Figure 6.

RLI1 Directly Binds to the BC1 and BU1 Promoters.

(A) EMSA analysis showing that RLI1 binds to the P1BS cis-element. The DNA fragments containing P1BS were incubated with RLI1-His recombinant proteins as indicated. 6xHis proteins were used as negative controls.

(B) RLI1 can bind to the core DNA sequences of NNAKATNC. EMSA detects the interaction between RLI1 and different P1BS mutant versions; wild-type P1BS (GAATATGC) was used as positive control.

(C) Logo analysis of RLI1 recognition bases. Logo analysis was performed at http://weblogo.threeplusone.com.

(D) Relative expression levels of BU1 and BC1 in the lamina joints of wild-type, rli1-1, RLI1-OE-3, and RLI1-OE-7 plants. Lamina joint tissues from 21-d-old plants were harvested for RNA extraction and RT-qPCR analyses. Values represent means ± sd (n = 3). Data significantly different from the control are indicated. P values were determined by Student’s t test. Mutants versus the wild type: #P < 0.05; overexpressors versus wild the type: **P < 0.01.

(E) and (F) ChIP-qPCR assays showing that RLI1 associates with the promoters of BU1 and BC1 in vivo. Lamina joint tissues of 4-week-old wild type and 35S:RLI1-FLAG (RLI1-OE-3) were harvested for ChIP analysis. Enriched DNA fragments in the BU1 and BC1 promoters were quantified using RT-qPCR. The red lines indicate the candidates of RLI1 recognition sites in the promoters of BU1 and BC1. Values represent means ± sd (n = 3). Data significantly different from the control are indicated. P values were determined by Student’s t test. RLI1-FLAG versus No RLI1-FLAG: **P < 0.01 and *P < 0.05.