Download A Sugar-Inducible Protein Kinase, VvSK1, Regulates Hexose

A Sugar-Inducible Protein Kinase, VvSK1, Regulates
Hexose Transport and Sugar Accumulation in
Grapevine Cells
Fatma Lecourieux*, David Lecourieux, Céline Vignault, and Serge Delrot
UMR Ecophysiology and Grape Functional Genomics, University of Bordeaux, INRA, Institut des Sciences de
la Vigne et du Vin, 33882 Villenave d’Ornon, France (F.L., D.L., S.D.); and FRE CNRS 3091, University of
Poitiers, 86022 Poitiers cedex, France (C.V.)
In grapevine (Vitis vinifera), as in many crops, soluble sugar content is a major component of yield and economical value. This
paper identifies and characterizes a Glycogen Synthase Kinase3 protein kinase, cloned from a cDNA library of grape Cabernet
Sauvignon berries harvested at the ripening stage. This gene, called VvSK1, was mainly expressed in flowers, berries, and
roots. In the berries, it was strongly expressed at postvéraison, when the berries accumulate glucose, fructose, and abscisic acid.
In grapevine cell suspensions, VvSK1 transcript abundance is increased by sugars and abscisic acid. In transgenic grapevine
cells overexpressing VvSK1, the expression of four monosaccharide transporters (VvHT3, VvHT4, VvHT5, and VvHT6) was upregulated, the rate of glucose uptake was increased 3- to 5-fold, and the amount of glucose and sucrose accumulation was more
than doubled, while the starch amount was not affected. This work provides, to our knowledge, the first example of the control
of sugar uptake and accumulation by a sugar-inducible protein kinase.
Efficient and coordinated transport of assimilates is
essential for plant growth, development, and productivity. Plant growth and plant productivity are not
limited by photosynthesis per se but rather by the
ability of the plant to export and to partition its
assimilates in a proper way. Increased crop yields are
paralleled by a better partitioning of assimilates between the harvestable part of the plant and the rest of
the plant (Gifford and Evans, 1981; Gifford et al., 1984;
Hoffmann-Thoma et al., 1996). Long-distance and intercellular transport of sugars is mediated by protoncoupled Suc transporters (belonging to the disaccharide
transporter family) and hexose and polyol transporters (belonging to the monosaccharide transporter
family). Many of these transporters have been identified in a wide range of species (Lemoine, 2000; Sauer
et al., 2004; Büttner, 2007). Disaccharide transporter
genes belong to small multigenic families (e.g. nine
members in Arabidopsis [Arabidopsis thaliana] and four
in tomato [Solanum lycopersicum]; Sauer et al., 2004;
Hackel et al., 2006) and can be clustered into four
groups regarding the homologies of the encoded protein sequences. The Arabidopsis genome has 53
* Corresponding author; e-mail fatma.lecourieux@bordeaux.
inra.fr.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Serge Delrot ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.109.149138
1096
homologous sequences encoding putative monosaccharide transporters, which are distributed into seven
distinct clusters (Büttner, 2007).
Control of the expression of these transporters in
specific cell types at precise stages of development
allows fine-tuning in the plant of its carbon distribution. Although many sugar transporters have now
been identified and characterized, little is known
about their regulation. Up-regulation of a Glc transporter gene has been observed after treatment of
Chenopodium rubrum cells with cytokinin (Ehness and
Roitsch, 1997). In Arabidopsis, the monosaccharide
transporter gene AtSTP4 may be induced by fungal
and bacterial elicitors (Truernit et al., 1996; Fotopoulos
et al., 2003), whereas the fungal elicitor cryptogein
rapidly and completely blocks Glc uptake in tobacco
(Nicotiana tabacum) cells (Bourque et al., 2002).
Accumulating evidence indicates that sugars may
control the expression and activity of their own transporters. The ability to sense altered sugar concentration is important and allows the plant to regulate its
metabolism and compartmentation processes in order
to face the demands of sink organs (Roitsch, 1999).
There is some discrepancy about the effect of sugars on
the control of these transporters. In C. rubrum cell
suspensions, monosaccharide transporter genes are
constitutively expressed and not regulated by sugar
(Roitsch and Tanner, 1994). Suc was reported to repress
the sugar beet (Beta vulgaris) Suc transporter BvSUT1
both at the transcriptional and translational levels
(Chiou and Bush, 1998; Vaughn et al., 2002; RansomHodgkins et al., 2003), whereas it up-regulates OsSUT1
in rice (Oryza sativa; Matsukura et al., 2000). Down-
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Regulation of Sugar Transport by a Protein Kinase
regulation of monosaccharide transport in response to
Glc supply was also observed in suspension-cultured
cells of olive (Olea europaea; Oliveira et al., 2002) and in
peach (Prunus persica) tree buds (Maurel et al., 2004).
By contrast, the expression of VvHT1 in grape (Vitis
vinifera) cell suspensions is induced by low concentration of Suc (Atanassova et al., 2003) and repressed at
high Suc concentration (Conde et al., 2006), suggesting
that the effect of sugars on transporter genes depends
on sugar concentration and on the physiological status
of the cells.
