1. Volume 10, Issue 4, Pages 605-618 (April 2017)
Arabidopsis CBL-Interacting Protein Kinases Regulate Carbon/Nitrogen-Nutrient Response by Phosphorylating Ubiquitin Ligase ATL31 
Shigetaka Yasuda, Shoki Aoyama, Yoko Hasegawa, Takeo Sato, Junji Yamaguchi 
Molecular Plant 
Volume 10, Issue 4, Pages (April 2017)
DOI: /j.molp
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2. Figure 1
Expression Levels of CIPK7, CIPK12, and CIPK14 Are Upregulated under High C/Low N Conditions.
(A) C/N-nutrient responses during the post-germination growth stage. WT seedlings were grown on MS medium containing the indicated concentrations of Glc and N. Photos were taken of 7-day-old plants.
(B–E) Expression levels of CIPK7, CIPK12, and CIPK14 under different C/N-nutrient conditions. qRT–PCR analysis of CHS (B), CIPK7 (C), CIPK12 (D), and CIPK14 (E) mRNAs in 7-day-old WT seedlings grown on MS medium containing the indicated concentrations of Glc and N. The level of 18S rRNA was used as an internal control. Relative expression levels were compared with those of WT seedlings grown on 0 mM Glc/60 mM N medium. Data shown are means. Error bars indicate SD (n = 4). Different letters above bars indicate significant differences assessed by one-way ANOVA with Turkey's post hoc test. Glc, glucose; N, nitrogen.
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
CIPK7, CIPK12, and CIPK14 Function as Negative Regulators of the C/N-Nutrient Response during the Post-germination Growth Stage.
C/N-nutrient responses during the post-germination growth stage. Indicated plants were grown on MS medium containing the indicated concentrations of Glc and N. Photos were taken of 7-day-old seedlings (A and C), and the percentages of seedlings with green cotyledons were scored (B and D). Data shown are means. Error bars indicate SD (n = 3). Different letters above bars indicate significant differences in each condition assessed by one-way ANOVA with Turkey's post hoc test. Glc, glucose; N, nitrogen.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3
CIPK7, CIPK12, and CIPK14 Function in the C/N-Nutrient Response during the Mature Growth Stage.
(A) Thirteen-day-old Arabidopsis plants grown on MS medium containing 30 mM Glc and 60 mM N were transferred to MS medium containing the indicated concentrations of Glc and N. Photos were taken 5 days after transfer. Scale bars, 10 mm.
(B) Fresh weight of shoots of plants treated as in (A). Data shown are means. Error bars indicate SD (n = 6).
(C) The anthocyanin content of shoots of plants treated as in (A). Data shown are means.
Error bars indicate SD (n = 10). Asterisks indicate significant difference compared with WT under the same conditions assessed by two-tailed t-test (*P < 0.05). Glc, glucose; N, nitrogen.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4 ATL31 Interacts with CIPK7, CIPK12, and CIPK14.
(A) Interactions of ATL31 with CIPK7, CIPK12, and CIPK14 in split-ubiquitin yeast two-hybrid assays. Yeast cells were transformed with ATL31C143S-Cub and -Nub, Nub-CIPK7, Nub-CIPK12, or Nub-CIPK14, and the transformants were streaked onto solidified medium supplemented with X-gal. Blue patches indicate positive interactions. Cub, the C-terminal half of ubiquitin; Nub, the N-terminal half of ubiquitin.
(B) Interactions of ATL31 with CIPK7, CIPK12, and CIPK14 in GFP pull-down assays. GFP, GFP-CIPK7, GFP-CIPK12, or GFP-CIPK14 bound anti-GFP beads were incubated with recombinant MBP-ATL31ΔTM. Input and precipitated proteins were analyzed by western blotting using anti-MBP and anti-GFP antibodies. ATL31ΔTM, the intracellular region of ATL31.
(C–E) Interactions of ATL31 with CIPK7, CIPK12, and CIPK14 in co-immunoprecipitation assays. ATL31C143S-FLAG and GFP-CIPK7 (C), FLAG-CIPK12 and ATL31C143S-GFP (D), and ATL31C143S-FLAG and GFP-CIPK14 (E) were transiently co-expressed in N. benthamiana leaves. Leaf extracts (Input) were subjected to immunoprecipitation with anti-FLAG beads. Inputs and immunoprecipitates (IP: α-FLAG) were analyzed by western blotting using anti-FLAG and anti-GFP antibodies. Asterisk indicates immunoglobulin heavy chain.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5
CIPK14 Phosphorylates ATL31 and Triggers Its Association with Protein In Vitro.
