Fig 1.
The shs1 mutant is an allele of CUC1.
(A-H), Aerial and (I-P), lateral views of flowers at stage 15a; and (Q-X), lateral view of mature green siliques. From: (A, I, Q), Columbia-0; (B, J, R), hws-1 (Columbia-0 background);(C, K, S), hws-2 (Ler background); (D, L, T), hws-1/shs+/-; (E, M. U), hws-1/shs1 (hws-1/cuc1-1D); and primary transformants of (F, N, V), Columbia-0; (G, O, W), hws-1; and (H, P, X), hws-2 complemented with a genomic region containing the CUC1pr::CUC1-1D gene. Scale bars: 1mm. Arrows show the sepal fusions. A petal in F and a sepal on P have been removed. * in N shows stamen fusion. (Y), Mapping strategy used to identify the cuc1-1D mutation. Structure of the gene and location of the transition substitution (G→A) 1,238bp from the ATG are included, intragenic regions are represented by thin lines and exons by black boxes.
Fig 2.
Mutations and constructs in CUC1, CUC2 and MIR164.
Schematic diagram of MIR164, MIR164 complementary binding sites in CUC1 and CUC2 mRNAs and CUC1, CUC2 proteins or their equivalent in generated constructs; (A), wild type (B), cuc1-1D mutation; (C), cuc1-1D mutation and MIR164 modified site introduced for complementation analyses; (D), cuc2-1D mutation (modified from [24]); (E), cuc1-1D silent version (cuc1-1D-SV). Mutations are underlined, the amino acid substitutions are identified in red/blue font, and changes in binding affinity from the MIR164 are indicated with a red dot. (F-K), Complementation analyses in primary transformants using a modified version of MIR164B; (F-G), aerial and (H-I), lateral view of flowers at stage 15a and (J-K), lateral view of mature siliques from complementation lines in cuc1-1D and hws-1/cuc1-1D backgrounds using the 35Spro::164B C→T construct, arrows show sepal fusion. Twenty-four primary independent transformants from each line were analysed. All transformants reverted or not the sepal fusion phenotype in the cuc1-1D and hws-1/cuc1-1D backgrounds respectively. Scale bars: 30 μm F-G and 1mm in H-K.
Fig 3.
Floral organ number is affected in single, double and triple mutants of hws-1, cuc1-1D and cuc2-1D.
Comparative phenotypic analyses of flowers at developmental stage 15a. (A-E), lateral view of flowers; (F-J), close up of sepal separation; (K-O), aerial view at stage 15a from: (A, F, K) cuc1-1D; (B, G, L), cuc2-1D; (C, H, M), hws-1/cuc2-1D; (D, I, N), cuc1-1D/ cuc2-1D and (E, J, O), hws-1/cuc1-1D/cuc2-1D. (P-W), dissected flowers at stage 15a from: (P) Columbia-0, (Q) hws-1, (R) cuc1-1D, (S) cuc2-1D, (T) hws-1/cuc1-1D, (U) hws-1/cuc2-1D, (V) cuc1-1D/ cuc2-1D and (W) hws-1/cuc1-1D/cuc2-1D. Scale bars: 1 mm in (A-J) and 300 μm in (K-W), * show misshapen organs. (X), Five flowers from six plants of each genotype were dissected and their floral organs quantified and statistically analysed by regression analyses using generalized linear models. Stars indicate a significant difference in the mean at P≤0.05 n = 30. Bars indicate SD.
Fig 4.
A silent mutation in CUC1 does not change floral organ numbers in a Columbia-0 background but induces extra floral organs in hws-1, cuc1-1D and hws-1/cuc1-1D.
(A, D, G, J, M), Lateral view; (B, E, H, K), Aerial view; (C, F, I, L), dissected flowers of primary transformants in the following backgrounds: (A-C), Columbia-0; (D-F), hws-1; (G-I), cuc1-1D; and (J-L), hws-1/cuc1-1D, note bifurcated anther inidicated with a white star in panel L. (M), Mature siliques showing suppression of sepal fusion in hws-1: left silique originated from a hws-1 mutant, right silique originated from a primary transformant hws-1 plant transformed with CUC1-SV. Scale bars: 1mm. Black and white stars show altered floral organs.
Fig 5.
HWS affect cell proliferation in petals.
Analyses of (A), petals size (mm2) and (B), petal cell size (μm2) in Columbia-0, hws-1, cuc1-1D, and hws-1/cuc1-1D. Five flowers from four independent plants from each genotype were dissected and their size and the size of petal cells were determined. (C), Relative petal and cell sizes compared to Columbia-0 (100%). Stars indicate a significant difference in the mean at P≤0.001 n = 80.
Fig 6.
Transcript levels of CUC1 CUC2, MIR164A, MIR164B and MIR164C genes are affected in single and double mutants and in the Pro35:HWS lines.
RT-qPCR measurements of (A), CUC1; (B), CUC2; (C), HWS; (D), MIR164A; (E), MIR164B; (F), MIR164C RNA levels in Columbia-0, hws-1, 35Spro:HWS, cuc1-1D, hws-1/cuc1-1D and cuc2-1D. Stars indicate a significant difference in the mean at P≤0.001. Relative expression values represent the mean ± SD of three biological replicates and two technical replicates from each sample (n = 30).