In contrast to the situation in bacteria, yeast, and
mammals, where sugar signaling pathways are extensively studied (Johnston, 1999), signaling cascades
involved in response to sugar in plants are still poorly
understood. However, the combination of mutant
isolation and pharmacological approaches reveals the
involvement of protein kinases, protein phosphatases,
and other signal transduction mediators such as Ca2+
and calmodulin (Smeekens, 2000). Among the protein
kinases identified in sugar sensing and signaling in
plants, the importance of sucrose nonfermenting-like
protein kinase complexes and interacting proteins
has been well established (Rolland et al., 2006). In
addition, the involvement of other protein kinases,
like calcium-dependent protein kinases (Ohto and
Nakamura, 1995) and mitogen-activating protein kinases
(Ehness et al., 1997), has also been suggested.
Glycogen Synthase Kinase3 (GSK3) is a constitutively active Ser/Thr kinase involved in diverse physiological activities and functions such as metabolism,
cell cycle, gene expression, and development (Rayasam
et al., 2009). In mammalian cells, GSK3 is an important
signaling player that is ubiquitously expressed and
has been implicated in the regulation of Glc transport
and metabolism (Grimes and Jope, 2001). The few
existing reports about regulation of Glc uptake by
GSK3 have produced different findings in different
cell systems (Nikoulina et al., 2002; Wieman et al.,
2007), implicating GSK3 in either insulin signaling or
non-insulin-stimulated conditions in cell types that are
not insulin sensitive. GSK3/SHAGGY-like kinases
(GSKs) are a large gene family in plants (Jonak and
Hirt, 2002). Recent functional studies indicate that
plant GSKs are involved in diverse important processes including hormone signaling, development,
and stress responses (Dornelas et al., 2000; Jonak
et al., 2000; Piao et al., 2001; Choe et al., 2002; Li and
Nam, 2002; Perez-Perez et al., 2002; Vert and Chory,
2006; Kempa et al., 2007).
In this work, we report the involvement of VvSK1,
a GSK3/SHAGGY-like kinase, in the regulation of
sugar accumulation in grape cell suspension in response to sugar. This GSK3-like kinase, whose expression is increased by sugars, probably acts through the
regulation of monosaccharide transporter gene expression. The analysis of its gene expression during grape
berry development also suggests that it may be involved in the control of sugar accumulation during
berry ripening.
RESULTS
Isolation and Sequence Analysis of VvSK1
Total RNA extracted from Cabernet Sauvignon
berry cells treated or not with 58 mM Suc was hybridized with 70-mer oligoarrays bearing a set of 14,562
unigenes (Qiagen Operon Array-Ready Oligo Set for
the Grape Genome Version 1.0). Among the genes that
were significantly up-regulated after 1 h of Suc treatment, a gene corresponding to the EST sequence
(TC46745) showed homology to the GSK3 family and
was selected for further investigation. This choice was
directed by previous data showing that members of
the GSK3 family are involved in the regulation of
carbohydrate metabolism in eukaryotic cells (Embi
et al., 1980; Woodgett and Cohen, 1984; Kempa et al.,
2007). The full-length cDNA was amplified by PCR
using a cDNA library prepared from postvéraison Cabernet Sauvignon berries and was named VvSK1 (GenBank accession no. XP_002272112; GSVIVT00026235001).
VvSK1 encodes a protein of 468 amino acids that
contains the 11 highly conserved domains characteristic of kinases, including the Tyr residue in the T-loop.
VvSK1 exhibits high amino acid sequence similarities
with GSKs from others species: 87% with NtSK91
(tobacco), 87% with LpSK6 (tomato), 87% with PSK6
(Petunia hybrida), and 86% with AtSK8 (Arabidopsis;
Fig. 1).
Analysis of the grape genome (Jaillon et al., 2007)
allowed the identification of six additional GSK3
members that were numbered from 2 to 7. Interestingly, VvSK1 only shares 44% to 67% identity with
other members of the grape GSK family. Based on
protein sequence homology, the VvSKs were grouped
into four classes (I–IV; Fig. 2). VvSK1 (XP_002272112)
belongs to class III. VvSK2 (XP_002279596), VvSK3
(XP_002280673), and VvSK4 (XP_002276754) are related to members of class I. VvSK5 (XP_002270455)
and VvSK6 (XP_002281788) are related to class II, and
VvSK7 (XP_002263398) is related to class IV.
Expression of VSK1 in Response to Sugar and
ABA Treatments
The expression profile of VvSK1 was assessed by
RNA blot using RNA samples extracted from Cabernet Sauvignon berry suspension cells collected 0, 1, 2,
4, 6, and 8 h after supply of 58 mM Suc as well as from
control cells without sugar supply. VvSK1 transcripts
accumulate from 1 h until the end of the experiment
(Fig. 3A). To exclude an osmotic effect of Suc, Cabernet
Sauvignon berry suspension cells were treated with
the same concentration of mannitol or polyethylene
glycol 600. None of these chemicals affected VvSK1
expression, indicating that the expression profile previously observed is not due to an osmotic stress but
rather to a Suc-specific effect (Fig. 3B).
In order to elucidate the signaling pathway involved
in the transcriptional regulation of this gene, we tested
different sugar analogs on Cabernet Sauvignon berry
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Lecourieux et al.
Figure 1. Sequence alignment of
VvSK1 with other GSK3-like protein kinases from various plant species. Identical residues are shown
in black, conserved residues in
dark gray, and similar residues in
light gray. GenBank accession numbers are as follows: VvSK1 (XP_
002272112), ASK8 (At4g00720),
PSK6 (AJ224164), NSK91 (AJ224163),
and LsPK6 (AY575716). The sequence alignment was done by the
Mcoffee program at http://tcoffee.
vital-it.ch/cgi-bin/Tcoffee/tcoffee_
cgi/index.cgi.
suspension cells (Fig. 3C). Cells were treated with
3-OMG (which is absorbed but poorly phosphorylated) and 2-DOG (which is absorbed and phosphorylated but not further metabolized) or with 58 mM Suc
in the presence of the hexokinase (HXK) inhibitor
mannoheptulose (MHL; Gibson, 2000). Blockage of
HXK activity in cells treated with 10 mM MHL resulted
in repressed levels of VvSK1 transcripts. Addition of
58 mM 3-OMG has no effect on VvSK1 expression. By
contrast, addition of 58 mM 2-DOG up-regulates
VvSK1, as was observed after Suc treatment. Taken
together, these results suggest that Suc up-regulates
VvSK1 expression through an HXK-dependent
pathway.
Hormones play a crucial role during grape berry
development. More particularly, endogenous ABA
levels increase after véraison, and treatments that
delay this increase delay the onset of ripening (Davies
et al., 1997; Deluc et al., 2009). In order to investigate a
potential effect of ABA on VvSK1 expression, Cabernet
Sauvignon berry cell suspensions were treated with 20
mM ABA and collected 0, 2, 4, 6, 8, and 12 h later. VvSK1
expression was monitored by quantitative reverse
transcription (RT)-PCR. The results revealed that
ABA increased the transcript abundance of VvSK1 by
2-fold within 2 h, and this amount of VvSK1 transcripts
was maintained for at least 10 h after ABA supply
(Fig. 3D).
Relative Expression Profile of VvSK1 in Grapevine
Organs and Berry Tissues
The relative transcript abundance of VvSK1 was
determined in different grapevine organs by RT-PCR
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Regulation of Sugar Transport by a Protein Kinase
Figure 2. Phylogenetic relationship between
GSK3 proteins from grape and Arabidopsis. The
phylogenetic tree was constructed using MEGA
version 4 (Tamura et al., 2007) using the Minimum Evolution method with 2,000 bootstrap
replicates. Names and GenBank accession numbers are as follows: VvSK1 (XP_002272112), VvSK2
(XP_002279596), VvSK3 (XP_002280673), VvSK4
(XP_002276754), VvSK5 (XP_002270455), VvSK6
(XP_002281788), VvSK7 (XP_002263398), ASK1-a
(At5g26751), ASK2-b (At3g61160), ASK3-g
(At3g05840), ASK4-d (At1g57870), ASK5-«
(At5g14640), ASK6-z (At2g30980), ASK7-BIN2-h
(At4g18710), ASK8-u (At4g00720), ASK9-i
(At1g06390), and ASK10-k (At1g09840).
with RNA extracted from Cabernet Sauvignon roots,
stems, leaves, flowers, and mature berries. VvSK1 was
predominantly expressed in roots, inflorescences, and
mature berries, whereas it was hardly detectable in
stems and leaves (Fig. 4A). VvSK1 transcript accumulation was also assessed during berry development.
VvSK1 was present in green berries, but the accumulation of the transcript increased mainly after véraison
stage and was maintained until the harvesting stage
(Fig. 4B). This expression profile is in agreement with
microarray data that were previously obtained from
Shiraz and Chardonnay developing berries (D. Glissant
and S. Delrot, unpublished data).
The level of expression of VvSK1 in the different
berry compartments at véraison (seeds, pulp, and
skin) was also analyzed by RT-PCR. As shown in
Figure 3C, VvSK1 showed ubiquitous expression in the
berry even though expression levels seemed higher in
skin and pulp than in seeds.
Overexpression of VvSK1 in Grapevine Cells Affects
Sugar Transport
In order to elucidate the function of VvSK1, transgenic grape cells overexpressing hemagglutinin (HA)tagged VvSK1 under the control of the cauliflower
mosaic virus 35S promoter were prepared. After stabilization of the cell suspension by subculture in
glycerol-maltose-naphthoxyacetic acid culture medium supplemented with paromomycin and cefotaxime, the expression of VvSK1-HA was tested by PCR
and western blotting. A DNA fragment of 1,408 bp
corresponding to VvSK1 was strongly amplified by
VvSK1-specific primers, in the cells expressing the
35S::VvSK1-HA construct, in comparison with cells
expressing the empty vector (Fig. 5A). In addition, the
recombinant protein VvSK1-HA with a mass of 57.2
kD was detected on a western blot with an anti-HA
antibody (Fig. 5B). To confirm that VvSK1 is a functional protein kinase, we performed a phosphorylation
assay using VvSK1 produced in Escherichia coli as a
glutathione S-transferase (GST) fusion protein. VvSK1
phosphorylated myelin basic protein as an artificial
substrate (Fig. 5C), showing that in addition to being
functional, VvSK1 exhibits a basal constitutive activity.
The expression level of different hexose transporter
genes in the 35S::VvSK1-HA grape cells was measured
in order to gain greater insights into sugar accumulation during grape berry development. Seven plasma
membrane monosaccharide transporter homologs
(VvHTs) and a plastidic Glc transporter (pGLT) have
been cloned from grape berries (Vignault et al., 2005;
Hayes et al., 2007). Specific primers were designed for
the sequences of VvHT1 (accession no. AJ001061),
VvHT2 (AY663846), VvHT3 (AY538259), VvHT4
(AY538260), VvHT5 (AY538261), VvHT6 (AY861386),
and VvpGLT (AY608701) and used to test the corresponding transcript amounts in 41B cells overexpressing VvSK1 (Fig. 6A). Compared with control cells,
overexpression of VvSK1 increased the accumulation
of VvHT3, VvHT4, VvHT5, and VvHT6 transcripts
when compared with control cells, whereas VvHT1,
VvHT2, and pGLT transcripts were not significantly
affected. Although transcript abundance was increased for all four hexose transporters, they were
up-regulated in a slightly different way. Thus, VvHT3
and VvHT4 transcript abundance was increased by
1.7-fold in the VvSK1-overexpressing line, whereas it
was increased by 3.4- and 2.8-fold for VvHT5 and
VvHT6, respectively. Interestingly, VvHT3, VvHT4,
VvHT5, and VvHT6 expression was increased after
supply of 58 mM Suc. Their transcripts accumulated
within 1 h and peaked 6 h after Suc addition (Fig. 6B).
These results suggest that Suc might affect the expression of these four VvHTs through a signaling cascade
involving VvSK1.
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Lecourieux et al.
increase in Glc and a 3.3-fold increase in Suc content as
compared with PFB8 control cells. By contrast, starch
amounts were not significantly different between both
cell lines. Fru amounts were too small to be quantified
and therefore could not be compared. For this experiment, three independent flasks of 41B cell suspension
cultures for each condition were used.
DISCUSSION
Figure 3. Analysis of VvSK1 expression in response to sugar and ABA
treatments in Cabernet Sauvignon berry cells. A, RNA gel-blot analysis
of VvSK1 accumulation after different times of treatment with 58 mM
Suc. B, Semiquantitative RT-PCR analysis of VvSK1 expression in
response to 58 mM mannitol and 58 mM polyethylene glycol (PEG)
treatment. The elongation factor VvEF1 was used as an internal control.
C, Effect of sugar analogs on VvSK1 accumulation determined by RNA
gel blot 4 h after treatment. Results are shown for nontreated control
cells (C) and cells treated with 58 mM Suc (S), 10 mM MHL (MHL), 58
mM Suc + 10 mM MHL (SMHL), 58 mM 2-DOG (2-DOG), and 58 mM
3-OMG (3-OMG). D, Quantitative RT-PCR of VvSK1 transcript accumulation after treatment with 20 mM ABA. Elongation factor VvEF1 and
Actin were used as internal controls. All data are means of three
replicates from independent experiments, with error bars indicating SE.
Sugars provide the reduced carbon needed for the
structural materials of the cell, storage compounds,
and all major primary and secondary metabolites.
They also influence plant growth and development by
acting as regulatory signals, which affect the expression of various genes involved in many processes of
the plant life cycle (Koch, 1996; Jiang and Carlson,
1997; Smeekens, 1998; Roitsch, 1999; Sheen et al., 1999).
Therefore, it is important to identify the molecular
players that may directly or indirectly control sugar
transport in plants. Although many sugar transporters
have now been identified, little is known about sugar
transport regulation at the molecular level (Delrot
et al., 2000).
Protein kinases are major components of intracellular signal transduction enabling plant cells to rapidly
respond to the environment. This paper shows that
VvSK1 is a sugar- and abscisic acid (ABA)-induced
GSK3 protein kinase that increases the transcript
As a consequence, VvSK1 might also affect sugar
transport activities in grape cells. To test this hypothesis, uptake of D-[14C]Glc was compared in control
cells and cells overexpressing VvSK1. The rate of Glc
accumulation by 41B cells overexpressing VvSK1 was
3- to 5-fold higher than in control cells during the time
course studied (Fig. 7).
Analysis of Sugar Content in Grape Cells
Overexpressing VvSK1
The stimulation of hexose transporter gene expression in 41B cells overexpressing VvSK1 and the enhanced ability of these cells to accumulate Glc
indicated that their sugar content might be altered by
VvSK1 expression. Soluble sugars (Glc, Fru, and Suc)
and starch contents were assayed 7 d after subculture
(Fig. 8). Comparison of the sugar profiles showed that
41B cells overexpressing VvSK1 displayed a 2-fold
Figure 4. Expression of VvSK1 in various tissues from Cabernet
Sauvignon plants. Expression of VvSK1 mRNA was determined by
semiquantitative RT-PCR, and elongation factor VvEF1 was used as an
internal control. A, VvSK1 expression in roots, stems, leaves, inflorescences, and mature berries. B, Expression of VvSK1 in developing
berries at 3 weeks (P3), 7 weeks (P7), 9 weeks (P9), 11 weeks (P11), and
15 weeks (P15) post flowering. C, Expression of VvSK1 in berry skin,
pulp, and seed at véraison stage. The experiment was repeated three
times independently with similar results.
