A silent mutation in <i>CUC1</i> does not change floral organ numbers in a Columbia-0 background but induces extra floral organs in <i>hws-1</i>, <i>cuc1-1D</i> and <i>hws-1/cuc1-1D</i>.

<p>(<b>A, D, G, J, M</b>), Lateral view; (<b>B, E, H, K</b>), Aerial view; (<b>C, F, I, L</b>), dissected flowers of primary transformants in the following backgrounds: (<b>A-C</b>), Columbia-0; (<b>D-F</b>), <i>hws-1</i>; (<b>G-I</b>), <i>cuc1-1D</i>; and (<b>J-L</b>), <i>hws-1/cuc1-1D</i>, note bifurcated anther inidicated with a white star in panel L. (<b>M</b>), Mature siliques showing suppression of sepal fusion in <i>hws-1</i>: left silique originated from a <i>hws-1</i> mutant, right silique originated from a primary transformant <i>hws-1</i> plant transformed with <i>CUC1-SV</i>. Scale bars: 1mm. Black and white stars show altered floral organs.</p

Phenotypic characterisation of <i>hst-24</i>.

<p>(<b>A</b>) Dissected flower from developmental stage 15a from <i>hst-24/hws-1</i>. (<b>B</b>) Comparative analyses of sepal and petal sizes from flowers (stage 15a) of Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i>. (<b>C</b>). Twenty-five flowers from six plants of Col-0, <i>hst-24/hws-1</i> and <i>hst-24</i> were dissected and their sepals and petals quantified and statistically analysed by regression analyses using generalized linear models. Stars indicate a significant difference in the mean at P≤0.001 n = 450. Bars indicate SD. (<b>D</b>) Rosettes, and (<b>E</b>) Dissected leaves from 22-day-old plants from Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i>. Bars in A, B = 1mm; and in D, E = 1 cm.</p

Mutations and constructs in <i>CUC1</i>, <i>CUC2</i> and <i>MIR164</i>.

<p>Schematic diagram of <i>MIR164</i>, <i>MIR164</i> complementary binding sites in <i>CUC1</i> and <i>CUC2</i> mRNAs and CUC1, CUC2 proteins or their equivalent in generated constructs; (<b>A</b>), wild type (<b>B</b>), <i>cuc1-1D</i> mutation; (<b>C</b>), <i>cuc1-1D</i> mutation and <i>MIR164</i> modified site introduced for complementation analyses; (<b>D</b>), <i>cuc2-1D</i> mutation (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185106#pone.0185106.ref024" target="_blank">24</a>]); (<b>E</b>), <i>cuc1-1D</i> silent version (<i>cuc1-1D-SV</i>). Mutations are underlined, the amino acid substitutions are identified in red/blue font, and changes in binding affinity from the <i>MIR164</i> are indicated with a red dot. (<b>F-K</b>), Complementation analyses in primary transformants using a modified version of <i>MIR164B</i>; (<b>F-G</b>), aerial and (<b>H-I</b>), lateral view of flowers at stage 15a and (<b>J-K</b>), lateral view of mature siliques from complementation lines in <i>cuc1-1D</i> and <i>hws-1/cuc1-1D</i> backgrounds using the <i>35S</i>_<i>pro</i>::<i>164B C→T</i> 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 <i>cuc1-1D</i> and <i>hws-1/cuc1-1D</i> backgrounds respectively. Scale bars: 30 μm F-G and 1mm in H-K.</p

<i>HWS</i> affect cell proliferation in petals.

<p>Analyses of (<b>A</b>), petals size (mm²) and (<b>B</b>), petal cell size (μm²) in Columbia-0, <i>hws-1</i>, <i>cuc1-1D</i>, and <i>hws-1/cuc1-1D</i>. Five flowers from four independent plants from each genotype were dissected and their size and the size of petal cells were determined. (<b>C</b>), Relative petal and cell sizes compared to Columbia-0 (100%). Stars indicate a significant difference in the mean at P≤0.001 n = 80.</p

The <i>shs-2</i> and <i>shs-3</i> mutants are alleles of <i>HST</i>.

<p>(<b>A-H</b>), Aerial and (<b>I-P</b>), lateral views of flowers at stage 15a; and (<b>Q-X</b>), lateral view of mature green siliques from wild type in Col-0, <i>hws-1</i>, <i>shs-2/hws-1</i> (<i>hst-23/hws-1</i>), <i>shs-2</i> (<i>hst-23)</i>, <i>shs-3/hws-1</i> (<i>hst-24/hws-1</i>), <i>shs-3</i> (<i>hst-24)</i>, <i>hws-1xhst-1</i>, <i>hst-1</i>. Bars = 1mm. (<b>Y</b>), Mapping strategy used to identify the <i>hst-23 and hst-24</i> mutations. Structure of the gene and location of the transition substitution (C.G→T.A) at positions 4.587 Kb and 5.517 Kb in <i>hst-23</i> and (G.C→A.T) at 0.583 Kb in <i>hst-24</i> from the ATG are included, intragenic regions are represented by thin lines and exons by dark boxes.</p

Mean of sepal and petal numbers in Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i> in Col-0 from the first 25 flowers of the inflorescences, (flowers n = 200).

