The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in <i>Sorghum bicolor</i> contains its co-expressed vacuolar MATE transporter

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters

CYP83B1 Is the Oxime-metabolizing Enzyme in the Glucosinolate Pathway in \u3ci\u3eArabidopsis\u3c/i\u3e

CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450 monooxygenase. We searched the genome of A. thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides. By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli. The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a “P450mox” that converted oximes into toxic compounds that the plant detoxified into glucosinolates

Clinical Study Echocardiographic Measures of Diastolic Function Are Preload Dependent during Triggered Positive Pressure Ventilation: A Controlled Crossover Study in Healthy Subjects

Background. The use of echocardiography in intensive care settings impacts decision making. A prerequisite for the use of echocardiography is relative resistance to changes in volume status and levels of positive pressure ventilation (PPV). Studies on indices of diastolic function report conflicting results with regard to dependence on volume status. Evidence is scarce on PPV. Methods. Ten healthy subjects were exposed to 6 levels of positive end-expiratory pressure (PEEP) and pressure support (PS) following a baseline reading. All ventilator settings were performed at three positions: horizontal, reverse-Trendelenburg, and Trendelenburg. Echocardiography was performed throughout. Results. During spontaneous breathing, early diastolic transmitral velocity (E) changed with positioning (P &lt; 0.001), whereas early diastolic velocity of the mitral annulus (e ) was independent (P = 0.263). With PPV, E and e proved preload dependent (P values &lt; 0.001). Increases in PEEP, PS, or a combination influenced E and e in reverse-Trendelenburg-and horizontal positions, but not in the Trendelenburg position. Discussion. The change towards preload dependency of e with PPV suggests that PPV increases myocardial preload sensitivity. The susceptibility of E and e to preload changes during PPV discourages their use in settings of volume shifts or during changes in ventilator settings. Conclusion. Positioning and PPV affect E and e

Vanillin formation from ferulic acid in <em>Vanilla planifolia</em> is catalysed by a single enzyme

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. (Résumé d'auteur