Figure 2.

RRBS cis-Elements in the RR4 and NSP2 Promoters Are Required for Cytokinin Regulation and Expression in Nodule Primordia.

(A) ChIP-qPCR of RR1 binding to RR4 and NSP2 promoters. The Actin11 ORF was used as a negative control, and IP values were normalized for each gene against the input genomic DNA. Ratios with Actin11 were calculated to visualize RR1 binding fold enrichment (n > 30 independent transgenic roots/construct).

(B) EMSA analysis of the RR1 binding to different 12-bp oligonucleotides labeled with [α-³²P] dATP (indicated by asterisk): the major SELEX consensus, boxes 1 to 4 (RR4 promoter), boxes 5 and 6 (NSP2 promoter), and a SELEX consensus where the central GAGA core was replaced by a ACTG sequence (SELEX mut.).

(C) Fluorometric quantification of GUS activity in roots expressing Pro_RR4:GUS (left) or Pro_NSP2:GUS (right) fusions after treatment with 10⁻⁷ M of BAP for 3 h. Error bars represent sd (n > 20 independent transgenic roots). A Kruskal-Wallis test was performed to assess significant differences (α < 0.05).

(D) Histochemical staining of GUS activity in nodule primordia expressing Pro_RR4:GUS (left) or Pro_NSP2:GUS (right) fusions. In [(C) and (D)], Pro_RR4-Δbox:GUS and Pro_NSP2-3XMut:GUS fusions were also used (see Methods for details).

Bars in (D) = 50 μm.