Leibniz Institute of Plant Biochemistry

Convergence and parallelism are contentious terms in evolutionary biology, but both denote essentially a ubiquitous phenomenon: The occurrence of similar phenotypes, in different evolutionary lineages, in a way that cannot be easily reconducted to descent from a shared ancestor. In this article, we trace the historical definitions of the two terms and the current conceptual frameworks to classify instances of repeated evolution, presenting the limits of these approaches in considering convergence and parallelism as a strict dichotomy rather than as part of a continuum along the spectrum of phenotypic similarity. We then present cases of convergence-broadly defined-from plant domestication and specialized metabolism, with the objective of understanding the intricacies between natural selection, constraints and drift underlying the recurrent appearance of complex traits.

The anticandidal activity of Monotes kerstingii leaf and root crude extracts was evaluated against five clinical Candida isolates: C. albicans, C. parapsilosis, C. krusei, C. glabrata and C. tropicalis. Extracts from both organs displayed Minimal Inhibitory Concentrations (MICs) ranging from 3.9 to 2000µg/mL. Out of the five Candida species, the leaf hydroethanolic extract (EMKL) was the most active with MIC values of 3.9, 15.6 and 31.5µg/mL on C. krusei, C. parapsilosis and C. albicans, respectively. The chemical investigation of these extracts led to the characterization of six previously undescribed metabolites, including a glycosylated stilbene: kerstingioside (1), three cis stilbene-coumarins: cis-kerstilbcoumarin A-C (2-4) among which two as inseparable cis/trans-mixtures, one flavanone: kerstingiiflavanone (5) and one fatty acid glycoside, monestoside B (6), alongside with 22 known compounds. The distribution of the annotated metabolites in the roots and leaves was confirmed by comprehensive RP-UHPLC-Orbitrap-MS and MS/MS analysis. In addition, all the isolated compounds were screened for their anticandidal activity. Stilbene 7 was the most active constituent, with MICs of 7.8, 15.6, and 15.6µg/mL against C. albicans, C. krusei, and C. parapsilosis, respectively, while compounds 14 and 15 exhibited only moderate activity.

Tubulin is a heterodimeric protein composed of α- and β-subunits, which polymerize to form the cell's microtubules. The latter are key components in mitotic spindle formation and essential targets in anticancer therapy. Compounds such as paclitaxel, tubulysins, dolastatins and synthetic analogues of these latter compounds, including cemadotin, exert their cytotoxic effects by disrupting microtubule dynamics. Previously, we reported the production and anticancer activity of a library of cemadotin analogues featuring a C-terminal tertiary amide functionalized with a variety of N-substituents, thus resulting in compounds occurring as a mixture of amide rotamers. Here we describe a comprehensive NMR and conformational study that provides new insights into the effect of the conformational equilibrium on the binding mode of the novel cemadotin analogues to the tubulin target. The conformational behavior of the isomer equilibrium of cemadotin's terminal amide bond was investigated by TOCSY and ROESY NMR experiments, which allowed the identification and quantification of individual rotamer populations. A slow interconversion between the s-cis and s-trans amide rotamers was observed under standard NMR conditions (25 °C), indicating a significant energy barrier and conformational rigidity. Molecular docking and saturation transfer difference (STD) NMR experiments were performed with a representative analogue and tubulin to assess the binding mode. The results revealed that the s-trans rotamer is the predominant conformer in solution and exhibits a more favorable interaction with tubulin compared to the s-cis isomer, thus helping to understand the conformational requirements for an improved tubulin binding and the inhibition of the polymerization process.

Data-driven modeling based on machine learning (ML) isbecominga central component of protein engineering workflows. This perspectivepresents the elements necessary to develop effective, reliable, andreproducible ML models, and a set of guidelines for ML developmentsfor protein engineering. This includes a critical discussion of softwareengineering good practices for the development and evaluation of ML-basedprotein engineering projects, emphasizing supervised learning. Theseguidelines cover all of the necessary steps for ML development, fromdata acquisition to model deployment. Additionally, the present perspectiveprovides practical resources for the implementation of the outlinedguidelines. These recommendations are also intended to support editorsand scientific journals in enforcing good practices in ML-based proteinengineering publications, promoting high standards across the community.With this, the aim is to further contribute to improved ML transparencyand credibility by easing the adoption of software engineering bestpractices into ML development for protein engineering. We envisionthat the wide adoption and continuous update of best practices willencourage informed use of ML on real-world problems related to proteinengineering.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX-type hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high-performance liquid chromatography (SAX-HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg2+ dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4-InsP7, while Subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher order mutants of Subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wild-type (WT) and Subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