<p>Mean of sepal and petal numbers in Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i> in Col-0 from the first 25 flowers of the inflorescences, (flowers n = 200).</p

miRNA pathway and co-suppression between <i>hws-1</i> and miRNA pathway mutants.

<p>Single (<b>A, G, J, M, P, S</b>) <i>hws-1</i>, (<b>D</b>) <i>hws-2</i>, (<b>B</b>) <i>ddl-2</i>, (<b>E</b>) <i>se-1</i>, (<b>H</b>) <i>hyl-1</i>, (<b>K</b>) <i>dcl1-9</i>, (<b>N</b>) <i>hen1-5</i>, (<b>Q</b>) <i>hst-1</i>, (<b>T</b>) <i>ago1-37</i>, and double (<b>C</b>) <i>hws-1Xddl-2</i>, (<b>F</b>) <i>hws-1Xse-1</i>, (<b>I</b>) <i>hws-1Xhyl-1</i>, (<b>L</b>) <i>hws-1Xdcl1-9</i>, (<b>O</b>) <i>hws-1Xhen1-5</i>, (<b>R</b>) <i>hws-1Xhst-1</i>, (<b>U</b>) <i>hws-1Xago1-37</i>, mutants showing co-suppression of phenotypes. Bars = 1mm. The (<b>V</b>) miRNA pathway (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189788#pone.0189788.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189788#pone.0189788.ref036" target="_blank">36</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189788#pone.0189788.ref061" target="_blank">61</a>]) has been included for reference.</p

The <i>shs1</i> mutant is an allele of <i>CUC1</i>.

<p>(<b>A-H</b>), Aerial and (<b>I-P</b>), lateral views of flowers at stage 15a; and (<b>Q-X</b>), lateral view of mature green siliques. From: (<b>A, I, Q</b>), Columbia-0; (<b>B, J, R</b>), <i>hws-1</i> (Columbia-0 background);(<b>C, K, S</b>), <i>hws-2</i> (<i>L</i>er background); (<b>D, L, T</b>), <i>hws-1/shs</i>+/-; (E, M. U), <i>hws-1/shs1 (hws-1/cuc1-1D)</i>; and primary transformants of (<b>F, N, V</b>), Columbia-0; (<b>G, O, W</b>), <i>hws-1</i>; and (<b>H, P, X</b>), <i>hws-2</i> complemented with a genomic region containing the <i>CUC1pr</i>::<i>CUC1-1D</i> 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. (<b>Y</b>), Mapping strategy used to identify the <i>cuc1-1D</i> 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.</p

Transcript levels of <i>CUC1 CUC2</i>, <i>MIR164A</i>, <i>MIR164B</i> and <i>MIR164C</i> genes are affected in single and double mutants and in the <i>Pro</i>_<i>35</i>:<i>HWS</i> lines.

<p>RT-qPCR measurements of (<b>A</b>), <i>CUC1</i>; (<b>B</b>), <i>CUC2</i>; (<b>C</b>), <i>HWS</i>; (<b>D</b>), <i>MIR164A</i>; (<b>E</b>), <i>MIR164B</i>; (<b>F</b>), <i>MIR164C</i> RNA levels in Columbia-0, <i>hws-1</i>, <i>35S</i>_<i>pro</i>:<i>HWS</i>, <i>cuc1-1D</i>, <i>hws-1/cuc1-1D</i> and <i>cuc2-1D</i>. 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).</p

Floral organ number is affected in single, double and triple mutants of <i>hws-1</i>, <i>cuc1-1D</i> and <i>cuc2-1D</i>.

<p>Comparative phenotypic analyses of flowers at developmental stage 15a. (<b>A-E</b>), lateral view of flowers; (<b>F-J</b>), close up of sepal separation; (<b>K-O</b>), aerial view at stage 15a from: (<b>A, F, K</b>) <i>cuc1-1D</i>; (<b>B, G, L</b>), <i>cuc2-1D</i>; (<b>C, H, M</b>), <i>hws-1/cuc2-1D</i>; (<b>D, I, N</b>), <i>cuc1-1D/ cuc2-1D</i> and (<b>E, J, O</b>), <i>hws-1/cuc1-1D/cuc2-1D</i>. (<b>P-W</b>), dissected flowers at stage 15a from: (<b>P</b>) Columbia-0, (<b>Q</b>) <i>hws-1</i>, (<b>R</b>) <i>cuc1-1D</i>, (<b>S</b>) <i>cuc2-1D</i>, (<b>T</b>) <i>hws-1/cuc1-1D</i>, (<b>U</b>) <i>hws-1/cuc2-1D</i>, (<b>V</b>) <i>cuc1-1D/ cuc2-1D</i> and (<b>W</b>) <i>hws-1/cuc1-1D/cuc2-1D</i>. Scale bars: 1 mm in (<b>A-J</b>) and 300 μm in (<b>K-W</b>), * show misshapen organs. (<b>X</b>), 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.</p