New cross talk between ROS, ABA and auxin controlling seed maturation and germination unraveled in APX6 deficient Arabidopsis seeds

<div><p>Successful execution of germination program greatly depends on the seeds’ oxidative homeostasis. We recently identified new roles for the H₂O₂-reducing enzyme ascorbate peroxidase 6 (APX6) in germination control and seeds’ stress tolerance. <i>APX6</i> replaces <i>APX1</i> as the dominant APX in dry seeds, and its loss-of-function results in reduced germination due to over accumulation of ROS and oxidative damage. Metabolic analyses in dry <i>apx6</i> seeds, revealed altered homeostasis of primary metabolites including accumulation of TCA cycle metabolites, ABA and auxin, supporting a novel role for APX6 in regulating cellular metabolism. Increased sensitivity of <i>apx6</i> mutants to ABA or IAA in germination assays indicated impaired perception of these signals. Relative suppression of <i>ABI3</i> and <i>ABI5</i> expression, and induction of <i>ABI4</i>, suggested the activation of a signaling route inhibiting germination in <i>apx6</i> seeds that is independent of ABI3. Here we provide additional evidence linking ABI4 with ABA- and auxin-controlled inhibition of germination and suggest a hypothetical model for the role of APX6 in the regulation of the crosstalk between these hormones and ROS.</p></div

Comparison of <i>de novo</i> assembly using Trinity and Velvet-oases programs.

*<p>Represents the number of contigs that at less 200 bp in length.</p>#<p>represents the result of TIGCL and Phrap for reduce the redundancy after Trinity with 25-mer assembly.</p

SNP statistical information based on mapping <i>C. frutescens</i> reads in reference to <i>C. annuum</i> contigs.

<p>SNP statistical information based on mapping <i>C. frutescens</i> reads in reference to <i>C. annuum</i> contigs.</p

The candidates on the relative capsaicinoids biosynthetic pathway.

<p>The blue and red bars represent new candidates and known candidates from Mazourek, 2009.</p

<em>De Novo</em> Transcriptome Assembly in Chili Pepper (<em>Capsicum frutescens</em>) to Identify Genes Involved in the Biosynthesis of Capsaicinoids

<div><p>The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (<em>Capsicum frutescens</em> L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for <em>de novo</em> assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (<em>C. annuum</em>) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in <em>C. frutescens</em> and <em>C. annuum</em> sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of <em>de novo</em> transcriptome assembly is applicable to a wide range of similar studies.</p> </div

Size distribution of the unigenes and CDS.

<p>The blue and red bars indicate unigene and CDS, respectively.</p

Illustrated the partly distribution (ratio of alignment/short no less than 0.8) of homologous length and aligned length.

<p>The X axis represents the ratio is length of pepper EST/unigene length, the Y axis is represents the ratio of alignment length/shorter between pepper EST and unigene.</p

Image8_Unexpected Dynamic Binding May Rescue the Binding Affinity of Rivaroxaban in a Mutant of Coagulation Factor X.JPEG

A novel coagulation factor X (FX) Tyr319Cys mutation (Y99C as chymotrypsin numbering) was identified in a patient with severe bleeding. Unlike the earlier reported Y99A mutant, this mutant can bind and cleave its specific chromogenetic substrate at a normal level, suggesting an intact binding pocket. Here, using molecular dynamics simulations and MM-PBSA calculations on a FX-rivaroxaban (RIV) complex, we confirmed a much stronger binding of RIV in Y99C than in Y99A on a molecular level, which is actually the average result of multiple binding poses in dynamics. Detailed structural analyses also indicated the moderate flexibility of the 99-loop and the importance of the flexible side chain of Trp215 in the different binding poses. This case again emphasizes that binding of ligands may not only be a dynamic process but also a dynamic state, which is often neglected in drug design and screening based on static X-ray structures. In addition, the computational results somewhat confirmed our hypothesis on the activated Tyr319Cys FX (Y99C FXa) with an impaired procoagulant function to bind inhibitors of FXa and to be developed into a potential reversal agent for novel oral anticoagulants (NOAC).</p

DataSheet1_Unexpected Dynamic Binding May Rescue the Binding Affinity of Rivaroxaban in a Mutant of Coagulation Factor X.ZIP

A novel coagulation factor X (FX) Tyr319Cys mutation (Y99C as chymotrypsin numbering) was identified in a patient with severe bleeding. Unlike the earlier reported Y99A mutant, this mutant can bind and cleave its specific chromogenetic substrate at a normal level, suggesting an intact binding pocket. Here, using molecular dynamics simulations and MM-PBSA calculations on a FX-rivaroxaban (RIV) complex, we confirmed a much stronger binding of RIV in Y99C than in Y99A on a molecular level, which is actually the average result of multiple binding poses in dynamics. Detailed structural analyses also indicated the moderate flexibility of the 99-loop and the importance of the flexible side chain of Trp215 in the different binding poses. This case again emphasizes that binding of ligands may not only be a dynamic process but also a dynamic state, which is often neglected in drug design and screening based on static X-ray structures. In addition, the computational results somewhat confirmed our hypothesis on the activated Tyr319Cys FX (Y99C FXa) with an impaired procoagulant function to bind inhibitors of FXa and to be developed into a potential reversal agent for novel oral anticoagulants (NOAC).</p

Image2_Unexpected Dynamic Binding May Rescue the Binding Affinity of Rivaroxaban in a Mutant of Coagulation Factor X.JPEG

A novel coagulation factor X (FX) Tyr319Cys mutation (Y99C as chymotrypsin numbering) was identified in a patient with severe bleeding. Unlike the earlier reported Y99A mutant, this mutant can bind and cleave its specific chromogenetic substrate at a normal level, suggesting an intact binding pocket. Here, using molecular dynamics simulations and MM-PBSA calculations on a FX-rivaroxaban (RIV) complex, we confirmed a much stronger binding of RIV in Y99C than in Y99A on a molecular level, which is actually the average result of multiple binding poses in dynamics. Detailed structural analyses also indicated the moderate flexibility of the 99-loop and the importance of the flexible side chain of Trp215 in the different binding poses. This case again emphasizes that binding of ligands may not only be a dynamic process but also a dynamic state, which is often neglected in drug design and screening based on static X-ray structures. In addition, the computational results somewhat confirmed our hypothesis on the activated Tyr319Cys FX (Y99C FXa) with an impaired procoagulant function to bind inhibitors of FXa and to be developed into a potential reversal agent for novel oral anticoagulants (NOAC).</p