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Regulation of Sugar Transport by a Protein Kinase
pressed in flowers (Tichtinsky et al., 1998; Charrier
et al., 2002; Wilson et al., 2004). Among these, ASK
(AtSK3-2) was shown to be involved in the control of
cell elongation in flower development and in response
to light (Claisse et al., 2007). Our data showed that
VvSK1 was not only expressed in flowers but also in
two other major sink organs, roots and berries, indicating that its role was not only restricted to flower
development in grapevine (Fig. 4A). In berries, VvSK1
transcript amounts transiently decreased after the
green stage and increased again after the véraison
stage (Fig. 4B), when both sugars and ABA accumulate
in this organ (Davies et al., 1997; Pan et al., 2005). Our
experiments showed that both sugar and ABA could
increase the transcript abundance of VvSK1 in grape
cell suspensions (Fig. 3, A and D). It is well known that
sugar signaling does not operate alone but rather is
integrated in cellular regulatory networks. Most notably, there is a tight interaction between sugar and
hormonal signaling, particularly for ABA (Smeekens,
Figure 5. Characterization of transgenic cells overexpressing VvSK1.
A, RT-PCR of control sample (PFB8) and VvSK1 overexpressors (35S::
VvSK1-HA) detecting VvSK1 transcript. Elongation factor VvEF1 was
used as an internal control. B, Protein extracts from cells overexpressing
VvSK1-HA were separated and immunodetected using a specific HA
antibody. C, GST-VvSK1 phosphorylates myelin basic protein (MBP) in
vitro. Top, Coomassie Brilliant Blue staining showing the recombinant
protein; bottom, myelin basic protein phosphorylation by VvSK1.
abundance of several monosaccharide transporters
and the soluble sugar content of grape cells. MsK4, a
GSK3 homolog from group IV, controls carbohydrate
metabolism in response to salt stress (Kempa et al.,
2007). MsK4 is a starch-associated protein kinase
that, when overexpressed, modulates carbohydrate
metabolism. Additionally, MsK4 acts as a signaling
component in the high-salinity response, linking stress
signaling to metabolic adaptation.
The Vitis GSK3 phylogenic tree revealed that VvSK1
was less related to the other VvSKs than to homologs
from other species. Indeed, VvSK1 shared highest
similarities with GSK3s from Solanaceae proteins
NSK91, NSK59, and LpSK6 (87%, 87%, 86.6%), PSK6
and PSK7 (87% and 84.3%), and Arabidopsis ASK2
and ASK8 (78% and 86%). These GSK3s, which belong
to the same clade (group III), are preferentially ex-
Figure 6. Overexpression of VvSK1 modifies the transcription of some
grape monosaccharide transporter genes. A, Quantitative RT-PCR
analysis of monosaccharide transporters in control (PFB8; gray bars)
and 35S::VvSK1-HA (black bars) grape cells. Expression levels of
VvHTs were evaluated in transgenic 41B cells. B, Kinetics of expression
of VvHT3 (white diamonds), VvHT4 (black circles), VvHT5 (black
triangles), and VvHT6 (white squares) assessed by quantitative RT-PCR
using total RNA extracted from Cabernet Sauvignon berry cells treated
with 58 mM Suc. Elongation factor VvEF1was used as an internal
control. All data are means of three replicates, with error bars indicating SE. The primer sequences are given in “Materials and Methods.”
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Lecourieux et al.
Figure 7. Activity of the monosaccharide transport system in VvSK1overexpressing cells. D-[14C]Glc uptake was measured in control cells
transformed with PFB8 empty vector (triangles) and 35S::VvSK1
(squares) cells. Cell aliquots were collected at different time periods
(0, 0.5, 2, and 4 h). Errors bars indicate SD. For this experiment, six
independent flasks of 41B cell suspension cultures for each condition
were used and analyzed. d.w, Dry weight.
2000; Finkelstein and Gibson, 2002; Léon and Sheen,
2003; Rolland et al., 2006). Also, it is known that ABA
enhances sugar accumulation in crop sink organs
(Kobashi et al., 2001), and data from the literature
show that both sugar and ABA act synergistically
(Smeekens, 2000; Léon and Sheen, 2003; Rolland et al.,
2006). In peach fruit flesh, sugar accumulation is
stimulated by ABA that in turn stimulates uptake of
sugars (Kobashi et al., 2001). ABA induces the expression of VvHT1 (Atanassova et al., 2003) and acts
synergistically with physiological concentrations of
monosaccharides to transiently induce VvHT1 transcription. VvHT1 responsiveness to both signals involves the presence in its promoter of a specific
overlapping configuration of the two sugar response
cis-elements. This configuration seems a target for the
fixation and the action of VvASR (for V. vinifera
abscisic acid, stress, ripening-induced) protein (also
called VvMSA; Cakir et al., 2003). VvASR expression is
strongly up-regulated by the combined effect of sugars
and ABA (Cakir et al., 2003). More recently, it was
shown that ABA increases invertase activities and
amounts during grape development in correlation
with soluble sugar concentration (Pan et al., 2005).
