1. Volume 7, Issue 2, Pages 422-436 (February 2014)
Characterization of Evolutionarily Conserved Motifs Involved in Activity and Regulation of the ABA-INSENSITIVE (ABI) 4 Transcription Factor 
Josefat Gregorio, Alma Fabiola Hernández-Bernal, Elizabeth Cordoba, Patricia León 
Molecular Plant 
Volume 7, Issue 2, Pages (February 2014)
DOI: /mp/sst132
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Structural Characteristics of the ABI4 Proteins.
(A) Schematic diagram of the ABI4 protein showing conserved domains. The PEST sequence, the AP2-associated motif, the AP2 domain (AP2), the serine/threonine-rich region (S/T), the LRP motif, and the acidic domain present in the carboxy terminal region are indicated. The bipartite sequences predicted as NLS are marked with the *. The homology of the PEST sequence and LRP motifs are shown for Arabidopsis thaliana (Ath), Arabidopsis lyrata (Aly), Capsella rubella (Cru), Brassica rapa (Bra), and Thellungiella halophila (Tha). The consensus motifs of the AP-associated motif and of the acidic domain obtained from the comparison of 31 ABI4 sequences are shown.
(B) The AP2-associated-like motif present in members of the A2 subgroup (A2) is shown. Members of the A2 subgroup include AT2G40340, AT2G40350, AT3G11020, AT5G05410, AT2G38340, AT1G75490, AT5G18450, and AT3G57600.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Function of the ABI4 Protein of Theobroma cacao as a Transcriptional Activator.
(A) Relative luciferase (LUC) activity from protoplasts transfected with the pABI4–CE1 as a reporter construct alone (black bar) or co-transfected with the Arabidopsis ABI4 (AtABI4; white bar) or the putative ABI4 protein from Theobroma cacao (TcABI4; gray-dotted bar). The LUC specific activity was corrected by GUS-specific activity. Relative units of LUC activity are displayed normalized to the levels in the pABI4–CE1 construction. Error bars indicate SD from biological triplicates. Germination and greening in 1X GM (B), 3 μM ABA (C), 7% glucose (D), and 150mM NaCl (E) of L1, L5, and L7 independent transgenic lines (abi4 35S::TcABI4), Col-0 wild-type, and abi4 mutant. Germination and greening were scored after 15 d for GM, 18 d for ABA and glucose media, and 24 d for NaCl conditions of transfer to the growth chamber.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Function of the AP2-Associated Motif.
(A) Schematic representation of the GFP fusions used in the subcellular localization analysis. The diagrams highlight some important elements such as the AP2-associated motif and NLS (gray boxes), the AP2 domain (black box), and the LRP motif (hatched box). The deletion of the AP2-associated motif and the mutation of the LRP are also indicated in the corresponding construct.
(B) Confocal images of GFP localization in protoplasts transformed with the full ABI4::GFP protein (ABI4–WT), an ABI4 fragment including the first 117 amino acids from the ATG (ABI4–NLS), a construct that lacks the AP2-associated motif that includes only the first 39 amino acids from the ATG (ABI4–∆NLS), a deletion of 14 amino acids corresponding to the AP2-associated motif (ABI4–∆APaM), and a mutation of the LRP motif (ABI4–mLRP). Scale bar: 10 μm.
(C) Protein levels of ABI4–WT and the ABI4–∆APaM proteins from transfected protoplasts. Total protein extracts were obtained after 15h of transfection. Each lane contains 80 μg of total protein extracts. Immunodetection was performed using a 1: dilution of the GFP antibody. The RbcL protein from the Ponceau-stained membrane was used as a loading control.
(D) The level of the fusion proteins detected in the Western blot in (C) was quantified by densitometric analysis and expressed in arbitrary units relative to the ABI4–WT (white bar) which was taken as 1. Each bar corresponds to the standard deviation of three biological independent experiments.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
The LRP Motif Is an Important Element for the ABI4 Transcriptional Activity.
(A) Relative LUC activity from Col-0 protoplasts transfected with the pABI4–CE1::LUC reporter construct alone (black bar) or co-transfected with the effector 35S:ABI4 (ABI4–WT, white bar) or the 35S::ABI4m-LRP, carrying a mutation in the LRP motif (ABI4–mLRP, dashed bar). Relative units of LUC activity are expressed as the fold-induction between the specific LUC activity in the presence of ABI4 and the activity without ABI4, taken as 1. Each bar corresponds to the standard deviation of at least three independent biological experiments. To correct for transfection efficiency, the LUC specific activity was corrected by GUS-specific activity.
(B) Protein levels of the ABI4–WT::GFP and the ABI4–mLRP::GFP were determined by Western blot analysis. Total protein extracts were obtained after 15h of transfection. Each lane contains 80 μg of total protein extracts. Immunodetection was performed using a 1: dilution of the GFP antibody. The negative control (Control) corresponds to the pEarlyGate103 destination vector. The RbcL protein from the Ponceau-stained membrane was used as a loading control. The protein gel shown is representative from three independent biological replicates.
(C) The amount of the fusion proteins detected in (B) was quantified by densitometric analysis, expressed as arbitrary units relative to the ABI4–WT (white bar), taken as 1.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 The PEST Sequence Is Involved in the ABI4 Protein Stability.
(A) The transactivation activities of the wild-type ABI4 protein (ABI4–WT) and the ABI4 with 19 amino acids corresponding to the PEST sequence deleted (ABI4–∆P) were determined measuring the relative LUC activity from Arabidopsis Col-0 protoplasts transfected with the pABI4–CE1::LUC reporter construct (black bar) or co-transfected with the effector 35S:ABI4 (white bar) or the 35S::ABI4–∆P (gray bar). Relative units of LUC activity are expressed as the fold-induction from the activity without the effector ABI4 plasmid (black bar), taken as 1. Each bar indicates the standard deviation of three independent biological experiments.
(B) GFP fluorescence of protoplasts transfected with the ABI4::GFP (ABI4–WT) or ABI4–∆P::GFP fusion proteins was followed using confocal microscopy. Transfection was performed using equal concentration of both constructs. Scale bar: 10 μm.
(C) Protein levels of ABI4–WT and ABI4–∆P fusions were analyzed by Western blot from total protein extracts obtained 15h after transfection. Each lane contains 80 μg of total protein extracts. Immunodetection was performed using a 1: dilution of the GFP antibody. The negative control (Control) corresponds to the pEarlyGate103 destination vector. The RbcL protein from the Ponceau-stained membrane was used as a loading control. The protein gel shown is representative of three independent biological replicates.
(D) The amount of the ABI4–Wt (white bar) or ABI4–∆P (gray bar) fusion proteins detected in the Western analysis (C) was quantified by densitometric analysis and expressed as arbitrary units relative to the ABI4–WT, taken as 1. Error bars indicate SD from three biological independent experiments.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Determination of the ABI4 Protein Half-Life in Arabidopsis Protoplast.
(A) The level of ABI4::GFP and ABI4–∆P::GFP fusions was determined by immunodetection 12h after protoplasts transformation, following the addition of the translation inhibitor cycloheximide. Total protein extracts were extracted at times 0, 2, 4, and 6h after the cycloheximide treatment. Extracts of each time point (80 μg) were resolved in a SDS–PAGE gel and subsequent immunoblotting membrane was performed using a 1: dilution of the GFP antibody. The negative control (Control) corresponds to the pEarlyGate103 destination vector. The RbcL protein from the Ponceau-stained membrane was used as a loading control.
(B) Protein half-life was determined using densitometric analysis. Data points represent the average and SD (error bars) of three biological independent experiments reported as relative units of densitometry normalized to the levels of ABI4–WT::GFP (circles) or ABI4–∆P::GFP (squares) at the beginning of the treatment, taken as 1. The half-life of the ABI4–WT::GFP protein is indicated by the dotted line.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
The ABI4 Protein Turnover Mediated by the PEST Motif Involves the 26S Proteasome Pathway.
The level of ABI4::GFP (A) and ABI4–∆P::GFP (B) fusion proteins was determined by immunodetection 16h after protoplast transformation, without any treatment, or after the addition of 50 μM cycloheximide (CHX) and CHX in combination with MG132. The negative control (CN) corresponds to the pEarlyGate103 destination vector. The RbcL protein from the Ponceau-stained membrane was used as a loading control. The ABI4::GFP (C) and ABI4–∆P::GFP (D) fusions in the different treatments were quantified by densitometry and are reported as relative units and normalized to the levels of ABI4–WT (A) or ABI4–∆P::GFP (B) without treatment, taken as 1. Error bars indicate standard deviation from three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

9. Figure 8 Glucose But Not ABA Affects ABI4::GFP Protein Accumulation.
(A) Protein accumulation of ABI4–WT::GFP fusion protein from protoplasts after 15h of transformation alone (–) or incubated in the presence of 1 μM ABA (+).
(B) Protein accumulation of ABI4–WT::GFP fusion protein from protoplasts after 15h of transformation alone (–) or incubated in the presence of 150mM glucose (+) were determined by immunodetection as described in Figure 5. The negative control (CN) corresponds to the pEarlyGate103 destination vector. The RbcL protein from the Ponceau-stained membrane was used as a loading control.
(C, D) Quantification of the ABI4::GFP protein levels was determined by densitometry and is reported as relative units normalized to the levels of ABI4–WT without treatment. Error bars indicate standard deviation from three independent experiments.
Molecular Plant 2014 7, DOI: ( /mp/sst132)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions