1. Volume 8, Issue 3, Pages 467-478 (March 2015)
Red-Light-Dependent Interaction of phyB with SPA1 Promotes COP1–SPA1 Dissociation and Photomorphogenic Development in Arabidopsis 
Xue-Dan Lu, Chuan-Miao Zhou, Peng-Bo Xu, Qian Luo, Hong-Li Lian, Hong-Quan Yang 
Molecular Plant 
Volume 8, Issue 3, Pages (March 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
SPA1 Interacts Specifically with the Red-Light-Activated Pfr Form of phyB and phyA in Yeast Cells.
(A and C) Yeast two-hybrid prey constructs. All full-length and truncated forms of phyB (A) and phyA (C) were fused with the GAL4-activation domain (AD).
(B and D) Yeast two-hybrid assays showing red-light-dependent interactions between phyB (B) or phyA (D) and SPA1. Full-length of SPA1 fused to a GAL4 binding domain was the bait construct. Yeast cells coexpressing the indicated combinations of constructs were grown on nonselective (SD-T-L) or selective media with 25 μM PCB (SD-T-L-H + PCB), in continuous red light (R, 3 μmol/m2/s), far-red light (FR, 1 μmol/m2/s), or darkness (D). Yeast growth on the selective media indicated interactions. The pBridge and pGADT7 empty vectors were used as negative control.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
phyB and phyA Colocalize with SPA1 to the Nucleus in Arabidopsis Protoplasts upon Red and Red/Far-red Light, Respectively.
(A) phyB translocalized into nucleus and colocalized with SPA1 under red light (20 μmol/m2/s) but not in darkness. Bars, 5 μm. The inner panels are enlarged portions of nucleus in their outer panels, respectively.
(B) phyA translocalized into nucleus and colocalized with SPA1 under far-red light (3 μmol/m2/s) and red light (20 μmol/m2/s) but not in darkness. Bars, 5 μm. The inner panels are enlarged portions of nucleus in their outer panels.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
phyB Interacts with SPA1 In Vivo and Red Light Enhances the phyB–SPA1 Interaction in Arabidopsis.
(A) BiFC assays of in vivo interactions of phyB–SPA1 in red light (20 μmol/m2/s) and darkness. The indicated constructs were cotransformed into tobacco leaf epidermal cells. The images show overlays of fluorescence and light views. The right-hand panels show the nuclei labeled by red boxes in the adjacent panels at high magnification, respectively. Bars, 20 μm.
(B) Coimmunoprecipitation assay showing that phyB interacts with SPA1 in Arabidopsis in a red-light-dependent manner. Seedlings overexpressing Myc-SPA1 in spa1 mutant background were grown in darkness for 5 d and then exposed to red light (20 μmol/m2/s) for 0, 10, 30, or 60 min. The endogenous phyB proteins were immunoprecipitated with anti-phyB antibody and the immunoblot was probed with anti-Myc and anti-phyB antibody.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
SPAs Are Epistatic to PHYB and PHYA to Regulate Photomorphogenesis under Red Light and Far-Red Light, Respectively.
(A and C) Seedlings were grown on MS medium under red light (7 μmol/m2/s) (A) and far-red light (0.5 μmol/m2/s) (C) for 5 d before hypocotyl lengths were measured. Wild-type (WT), phyB, spa1 spa2 (spa12), phyB spa1 spa2 (phyB spa12), spa1 spa2 spa4 (spa124), phyB spa1 spa2 spa4 (phyB spa124), spa1 spa2 spa3 spa4 (spa1234), phyB spa1 spa2 spa3 spa4 (phyB spa1234), phyA, phyA spa1 spa2 (phyA spa12), phyA spa1 spa2 spa4 (phyA spa124), phyA spa1 spa2 spa3 spa4 (phyA spa1234). Bars, 5 mm.
(B and D) Quantification data of (A) and (C). Numbers indicate the hypocotyl lengths of the indicated plants. Data are means ± SD (n = 30). a to g indicate statistically significant differences between means for hypocotyl lengths of the indicated genotypes, as determined by Tukey’s LSD test (P ≤ 0.01).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5
phyB Suppresses COP1–SPA1 Interaction in Response to Red Light In Vivo.
(A) Transgenic seedlings overexpressing Myc-SPA1 in spa1 mutant background (phyB present) were grown in continuous darkness for 5 d and then exposed to red light (70 μmol/m2/s) for the indicated time. COP1, pulled down by immunoprecipitated Myc-SPA1, was progressively reduced with prolonged exposure to red light. The membrane was stripped and reprobed with anti-phyB antibody to detect the phyB protein levels. Relative band intensities were normalized for each panel and shown under each panel.
(B) 5 d dark-grown transgenic seedlings overexpressing Myc-SPA1 in phyB mutant background (phyB absent) were exposed to red light (70 μmol/m2/s) for the indicated time. The abundance of COP1 coimmunoprecipitated by Myc-SPA1 remained similar. Relative band intensities were normalized and are shown under each panel.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
Red-Light-Activated phyB Inhibits the COP1–SPA1 Interaction in Yeast Cells.
(A) Yeast three-hybrid constructs expressing both the bait and the bridge proteins. COP1 or CC1 were fused with GAL4 binding domain (BD), respectively. PMet25, inducible promoter driving the expression of the bridge protein phyB. COP1 or CC1 fused to BD domain without bridge proteins were used as negative controls in yeast three-hybrid assays. CC1 fused to BD was also the bait construct of yeast two-hybrid assays.
(B) Quantitative yeast three-hybrid analyses show that red-light-activated phyB repressed the interaction of COP1–SPA1. Yeast cells were cultured in SD-T-L-M medium with 25 μM PCB in darkness for 18 h (Dark), in darkness for 17 h and then exposed to red light (40 μmol/m2/s) for 1 h (Red 1 h) or in darkness for 9 h and then exposed to red light for 9 h (Red 9 h). The interaction values (Miller units) in darkness were set to 100%, and the relative interactions under red light were expressed as the percentage of the dark value. Data are mean ± SD (n = 3).
(C) Yeast two-hybrid assays show that CC1 did not interact with phyB in either red light (R, 3 μmol/m2/s) or darkness (D) in yeast cells.
(D) Quantitative yeast three-hybrid analyses showing the red-light-dependent phyB inhibition of the CC1–SPA1 interaction. In the presence of phyB, exposure to red light (40 μmol/m2/s) for 2 h (Red 2 h) or 7 h (Red 7 h) resulted in a dramatic reduction of CC1–SPA1 interaction compared with that in darkness (Dark). In the absence of phyB, the CC1–SPA1 interaction was not affected by indicated red light treatment.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7
Red-Light-Activated phyB Interacts with SPA1 to Suppress the Formation and Activity of COP1–SPA1 Complex.
(A) In darkness, the Pr form of phyB localizes to the cytoplasm while SPA1 interacts with COP1 in nucleus and facilitates the COP1-mediated ubiquitination and degradation of photomorphogenesis-promoting transcription factor HY5, inhibiting target gene expression and promoting skotomorphogenesis.
(B) Upon red light irradiation, phyB converts to its biologically active Pfr form and translocates into nucleus while COP1 is exported to cytoplasm slowly. Pfr-phyB interacts with SPA1 in nucleus and impairs the interaction of SPA1 with COP1, alleviating the degradation of HY5 and initiating the photomorphogenic gene expression and photomorphogenesis.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions