HAWAIIAN SKIRT, and F-box gene from Arabidopsis, is a new player in the microRNA pathway

F-box proteins belong to a multi-protein E3 ubiquitin ligase complex (SCF) that target proteins for degradation via the proteasome.We demonstrated that HAWAIIAN SKIRT(HWS), an Arabidopsis ubiquitin protein ligase (SCFHWS), regulates organ growth, flower development and timing of abscission. Mutants of this gene (hws-1) are pleiotropic and the most obvious phenotype is the fusion of its floral organs, a phenotype shared with the cuc1/cuc2 double mutants and over-expressing lines of MIR164B. To understand the molecular mechanisms of HWS during plant development, an ethylmethylsulphonate mutagenized population of hws-1 seeds was generated and screened for mutations suppressing the hws-1 sepal fusion. We isolated shs-1/hws-1, shs-2/hws-1, and shs-3/hws-1, (suppressor of hws-1) mutants. Mapping analyses shown that shs1 is mutated in the miRNA164 binding site of CUPSHAPED COTYLEDON1 (CUC1) mRNA; while shs-2 and shs-3 are novel alleles of the plant homolog of Exporting-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Consequently, we renamed them cuc1-1D, hst23 and hst24, respectively. We demonstrated that transcript levels of CUC1 and CUPSHAPED COTYLEDON 2 (CUC2), and MIR164 change in cuc1-1D and in hws-1 mutants; analyses revealed a role for HWS in cell proliferation and control of floral organ number. Additional genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway were performed and double mutants obtained shown restoration of the hws-1 sepal fusion phenotype. Our data propose HWS as a new regulator in miRNA pathway and reveal a role for HWS to control floral organ number and cell proliferation

Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development

The ERECT PANICLE 3 gene of rice encodes a peptide that exhibits more than 50% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Ectopic expression of the Os02g15950 coding sequence, driven by the HWS (At3g61950) promoter, rescued the hws-1 flower phenotype in Arabidopsis confirming that EP3 is a functional orthologue of HWS. In addition to displaying an erect inflorescence phenotype, loss-of-function mutants of Os02g15950 exhibited a decrease in leaf photosynthetic capacity and stomatal conductance. Analysis of a range of physiological and anatomical features related to leaf photosynthesis revealed no alteration in Rubisco content and no notable changes in mesophyll size or arrangement. However, both ep3 mutant plants and transgenic lines that have a T-DNA insertion within the Os02g15950 (EP3) gene exhibit smaller stomatal guard cells compared with their wild-type controls. This anatomical characteristic may account for the observed decrease in leaf photosynthesis and provides evidence that EP3 plays a role in regulating stomatal guard cell development

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway, also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth

HAWAIIAN SKIRT, and F-box gene from Arabidopsis, is a new player in the microRNA pathway

F-box proteins belong to a multi-protein E3 ubiquitin ligase complex (SCF) that target proteins for degradation via the proteasome.We demonstrated that HAWAIIAN SKIRT(HWS), an Arabidopsis ubiquitin protein ligase (SCFHWS), regulates organ growth, flower development and timing of abscission. Mutants of this gene (hws-1) are pleiotropic and the most obvious phenotype is the fusion of its floral organs, a phenotype shared with the cuc1/cuc2 double mutants and over-expressing lines of MIR164B. To understand the molecular mechanisms of HWS during plant development, an ethylmethylsulphonate mutagenized population of hws-1 seeds was generated and screened for mutations suppressing the hws-1 sepal fusion. We isolated shs-1/hws-1, shs-2/hws-1, and shs-3/hws-1, (suppressor of hws-1) mutants. Mapping analyses shown that shs1 is mutated in the miRNA164 binding site of CUPSHAPED COTYLEDON1 (CUC1) mRNA; while shs-2 and shs-3 are novel alleles of the plant homolog of Exporting-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Consequently, we renamed them cuc1-1D, hst23 and hst24, respectively. We demonstrated that transcript levels of CUC1 and CUPSHAPED COTYLEDON 2 (CUC2), and MIR164 change in cuc1-1D and in hws-1 mutants; analyses revealed a role for HWS in cell proliferation and control of floral organ number. Additional genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway were performed and double mutants obtained shown restoration of the hws-1 sepal fusion phenotype. Our data propose HWS as a new regulator in miRNA pathway and reveal a role for HWS to control floral organ number and cell proliferation

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

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