VvSK1 was induced by the addition of exogenous
Suc, and this induction was not due to an osmotic
effect (Fig. 3C). This indicates that VvSK1 was involved downstream of sugar perception. In plants,
two signaling pathways relaying sugar sensing have
been described. The first one is HXK dependent, and
the second is independent of HXK activity (Smeekens,
2000). To investigate the role of HXK in the Suc
induction of VvSK1, we tested the effect of Glc analogs
and an inhibitor of HXK activity. The results showed
that the HXK inhibitor MHL blocked VvSK1 induction
by Suc, whereas 3-OMG had no effect and 2-DOG
stimulated VvSK1 expression in grape cells (Fig. 3B).
This indicates that an HXK-dependent pathway may
be involved in the sugar control of VvSK1 expression
in grape cells.
VvSK1 overexpression in grape cell suspension affects the level of expression of some sugar transporter
genes expressed in grape berries (Fig. 6A). The VvHTs
whose expressions were affected by VvSK1 overexpression were also affected at the transcriptional level
by sugars and ABA (Fig. 6B; data not shown). This
result was in accordance with the observation that
VvSK1 overexpression increased by more than 3-fold
the uptake rate of radiolabeled Glc in comparison
with control cells (Fig. 7). The involvement of VvSK1
in the control of sugar compartmentation was further
strengthened by the fact that cells overexpressing
VvSK1 accumulate twice more Glc and three times
more Suc than the control, whereas the starch content
remains unaffected. Sugar levels of suspension cells
significantly differ from those in berries, where monosaccharide concentrations can reach 1.5 M, whereas Suc
is poorly detectable (Fillion et al., 1999; Liu et al., 2006).
In this context, the role of VvSK1 on sugar transport
and accumulation in grape berries has to be further
investigated by altering VvSK1 expression in berries.
In this work, VvSK1 was identified as a potential
regulator of hexose uptake and sugar accumulation in
grapevine cells. As a functional protein kinase, VvSK1
can regulate its targets by direct phosphorylation.
Protein phosphorylation is one of the most important
and best characterized posttranslational protein modifications regulating cellular signaling processes. In
plants, previous work suggested that protein phosphorylation plays a direct role in the regulation of
Figure 8. Sugar content of VvSK1-overexpressing cells. Left y axis
shows Glc (dark gray bars) content, and right y axis shows Suc (black
bars) and starch (light gray bars) contents. Levels of sugars were
analyzed in control cells (PFB8) and 35S::VvSK1 transgenic cells 7 d
after subculture. Errors bars indicate SD. For this experiment, three
independent flasks of 41B cell suspension cultures for each condition
were used and analyzed. d.w, Dry weight.
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Regulation of Sugar Transport by a Protein Kinase
sugar transporter transcription and sugar transport
activity (Roblin et al., 1998; Ransom- Hodgkins et al.,
2003). Key enzymes from carbon metabolism may also
be directly regulated by phosphorylation (Sugden
et al., 1999; Ikeda et al., 2000). Furthermore, in yeast,
the transcription factor RGT1 that functions as a repressor in the absence of Glc becomes a transcriptional
activator required for maximal induction of HXT1
gene expression upon phosphorylation in the presence
of high concentrations of Glc (Mosley et al., 2003).
Therefore, one can hypothesize that VvSK1 can affect
sugar uptake and accumulation in grapevine through
the regulation of hexose transporter expression. This
regulation might be mediated through VvSK1 phosphorylation events affecting the activity and/or
stability of specific transcription factors, sugar transporters, and/or enzymes from sugar metabolism. This
still has to be demonstrated.
In grapevine, one of the main features of berry
development is the accumulation of hexose sugars (i.e.
Glc and Fru), which begins at véraison and continues
throughout the ripening process. In addition to participating in the regulation of sugar homeostasis in
grape cells, VvSK1 may play a role in grape berry
development by affecting sugar compartmentation.
This hypothesis is further supported by the capacity of
VvSK1 to up-regulate the expression of several VvHT
genes and more particularly that of VvHT3 and
VvHT6. VvHT6 transcript abundance correlates well
with hexose accumulation in the berries and was
recently proposed to play a significant role in hexose
accumulation during berry ripening (Deluc et al.,
2007). If VvHT6, which is highly homologous to the
tonoplast Glc transporter AtTMT2 (Wormit et al.,
2006), acts as a tonoplast sugar transporter, it would
only favor indirectly Glc uptake across the plasma
membrane by decreasing the cytosolic Glc concentration. VvHT3 transcript accumulates in young berries
but also later during the phase of sugar storage (Hayes
et al., 2007). VvHT4 expression shows little change
throughout berry development, whereas VvHT5 transcript amounts increased by 3-fold at 14 weeks post
flowering. As both genes are very weakly expressed in
Cabernet Sauvignon berry flesh, these transporters,
unlike VvHT3 and VvHT6, may not contribute significantly to sugar import during berry development
(Hayes et al., 2007). Nevertheless, the authors did not
rule out the possibility that these VvHTs could be
better expressed in more specific pulp cell types.
Furthermore, as VvSK1 expression in not restricted to
the berry, the role of the kinase in the regulation of
these two VvHTs in other plant organs is possible if
they are expressed in the same organs.
