Dietrich Ober

The dried herb of blue fenugreek is used as a spice in the alpine region for the preparation of traditional bread and cheese. After drying, the herb is stored for a period of six to twelve months. During this time, the herb is expected to undergo changes in the compositions of the major flavor- and odor-determining compounds. To identify eventual biochemical processes, we applied different growing (conventional and sterile) and drying (air- and freeze drying) conditions and subsequently conducted periodical analysis of key aroma constituents (α-keto acids and volatile compounds) by LC-MS and GC-MS. The amount of glyoxylic acid was drastically increased in the air-dried sample, while the freeze-dried sample showed significantly higher amounts of α-keto-glutaric acid and pyruvic acid, respectively. During storage, a decrease in sulfuric compounds and an increase in alkane aldehydes were observed when comparing conventional and sterile samples. However, this increase was even greater for monoterpenes (especially camphor and p-cymene), showing thrice as high amounts after storage. Interestingly, both compounds were only formed significantly during the storage under conventional conditions, indicating that their production is induced/caused by microbial organisms.

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites found in several plant lineages with independent evolutionary origins. In comfrey ( Symphytum officinale ), two independent homospermidine oxidase (HSO) paralogs are responsible for oxidation of homospermidine (Hspd) to the bicyclic pyrrolizidine in roots and young leaves. As PA biosynthesis in S. officinale and Heliotropium indicum (Indian heliotrope) share a common ancestor, we tested whether H. indicum is also able to synthesize PAs, not only in aerial parts but also in roots. H. indicum constitutively synthesizes not only complex PAs, in aerial parts but also simple PAs in roots. Of five copper‐containing amine oxidases (CuAOs) identified in H. indicum , three have the ability to convert Hspd to the bicyclic pyrrolizidine in vitro . CRISPR/Cas9 genome editing confirmed that, in planta , only one of these CuAOs is involved in PA biosynthesis in roots, which is identical to the HSO involved in PA biosynthesis in leaves. PA biosynthesis in roots is less efficient than in aerial parts, an observation that allowed the analysis of various pathway intermediates using tracer feeding experiments. The CuAO phylogeny, together with comparative gene structure analyses, suggest a common evolutionary origin of PA‐producing CuAOs. However, independent scenarios of PA metabolism were observed in Indian heliotrope and comfrey, adding a further aspect of diversity in the regulation of PA biosynthesis.

Als eine der ältesten Einrichtungen der Christian-Albrechts-Universität zu Kiel ist der Botanische Garten Kiel mit seinen über 355 Jahren ein Ort der Wissenschaft, der universitären Bildung und der Erholung für die Bürgerinnen und Bürger der Stadt Kiel und des Landes Schleswig-Holstein. Im Verbund mit den Botanischen Gärten der Welt stellt sich der Botanische Garten Kiel mit seinen umfangreichen und wertvollen Pflanzensammlungen dem immer schneller fortschreitenden Verlust der natürlichen Vielfalt entgegen und engagiert sich nicht nur für die Sensibilisierung der Öffentlichkeit für die Herausforderungen der Zukunft, sondern auch für die Aus- und Weiterbildung der Verantwortlichen von morgen.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways. Graphical Abstract

SUMMARY: Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey ( Symphytum officinale , Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper‐containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, So CuAO1 is expressed in young leaves, while So CuAO5 is expressed in roots. CRISPR/Cas9‐mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA‐free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of So CuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species‐specific, tight spatio‐temporal regulation, and independent evolutionary origins in multiple plant lineages.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive posttranslational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in‐depth biochemical studies of this enzyme, we report here the development of a simple and robust in vitro enzyme assay. It involves pre‐column derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid‐producing plant Senecio vernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs.

Plants, fungi and algae are important components of global biodiversity and are fundamental to all ecosystems. They are the basis for human well-being, providing food, materials and medicines. Specimens of all three groups of organisms are accommodated in herbaria, where they are commonly referred to as botanical specimens.The large number of specimens in herbaria provides an ample, permanent and continuously improving knowledge base on these organisms and an indispensable source for the analysis of the distribution of species in space and time critical for current and future research relating to global biodiversity. In order to make full use of this resource, a research infrastructure has to be built that grants comprehensive and free access to the information in herbaria and botanical collections in general. This can be achieved through digitization of the botanical objects and associated data.The botanical research community can count on a long-standing tradition of collaboration among institutions and individuals. It agreed on data standards and standard services even before the advent of computerization and information networking, an example being the Index Herbariorum as a global registry of herbaria helping towards the unique identification of specimens cited in the literature.In the spirit of this collaborative history, 51 representatives from 30 institutions advocate to start the digitization of botanical collections with the overall wall-to-wall digitization of the flat objects stored in German herbaria. Germany has 70 herbaria holding almost 23 million specimens according to a national survey carried out in 2019. 87% of these specimens are not yet digitized. Experiences from other countries like France, the Netherlands, Finland, the US and Australia show that herbaria can be comprehensively and cost-efficiently digitized in a relatively short time due to established workflows and protocols for the high-throughput digitization of flat objects.Most of the herbaria are part of a university (34), fewer belong to municipal museums (10) or state museums (8), six herbaria belong to institutions also supported by federal funds such as Leibniz institutes, and four belong to non-governmental organizations. A common data infrastructure must therefore integrate different kinds of institutions.Making full use of the data gained by digitization requires the set-up of a digital infrastructure for storage, archiving, content indexing and networking as well as standardized access for the scientific use of digital objects. A standards-based portfolio of technical components has already been developed and successfully tested by the Biodiversity Informatics Community over the last two decades, comprising among others access protocols, collection databases, portals, tools for semantic enrichment and annotation, international networking, storage and archiving in accordance with international standards. This was achieved through the funding by national and international programs and initiatives, which also paved the road for the German contribution to the Global Biodiversity Information Facility (GBIF).Herbaria constitute a large part of the German botanical collections that also comprise living collections in botanical gardens and seed banks, DNA- and tissue samples, specimens preserved in fluids or on microscope slides and more. Once the herbaria are digitized, these resources can be integrated, adding to the value of the overall research infrastructure. The community has agreed on tasks that are shared between the herbaria, as the German GBIF model already successfully demonstrates.We have compiled nine scientific use cases of immediate societal relevance for an integrated infrastructure of botanical collections. They address accelerated biodiversity discovery and research, biomonitoring and conservation planning, biodiversity modelling, the generation of trait information, automated image recognition by artificial intelligence, automated pathogen detection, contextualization by interlinking objects, enabling provenance research, as well as education, outreach and citizen science.We propose to start this initiative now in order to valorize German botanical collections as a vital part of a worldwide biodiversity data pool.

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 – 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.