Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in Arabidopsis thaliana via the THESEUS1 Pathway

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion

Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in <i>Arabidopsis thaliana</i> via the THESEUS1 Pathway

<div><p>Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in <i>Arabidopsis thaliana</i>, with loss-of-function <i>atkin13a</i> mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. <i>AtKIN13A</i> is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, <i>AtKIN13A</i> interacts genetically with <i>ANGUSTIFOLIA</i>, encoding a regulator of Golgi dynamics. Reduced <i>AtKIN13A</i> activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the <i>THESEUS1</i>-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the <i>atkin13a</i> mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.</p></div

Petal phenotypes of <i>atkin13a</i> and <i>atkin13b</i> loss-of-function mutants.

<p>(A) Photographs of petals of the indicated genotypes. Scale bar is 1 mm. (B) Schematic representation of the <i>AtKIN13A</i> and <i>AtKIN13B</i> loci and position of mutant alleles. Open rectangles represent UTRs, filled rectangles show the protein-coding region, and thick connecting lines show introns. (C–I) Measurements of petal parameters for the indicated genotypes. Numbers in C and D indicate relative parameter values with respect to the corresponding wild-type values. Asterisk indicates significant difference from wild-type at p<0.05 (with Bonferroni correction for comparisons to Col-0). (C) Petal size. (D) Petal-cell size. (E) Petal length. (F) Petal cell number in the longitudinal direction. (G) Petal width. (H) Petal cell number along the petal width. (I) Petal index, i.e. length divided by width. (J) Gel prints (top) and representative cells (bottom) from petals of the indicated genotypes. Scale bars are 1 mm (top panel) and 100 µm (bottom panel). Values are mean + SD of 12 petals (C,E–I) or of 50 petal cells each from more than 10 petals (D).</p

Increased postmitotic cell expansion in <i>atkin13a</i> mutants.

<p>(A) Developmental series of petal size for the indicated genotypes. Flower 1 represents the youngest open flower, while flower −1 is the oldest unopened flower bud. Values are normalized to the size of the wild-type petals from the oldest measured flowers. Values represent mean ± SEM (n<10 petals). (B) Gel-print images of wild-type petal cells from the indicated flower stages (after <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004627#pgen.1004627-Smyth1" target="_blank">[71]</a>). (C) Petal-cell size from stage 10–11 buds is not different between wild type and <i>atkin13a-3</i> mutants. Values are mean ± SD of 50 petal cells each from more than 6 petals. (D) Etiolated hypocotyls are longer in <i>atkin13a-3</i> mutants than in wild type. Values are mean ±SD of 10 plants. 8-day old seedlings were measured. (E) Toluidine-blue stained cross section through a mature wild-type (left) and a mature mutant petal (right). Adaxial side is to the right in both images. (F) Cross-sectional cell area, cell height and cell width from petals of wild type and <i>atkin13a-3^EMS</i> mutants. Values are normalized to wild-type values and represent mean ± SD of 50 petal cells. (G) Thickness of petal-cell walls as measured from scanning-electron micrographs. Values are mean ± SEM of 100 petal cells from 10 petals, normalized to the wild-type value. (H) Scanning-electron micrographs of wild-type petals before (top left) and after (top right and bottom) freeze fracturing. Bottom image also indicates how measurements of cell-wall thickness were taken (pairs of white crosses). Length of scale bars is indicated. (I) Petal dry weight of the indicated genotypes. Values are mean + SD from 3 replicates of 50 petals each, normalized to the respective wild-type values. Differences between the three <i>atkin13a</i> mutant alleles are not statistically significant (n.s.). Asterisk indicates significant difference from wild-type at p<0.05 (with Bonferroni correction where appropriate).</p

Redundant function of <i>AtKIN13A</i> and <i>AtKIN13B</i>.

<p>(A,B) Lower-magnification (A) and higher-magnification view of opened siliques of an <i>atkin13a-3 +/atkin13a-3 atkin13b-1</i> plant to show seed abortion (arrowheads in (A)) and unfertilized ovules (arrows in (B)). (C) Quantification of seed abortion. Values are based on more than 100 seeds per genotype. (D) Whole-plant photographs of the indicated genotypes after EtOH-induction before (early) and during bolting (late). (E) Overlays of light and CFP fluorescence micrographs of flowers from the indicated genotypes after EtOH-induction. CFP fluorescence indicates expression of the amiRNA transgene. The two images on the right show a single flower and an overview of an inflorescence from an <i>amiKIN13B</i>-expressing <i>atkin13a-3</i> mutant plant. (F) Petal sizes of indicated genotypes. Values are mean ±SD of 12 petals from more than 8 plants. (G) Petal-cell sizes of the indicted genotypes. Values are mean ± SD of 50 petal cells from more than 16 petals. (H) Relative expression of <i>AtKIN13A</i> (left) and <i>AtKIN13B</i> (right) in plants of the indicated genotypes after EtOH induction. Values are mean ± SD of three technical replicates. A biological replicate experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004627#pgen.1004627.s008" target="_blank">Fig. S8D</a>. Asterisk indicates significant difference from wild-type at p<0.05 (with Bonferroni correction).</p

Monosaccharide composition of cell walls from 20-days old leaves.

<p>Alcohol-insoluble residues (AIR) were prepared from 20-days old leaves of the indicated genotypes. The results are given as average (µg/mg of AIR) of three independent biological replicates (±SE). Numbers in bold indicate significant differences between the respective mutant and Col-0 wild type (Student's t-test, p<0.05).</p><p>Monosaccharide composition of cell walls from 20-days old leaves.</p

Genetic interaction between <i>an</i> and <i>atkin13A</i>.

<p>(A) Photographs of inflorescences of the indicated genotypes. As the <i>atkin13a-3</i> mutant is from the L<i>er</i> background and <i>an-1</i> from the Col-0 background, double and single mutants were selected either with (top row) or without functional <i>ERECTA</i> (bottom row). Scale bar is 5 mm. (B) Photographs of rosettes Scale bar is 10 mm. (C) Photographs of leaf trichomes of the indicated genotypes. (D–J) Measurements of petal parameters for the indicated genotypes. (D) Petal size. (E) Petal length. (F) Petal width. (G) Petal index, i.e. length divided by width. (H) Petal-cell size. (I) Petal cell number in the longitudinal direction. (J) Petal cell number along the petal width. Values are mean ± SD of 12 petals from more than 8 plants (A–F,H,I) or of 50 petal cells from more than 8 petals (G). Asterisk indicates significant difference at p<0.05.</p

Excess cell expansion in <i>atkin13a</i> mutants requires <i>THE1</i> activity.

<p>(A,D–I) Measurements of petal parameters for the indicated genotypes. (A) Petal size. (B) Whole-flower photographs of the indicated genotypes. (C) Photographs of petals of the indicated genotypes. Scale bars are 1 mm in (B,C). (D) Petal-cell size. (E) Petal index, i.e. length divided by width. (F) Petal length. (G) Petal cell number in the longitudinal direction. (H) Petal width. (I) Petal cell number along the petal width. Values are mean ± SD of more than 12 petals from 8 plants (A,E–I) or of 50 petal cells from more than 8 petals (D). Asterisk indicates significant difference at p<0.05.</p