In grape berries, as in other sink organs, sugar
accumulation significantly impacts crop yield and
economic value. To our knowledge, this work provides
the first demonstration that soluble sugar content may
be significantly affected by the manipulation of a
single gene involved in signaling pathways. VvSK1
transgenic cells may provide a useful model to inves-
tigate the metabolic and physiological consequences
of a deregulation of sugar uptake and accumulation.
Analysis of the effect of Glc analogs and the involvement of ABA place VvSK1 downstream to the
HXK-dependent sugar signaling cascade, ABA being
involved downstream of HXK1 (Zhou et al., 1998;
Arenas-Huertero et al., 2000). The expression profiles
of VvSK1 in sink organs (roots and fruits) and during
berry development indicate a key role of this protein
kinase in the maturation process that still remains to be
demonstrated in planta. Overall, our data provide a
first trail for the identification of potential signaling
components affecting sugar transport and accumulation during grape berry development and ripening.
MATERIALS AND METHODS
Isolation of GSK cDNA
Grapevine (Vitis vinifera) VvSK1 was isolated from an EST (accession no.
TC46745 [new TC92799]) collected at the Dana-Farber Cancer Institute Vitis
vinifera Gene Index and containing the entire open reading frame. For expression analysis, the entire clone was generated from a cDNA library of grape
Cabernet Sauvignon berries (ripening stage) by PCR using synthetic oligonucleotide primers designed to begin and end at the start and stop codons of the
open reading frame (forward primer, 5#-TACCCGGGACTCGAGATGAATGTGATGCGTCGTCTCAAGAGCATT-3#; reverse primer, 5#-ATGGATCCTCAGCGGCCGCCTTTCCTTGCATGCTCAGGAATGAGA-3#). The VvSK1 cDNA
was cloned into the pGEM-T Easy vector (Promega) for DNA sequencing prior
to HA tagging and subcloning into the vector pFB8 downstream of the 35S
promoter of cauliflower mosaic virus.
RNA Isolation and cDNA Production
After collection, the cells were immediately frozen in liquid nitrogen and
stored at –80°C until RNA extraction. The samples were ground under liquid
nitrogen with a mortar and pestle, and RNA was isolated as described
previously (Reid et al., 2006). Briefly, the extraction buffer contained 300 mM
Tris-HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 2% cetyltrimethylammonium
bromide, 2% polyvinylpolypyrrolidone, 0.05% spermidine trihydrochloride,
and 2% b-mercaptoethanol. Fifteen milliliters of prewarmed (65°C) extraction
buffer was added to the ground tissue. Samples were incubated at 65°C for 10
min and then extracted twice with chloroform. The final aqueous layer was
centrifuged at 30,000g for 20 min at 4°C, then 0.1 volume of 3 M NaOAc (pH
5.2) and 0.6 volume of isopropanol were added to the supernatant and stored
overnight at 280°C. Samples were pelleted by centrifugation (3,500g, 30 min)
the next day and resuspended in 0.7 mL of Tris-EDTA buffer (pH 7.5). To
selectively precipitate the RNA, 0.3 volume of 8 M LiCl was added and the
sample was stored overnight at 4°C. RNA was pelleted the next day, washed
with ice-cold 70% ethanol, air dried, and dissolved in diethyl pyrocarbonatetreated water. RNA isolation was followed by DNaseI treatment. Total RNA
was quantified, and the quality was evaluated on 1% agarose gels.
For RT-PCR analysis, 2 mg of total RNA were reverse transcribed with
oligo(dT)12-18 in a 20-mL reaction mixture using the Moloney murine leukemia
virus reverse transcriptase (Promega) according to the manufacturer’s instructions. After heat inactivation of the reaction mixture, PCR was performed
using 2.5 mL of the first-strand cDNA sample with 20 pmol of the primers in a
50-mL reaction.
Real-time PCR expression analysis was performed using a commercial IQ
SYBR-Green Supermix kit (Bio-Rad) with the CFX96 Real-Time PCR Detection
system (Bio-Rad). Gene transcripts were quantified upon normalization to EF1
and Actin as internal standards by comparing the cycle threshold of the target
gene with those of standard genes. All biological samples were tested in
triplicate, and SE values of means were calculated using standard statistical
methods. Specific oligonucleotide primer pairs were designed with Beacon
Designer 7 software (Premier Biosoft International). Specific annealing of the
oligonucleotides was controlled by dissociation kinetics performed at the end
of each PCR run. The efficiency of each primer pair was measured on a PCR
Plant Physiol. Vol. 152, 2010
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Lecourieux et al.
product serial dilution. The primers used for amplification were as follows:
VvSK1-For, 5#-GGTCGATCAAGAAGCAGAACAG-3#, and VvSK1 -Rev,
5#-GCAGCAGTTTCCTGTGATGTAG-3#; VvHT1-For, 5#-CGTTGTTCACATCGTCGCTTTATC-3#, and VvHT1-Rev, 5#-GAGCAGTCCTCCGAATAGCATTG-3#; VvHT2-For, 5#-GGCATAGGGGTGGTGGTAG-3#, and VvHT2-Rev,
5#-TCGGGCTTTACGGAGAGAG-3#; VvHT3-For, 5#-GCTACTCTCTACTCGTCCGCTTTG-3#, and VvHT3-Rev, 5#-CCCGTCATTGCTTCCGAACTTG-3#;
VvHT4-For, 5#-GGGCTGGCGAGTTTCTCTAG-3#, and VvHT4-Rev, 5#-AGTTCTGCTTGGACATCGTTTG-3#; VvHT5-For, 5#-CAGGCTGTTCCACTGTTCTTATCG-3#, and VvHT5-Rev, 5#-AGTTAGGAGGACCGCAGGAATC-3#;
VvHT6-For, 5#-TTTATTTGCATGAGGAGGGAGTC-3#, and VvHT 6-Rev,
5#-GAGCAGCAGCCTGGATATAATC-3#; VvpGLT-For, 5#-CAGTGGTTATGGGAGCCGAGTTTG-3#, and VvpGLT-Rev, 5#-TCCACCAGAAGCTCTAGCCTTGAC-3#.