(A) In vitro phosphorylation of ATL31 by recombinant CIPK14. MBP-ATL31ΔTM and MBP-ATL31ΔTM/T209A were incubated with recombinant GST-CIPK14CA in reaction buffer for the indicated time period. Proteins were subjected to Phos-tag SDS–PAGE (Phos-tag; top panel) or normal SDS–PAGE followed by western blotting using anti-MBP (WB: α-MBP), anti-pT209, (WB: α-pT209), and anti-GST (WB: α-GST) antibodies.
(B) Far-western blotting analysis of the interaction of ATL31 with λ. Recombinant MBP-ATL31ΔTM was incubated with recombinant GST-CIPK14CA in reaction buffer for the indicated time period, followed by western blotting using anti-MBP (WB: α-MBP), anti-pT209 (WB: α-pT209), and anti-GST (WB: α-GST) antibodies. Far-western blotting was performed using His λ as a probe (Far-WB: His λ), and bound His λ was detected by western blotting with anti-His antibody.
(C) CBL8 enhances CIPK14-mediated ATL31 phosphorylation in a Ca2+-dependent manner. Indicated recombinant proteins were incubated in reaction buffer containing 1 mM CaCl2 or 10 mM EGTA for 60 min, followed by western blotting using anti-pT209 (WB: α-pT209), anti-MBP (WB: α-MBP), anti-GST (WB: α-GST), and anti-His (WB: α-His) antibodies.
ATL31ΔTM, the intracellular region of ATL31; CIPK14CA, a constitutively active form of CIPK14.
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
CIPK7, CIPK12, and CIPK14 Are Critical for the Function of ATL31 in the C/N-Nutrient Response.
(A) Expression levels of ATL31 mRNA in the indicated plants. Total RNA was extracted from 7-day-old seedlings grown on MS medium. Actin was used as an internal control.
(B and C) C/N-nutrient responses during the post-germination growth stage. Indicated plants were grown on MS medium containing the indicated concentrations of Glc and N. Photos were taken at 7-day-old (B), and the percentage of 7-day-old seedlings with green cotyledons was scored (C). Data shown are means. Error bars indicate SD (n = 3). Different letters above bars indicate significant differences in each condition assessed by one-way ANOVA with Turkey's post hoc test.
(D) Accumulation of proteins in the indicated plants under different C/N-nutrient conditions. Proteins were extracted from 7-day-old seedlings grown on MS medium containing the indicated concentrations of Glc and N. The level of  proteins was analyzed by western blotting using anti  antibody. Coomassie brilliant blue (CBB) staining was used as a loading control. The numbers below the blot indicate the relative signal intensity of  proteins compared with WT in each condition. Data shown are means. Error bars indicate SD (n = 3). Asterisk indicates significant difference compared with WT assessed by one-way ANOVA with Dunnett's post hoc test (*P < 0.05). Glc, glucose; N, nitrogen.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

8. Figure 7
CIPK7, CIPK12, and CIPK14 Are Essential for the Phosphorylation and Stabilization of ATL31 under High C/Low N Conditions.
(A) Total protein and Thr-209 phosphorylation levels of ATL31 after treatment under different C/N-nutrient conditions. WT and 35S-ATL31 in WT seedlings were grown in liquid MS medium, and 10-day-old seedlings were treated under the indicated C/N-nutrient conditions for 3 h. The extracts (Input) were subjected to immunoprecipitation with anti-FLAG beads (IP: α-FLAG). Immunoprecipitated ATL31-FLAG was analyzed by western blotting using anti-FLAG (WB: α-FLAG) and anti-pT209 (WB: α-pT209) antibodies. Equal protein loading and elution on immunoprecipitation were verified by CBB staining and eluted immunoglobulin (IgG) light chains, respectively. Total RNA was extracted from the same plant materials, and the expression levels of ATL31-FLAG mRNA were analyzed by RT–PCR. Actin was used as an internal control.
(B) Analysis of total protein and Thr-209 phosphorylation levels of ATL31 under high C/low N conditions in cipk7/12/14 mutant background. WT, 35S-ATL31 in WT, and 35S-ATL31 in cipk7/12/14 seedlings were treated under the indicated C/N-nutrient conditions. Total ATL31-FLAG protein, Thr-209 phosphorylated ATL31-FLAG protein, and ATL31-FLAG mRNA levels were analyzed as described in (A). Glc, glucose; Sor, sorbitol; N, nitrogen.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

9. Figure 8
EGTA Treatment Impairs the Function of ATL31 in the C/N-Nutrient Response.
(A and B) Effects of EGTA on the C/N-nutrient response. WT (A) and 35S-ATL31 in WT (B) were grown on MS medium containing the indicated concentrations of Glc and N with or without 5 mM EGTA. Photos were taken of 7-day-old seedlings, and the percentage of seedlings with green cotyledons was scored. Data shown are means. Error bars indicate SD (n = 3). Asterisks indicate significant difference compared with 0 mM EGTA in each condition assessed by Student's t-test (*P < 0.05, **P < 0.01).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions