Figure 1. Sequence alignment of VvSK1 with other GSK3 like PK from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar
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residues in light gray. GeneBank accession numbers are as follows : VvSK1 (XP_0022721 12), 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.
Figure 2. Phylogenetic relationship between GSK3 proteins from grape and Arabidopsis. A 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_0022721 12, GSVIVT00026235001), VvSK2 (XP_002279596, GSVIVT0002 8178001), VvSK3 (XP_002280673, GSVIVT0002254400 1), VvSK4 (XP_002276754, GSVIVT000207 13001), VvSK5 (XP_002270455, GSVIVT00035848001), VvSK6 (XP_002281788, GSVIVT00018774001), VvSK7 (XP_002263398, GSVIVT00035285001), ASK1-alpha (At5g26751), ASK2-beta (At3g6 1160), ASK3 -gamma (At3g05 840), ASK4-delta (At 1 g57 870), ASK5-epsilon (At5g14640), ASK6-zeta (At2g30980), ASK7-BIN2-eta (At4g1 8710), ASK8-theta (At4g00720), ASK9-iota (At1g06390), and ASK1 0-kappa (At1g09840).
Figure 3. Analysis of VvSK1 expression in response to sugar and ABA treatments in CSB cells. A, RNA gel blot analysis of VvSK1 accumulation after different times of treatment with 58 mM sucrose. B, Semi-quantitative RT-PCR analysis of VvSK1 expression in response to 58 mM mannitol and 58 mM PEG treatment. The elongation factor VvEF1was used as an internal control. C, Effect of sugar analogs on VvSK1 accumulation determined by RNA gel blot 4h after treatment. Results are shown for: non treated control cells (C), and cells treated with 58 mM sucrose (S), 10 mM MHL (MHL), 58 mM sucrose + 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 1&M ABA. Elongation factor VvEF1and actin were used as internal controls. All data are means of three replicates from independent experiments, with error bars indicating SE.
Figure 4. Expression of VvSK1 in various tissues from cabernet sauvignon plants. Expression of VvSK1 mRNA was determined by semi-quantitative RT-PCR and elongation factor VvEF1was used as an internal control. A, VvSK1 expression in roots, stems, leaves, inflorescences and mature berries. B, Expression of VvSK1 in developing berries: 3 weeks (P3), 7 weeks (P7), 9 weeks (P9), 11 weeks (P11) and 15 weeks (P15) post-flowering. C,Downloaded on November 17, 2020. - Published by https://plantphysiol.org
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Expression of VvSK1 in berry skin, pulp and seed at véraison stage. The experiment was repeated 3 times independently with similar results.
Figure 5. Characterization of transgenic cells over-expressing VvSK1. A, RT-PCR of control (PFB 8) and VvSK1 over-expressors (35 S: :VvSK1 -HA) detecting VvSK1 transcript.
Elongation factor VvEF1was 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. Upper panel, Coomassie blue staining showing the recombinant protein; down panel, MBP phosphorylation by VvSK1.
Figure 6. Over-expression of VvSK1 modifies the transcription of some grape monosaccharide transporter genes. A, Quantitative RT-PCR analysis of monosaccharide transporters in control (PFB8, grey bars) and 35S:: VvSK1-HA (black bars) grape cells.
Expression levels of VvHTs were evaluated in transgenic 41B cells. B, Kinetic of expression of VvHT3 (K y), VvHT4 (•), VvHT5 (A), and VvHT6 (0) assessed by quantitative RT-PCR using total RNA extracted from CSB cells treated with 58 mM sucrose. 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.
Figure 7. Activity of the monosaccharide transport system in VvSK1 over-expressing cells. D-[14C]Glucose uptake was measured in control cells transformed with PFB8 empty vector (
A
)and 35S:: VvSK1 cells (
•
). Cell aliquots were collected at different time periods (0, 0.5, 2, and 4h). Errors bars indicate SD. For this experiment, 6 independent flasks of 41 B cell suspension cultures for each condition were used and analyzed.Figure 8. Sugar content of VvSK1 over-expressing cells. Left Y axis shows Glucose (grey bars) content and right Y axis shows sucrose (black bars) and starch (doted bars) contents.
Levels of sugars were analyzed in control cells (PFB8) and 35S:: VvSK1 transgenic cells, 7 days after subculture. Errors bars indicate SD. For this experiment, 3 independent flasks of 41 B cell suspension cultures for each condition were used and analyzed.
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Figure 1. Sequence alignment of VvSK1 with other GSK3 like PK from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar residues in light gray. GeneBank 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.
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Figure 2. Phylogenetic relationship between GSK3 proteins from grape and Arabidopsis. A 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, GSVIVT00026235001), VvSK2 (XP_002279596, GSVIVT00028178001), VvSK3 (XP_002280673, GSVIVT00022544001), VvSK4 (XP_002276754, GSVIVT00020713001), VvSK5 (XP_002270455, GSVIVT00035848001), VvSK6 (XP_002281788, GSVIVT00018774001), VvSK7 (XP_002263398, GSVIVT00035285001), ASK1-alpha (At5g26751), ASK2-beta (At3g61160), ASK3-gamma (At3g05840), ASK4-delta (At1g57870), ASK5-epsilon (At5g14640), ASK6-zeta (At2g30980), ASK7-BIN2-eta (At4g18710), ASK8-theta (At4g00720), ASK9-iota (At1g06390), and ASK10-kappa (At1g09840).
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58 mM Sucrose
Figure 3. Analysis of VvSK1 expression in response to sugar and ABA treatments in CSB cells. A, RNA gel blot analysis of VvSK1 accumulation after different times of treatment with 58 mM sucrose. B, Semi-quantitative RT-PCR analysis of VvSK1 expression in response to 58 mM mannitol and 58 mM PEG treatment. The elongation factor VvEF1α was used as an internal control. C, Effect of sugar analogs on VvSK1 accumulation determined by RNA gel blot 4h after treatment. Results are shown for: non treated control cells (C), and cells treated with 58 mM sucrose (S), 10 mM MHL (MHL), 58 mM sucrose + 10 mM MHL (SMHL), 58 mM 2-DOG (2-DOG) and, 58 mM 3-OMG (3-OMG). D, Kinetic of accumulation of VvSK1 transcript after treatment with 20 µM ABA analyzed by quantitative RT-PCR. Elongation factor VvEF1γ and actin were used as internal controls. All data are means of three replicates, with error bars indicating SE.
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Mature berries
VvSK1
roots stems leaves Inflorescences
VvEF1
α
A
VvSK1 VvEF1
α
Green Véraison Ripening
P3 P7 P9 P11 P15
B
Véraison
Skin Pulp Seed VvEF1
α
VvSK1
C
Figure 4. Expression of VvSK1 in various tissues from cabernet sauvignon plants. Expression of VvSK1 mRNA was determined by semi-quantitative RT-PCR and elongation factor VvEF1α was used as an internal control. A, VvSK1 expression in roots, stems, leaves, inflorescences and mature berries. B, Expression of VvSK1 in developing berries: 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 3 times independently with similar results.
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PFB8 35S::
VvSK1-HA
VvSK1 VvEF1
α
A
VvSK1-HA
B
VvSK1-GST
GST
MBP VvSK1-GST GST
C
MW marker
Figure 5. Characterization of transgenic cells over-expressing VvSK1. A, RT-PCR of control (PFB8) and VvSK1 over-expressors (35S::VvSK1-HA) detecting VvSK1 transcript. Elongation factor VvEF1α 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. Upper panel, Coomassie blue staining showing the recombinant protein; down panel, MBP phosphorylation by VvSK1.
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A
VvHT1 VvHT2 VvHT3 VvHT4 VvHT5 VvHT6 VvpGLT
Relative expression level Relative expression level
Figure 6. Over-expression of VvSK1 modifies the transcription of some grape monosaccharide transporter genes. A, Quantitative RT-PCR analysis of monosaccharide transporters in control (PFB8, grey bars) and 35S::VvSK1-HA (black bars) grape cells. Expression levels of VvHTs were evaluated in transgenic 41B cells. B, Kinetic of expression of VvHT3 (), VvHT4 (), VvHT5 (), and VvHT6 () assessed by quantitative RT-PCR using total RNA extracted from CSB cells treated with 58 mM sucrose.
Elongation factor VvEF1γ and actin were used as internal controls. All data are means of three replicates, with error bars indicating SE. The primer sequences are given in materials and methods.Downloaded on November 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.