Figure 8.

MLK2 Antagonizes the Function of RGA to Bind CCA1 in Vitro and in Vivo.

(A) Competitive pull-down of CCA1 with RGA and MLK2. Beads containing GST-fused CCA1 were assayed for their ability to bind a soluble His-fused RGA with increased dosage of MBP-MLK2.

(B) Co-IP between RGA and CCA1 with or without MLK1/2. FLAG-CCA1 and HA-RGA were cotransformed with or without MYC-MLK1/2 into Arabidopsis protoplasts, immunoprecipitated using an anti-FLAG antibody, and then detected with anti-Flag and anti-HA. The cells were harvested at 2 h after lights-on zeitgeber time (ZT2).

(C) The vectors used in the GUS activity assay.

(D) The GUS activity from the Pro_DWF4:GUS reporter construct for cells transformed with CCA1, RGA, and MLK1/2. The x axis shows the relative GUS activity compared with the internal luciferase control (35S:LUC). Asterisks indicate P < 0.05 by t test.

(E) Gel shift assay with CCA1 and fragments of the DWF4 promoter region with increasing dosage of RGA. The binding ability of CCA1 to fragments of the DWF4 promoter labeled with ³²P was assessed with increasing amounts of RGA (1, 4, and 10 µg).

(F) The relative enrichment of CCA1 at the DWF4 promoter was assessed using ChIP-PCR in rga28 and mlk1 mlk2 mutants. Experiments were repeated at least three times, and each experiment shown indicates the mean ± se, n = 3 replicates. The leaves were cross-linked at 2 h after lights-on zeitgeber time (ZT2).