13 research outputs found

    CpLEPA Is Critical for Chloroplast Protein Synthesis Under Suboptimal Conditions in <em>Arabidopsis thaliana</em>

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    <div><p>LEPA is one of the most conserved translation factors and is found from bacteria to higher plants. However, the physiological function of the chloroplast LEPA homolog in higher plants remains unknown. Herein, we demonstrate the physiological role of cpLEPA in enabling efficient photosynthesis in higher plants. The <em>cplepa-1</em> mutant displays slightly high chlorophyll fluorescence and pale green phenotypes under normal growth conditions. The growth of the <em>cplepa-1</em> mutant is reduced when grown on soil, and greater reduction is observed under intense light illumination. Photosynthetic activity is impaired in the <em>cplepa-1</em> mutants, which is reflected in the decreased steady-state levels of chloroplast proteins. <em>In vivo</em> protein labeling experiments explained the decrease in the steady-state levels of chloroplast proteins. An abnormal association of the chloroplast-encoded mRNAs with ribosomes suggests that the protein synthesis deficiencies in <em>cplepa-1</em> are due to defects in translation initiation in the chloroplasts. The cpLEPA protein appears to be an essential translation factor that promotes the efficiency of chloroplast protein synthesis.</p> </div

    Immunolocalization and Expression of <i>cpLEPA</i>.

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    <p>A: Immunolocalization analysis of cpLEPA. The chloroplast, thylakoid, stroma and envelope fractions were subjected to immunoblot analysis with specific antisera against cpLEPA. Equal amounts of protein (20 µg) were loaded in each lane. The lanes marked <i>cplepa-1</i>, <i>cplepa-2</i> and <i>cplepa-1/</i>35s::cpLEPA were loaded with equal amounts of total protein (20 µg). B: Salt washing of the membranes. The thylakoid membranes were incubated with 250 mM NaCl, 200 mM Na<sub>2</sub>CO<sub>3</sub>, 1 M CaCl<sub>2</sub> and 6 M urea for 30 min at 4°C. Then, the thylakoid proteins were separated by SDS-PAGE and immunoblotted with anti-LEPA, anti-RbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and anti-CP47 antibodies. RbcL and CP47 were used as markers. Thylakoid membrane preparations that had not been subjected to treatment were used as controls. C: Expression patterns of <i>cpLEPA.</i> Upper panel: <i>cpLEPA</i> expression levels in different organs of <i>Arabidopsis</i>, as determined by RT-PCR analysis. RNA samples isolated from seedlings, rosettes, flowers, roots, petiole, cauline tissue and siliques of wild-type plants were reverse-transcribed and subjected to PCR analysis. Middle panel: Transcript levels of <i>cpLEPA</i> in <i>Arabidopsis</i> leaves at 5, 15, 25, 35 and 45 d. Bottom panel: Light-induced accumulation of <i>cpLEPA</i> transcripts. Three-week-old plants grown under medium light (120 µmol m<sup>−2</sup> s<sup>−1</sup>), low light (40 µmol m<sup>−2</sup> s<sup>−1</sup>) or high light (500 µmol m<sup>−2</sup> s<sup>−1</sup>) were used. <i>ACTIN</i> is shown as a control.</p

    Identification and Phenotyping of the <i>cplepa</i> Mutants.

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    <p>A: T-DNA insertion sites in the <i>cpLEPA</i> gene. Exons are indicated by black boxes, introns by lines, and the T-DNA insertions by vertical arrows. The horizontal arrows illustrate the primers used for T-DNA insertion verification and RT-PCR. The scale bar indicates 500 bp. B: RT-PCR analysis. RT-PCR was performed using specific primers for <i>cpLEPA</i> or <i>ACTIN</i>. C: Two-week-old WT and <i>cplepa-1</i> mutants grown on MS medium supplied with 0, 1% sucrose and 2% sucrose. D: Complementation of the <i>cplepa-1</i> mutant. The cDNA of the <i>cpLEPA</i> gene was cloned into a binary plant transformation vector and used for complementation of the <i>cplepa-1</i> mutant (<i>cplepa-1</i>/35S::<i>cpLEPA</i>). Four-week-old WT, <i>cplepa-1, cplepa-2</i> and <i>cplepa-1</i>/35S::<i>cpLEPA</i> plants were grown on soil. Fluorescence was measured with a CF Imager and visualized using a pseudocolor index, as indicated at the bottom, <i>Fm</i> and <i>Fv/Fm</i> value were presented. E: Growth of wild-type and <i>cplepa-1</i> mutant plants on soil at 120 µmol m<sup>−2</sup> s<sup>−1</sup>. The values shown are averages ± s.e. (n = 6).</p

    Photosensitivity Analysis of <i>cplepa-1</i> Plants.