Calcium (Ca2+) signalling plays a central role in plant immunity, as underscored by recent findings showing that many disease resistance mechanisms result in formation of Ca2+-permeable pores, and that optogenetic activation of Ca2+ influx is sufficient to trigger immune responses. This review emphasizes on Ca2+ decoding, i.e. how diverse intracellular proteins interpret Ca2+ signals to drive cellular reactions. States of "Ca2+ responsiveness" - defined by the distinct sensitivities of various decoders and additional sensitization mechanisms - contribute to the regulation of immunity, possibly including the mutual potentiation of pattern- and effector-triggered immunity pathways. Additionally, the "PRIMER-bystander" model of immune signalling is interpreted within this decoding framework. Here, infected cells are proposed to enter a primed (PRIMER) immune state through strong Ca2+ signals derived from resistosome pores, while adjacent bystander cells respond to spreading signalling molecules from their neighbours. Through this spatial arrangement, coordination is achieved between cell-autonomous (autocrine) responses and non-autonomous (paracrine or endocrine) signalling, allowing robust immune propagation across plant tissues. By framing plant immunity through this Ca2+ "sense and sensitivity" paradigm, insights are provided into immune system robustness, and potential targets may be identified for future development of disease-resistant, climate-resilient crops.

SUMMARYThe decoding of calcium signals by plant calcium‐dependent kinases (CPKs) is not fully understood yet. Based on kinetic in vitro measurements of the activity of several CPK proteins, their individual activity profile was modeled and coupled to cytosolic calcium concentration changes from in vivo measurements of guard cells and epidermal leaf cells. In addition, computationally produced surrogate data were used. It was analyzed how individual activity profiles of CPKs will change in response to the different calcium time courses. In addition, the impact of modeling explicitly individual elementary reaction steps for binding and unbinding in comparison to using a heuristic Hill equation for the binding process was investigated.

We developed OpenStats, a user-friendly web application that brings the power of the R language to researchers through a high-level interface and broad support for statistical methods such as t-tests and ANOVA. OpenStats was integrated into our electronic lab notebook Chemotion ELN via its third-party API, enabling direct data exchange with it. A pivotal feature of OpenStats is its ability to record each analysis step in a structured history. This record allows users to retrace their work and enables automatic replay of the entire analysis, promoting reproducibility and long-term data integrity. In the modern research landscape, Research Data Management (RDM) tools like electronic lab notebooks (ELNs) are crucial for generating reproducible, repeatable, and transparently documented data. However, integrating statistical analysis tools into RDM systems remained a challenge. We demonstrated how OpenStats can be seamlessly linked to Research Data Management (RDM) systems, using Chemotion ELN as a reference implementation, by our application to a standard dose-response assay scenario. This linkage allows the integration of statistical data analysis in the form of a closed, traceable workflow into the RDM world. It therefore complements systems that are aiming for reproducible and standardized workflows in the scientific environment.

Hemicelluloses are important dietary fibers and a key component of lignocellulosic biomass. Despite numerous observations for fluorescently tagged cellulose synthases, the subcellular journeys and biochemical activities of intracellular cellulose synthase-like enzymes such as β-mannan synthases (ManS) remain largely unexplored. This study identifies C-terminal fluorescent protein tags that maintain ManS activity in yeast to accelerate the Design, Build, Test, Learn cycles for polysaccharide biosynthesis. Using the Amorphophallus konjac ManS as a case study, we demonstrate that the enzyme colocalizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in Pichia pastoris. The ManS first transmembrane domain was found to be critical for the punctate localization of the enzyme, its overall expression level and its function. Additionally, we explored how fluorescently tagged ManS is influenced by genetic or chemical perturbations of native yeast cell wall components, such as reducing protein mannosylation and severely disrupting β-1,3-glucans. Finally, we identified alternative feeding strategies and episomal vectors for Pichia, which were extended to Saccharomyces cerevisiae, to accelerate hemicellulose research. We propose that expanding the Plant MoClo-compatible plasmid repertoire is essential to swiftly prototype carbohydrate-active enzymes in yeast before proceeding with more time-intensive analyses in plants. Requiring only hours or days instead of weeks or months for plant transformation/regeneration, our yeast prototyping strategies can derisk the bioengineering of carbohydrate-active enzymes.