Fig 1. miR172 promotes flowering in wheat by repressing AP2L genes.

(A) Six-week-old wild type Kronos plants (Wt) compared to UBI_pro:miR172 and MIM172 transgenic plants in the same genetic background grown under LD. Scale bar = 10 cm. (B) Box plots showing miR172 expression levels in the 5^th leaf of UBI_pro:miR172, wild type Kronos (Wt) and MIM172 plants grown under LD. Mature miR172 levels were determined by qRT-PCR using the small nucleolar RNA 101 (SnoR) as internal reference. Data correspond to four independent biological replicates. (C-D) Box plots showing days to heading (C; n ≥ 7) and number of leaves produced by the main tiller (D; n ≥ 7) in the same genotypes and conditions as in B. (E) Schematic diagrams showing the gene structures of AP2L5 and AP2L1. Exons are indicated as dark grey boxes, AP2 domains are indicated in light grey, and the miR172-target sites in red. The positions of the TILLING mutations K3946 (AP2L5) and K863 (AP2L1) in the A homeologs are indicate above the gene structures, and the natural variant with 2 nucleotides deletion (AP2L5) and CAD161 splicing mutant (AP2L1) in the B homeologs below. (F) Box plots showing days to heading under LD conditions for wild type (Wt), plants harboring mutations in only one AP2L1 homeolog (aaBB and AAbb), in both AP2L1 homeologs (ap2l1-null = aabb), ap2l5-null mutants, and apl2l1 ap2l5 combined null mutants (n ≥ 8). (G) Box plots showing days to heading under LD conditions for an F₂ population segregating for the K4254 mutation in the miR172-target site of AP2L-A1 gene (n ≥ 10).; Wt = homozygous wild type plants, mut = homozygous K4254 mutants, het = heterozygous plants. The interaction between the wild type miR172-target site and miR172 is shown in the right. The TILLING line K4254 has a G>A mutation in the miR172 target site that reduces the free energy of interaction with miR172. (H-I) Box plots showing the expression levels of AP2L1 in the 5^th leaves (H; n = 4) and days to heading (I; n ≥ 5) for wild type Kronos (Wt) and three independent UBI_pro:AP2L-B1 transgenic lines grown under LD. (J-M) Wild type Kronos (Wt), UBI_pro:AP2L1 (transgenic line #5), MIM172 and MIM172 UBI_pro:AP2L-B1#5 plants grown under LD. (J) Eight-week-old plants. Scale bar = 10 cm. (K-M) Box plots showing days to heading (K; n ≥ 6), number of leaves produced by the main tiller (L; n ≥ 5) and expression levels of AP2L1 in the 5^th leaves (M). Different letters above the box plots indicate significant differences based on Tukey tests (P < 0.05), except for panels (D), (F) and (L) where non-parametric Kruskal-Wallis tests were used (Data A in S1 Data).