at 60°C. The NADPH2 released by the enzymatic reactions was measured
spectrophotometrically at 340 nm with an absorbance microplate reader
(ELx800UV; Bio-Tek Instruments; Grechi et al., 2007).
Grapevine Transformation and Culture Conditions
ACKNOWLEDGMENTS
Grapevine transformations were made in 41B rootstock (V. vinifera ‘Chasselas’ 3 Vitis berlandieri). An embryogenic cell suspension culture was
initiated as described previously (Coutos-Thévenot et al., 1992a, 1992b).
This cell suspension was subcultured weekly in 25 mL of glycerol-maltose
culture medium (Coutos-Thévenot et al., 1992b) supplemented with synthetic
auxin (naphthoxy acetic acid at 1 mg L–1) in the dark. Embryogenic cells were
transformed using an Agrobacterium tumefaciens cocultivation method (Mauro
et al., 1995), and after selection, the transgenic cells were subcultured in
the same condition in a medium supplemented with paromomycin at 2 mg
mL–1 (final concentration) and cefotaxime at 200 mg mL–1 (Duchefa).
We thank Christian Kappel for assistance in bioinformatics, Messaoud
Meddar for preparing the cell culture media, Cécile Gaillard for assistance in
grape cell transformation, Ghislaine Hilbert and Christelle Renaud for help in
sugar content analysis, Prof. Heribert Hirt for critical reading of the manuscript, and Prof. Grant Cramer for improving the style of the manuscript.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers XM_002272076 (VvSK1), XM_002279560
(VvSK2), XM_002280637 (VvSK3), XM_002276718 (VvSK4), XM_002270419
(VvSK5), XM_002281752 (VvSK6), XM_002263362 (VvSK7), At5g26751 (ASK1-a),
At3g61160 (ASK2-b), At3g05840 (ASK3-g), At1g57870 (ASK4-d), At5g14640
(ASK5-«), At2g30980 (ASK6-z), At4g18710 (ASK7-BIN2-h), At4g00720 (ASK8-u),
At1g06390 (ASK9-i), At1g09840 (ASK10-k), AJ001061 (VvHT1), AY663846
(VvHT2), AY538259 (VvHT3), AY538260 (VvHT4), AY538261 (VvHT5),
AY861386 (VvHT6), AY854146 (VvHT7), and AY608701 (pGLT).
Received October 9, 2009; accepted November 10, 2009; published November
18, 2009.
LITERATURE CITED
Western-Blot Analysis
Western blotting was performed with 30 mg of protein extracts separated
by SDS-PAGE, immunoblotted to poly(vinylidene difluoride) membranes
(Millipore), and probed with –HA antibody (Sigma) in Tris-buffered saline,
pH 7.4, containing 0.05% Tween 20. Peroxidase-conjugated goat anti-rabbit
IgG was used as secondary antibody, and the reaction was revealed by
fluorography (ECL; Amersham Biosciences).
In Vitro Kinase Assay
The open reading frame of VvSK1 was cloned into GST fusion protein
expression vector pGEX4T-1 (Pharmacia). Expression of the GST fusion
protein and affinity purification were performed as described previously
(Kiegerl et al., 2000). Kinase reactions were performed in 15 mL of kinase
buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 5 mM EGTA, and 1 mM
dithiothreitol) containing 5 mg of GST fusion protein, 5 mg of myelin basic
protein, 0.1 mM ATP, and 2 mCi of [32P]ATP. The protein kinase reactions were
performed at room temperature for 30 min, and the reactions were stopped by
adding 43 SDS loading buffer and heating for 5 min at 95°C. The phosphorylation of myelin basic protein was analyzed by autoradiography after
separation by 12.5% SDS-PAGE.
Glc Uptake Studies
Transgenic cells overexpressing VvSK1 were collected 10 d after subculture
and washed three times with culture medium lacking sugar at pH 5.0. Cells
were then resuspended in the same fresh medium and equilibrated for 2 h at
120 rpm. Uptake was initiated by the addition of D-[14C]Glc (final concentration, 0.133 MBq g21 fresh weight). One-milliliter aliquots of the cell suspension
were collected after 0, 0.5, 2, and 4 h of incubation with the radioactive sugar.
The reaction was stopped by the addition of 23 5 mL of culture medium
without sugar, and the cells were collected by filtration on GF/C Whatman
membranes. The filters were dried, and their radioactivity was measured in a
Packard Tri-Carb 1900 TR liquid scintillation counter (Packard Instruments).
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