Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam.

Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1

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Scatter plots for stomatal regulation in <i>crk</i> mutants.

<p><b>(A)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to ABA (x-axis) and flagellin (flg22; y-axis). <b>(B)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to ABA (x-axis) and chitin (y-axis). <b>(C)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to chitin (x-axis) and flg22 (y-axis). Black dashed lines indicate the cut-off for reduced, normal, and high response with respect to Col-0. Grey dashed lines show regression fit with correlation (R), coefficient of determination (R²) and significance reported in lower right corner in each plot. Reduced or increased responses were statistically significant in the majority of mutants (see respective barplots in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s015" target="_blank">S15A</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s023" target="_blank">S23</a> Figs).</p

Phenotypic analysis of the <i>Arabidopsis thaliana</i> CRK protein family.

<p>A T-DNA insertion collection for the CRK family was compiled and subjected to phenotyping addressing aspects of plant development, biotic and abiotic stress responses, photosynthesis as well as stomatal regulation. Length of red and blue bars in the five phenotyping sections is representative of the number of <i>crk</i> lines found to have phenotypes in the thematic area. Information about the sections in the pie chart is displayed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>. The red outline in the pie chart highlights the lines included in the analyses and figures throughout the manuscript. The gray scale bar serves as a reference for comparison. The length of the scale bar corresponds to ten lines.</p

Phylogenetic clustering of the <i>Arabidopsis thaliana</i> CRK group of RLKs and summary of the <i>crk</i> T-DNA insertion collection.

<p><b>(A)</b> The coding region of the CRKs of <i>Arabidopsis thaliana</i> (including the truncated <i>CRK9 At4g23170</i> and the putative pseudogene <i>CRK35 At4g11500</i>) was aligned using Muscle. The maximum-likelihood phylogenetic tree was estimated in MEGA6 using all sites (no gap penalty). The initial guide tree was constructed using maximum parsimony. Values at branch nodes represent bootstrap values (1000 replicates). CRK43 (At1g70740), CRK44 (At4g00960) and CRK45 (At4g11890) lack signal peptide, CRK ectodomain (ED) and transmembrane domain. <b>(B)</b> Information on T-DNA insertion lines for corresponding <i>crk</i> mutants is summarized: location of the T-DNA insertion in the gene (detailed information in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s003" target="_blank">S3 Fig</a>), number of T-DNA insertions per line (determined by quantitative PCR; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>) and transcript level of the corresponding <i>crk</i> mutant (according to semi-quantitative RT-PCR and qPCR; detailed information in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>). For two additional <i>crk10</i> alleles (<i>crk10-1</i> and <i>crk10-3</i>) information can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>.</p

Scatter plots of stomatal regulation in <i>crk</i> mutants.

<p>The <i>crk5</i> was insensitive to all studied stimuli, whereas <i>crk31</i> was particularly insensitive to O₃. The lines <i>crk19-1</i> and <i>crk22</i> were more sensitive to the analysed stimuli. <b>(A)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to O₃ (x-axis) and CO₂ (y-axis). (<b>B</b>) Scatter plot of stomatal responses of <i>crk</i> mutants to O₃ (x-axis) and darkness (y-axis) (<b>C</b>) Scatter plot of stomatal responses of <i>crk</i> mutants to CO₂ (x-axis) and darkness (y-axis). Dashed lines indicate the cut-off for reduced, normal, and high response with respect to Col-0. Grey dashed lines show regression fit with correlation (R), coefficient of determination (R²) and significance reported in lower right corner in each plot. Reduced or increased responses were statistically significant in the majority of mutants (see respective barplots in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s017" target="_blank">S17</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s018" target="_blank">S18</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s019" target="_blank">S19</a> Figs).</p

Plant development is affected in several <i>crk</i> mutants.

<p><b>(A)</b> Representative pictures of 17-day old seedlings of Col-0 wild type and <i>crk2</i>. Complementation of <i>crk2</i> with 35S::<i>CRK2-CDS</i>:YFP rescued the growth defect of the mutant. Plants were grown under the following conditions: 250 μmol m^-2 s^-1 light intensity under 12 h-day length (day: 23°C, 70% relative humidity; night: 18°C, 90% relative humidity). Bar = 1 cm. Pictures are representative of three independent experiments. <b>(B)</b> A selection of <i>crk</i> mutant lines showing earlier senescence compared to Col-0 wild type. Results are means ± SE (<i>n</i> = 8). <b>(C)</b> Several <i>crk</i> mutants flowered earlier compared to wild type while <i>crk2</i> flowered later. Results are means ± SE (<i>n</i> = 8). <b>(D)</b> Time course analysis of endosperm rupture showed delayed germination in several <i>crk</i> mutants compared to wild type. Results represent means from three independent biological experiments (<i>n</i> = 30). Testa and endosperm rupture were assessed every 5 hours up to 51 hours of imbibition. A seed was considered as germinated when the radicle protruded through both envelopes. <b>(E)</b> Several <i>crk</i> mutants exhibit a lower pavement cell density (number of pavement cells / mm²) in cotyledons. Results are means ± SE (<i>n</i> = 15). <b>(F)</b> Three <i>crks</i> showed slightly longer roots compared to wild type (measured eight days after stratification). Results are means ± SE (<i>n</i> = 16). (<b>B-F)</b> Differences between mutants and Col-0 wild type were compared and analysed using one-way-ANOVA (<i>post hoc</i> Dunnett, asterisks indicate statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) for <b>(B, C, E</b>) and linear model with single step p-value adjustment (<b>F</b>). All experiments were repeated three times with similar results.</p

Stomatal development and responses are impaired in specific <i>crks</i>.

<p><b>(A)</b> A subset of the <i>crk</i> mutants showed altered water loss (shown as decrease of fresh weight) compared to Col-0 wild type plants after detachment of shoots from roots as evaluated from rosette weight. Complementation of the <i>crk2</i><b>(B)</b>, <i>crk5</i><b>(C)</b> or <i>crk45</i><b>(D)</b> mutants restored a wild type-like water loss phenotype as interpreted from decrease of fresh weight of excised rosettes. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results. <b>(E)</b> Stomatal apertures were measured 2 h after abscisic acid (ABA) treatment. Some <i>crk</i> mutants are impaired in stomatal closure 2 h after treatment with 10 μM ABA. Results are means of % stomatal aperture ratio (width/length) ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control and ABA treatment at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (linear model, single-step p-value adjustment). Lowercase letters indicate statistical significance between wild type Col-0 and <i>crk</i> mutant at <i>P</i><0.05 (a), <i>P</i><0.01 (b) and <i>P</i><0.001 (c) according to one-way ANOVA with <i>post hoc</i> Dunnett’s test. <b>(F)</b> Stomatal density (number of stomata/mm²) is correlated with stomatal length (μm). Most of the <i>crks</i> exhibit a smaller stomata density which correlates with longer stomata (Pearson correlation -0.69, p-value = 0.04). Results are means (average number of stomata measured = 500). <b>(G-I)</b> Time courses of stomatal conductance (relative units) in response to a 3 min pulse of 500–600 ppb of O₃ (<b>G</b>), darkness (<b>H</b>) and elevation of CO₂ from 400 ppm to 800 ppm <b>(I)</b> in a subset of <i>crk</i> mutants and Col-0. Stimuli were applied at 0 time point, which is indicated by an arrow; pre-treatment stomatal conductance was used for normalization. Graph shows the mean of two experiments (<i>n</i> = 6). (<b>J</b>) Overexpression of CRK5 led to lower stomatal conductance compared to Col-0 wild type. (<b>K</b>) Complementation of the <i>crk5</i> mutant restored wild type-like phenotype in the response to a 3-min pulse of 500–600 ppb of O₃, darkness, and elevating CO₂ from 400 to 800 ppm. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results.</p