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Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.

Die Forschung an ribosomal synthetisierten und posttranslational modifizierten Peptiden (RiPPs) hat das Verstandnis von Biosynthesemechanismen zum Aufbau dieser Peptide weit vorangetrieben. Wahrend die meisten Beispiele fur RiPPs von Bakterien stammen, gibt es auser den Amanitinen und Phalloidinen nur wenig bekannte Vertreter, die von Pilzen produziert werden. Das pilzliche Cyclopeptid Omphalotin A tragt mehrere N-Methylierungen des Peptidruckgrats, eine Modifikation die so bisher nur fur nicht-ribosomal synthetisierte Peptide beschrieben wurde. Bei Untersuchungen des Genoms eines Omphalotin-produzierenden Pilzes konnten zwei Gene identifiziert werden, die an der Biosynthese beteiligt sind. Ein Gen kodiert fur die Methyltransferase OphMA, die eine Selbstmethylierung am eigenen C-Terminus durchfuhrt und anschliesend durch eine von einem zweiten Gen kodierte Prolyl-Oligopeptidase (OphP) erkannt, abgespalten und cyclisiert wird. Diese Arbeit zeigt einen neuen Biosynthesemechanismus fur RiPPs auf, bei dem das Vorstufenpeptid Bestandteil des posttranslational modifizierenden Enzyms ist.

Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation is presented. SHG-based three-dimensional reconstructions of Chlamydomonas cells containing starch granules are shown.