Elisabeth Kaltenegger

Due to climate change, extreme weather events such as droughts are becoming more frequent and intense. This has a profound impact on plant performance and ecological interactions, including those involving herbivorous insects. The combined impact of drought stress and insect herbivory on plant metabolism has rarely been studied, particularly in woody plants. In this study, we investigated the influence of varying degrees of drought, both alone and in combination with herbivory by the leaf beetle Chrysomela tremulae , on the morphological and chemical characteristics of black poplar ( Populus nigra ) trees using a full factorial experimental design. We quantified morphological traits, volatile organic compound (VOC) emissions, phytohormone and amino acid concentrations, and phenolic profiles. Drought conditions increased the concentrations of salicylic acid (SA) and abscisic acid (ABA), while feeding induced ABA and SA. Amino acid profiles shifted significantly under drought conditions, particularly in beetle-infested plants. In contrast, salicinoids, which are the most important phenolic defense compounds in poplars, remained relatively stable. We also observed significant compound-specific effects on both constitutive and herbivore-induced VOC emissions. Our results demonstrate that drought and insect herbivory exert a joint influence on the chemical responses of P. nigra across multiple metabolic pathways. These findings highlight how the interaction between abiotic and biotic stresses can influence the defense chemistry of trees, which will consequently affect ecological interactions in forest ecosystems in the face of climate change.

Pollinators share the complex information and resource landscape of their host plants with herbivores. Yet, how sap feeders affect floral attractiveness to pollinators remains poorly understood, despite the critical role of this tripartite interaction in natural and agricultural ecosystems. In dioecious plant species, which display pronounced sexual dimorphism, these intricate interactions may vary in magnitude and direction between females and males, with significant implications for plant population dynamics and species co-evolution. In this study, we examined how infestation by the oligophagous aphid Brachycaudus lychnidis affects sex-specific interactions between the dioecious plant Silene latifolia and its specialist moth pollinator Hadena bicruris. We exposed male and female plants to aphid herbivory and evaluated its effects on floral traits (visual cues, floral scent, and nectar chemistry) and pollinator behaviour. While aphid infestation affected some floral traits equally in both sexes and others more strongly in males or in females, we observed stronger declines in female attractiveness to pollinators, which were mainly linked to nectar compounds potentially acting as feeding cues or behavioural modulators. We discuss our results in the light of sexual selection and plant defence theory while emphasizing the complementarity of female and male traits in stabilizing this specialized plant–pollinator–herbivore system.

Pollinators share the complex information and resource landscape of their host plants with herbivores. Yet, how sap feeders affect floral attractiveness to pollinators remains poorly understood, despite the critical role of this tripartite interaction in natural and agricultural ecosystems. In dioecious plant species, which display pronounced sexual dimorphism, these intricate interactions may vary in magnitude and direction between females and males, with significant implications for plant population dynamics and species co-evolution. In this study, we examined how infestation by the oligophagous aphid Brachycaudus lychnidis affects sex-specific interactions among the dioecious plant Silene latifolia and its specialist moth pollinator Hadena bicruris . We exposed male and female plants to aphid herbivory and evaluated its effects on floral traits (visual cues, floral scent, and nectar chemistry) and pollinator behaviour. While aphid infestation affected some floral traits equally in both sexes and others more strongly in males or in females, we observed stronger declines in female attractiveness to pollinators, which were mainly linked to nectar compounds potentially acting as feeding cues or behavioural modulators. We discuss our results in the light of sexual selection and plant defence theory while emphasizing the complementarity of female and male traits in stabilizing this specialized plant-pollinator-herbivore system. Highlight: Aphid infestation alters multiple visual and chemical floral traits in a plant sex-specific manner, leading to reduced attractiveness to moth pollinators in female plants, but not in males. Graphical Plant-sex specific effect of aphid infestation on floral traits (number, size, colour, scent composition, nectar quantity and composition) and pollinator behaviour.

Premise: Enzymes that are encoded by paralogous genes and produce identical specialized metabolites in distantly related plant lineages are strong evidence of parallel phenotypic evolution. Inference of phenotypic homology for metabolites produced by orthologous genes is less straightforward, since orthologs may be recruited in parallel into novel pathways. In prior research on pyrrolizidine alkaloids (PAs), specialized metabolites of Apocynaceae, the evolution of homospermidine synthase (HSS), an enzyme of PA biosynthesis, was reconstructed and a single origin of PAs inferred because HSS enzymes of all known PA‐producing Apocynaceae species are orthologous and descended from an ancestral enzyme with the motif (VXXXD) of an optimized HSS.Methods: We increased sampling, tested the effect of amino acid motif on HSS function, revisited motif evolution, and tested for selection to infer evolution of HSS function and its correlation with phenotype.Results: Some evidence supports a single origin of PAs: an IXXXD HSS‐like gene, similar in function to VXXXD HSS, evolved in the shared ancestor of all PA‐producing species; loss of HSS function occurred multiple times via pseudogenization and perhaps via evolution of an IXXXN motif. Other evidence indicates multiple origins: the VXXXD motif, highly correlated with the PA phenotype, evolved two or four times independently; the ancestral IXXXD gene was not under positive selection, while some VXXXD genes were; and substitutions at sites experiencing positive selection occurred on multiple branches in the HSS‐like gene tree.Conclusions: The complexity of the genotype‐function‐phenotype map confounds the inference of PA homology from HSS‐like gene evolution in Apocynaceae.

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

In plants, homospermidine synthase (HSS) is a pathway‐specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA‐producing Convolvulaceae (“morning glories”), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function—the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

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.

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.

It is thought that after whole-genome duplications (WGDs), a large fraction of the duplicated gene copies is lost over time while few duplicates are retained. Which factors promote survival or death of a duplicate remains unclear and the underlying mechanisms are poorly understood. According to the model of gene dosage balance, genes encoding interacting proteins are predicted to be preferentially co-retained after WGDs. Among these are genes encoding proteins involved in complexes or in signal transduction. We have investigated the way that repeated WGDs during land plant evolution have affected cytokinin signaling to study patterns of gene duplicability and co-retention in this important signal transduction pathway. Through the integration of phylogenetic analyses with comparisons of genome collinearity, we have found that signal input mediated by cytokinin receptors proved to be highly conserved over long evolutionary time-scales, with receptors showing predominantly gene loss after repeated WGDs. However, the downstream elements, e,g. response regulators, were mainly retained after WGDs and thereby formed gene families in most plant lineages. Gene dosage balance between the interacting components indicated by co-retention after WGDs seems to play a minor role in the evolution of cytokinin signaling pathway. Overall, core genes of cytokinin signaling show a highly heterogeneous pattern of gene retention after WGD, reflecting complex relationships between the various factors that shape the long-term fate of a duplicated gene.