Identification and characterisation of a mads-box gene from Rafflesia Cantleyi Solms-Laubach (Rafflesiaceae)

Master'sMASTER OF SCIENC

Identification and Characterization of RcMADS1, an AGL24 Ortholog from the Holoparasitic Plant Rafflesia cantleyi Solms-Laubach (Rafflesiaceae)

10.1371/journal.pone.0067243PLoS ONE86-POLN

Complementation analysis of <b><i>svp-41</i></b><b> mutant and rescue of </b><b><i>FRI</i></b><b> phenotype by </b><b><i>RcMADS1</i></b><b> expression.</b>

<p>(A) <i>RcMADS1</i> failed to rescue <i>svp-41</i>. WT (left), <i>svp-41</i> mutant (middle) and <i>svp-41+RcMADS1</i> (right). (B) The flowering time of plants in (A) represented by rosette leaf numbers at bolting (mean ± s.d.). (C) Rescuing of <i>FRI</i> phenotype. WT (left), <i>FRI</i> containing Col line (middle) and <i>FRI</i> containing Col line <i>+RcMADS1</i> (right). (D) The flowering time of plants in (C) represented by rosette leaf numbers (mean ± s.d.). Asterisks indicate that means are significantly different (P≤0.05) according to Student’s <i>t</i>-Test. Scale bars = 1cm.</p

Ectopic expression of <i>RcMADS1</i> in <i>Arabidopsis thaliana</i> causes conversion of carpels into inflorescence-like structures.

<p>The stronger phenotypic lines are shown (A) Comparison of <i>35S::RcMADS1</i> (right) with WT (left). (B) Close-up of one inflorescence stem of <i>35S::RcMADS1</i> (right) and WT (left). (C). A wild-type <i>Arabidopsis</i> flower. (D) A <i>35S::RcMADS1</i> flower showing conversion of sepals and petals into leaf-like structures bearing conspicuous trichomes. (E) A <i>35S::RcMADS1</i> flower showing the conversion of carpel into an inflorescence-like structure. (F) A <i>35S::RcMADS1</i> flower with a secondary inflorescence developing from the axil of a sepal (red arrowhead). (G) <i>35S::RcMADS1</i> flowers displaying the three different severities of phenotypes. Weak: flower from EL4 similar to wild-type (left), Medium: flower from EL2 exhibiting conversion of sepals and petals into leaf-like structures while the carpel develops into a silique (middle), Strong: flower from EL8 exhibiting conversion of sepals and petals into leaf-like structures and the carpel is converted into inflorescence-like structure with secondary inflorescences (right). (H) Expression levels of <i>RcMADS1</i> in the three different transgenic lines exhibiting weak, medium and strong phenotypes. Expression levels were normalized against the expression of <i>TUB2</i>. Error bars indicate s.d. (I) Flowers of <i>35S::AGL24</i> (right), <i>35S::RcMADS1</i> (middle) and <i>35S::SVP</i> (left). Scale bars A, B = 1cm, C, D, E, F = 1 mm, G and I = 2 mm.</p

Ectopic expression of <i>RcMADS1</i> causes early flowering in <i>Arabidopsis</i>.

<p>(A) Representative plants from independent transgenic lines showing different flowering times (plant1 WT, plant2 EL4, plant3 EL2 and plant4 EL8). (B) The flowering time of plants in (A) indicated by rosette leaf numbers at bolting. Data are mean ± s.d. from 20 plants of each line. (C) Flowering time comparison of EL8 with WT, <i>35S::AGL24</i> and <i>35S::SVP.</i> Data are mean ± s.d. from 20 plants of each genotype. Asterisks indicate significantly different means (P≤0.05) according to Student’s <i>t</i>-Test. Scale bar = 1cm.</p

Phylogenetic tree of selected MADS-box genes from StMADS11 clade.

<p>(A) The tree was constructed based on the deduced amino acid sequences using the Phylogeny.fr with one click mode. (B) Phylogenetic tree of RcMADS1 with only AGL24, SVP and StMADS11. In both the trees RcMADS1 is nested closer to AGL24 than to SVP. AGL24 and SVP from <i>Arabidopsis thaliana</i>; BnAGL24 from <i>Brassica napus</i>; BjSVP from <i>Brassica juncea</i>; INCOMPOSITA from <i>Antirrhinum majus</i>; IbMADS3 and IbMADS4 from <i>Ipomoea batatas</i>; JOINTLESS from <i>Solanum lycopersicum</i>; MPF2 from <i>Physalis pubescens</i>; MPP3 from <i>Physalis peruviana</i>; OsMADS22, OsMADS47, OsMADS55 from <i>Oryza sativa</i>; PtMADS1 from <i>Populus tomentosa</i>; RcMADS1 from <i>Rafflesia cantleyi</i>; StMADS16 and StMADS11 from <i>Solanum tuberosum</i>; TaVRT2 from <i>Triticum aestivum</i>; ZmMADS22 and ZmMADS26 from <i>Zea mays</i>. The numbers next to the nodes are bootstrap percentages. The scale bars denote a divergence of 0.2 amino acid substitutions per site.</p

Complementation analysis of <i>agl24-1</i> mutant by <i>RcMADS1</i> expression driven by two different promoters.

<p>(A) Functional complementation of <i>agl24-1</i> by <i>35S::RcMADS1</i>. WT (left), <i>agl24-1</i> mutant (middle) and <i>agl24-1+35S::RcMADS1</i> (right). (B) The flowering time of plants in (A) represented by rosette leaf numbers at bolting (mean ± s.d.). (C) Functional complementation of <i>agl24-1</i> by <i>AGL24::RcMADS1</i>. WT (left), <i>agl24-1</i> mutant (middle) and <i>agl24-1+ AGL24::RcMADS1</i> (right). (D) The flowering time of plants in (C) represented by rosette leaf numbers at bolting (mean ± s.d.). Asterisks indicate that means are significantly different (P≤0.05) according to Student’s <i>t</i>-Test. Scale bars = 1cm.</p

<i>Rafflesia cantleyi</i> Solms-Laubach buds and flower.

<p>(A) Bud of approximately 4 cm developing on a <i>Tetrastigma</i> sp. vine in the forest in Pulau Tioman, Malaysia. (B) A longitudinal section of an older bud approximately 8 cm in diameter. (C) An open flower of <i>Rafflesia</i> sp. (approximately 1 m in diameter). Scale bars A = 2 cm, B = 1 cm and C = 15 cm.</p