru. Thus, FK3 in potato may correspond to Frk1 in tomato,
and together they may represent a class of ubiquitous fructokinases
distributed in both sink and source organs, which are not inhibited by
high concentrations of Fru.
Invertase and Suc synthase, which metabolize Suc in the first step of C
assimilation by sink tissues, are sugar-modulated genes (Koch et al.,
1992; Xu et al., 1996). All of the genes encoding enzymes involved in
the conversion of Suc to starch, except fructokinase, have been shown
to be regulated by sugar levels (Quick and Schaffer, 1996). Here we
have shown that expression of genes encoding fructokinase is also
enhanced by the addition of exogenous sugar. The mRNAs encoding
invertase, Suc synthase, and fructokinase can all be significantly
increased by 4 to 10 mm sugar and are almost maximal at 20 to 30 mm sugar. These similar responses to sugar at the level of gene expression may serve to coordinate the metabolism of
imported sugars and to initiate efficient Suc utilization in sink
tissues. Some storage proteins and starch-related genes are also sugar
modulated. However, these genes appear to be less sensitive to sugar
than to fructokinases, which were induced within 1.7 h and showed
maximum mRNA levels at 20 mm sugar. In contrast, patatin
required 2 d and 300 to 400 mm sugar to induce maximal mRNA levels (Wenzler et al., 1989), and starch synthase and sporamin required approximately 200 mm sugar and at least 6 h
to significantly induce mRNA levels (Nakamura et al., 1991; Visser et
al., 1991). Our results suggest that enzymes involved in primary
metabolism in sink tissue, such as fructokinase, are induced by
comparatively low sugar concentrations and over shorter time periods
than are genes related to storage-product biosynthesis. Because the
sugar concentration in tomato fruit apoplast is more than 20 mm (Damon et al., 1988; Schaffer and Petreikov, 1997b),
fructokinase gene expression may be maximally induced in tomato fruit
tissues, and both Frk1 and Frk2 mRNAs are
detectable at almost all the stages of fruit development (Kanayama et
al., 1997). However, developmental expression patterns are quite
different for Frk1 and Frk2 genes, suggesting
that they are regulated during fruit development by other factor(s) in
addition to sugar.
Frk1 mRNA is distributed ubiquitously in tomato pericarp
cells and is present in fruit at a relatively constant level at all stages of fruit development (Kanayama et al., 1997). These results suggest that Frk1 may play a primary role in carbohydrate metabolism in
all plant cells, essentially acting as a housekeeping enzyme supplying
glycolysis with F6P. Frk2 mRNA is present at a very low
abundance in leaves and increases to very high levels at the early
stage of fruit development, a period of starch deposition (Kanayama et
al., 1997). In situ hybridization showed that Frk2 mRNA was
also localized in cells of inner pericarp, where starch storage is
predominant. These data suggest that Frk2 may play a particularly
important role in starch synthesis in tomato fruit. The hypothesis that
fructokinase is important in starch synthesis in sink tissue has been
proposed in tomato (Kanayama et al., 1997; Schaffer and Petreikov,
1997a) and in potato by Ross et al. (1994). Fructokinase and
phosphoglucose isomerase may supply F6P to the starch biosynthetic
pathway and may reduce Fru-inhibition of Suc synthase by decreasing
cytosolic Fru accumulation. The role of fructokinase in contributing to
starch biosynthesis has also been implicated in potato tubers, in which
Fru is the preferred substrate, resulting in the low ratio of Fru to
Glc (Davies and Oparka, 1985). The differential inhibition of Frk2
relative to Frk1 is also consistent with its coordinate action in
starch biosynthesis with Suc synthase, an enzyme that shows a similar
pattern of Fru inhibition.
The results presented here suggest that Frk1 and Frk2 do not represent
redundant enzyme activities in tomato fruit but are likely to be active
in vivo in different cells and under different physiological
conditions, especially relative to tissue Fru concentration.
| |
FOOTNOTES |
1
This research was supported by grants from the
Binational Agricultural Research and Development Fund (no. US-2451-94)
and the University of California BIO-STAR program (no. S96-17).
2
Present address: Faculty of Agriculture, Tohoku
University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981, Japan.
*
Corresponding author; e-mail, abbennett{at}ucdavis.edu; fax
1-530-752-4554.
Received October 21, 1997;
accepted February 8, 1998.
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ABBREVIATIONS |
Abbreviation:
URA, uracil.
| |
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Abstract
Introduction