Hypocotyl lengths and cross sections of samples tested with experimental setup 1 (horizontal test, water vapour).

<p>For each series n > 18 replicas were analyzed. Error bars depict standard deviation.</p

List of genes encoding the selected TFs.

<p>The genes in Table 1 were selected on the basis of co-expression in gene vicinity networks for <i>MYB46</i> and <i>CESA7</i>, <i>CESA8</i> and <i>CESA4</i>. Locus, common name, T-DNA insertion line used for functional studies, and description of structural domains or type of DNA binding domain found in each TF are indicated. T-DNA insertion mutants in the selected group of genes were further characterized in their response towards <i>B</i>. <i>cinerea</i> and <i>P</i>. <i>cucumerina</i>.</p><p>List of genes encoding the selected TFs.</p

Co-localization of PROVIR proteins in <i>N</i>. <i>benthamiana</i> leaves.

<p>Co-expression of PROVIR 8, 9, and 11 proteins tagged to GFP with respective PROVIR 7, 8, and 11 proteins tagged to mCherry allowed identification of co-localization sites for PROVIR factors along or at discrete plasma membrane domains. Bright field left panel: scale bars are 8 μm. Fluorescent panels: scale bars are 2 μm, which provides a magnified detail of the boxed sectors in the left panel.</p

Density and cellulose content of dark grown hypocotyls.

<p>A) Density is analyzed as dry weight per wet volume (seed batch 1, n > 14). B) Cellulose content measured as μg hexoses per cell wall material (CWM). * P < 0.05, ** P < 0.01, n.s.: no significant difference.</p

PROVIR1-to-13 overexpression confers enhanced disease susceptibility to <i>P</i>. <i>cucumerina</i> infection.

<p>(<b>A</b>) Western blots with anti-GFP antibodies of crude protein extracts derived from T3 homozygous Col-0 plants expressing either a 35S::GFP construct or the respective PROVIR1-to-13::GFP constructs. Two independent lines for each PROVIR::GFP construct were used and the accumulation of the encoded fusion protein was compared to that of free GFP. Equal protein loading was check by Ponceau staining of the nitrocellulose filter. (<b>B</b>) Resistance response to <i>P</i>. <i>cucumerina</i> of Col-0 and two independent homozygous lines expressing each of the respective PROVIR::GFP proteins shown in panel <b>A</b>. Disease was evaluated 11 d.p.i. by determining the average lesion diameter on three leaves per plant from 15 plants per genotype. Data points represent average lesion size ± SE of measurements. An ANOVA was conducted to assess significant differences in disease symptoms, with a priori <i>P</i> < 0.05 level of significance; significant differences are indicated with letters. (<b>C</b>) Accumulation levels of endogenous <i>PROVIR7</i>, <i>PROVIR9</i>, <i>PROVIR12</i>, and <i>GRP3</i> transcripts measured in comparatively healthy Col-0 plants (left bars) and in T-DNA <i>provir7</i>, <i>provir9</i>, <i>provir12-1</i>, <i>provir12-2</i>, and <i>grp3</i> T-DNA insertion mutants (right black bars). Data represent mean ± SD; n = 3 biological replicates. Expression was normalized to the constitutive <i>ACT2</i> gene, then to expression in Col-0 plants. (<b>D</b>) Resistance response to <i>P</i>. <i>cucumerina</i> of Col-0 and <i>provir7</i>, <i>provir9</i>, <i>provir12-1</i>, <i>provir12-2</i>, and <i>grp3</i> plants. Disease was evaluated as in <b>B</b>.</p

Characterization of <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i> and <i>at1g66810</i> mutants.

<p>(<b>A</b>) Oregon Green 488 dextran-derived fluorescence upon infiltration of the full expanded leaves with the apoplastic pH indicator. In comparison to Col-0, more intense fluorescence emission was observed in the four mutants, and was particularly intense in <i>pap2</i> plants, indicative of enhanced alkalinization of the apoplast in mutants compared to Col-0 plants. (<b>B</b>) Estimation of plasma membrane-anchored (GPI-AGP) and free AGP content in Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> insertion mutant plants using β-d-Glucosyl Yariv reagent. AGP content was calculated with respect to a regression curve obtained by a radial diffusion assay in agarose plates containing Yariv regent and increasing amounts of gum Arabic (calibrating curve on the left). (<b>C</b>) Scanning electron microscopy (SEM) of leaf epidermis in Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> mutants. Only <i>pap2</i> exhibited a slight increase in epidermal cell size which was variable among different leaves of different plants. (<b>D</b>) Histochemical detection of lignin in proximal stem sections of Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i> and <i>at1g66810</i> mutant stems. Stem sections were stained with phloroglucinol-HCl (red color) for lignin detection in the interfascicular fiber walls and xylem cells, as observed with light microscopy. if, Interfascicular fibers; pi, Pith parenchyma; x, Xylem. (<b>E-F</b>) Cotyledon vein patterns were altered in <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> insertion mutants. <b>E</b>, magnified pictures showing pattern defects exhibited by some mutants (e.g., 2 loops; right picture) compared to the most common 4 loop pattern observed in Col-0 (left picture). <b>F</b>, columns 2–7: number of cotyledons displaying the venation pattern depicted at the top, which ranged from the most common 4 loops observed in Col-0 to the less common phenotype of 2+1 or 2 loops, but prevalent in the <i>pap2</i>, <i>bhlh99</i>, or <i>zfp2</i> mutants. “n” = total number of scored cotyledons.</p

Age Effects on Hypocotyl Mechanics - Fig 6

<p>Comparison of the results of the micromechanical tests with samples tested submerged in an osmotic liquid (green, Setup 2) and in air, kept in the native state by a humidifier (blue, Setup 1) for tensile stiffness (A) and fracture stress (B). The change in diameter from the initial value before the tensing to the point of maximal stress in the submerged setup (C).</p

Clamping of hypocotyls in microtensile testing devices and schematic stress-strain curve.

<p>Clamping of hypocotyls in microtensile testing devices and schematic stress-strain curve.</p

Comparative transcriptome analysis in <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i> and <i>at1g66810</i> insertion mutants.

<p><i>(A-B)</i> Comparisons of gene expression in <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> insertion mutant lines depicted using Venn diagrams. Sets of genes were selected using criteria described in Material and Methods. The gene number in each mutant for each set is displayed within an ellipsoidal circle. Genes common in two, three, or four mutants are indicated in the intersection of circles, so that the sum of the numbers within a cycle for each mutant represents the total number of genes deregulated in each mutant. (<b>A</b>) Intersection of genes up-regulated in each mutant with those up-regulated in three other mutants. (<b>B</b>) Intersection of down-regulated genes in each mutant with those down-regulated in the three other mutants. (<b>C</b>) Heat map clustering of up-regulated genes commonly expressed in the four mutants. (<b>D</b>) Clustering of selected gene groups encoding other signaling peptides family members, which are different to those shown in C, but are present in at least one, two, or three mutants but not in the four mutants. The description of genes that fall into each cluster is indicated on the right. Provisionally, some genes annotated as unknown have been coined as <i>PROVIR1</i> to <i>PROVIR13</i>.</p

Localization of different PROVIR-GFP proteins in <i>N</i>. <i>benthamiana</i> leaves and protein co-localization with the plasma membrane marker protein PIP-mCherry by confocal microscopy.

<p>Expression of PROVIR1-to-13-GFP (72 h.p.i.) resulted in distinct pericellular fluorescence patterns distributed either linearly along the plasma membrane or forming punctuated foci resembling small membrane clusters. Co-expression of PROVIR-GFP with the plasma membrane marker PIP-mCherry facilitated tracing the plasma membrane. Scale bars are 8 μm, except for the right longitudinal panel where scale bars are 2 μm. This provides a magnified detail of the boxed sector in the intermediate panel merging GFP and mCherry-derived fluorescence.</p