Target assignment to the chloroplast genome.

<p>Six PPR proteins were used for target assignment against the <i>Arabidopsis</i> chloroplast genome (154,478 bp) with the PPR code for 3 NSRs (residues 1, 4, and ii) extracted from 327 PPR motifs in <i>Arabidopsis</i> (At; Table S3), or from 464 PPR motifs in <i>Arabidopsis</i> and <i>Physcomitrella patens</i> (At + Pp; Table S4). The assignment was also performed with the PPR code excluding residue 1 (only residues 4 and ii; 2 NSRs and 1 NSR of “At + Pp” code).</p

Computational target assignment for PPR proteins.

<p>The targets for <i>Physcomitrella patens</i> PPR proteins (PpPPR_56, 71, 77, 78, 79, and 91) were computationally assigned against 13 editing sites in <i>Physcomitrella patens</i>, using a probability matrix (Table S3) and the FIMO program. Diamonds represent <i>P</i>-values indicating a matching score for the respective editing site. Correctly identified editing sites are highlighted in red. The analysis was also performed for an uncharacterized <i>Arabidopsis</i> PPR protein, AHG11, against 530 editing sites in <i>Arabidopsis</i> mitochondria and chloroplasts.</p

Elucidation of the RNA Recognition Code for Pentatricopeptide Repeat Proteins Involved in Organelle RNA Editing in Plants

<div><p>Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA-binding proteins that are commonly found in plants. Organelle transcript processing and stability are mediated by PPR proteins in a gene-specific manner through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. However, the sequence-specific RNA recognition mechanism of the PPR protein remains largely unknown. Here, we show the principle underlying RNA recognition for PPR proteins involved in RNA editing. The distance between the PPR-RNA alignment and the editable C was shown to be conserved. Amino acid variation at 3 particular positions within the motif determined recognition of a specific RNA in a programmable manner, with a 1-motif to 1-nucleotide correspondence, with no gap sequence. Data from the decoded nucleotide frequencies for these 3 amino acids were used to assign accurate interacting sites to several PPR proteins for RNA editing and to predict the target site for an uncharacterized PPR protein.</p> </div

Principles underlying PPR-RNA recognition.

<p>(<b>A</b>) Consensus amino acids for the PPR motif. The sequence logo was derived from 5668 individual PPR motifs (PROSITE accession no. PS51375). Residues 1, 4, and “ii” (position -2) were determined to be nucleotide-specifying residues (NSRs; asterisks). Previously identified residues for RNA recognition (6 and 1′, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057286#pone.0057286-Barkan1" target="_blank">[20]</a>) are also shown according to the PROSITE model. The last PPR motif was located 4 nucleotides before the editable C residue (Aln4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057286#pone-0057286-g001" target="_blank">Figure 1</a>). (<b>B</b>) NSR-deduced nucleotide frequencies. Nucleotide frequencies, determined according to the NSRs, are displayed in a logo (<i>left panel</i>). * indicates any amino acid; “n” indicates the occurrence frequencies of the NSRs in 327 <i>Arabidopsis</i> PRR-motifs. The nucleotide-specifying capacity of a residue was deduced from the low variability in the association between the individual amino acids and nucleotides and is presented as a <i>P</i>-value (<i>right panel</i>). The analysis was conducted for specific nucleotides (i.e., A, U, G, or C), purine/pyrimidine (R or Y), presence or absence of hydrogen bond groups (W or S), and presence or absence of amino/keto (M or K) groups, in alignment 4 (Aln4). (<b>C</b>) Example for deduced nucleotide frequencies by various combinations of NSRs.</p

Strategy for screening nucleotide specifying residues (NSRs) for the PPR motif.

<p>Potential NSRs were computationally surveyed by estimating the low variability in the association between amino acids in PPR motifs and nucleotides surrounding editing sites. (<b>A</b>) The RNA-editing PPR protein contains tandem repeats of the PPR motif (box) and an E motif (diamond) at the C-terminus. The PPR motifs of the editing-type PPR protein were aligned with the corresponding target RNA sequence in various positions, using a 1-motif to 1-nucleotide correspondence, in a contiguous linear manner. Alignment 1 (Aln1) was registered by fitting the last PPR motif of the protein to 1 nucleotide upstream of the editable cytosine residue (shown in blue). The nucleotide sequence was then moved toward the right, 1 nucleotide at a time, for Aln2–6. The association between amino acids in the PPR motif and the corresponding nucleotides was determined for each alignment. (<b>B</b>) Expected results for the statistical analysis. If the amino acids at particular positions are responsible for RNA recognition (e.g., Ser is observed at the residue 4 in the second, sixth, and ninth motifs), reduced variability should be observed between the type of amino acid and the specific nucleotide in a particular alignment (<i>left panel</i>; Aln4); if not, high variability would be expected (<i>right panel</i>; Aln1).</p

Flowchart for computational target assignment for PPR proteins.

