1. Volume 10, Issue 9, Pages 1206-1223 (September 2017)
The G-Protein β Subunit AGB1 Promotes Hypocotyl Elongation through Inhibiting Transcription Activation Function of BBX21 in Arabidopsis 
Dong-bei Xu, Shi-qing Gao, Ya-nan Ma, Xiao-ting Wang, Lu Feng, Lian-cheng Li, Zhao-shi Xu, Yao-feng Chen, Ming Chen, You-zhi Ma 
Molecular Plant 
Volume 10, Issue 9, Pages (September 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1 BBX21 Interacts with the Coiled-Coil Domain of AGB1.
(A) Schematic of the domain structure of AGB1 and truncated AGB1 proteins used in this study. Numbers indicate amino acid positions of AGB1.
(B) Yeast two-hybrid interactions between the AGB1 and BBX21. Co-transformed yeast cells were placed on control medium (SD/-Leu/-Trp) and selection medium (SD/-Leu/-Trp/-His/-Ade + X-α-gal). The empty vectors, AD and BD, were used as negative controls, and BBX21 and HY5 was used as a positive control.
(C) BiFC analysis of AGB1 and BBX21 in Arabidopsis protoplasts. Full-length AGB1, truncated AGB1, and BBX21 were fused to the split N- or C-terminal (YFPN or YFPC) fragments of YFP, respectively. Constructs without fusion partners comprised of the N-terminal (YFPN) or C-terminal (YFPC) YFP fragments were used as negative controls. DAPI staining was used to label nuclei. Scale bars, 20 μm.
(D) In vitro GST pull-down assay of His-BBX21 with GST-AGB1 (a) and GST-AGB1ΔCoil (b), respectively. His-BBX21 fusion proteins were detected using an anti-His antibody. GST alone was used as a negative control. All experiments were performed in triplicate and representative results are shown.
(E) In planta interaction of full-length BBX21 and AGB1 in N. benthamiana leaf cells. N. benthamiana leaves were transformed by agro-infiltration of Agrobacterium GV3101 cells. Leaf tissue was co-transformed with Pro35S:BBX21-Myc and Pro35S:AGB1-FLAG together, or the Pro35S:AGB1-FLAG plasmid alone. Total proteins were immunoprecipitated (IP) with an anti-Myc monoclonal antibody, and immunoblots were probed with anti-Myc or anti-FLAG monoclonal antibodies, respectively.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2
AGB1 Physically Interacts with the C-Terminal Region of BBX21.
(A) Schematic representations of the domain structure of BBX21 and the truncated BBX21 proteins used in this study. Numbers indicate amino acid positions of full-length BBX21 and the truncated BBX21 variants (S1–S4).
(B) Yeast two-hybrid interactions between BD-AGB1 and full-length BBX21 or truncated BBX21 variants cloned into the pGADT7. Co-transformed yeast cells were inoculated in liquid medium and then placed on control medium DDO (SD/-Leu/-Trp) and selection medium QDO (SD/-Leu/-Trp/-His/-Ade). Empty vectors (AD and BD) were transformed into yeast and used as negative controls.
(C) Interaction of AGB1 with the C-terminal region of BBX21 (S3). In vitro GST pull-down assays were performed to study the interaction between (a) AGB1 and the C-terminal fragment of BBX21 (TF-His-S3), or interaction AGB1 with TF-His (b). TF, trigger factor in pCold TF vector. Experiments were carried out three times, and representative results are shown.
(D) Interaction of AGB1 with the C-terminal fragment of BBX21 (S3) was verified by BiFC assays in N. benthamiana leaf cells. AGB1 and S3 region of BBX21 were fused with YFPN or YFPC, respectively. YFPN-only and YFPC-only refer to the empty vectors used as negative controls. DAPI staining marked the positions of nuclei. Scale bars, 20 μm.
(E) Interaction of AGB1 and the C-terminal region of BBX21 (S3) in N. benthamiana leaf cells. N. benthamiana leaf cells were co-transformed Pro35S:S3-Myc and Pro35S:AGB1-FLAG, or Pro35S:AGB1-FLAG alone. Total proteins were immunoprecipitated (IP) with an anti-Myc monoclonal antibody, and immunoblots were probed with anti-Myc or anti-FLAG monoclonal antibodies, respectively.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3
The Short Hypocotyl Phenotype of agb1-2 Is Partially Suppressed by bbx21-1 in the Dark or under Different Monochromatic Light Conditions.
(A, C, E, G, and I) The hypocotyl phenotypes of different mutant seedlings grown in the dark for 5 days (A), or under white light (100 μmol/m2/s) (C), red light (17.3 μmol/m2/s) (E), far-red light (68 μmol/m2/s) (G), and blue light (16.8 μmol/m2/s) (I) treatments for 3 days.
The white dashed lines indicated hypocotyl position in (A). Scale bars, 2 mm.
(B, D, F, H, and J) Hypocotyl lengths of different mutant seedlings grown in the dark for 5 days (B), or under white light (100 μmol/m2/s) (D), red light (17.3 μmol/m2/s) (F), far-red light (68 μmol/m2/s) (H), and blue light (16.8 μmol/m2/s) (J) treatments for 3 days. Data are presented as means ± SE (n = 30), and significant differences were analyzed by Duncan's multiple range test (P < 0.05 or P < 0.01).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4
The Phenotype of the Hypocotyls from the Seedlings Grown under Different Monochromatic Light Conditions.
(A–C) Hypocotyl phenotypes of wild-type (Col-0), agb1-2, OE-BBX21-GFP-6:agb1-2, bbx21-1, and OE-BBX21-GFP-2:Col-0 cultivated under red (79 μmol/m2/s) (A), far-red (33.5 μmol/m2/s) (B), and blue light (9.8 μmol/m2/s) (C) for 3 days. Scale bars, 2 mm.
(D–F) Hypocotyl lengths corresponding to lines shown in (A) to (C). Data are means of three independent experiments, and error bars represent SD (n = 30). Significant differences were analyzed by Duncan's multiple range test (P < 0.05).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5
Subcellular Localization and Transcriptional Activation Activity of BBX21 Variants.
(A) Subcellular localization of full-length BBX21 and truncated BBX21 proteins in Arabidopsis. Capital letters (GFP-S1 to GFP-S4) in the left border indicate the truncated BBX21 proteins corresponding to (B). GFP alone was used as a control. Scale bars, 20 μm.
(B) Schematic representations of the BBX21 variants. Numbers indicate amino acid positions of BBX21 and truncated BBX21 fragments (S1–S4) used in this study.
(C) Transcriptional activation assay of BBX21 variants in yeast. The GAL4 DNA binding domain-fused BBX21 variants were expressed in the yeast strain AH109 to analyze transcriptional activation. Serial dilutions were plated onto synthetic dextrose medium including SD/-Trp, SD/-Trp-Ade, and SD/-Trp-His-Ade, respectively. For each construct, three individual colonies were assessed. All experiments were performed three times and representative results are shown.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6 AGB1 Negatively Regulates the Activity of BBX21.
(A–D) The recombinant constructs pBridge-BBX21-AGB1, pBridge-BBX21, and empty vector pBridge controls were transformed into yeast (AH109). Transformed yeast cells were inoculated into liquid medium and plated on control medium SD/-Trp (A), selection medium SD/-Trp-Ade (B), SD/-Trp-Ade-Met (C), and SD/-Trp-Ade-Met plus 40 μg/mL X-α-gal (D). Experiments were performed three times.
(E) β-Galactosidase (β-gal) activity, representing the transcriptional activation of BBX21, was analyzed. Data represents the means of three independent replicates ± SD (n = 3). Significant differences were analyzed by Duncan's multiple range test (P < 0.05).
(F) Schematic representation of constructs used in the transient transfection assays in Arabidopsis protoplasts. Expression of various effector genes (AGB1 and BBX21) and an internal control gene (GUS) were driven by either the 35S or the Ubiquitin (Ubi) promoter, respectively. The region 2.0 kb upstream of the BBX21 gene start codon was fused to the firefly luciferase gene to generate the reporter construct.
(G) Activation of BBX21pro:LUC by the indicated combinations of proteins under dark and light treatment. Error bars represent means ± SE (n = 3). Significant differences were analyzed by Duncan's multiple range test (P < 0.01).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

8. Figure 7
AGB1 Negatively Regulates the Expression of BBX21 and Its Target Genes.
(A) qRT–PCR analysis of BBX21 expression in wild-type (Col-0) and agb1-2 seedlings. Four-day-old dark-grown seedlings were treated with white light (80 μmol/m2/s) for the indicated periods of time (h) prior to gene expression analysis. Control seedlings were kept in the dark. The agb1-2/Col-0 ratio indicates relative expression levels of BBX21 in agb1-2 under light treatment compared with wild-type plants. Error bars represent the means ± SE (n = 3).
(B) qRT–PCR analysis of BBX22 expression in wild-type (Col-0), agb1-2, and bbx21-1. Four-day-old dark-grown seedlings treated with red (R: 79 μmol/m2/s), far-red (FR: 33.5 μmol/m2/s), blue (9.8 μmol/m2/s), and white light (WL: 80 μmol/m2/s) for 0 h and 24 h. Control plants were kept in the dark for 4 days. Error bars represent means ± SE (n = 3).
(C) The expression levels of GA2ox1 in 5-day-old dark-grown wild-type (Col-0), agb1-2, bbx21-1, and OE-BBX21-GFP-2:Col-0 seedlings after plants were cultivated continually in the dark, or transferred to white light (WL: 80 μmol/m2/s) for 6 h. Data represent the means ± SD (n = 3). Amplification of the ACT2 gene under identical conditions served as a control.
(D) Schematic of the BBX21, BBX22, and GA2ox1 gene constructs, including the predicted promoter regions, used in ChIP assays. G-box like and G-box represent light response elements. Lines indicate the sequences detected by ChIP assays.
(E) ChIP assays showing that BBX21 associates with BBX21, BBX22, and GA2ox1 promoter sequences in vivo. ChIP was performed with anti-GFP monoclonal antibodies, and the ChIP DNA was analyzed by real-time qPCR. Error bars represent means ± SD of three technical replicates.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

9. Figure 8
A Working Model Depicting How AGB1 Regulates Hypocotyl Elongation by Controlling Light-Activated BBX21.
A model showing the role of AGB1 in hypocotyl elongation in Arabidopsis. AGB1 acts as a molecular switch through interaction with the light-activated transcription factor, BBX21, repressing transcriptional activation and self-activation. BBX21 functions as a positive regulator by directly activating itself and the expression of its target genes (BBX22, GA2ox1, and HY5) in the light. BBX21, BBX22, GA2ox1, and HY5 may play a positive role in the inhibition of hypocotyl elongation.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions