1. Volume 3, Issue 2, Pages 361-373 (March 2010)
The Arabidopsis bZIP1 Transcription Factor Is Involved in Sugar Signaling, Protein Networking, and DNA Binding 
Kang Shin Gene , Price John , Lin Pei-Chi , Hong Jong Chan , Jang Jyan-Chyun  
Molecular Plant 
Volume 3, Issue 2, Pages (March 2010)
DOI: /mp/ssp115
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Hexokinase-dependent glucose repression of AtbZIP1.
Shown are results of RNA gel-blot analyses.
(A) AtbZIP1 is repressed by low levels of glucose.
(B) Glucose repression of AtbZIP1 is rapid.
(C) Glucose repression can be reversed when glucose is removed from the culture medium in the dark condition (D-glc). Compare to the dark condition (D+glc), light (L+glc) appears to enhance glucose repression. Although cycloheximide (CHX) enhances AtbZIP1 expression, it does not block glucose repression.
(D) AtbZIP1 is repressed by sugars that can be taken up by the cells and phosphorylated efficiently by hexokinase.
(E) ABA alone or together with glucose does not affect glucose repression of AtbZIP1.
(F) Glucose repression of AtbZIP1 is compromised in HXK1 knockout (gin2) or antisense-HXK1. Indicated by arrows are the incompletely repressed AtbZIP1 transcripts. Note that the overexpression and anti-sense HXK plants are in BE background; abi4-1 and aba2-1 are in Col-0 background, and gin2-1 (AtHXK1 KO) is in Ler background. All experiments use 110 mM glucose (+glc) unless indicated otherwise.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Sugar Repression of AtbZIP1 in Transgenic Plants with a Promoter–Reporter Fusion Gene PAtbZIP1:GUS.
(A) The expression of the reporter gene can be suppressed by exogenous sucrose or glucose.
(B) Results of RNA gel-blot analyses showing that the endogenous AtbZIP1 is repressed by glucose normally in the WT and various reporter lines. Consistent with the endogenous AtbZIP1 gene, the PAtbZIP1:GUS reporter gene is repressed by 2% glucose.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
AtbZIP1 Acts as a Negative Regulator of Early Seedling Growth in the Absence of Exogenous Glucose (2%).
(A, B) Compared to the WT (Col-0), while the AtbZIP1 KO plants have higher rates, the overexpression (OX) plants have lower rates of true leaf development.
(C) Results of RNA gel-blot analyses confirm the KO and overexpression of AtbZIP1 in KO and OX plants, respectively.
(D) Dominant suppressing AtbZIP1–SRDX seedlings develop faster than the WT (Col-0) on sugar-free MS plates.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 AtbZIP1 can mediate the expression of sugar responsive genes.
(A) Microarray analysis reveals 98 putative AtbZIP1 repressible genes by Vann Diagram selection of 256 genes up regulated in AtbZIP1 KO plants and 249 genes down regulated in AtbZIP1 OX plants.
(B) Similar analysis was carried out to identify 46 putative AtbZIP1 inducible genes by comparing 260 down regulated genes in KO plants and 221 up regulated genes in OX plants. For the comparison of WT vs. KO, both received sugar-depletion treatment before RNA extraction. For the comparison of WT vs. OX, both were treated with 2% glucose before RNA extraction. Using a list of 2,348 glucose responsive genes identified from previous microarray analyses (Price et al., 2004), numbers of glucose responsive genes (in red) and the way these genes responding to glucose from each pool (in blue) are indicated.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 The AtbZIP1 interacting network.
(A) Yeast two-hybrid screen identifies AtbZIP11, AtbZIP44, AtbZIP10, AtbZIP25, and AtbZIP63 as AtbZIP1 interacting partners. Strength of each interaction is determined by colony growth on normal (-Leu-Trp-His) and high (-Leu-Trp-His-Ade) stringency selection plates, and as indicated by line thickness in the model.
(B-C) Results of bimolecular fluorescence complementation (BiFC) analyses indicate that AtbZIP1 interacts extensively with both S-group (AtbZIP44, AtbZIP53) and C-group (AtbZIP9, AtbZIP10, AtbZIP25, AtbZIP63) AtbZIPs in maize (B) and Arabidopsis (C) transient expression system. Symbols of cross-lined circles indicate negative interactions.
(D) Model depicting the AtbZIP1 interacting network based on the results of Y-2-H and BiFC. Strength of interaction is indicated by the line thickness. Red lines denote new interactions not being reported previously. Dotted lines indicate interactions found in Ehlert et al. 2006, but not in current study.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 AtbZIP1 can bind ACGT motif-based C- or G-box.
(A-B) EMSA results showing that AtbZIP1 can bind Hex, C-box, and G-box motif, respectively. The AtbZIP10 (A) and AtbZIP63 (B) can interfere with the binding as evidenced by the super-shift and reduction of the DNA-AtbZIP1 complexes, respectively.
(C) AtbZIP1 binding to the Hex motif is specific, as DNA-protein complex cannot be formed with mutant probe.
(D) AtbZIP1, but not AtbZIP10 or 25 can bind strong C- or G-box. The maltose binding protein is used as a background negative control.
(E) AtbZIP 63 seems to enhance DNA-AtbZIP1 complex formation, and AtbZIP1 causes a super-shift of the complex. Signals in the bottom of each gel are generated by free DNA probes.
Molecular Plant 2010 3, DOI: ( /mp/ssp115)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions