The relationships between PnLRR-RLK protein kinases with other eukaryotic genomes.

<p>The kinase domain amino acid sequences of PnLRR-RLKs genes and LRR-RLK kinases family representatives from <i>Arabidopsis</i>, <i>Oryza sativa</i> and human were used for generating the neighboring-joining tree with 1,000 bootstrap replicates. The PnLRR-RLKs family forms a close relationship to <i>Arabidopsis</i>, <i>Oryza sativa</i> kinases and <i>Homo sapiens</i> receptor Tyr kinase HsMLK1.</p

PnLRR-RLK27 contributes to the salt tolerance in transgenic <i>Physcomitrella patens</i> and <i>Arabidopsis</i>.

<p>(A) RT-PCR analysis revealed that the <i>PnLRR-RLK27</i> was successfully transcribed in <i>Physcomitrella patens</i>. (B) The size of transgenic <i>Physcomitrella patens</i> gametophyte plants was significantly larger than that of the wild type under salt stress conditions (4-week-old plants). (C) Statistical analysis of gametophyte size as shown in (B). (D) RT-PCR analysis revealed that the <i>PnLRR-RLK27</i> was successfully transcribed in <i>Arabidopsis</i>. <i>Tubulin</i> gene was used as internal references. (E) The seed germination rate of transgenic seedlings was significantly higher than that of the wild type under salt stress conditions (6 days' seed germination). (F) Statistical analysis of seed germination rates as shown in (E). Seed germination rates were calculated by counting the proportion of the WT plants and the transgenic seedlings bearing open green cotyledons. (G) PnLRR-RLK27 promotes the growth of <i>Arabidopsis</i> seedling after salinity treatment. (H) Measurement of root length of salinity-stressed <i>Arabidopsis</i> seedlings shown in (G). Vertical bars are presented as means ±SE, and asterisks (*) indicate significant differences of means between the transgenic lines and the WT plants at <i>P</i><0.05. <i>Bar</i> 10 mm.</p

Expression patterns of PnLRR-RLK27 in the Antarctic moss <i>Pohlia nutans</i> in response to different stress treatments analyzed by real-time PCR.

<p>(A) 200 mM NaCl treatment; (B) Cold treatment (4°C); (C) 20% PEG6000 treatment for simulated drought stress; (D) 50 μM abscisic acid (ABA) treatment. In each stress treatment, the different time point samples were all from the same plant pot cultures. Vertical bars indicate mean ±SE of three replicates of the sample. Asterisks (*) indicate statistically significant differences with the control group at <i>P</i><0.05.</p

Sequence alignment and subcellular localization of PnLRR-RLK27 in <i>Physcomitrella patens</i>.

<p>(A) Amino acid sequence alignment between PnLRR-RLK27 and other LRR-RLK family members <i>Physcomitrella patens</i> subsp. <i>patens</i> (PpSERK2), <i>Arabidopsis thaliana</i> (AtSERK2), <i>Oryza sativa</i> (OsSERK1), <i>Zea mays</i> (ZmSERK3). Multiple sequence alignments were conducted using ClustalW. Black boxes show identical amino acid residues, and gray shades show similar residues. Deletions are indicated by dashes to allow maximum alignment. (B and C) The localization of p35S:PnLRR-RLK27:GFP in <i>Physcomitrella patens</i> and <i>Arabidopsis</i> protoplasts. (D and F) The localization of p35S:PnLRR-RLK27:GFP in <i>Physcomitrella patens</i> and <i>Arabidopsis</i> protoplasts in the presence of FM4-64. (E and G) The colocalization of p35S:PnLRR-RLK27:GFP and H⁺-ATPase:RFP in <i>Physcomitrella patens</i> and <i>Arabidopsis</i> protoplasts. (B to G I and V) Green fluorescence of PnLRR-RLK27-GFP fusion protein or GFP protein; (B to C II and VI) Red autofluorescence of chloroplasts. (D to F II and VI) Red autofluorescence of plasma membrane in the presence of FM4-64. (E to G II and VI) Red autofluorescence of H⁺-ATPase:RFP fusion protein. (B to G III and VII) Merged image of green fluorescence, bright field, and red autofluorescence. (B to C IV and X) The protoplast in bright field. <i>Bar</i> 10 μm.</p

PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss <i>Pohlia nutans</i>, positively regulates salinity and oxidation-stress tolerance

<div><p>Leucine-rich repeats receptor-like kinases (LRR-RLKs) play important roles in plant growth and development as well as stress responses. Here, 56 LRR-RLK genes were identified in the Antarctic moss <i>Pohlia nutans</i> transcriptome, which were further classified into 11 subgroups based on their extracellular domain. Of them, PnLRR-RLK27 belongs to the LRR II subgroup and its expression was significantly induced by abiotic stresses. Subcellular localization analysis showed that PnLRR-RLK27 was a plasma membrane protein. The overexpression of PnLRR-RLK27 in <i>Physcomitrella</i> significantly enhanced the salinity and ABA tolerance in their gametophyte growth. Similarly, PnLRR-RLK27 heterologous expression in <i>Arabidopsis</i> increased the salinity and ABA tolerance in their seed germination and early root growth as well as the tolerance to oxidative stress. PnLRR-RLK27 overproduction in these transgenic plants increased the expression of salt stress/ABA-related genes. Furthermore, PnLRR-RLK27 increased the activities of reactive oxygen species (ROS) scavengers and reduced the levels of malondialdehyde (MDA) and ROS. Taken together, these results suggested that PnLRR-RLK27 as a signaling regulator confer abiotic stress response associated with the regulation of the stress- and ABA-mediated signaling network.</p></div

PnLRR-RLK27 enhances the oxidative tolerance and salt tolerance by promoting ROS-scavenging capacity and the expression levels of antioxidative-responsive genes in transgenic <i>Arabidopsis</i>.

<p>(A) The main root of transgenic lines were significantly longer than that of the control plants after 0.5 μM and 1.0 μM H₂O₂ treatment (10 days' seed germination). (B) Statistical analysis of the transgenic <i>Arabidopsis</i> seedling root length shown in (A). (C) Statistical analysis of the transgenic <i>Arabidopsis</i> seedling lateral root numbers shown in (A). (D) H₂O₂ levels of <i>Arabidopsis</i> by DAB staining in 4-week-old <i>Arabidopsis</i> plants after 200 mM NaCl treatment for 24 h. (E)and (F) POD and SOD activities in 4 week-old <i>Arabidopsis</i> plants after 200 mM NaCl treatment for 24 h. (G) the MDA levels in plants in 4-week-old <i>Arabidopsis</i> plants after 200 mM NaCl treatment for 24 h. (H) The content of proline in 2-week-old <i>Arabidopsis</i> seedlings after 200 mM NaCl treatment for 2 h. (I, J, K and L) The expression levels of antioxidative-responsive genes in 2-week-old <i>Arabidopsis</i> seedlings after 200 mM NaCl treatment for 2 h. Data are presented as means ±SE, and asterisks (*) indicate significant differences of means between the transgenic lines and the WT plants (<i>P</i><0.05). <i>Bar</i> 10 mm.</p

The stress-responsive genes expression pattern in PnLRR-RLK27 transgenic <i>Arabidopsis</i> and <i>Physcomitrella patens</i>.

<p>The expression levels of several abiotic stress/ABA-related genes were measured by qPCR analysis using the SYBR Green master mix (DBI). The <i>Arabidopsis</i> actin gene and <i>Physcomitrella patens</i> tubulin gene were served as normalization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172869#pone.0172869.s001" target="_blank">S1 Table</a>). Gene expression was analyzed using comparative Ct (2^-ΔΔCt) method. The reactions were performed in triplicate.</p

PnLRR-RLK27 reduces the ABA sensitivity in transgenic <i>Physcomitrella patens</i> and <i>Arabidopsis</i>.

<p>(A) The size of transgenic <i>Physcomitrella patens</i> gametophyte plants was significantly larger than that of the wild type after ABA treatment (4 week-old plants). (B) Statistical analysis of gametophyte size as shown in (A). (C) Seed germination of transgenic lines were significantly higher than that of the wild type under different concentrations of ABA (4 days' seed germination). (D) Statistical analysis of the transgenic <i>Arabidopsis</i> seedling greening shown in (C). (E) PnLRR-RLK27 promotes the growth of <i>Arabidopsis</i> seedling after ABA treatment. (F) Statistical analysis of the root length in transgenic <i>Arabidopsis</i> shown in (E). Vertical bars are means ±SE, and asterisks (*) indicate significant differences of means between the transgenic lines and the WT plants at <i>P</i><0.05. <i>Bar</i> 10 mm.</p