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    <p>A: The phenotypes of wild-type (WT) and <i>cplepa-1</i> mutant plants grown in a growth chamber at 120 µmol m<sup>−2</sup> s<sup>−1</sup> in the first two weeks, then transferred to low light (40 µmol m<sup>−2</sup> s<sup>−1</sup>) or high light (500 µmol m<sup>−2</sup> s<sup>−1</sup>) for another two weeks. B: The <i>Fv/Fm</i> ratio was measured for detached leaves from wild-type (WT) plants (red circles) and <i>cplepa-1</i> mutant plants (black squares) following high-light illumination (1,000 µmol m<sup>−2</sup> s<sup>−1</sup>) in the absence of lincomycin (Lin). C: The <i>Fv/Fm</i> ratio was measured for detached leaves from wild-type (WT) plants (red circles) and <i>cplepa-1</i> mutant plants (black squares) following high-light illumination (1,000 µmol m<sup>−2</sup> s<sup>−1</sup>) in the presence of lincomycin (Lin).</p

    CpLEPA Protein Sequence Alignment.

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    <p>The amino acid sequence of cpLEPA was compared with the sequences of homologous proteins from mitochondria in <i>Arabidopsis</i>, <i>Oryza sativa, Glycine max, Physcomitrella patens</i>, <i>Hordeum vulgare, Micromonas pusilla, Synechococcus, Microcystis aeruginosa,</i> and <i>Bacillus cereus</i>. The black boxes indicate strictly conserved amino acids, and the gray boxes indicate closely related residues. The predicted chloroplast transmembrane peptides are underlined in green, The LEPA domains are underlined in red, and the LEPA-II domain is underlined in blue. LEPA-C is underlined in purple, and the CTD is underlined in yellow.</p

    Chlorophyll Contents in Wild-Type and <i>cplepa-1</i> Plants.

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    <p>Chlorophyll Contents in Wild-Type and <i>cplepa-1</i> Plants.</p

    Northern Blot Analysis for Chloroplast Transcripts in Wild-Type and <i>cplepa-1</i> Plants.

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    <p>Northern blot analysis of the chloroplast transcripts <i>psbA</i>, <i>psbB</i>, <i>psbD</i>, <i>atpB</i>, <i>psaA</i>, <i>petB</i>, <i>rbcL</i>, <i>rpoA</i>, <i>rpoB</i> and <i>rrn23</i> in wild-type and <i>cplepa-1</i> mutant plants. Each lane was loaded with 10 µg of total RNA. The plants were grown on soil for 3 weeks under 120 µmol m<sup>−2</sup> s<sup>−1</sup> illumination. Additionally, 25S rRNA stained with EtBr was loaded as a control. The size of the transcript (in kb) is shown.</p

    Accumulation and Synthesis of Chloroplast Proteins in <i>cplepa-1</i> Plants.

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    <p>A:Immunoblot analysis of total protein extracts from wild-type and <i>cplepa-1</i> plants. Wild-type and <i>cplepa-1</i> plants grown on soil at a photon flux density of 120 µmol m<sup>−2</sup> s<sup>−1</sup> were used. For wild-type and <i>cplepa-1</i> plants, 10 µg of total proteins were loaded. The antibodies used are indicated on the right. Actin served as a control to normalize the protein levels. Similar results were obtained in two additional independent experiments. B: Pulse labeling of thylakoid proteins. Primary leaves of 12-day-old plants were radiolabeled with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049746#pone.0049746-Sun1" target="_blank">[<sup>35</sup>]</a> S-methionine in the presence of cycloheximide for 20 min. The thylakoid membranes were isolated, separated by SDS-urea-PAGE and visualized autoradiographically, lanes were loaded with equal protein contend. C: A coomassie blue-stained gel is presented to show that equal amounts of proteins were loaded.</p

    Polysome Association Analysis for Chloroplast Transcripts in Wild-Type and <i>cplepa-1</i> Plants.

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    <p>The association of <i>psbA</i>, <i>psbB</i>, <i>atpB</i>, <i>psaA</i> and <i>rrn23</i> transcripts with polysomes. Total extracts from wild-type and <i>cplepa-1</i> leaves grown on soil for 3 weeks at 120 µmol m<sup>−2</sup> s<sup>−1</sup> were fractionated on 15%–55% sucrose gradients. Ten fractions of equal volume were collected from the top to the bottom of the sucrose gradients, and equal proportions of the RNA purified from each fraction were analyzed by northern-blot analysis. The rRNAs were detected by ethidium bromide (EtBr) staining. The size of the transcript (in kb) is shown.</p

    Screenshot of ASDCD web interface is shown.

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    <p>The number of individual drugs, drug target interactions, and synergistic drug combination, relevant history and the significance of synergistic drug combination, some basic information about ASDCD and an easy-to-use search tool are provided.</p
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