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. 2013 Nov 22;25(11):4405–4420. doi: 10.1105/tpc.113.116590

Figure 4.

Figure 4.

CIB1 Is a Transcription Activator Regulated by CRY2a in Response to Blue Light.

(A) A diagram showing the structure of the E-box–driven dual-luciferase reporter gene and DNA sequence of the recombinant E-box elements. The DNA sequences (Ec, Ef, and Ek) containing four tandem-repeat E-box derived from the c, f, and k regions of WRKY53b chromatin (see Figure 7B). The 35S promoter (black arrow), 35S minimum promoter (white arrowhead), Renilla luciferase (REN), firefly luciferase (LUC), and T-DNA (left border [LB] and right border [RB] are indicated.

(B) Images showing the LUC activities of N. benthamiana leaves infiltrated with the Agrobacteria strain harboring the indicated reporter (Ec, Ef, or Ek), in the presence (+) or absence (−) of the cotransfecting Agrobacteria strain harboring the plasmid expressing CIB1. After Agrobacteria infiltration, the plants were kept in white light for 3 d before photographs were taken.

(C) Dual-luciferase assay of relative reporter activity of samples shown in (B). The relative LUC activities normalized to REN activity are presented as relative expression units (REUs). The sd is shown (n = 3). The P values of CIB1-dependent activation of the reporter expression of the Ec, Ef, or Ek recombinant promoters are 0.014, 0.003, or 0.006, respectively (Student’s t test).

(D) Results of EMSA assay showing the inhibitory effect of CRY2a on the DNA binding activity of CIB1 to E-box DNA in response to blue light. The E-box DNA (Ewt) was mixed with effectors, which are the insect cell lysates expressing 6His-CIB1-Flag (CIB1) fusion protein and increased amount (1 to 8×) of lysates of insect cells expressing 6His-CRY2a (CRY2a). The mixtures containing the indicated components were incubated under blue light (25 μmol m−2 s−1) or in darkness at 4°C for 2 h. The mixture was mixed with agarose beads conjugated with anti-Flag antibody and washed five times with binding buffer, and the bound DNA was eluted by elution buffer and subjected to quantitative PCR. RBUs are defined in Methods. The significance of the CRY2a-dependent effects of the affinity of CIB1 for DNA in the presence or absence of blue light are examined by the Jonckheere-Terpstra Trend test; P = 0.912 or 0.003 of the dark-treated or the light-treated samples, respectively.