HAWAIIAN SKIRT controls size and floral organ number by modulating CUC1 and CUC2 expression

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT (HWS) affects organ growth and the timing of floral organ abscission. The loss-of-function hws-1 mutant exhibits fused sepals and increased organ size. To understand the molecular mechanisms of HWS during plant development, we mutagenized hws-1 seeds with ethylmethylsulphonate (EMS) and screened for mutations suppressing hws-1 associated phenotypes. We isolated the shs1/hws-1 (suppressor of hws-1) mutant in which hws-1 sepal fusion phenotype was suppressed. The shs1/hws-1 mutant carries a G→A nucleotide substitution in the MIR164 binding site of CUP-SHAPED COTYLEDON 1 (CUC1) mRNA. CUC1 and CUP-SHAPED COTYLEDON 2 (CUC2) transcript levels were altered in shs1, renamed cuc1-1D, and in hws-1 mutant. Genetic interaction analyses using single, double and triple mutants of cuc1-1D, cuc2-1D (a CUC2 mutant similar to cuc1-1D), and hws-1, demonstrate that HWS, CUC1 and CUC2 act together to control floral organ number. Loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the CUC1 gene

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

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

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

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