<p>The analysis was initiated from the established PPR motif model of a protein of interest, PpPPR_71 for example. Information regarding the nucleotide-specifying residues (NSRs; residues 1, 4, and “ii”) was extracted from the PPR motifs and converted into a probability matrix that indicated the decoded nucleotide frequency for each of the 3 NSRs (residues 1, 4, and ii; listed in Table S3). If a PPR motif did not coincide with all 3 NSRs, it was converted into a matrix for 2 NSRs (residues 4 and ii). The single NSR (residue 4) was used for any remaining PPR motifs. In parallel, target RNA sequences for RNA editing were prepared, corresponding to alignment 4 (Aln4; the sequence upstream from the -4 nucleotide for the editable cytosine residue; highlighted in red). The target sequence for RNA editing (ccmFCeU122SF) by the PpPPR_71 protein is shown as an example. The PPR decoded nucleotide frequency matrix for the protein and nucleotide sequence were analyzed by the FIMO program, which calculated a <i>P</i>-value as a measure of the pattern matching score between the PPR protein and the nucleotide sequence.</p

Application of Trehalose Mitigates Short-Styled Flowers in Solanaceous Crops

Trehalose is a disaccharide and is often foliar applied by farmers aiming at increasing stress resistance or crop production. However, the physiological effect of exogenously applied trehalose on crops remains obscure. Here, we explored the effect of foliar trehalose application on style length of solanaceous crops, Solanum melongena and S. lycopersicum. Trehalose application promotes pistil to stamen ratio by gaining style length. Another disaccharide consisting of two glucose molecules, maltose, showed the same effect on style length of S. lycopersicum, while monosaccharide glucose did not. Trehalose is found to affect style length through uptake via roots or interaction with rhizosphere but not through absorption by shoots in S. lycopersicum. Our study suggests that yield improvement of solanaceous crops by trehalose application under stressed conditions is brought about by suppression of the occurrence of short-styled flowers. This study suggests that trehalose holds potential to act as a plant biostimulant in preventing short-styled flowers in solanaceous crops

Additional file 1: of Disruption of ureide degradation affects plant growth and development during and after transition from vegetative to reproductive stages

Figure S1. Three-week-old aah mutants grown on gellan gum medium. Two WAG seedlings grown on half-strength Murashige and Skoog medium containing 0.3% gellan gum were carefully removed to avoid damaging roots, transplanted to new medium and grown for an additional week. (PDF 1951 kb

Ultrastructure of dying cells in forceps minor of corpus callosum and ventromedial prefrontal cortex of the male pups at 24 h after delivery.

(A–D) Electron micrographs of forceps minor (FMI) of the male pups at 24 h after delivery of the OXT (A–C) and PBS (D) groups. (A) In the OXT group, cells containing pyknotic nuclei and debris of dying cells were abundant (asterisks). (B) An enlarged image of the phagocytic cell (P) shown in A containing many pyknotic nuclei and debris of dying cells. (C) A dying cell with chromatin condensation observed in the OXT group. (D) An enlarged image of the phagocytic cell (P) containing pyknotic nuclei and debris of dying cells in the PBS group. (E–G) Electron micrographs of ventromedial prefrontal cortex (vmPFC) of the male pups at 24 h after delivery of the OXT group. Dying cells with pyknotic nuclei indicated by an arrow and an arrowhead in E were enlarged and shown in F and G, respectively. (F) A dying cell with pyknotic nuclei that was not phagocytosed (arrow). (G) A dying cell with pyknotic nuclei that was encircled by the cytoplasm of the phagocytic cell (arrow). Scale bars: 4 μm (A–E) and 2 μm (F, G).</p

Additional file 8: of Disruption of ureide degradation affects plant growth and development during and after transition from vegetative to reproductive stages

Figure S7. Growth of representative ups1 and ups2 mutants. Plants were grown at 23 °C in soil for 7 weeks under long-day conditions. (PDF 257 kb