Making reproducible, auditable and scalable data-processing analysis workflows is an important challenge in the field of bioinformatics. Recently, software containers and cloud computing introduced a novel solution to address these challenges. They simplify software installation, management and reproducibility by packaging tools and their dependencies. In this work we implemented a cloud provider agnostic and scalable container orchestration setup for the popular Galaxy workflow environment. This solution enables Galaxy to run on and offload jobs to most cloud providers (e.g. Amazon Web Services, Google Cloud or OpenStack, among others) through the Kubernetes container orchestrator. Availability: All code has been contributed to the Galaxy Project and is available (since Galaxy 17.05) at https://github.com/galaxyproject/ in the galaxy and galaxy-kubernetes repositories. https://public.phenomenal-h2020.eu/ is an example deployment.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major economically important legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Obviously, changes in seed protein patterns might directly affect both of these aspects. Thus, here we address the pea seed proteome in more detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. Accordingly, 1938 and 1989 non-redundant proteins were identified in yellow and green pea seeds, in total. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP) potentially indicating the high efficiency of our experimental workflow. In total 981 protein groups could be assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.

C—H insertion reactions with organometallic and enzymatic catalysts based on earth-abundant iron complexes remain one of the major challenges in organic synthesis. In this report, we describe the development and application of these iron-based catalysts in the reaction of two different carbene precursors with N-heterocycles for the first time. While FeTPPCl showed excellent reactivity in the Fe(III) state with diazoacetonitrile, the highest activities of the YfeX enzyme could be achieved upon heme-iron reduction to Fe(II) with both diazoacetonitrile and ethyl diazoacetate. This highlights unexpected and subtle differences in reactivity of both iron catalysts. Deuterium labeling studies indicated a C—H insertion pathway and a marked kinetic isotope effect. This transformation features mild reaction conditions, excellent yields or turnover numbers with broad functional group tolerance, including gram-scale applications giving a unique access to functionalized N-heterocycles.

Toxic proteins are prime targets for molecular farming and efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. Achieving conditional activity of cytotoxins and their maintenance in form of stably transformed transgenes is challenging. We demonstrate here a switchable version of the highly cytotoxic bacterial ribonuclease barnase by using efficient temperature-dependent control of protein accumulation in living multicellular organisms. By tuning the levels of the protein, we were able to control the fate of a plant organ in vivo. The on-demand-formation of specialized epidermal cells (trichomes) through manipulating stabilization versus destabilization of barnase is a proof-of-concept for a robust and powerful tool for conditional switchable cell arrest. We present this tool both as a potential novel strategy for the manufacture and accumulation of cytotoxic proteins and toxic high-value products in plants or for conditional genetic cell ablation.

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well-studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development is completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes.

The exocyst is a conserved hetero-octameric complex that mediates early tethering of post-Golgi vesicles during exocytosis. Its Exo70 subunit functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst functions remains challenging. Exo70B2 localized to dynamic foci at the plasma membrane and transited through Brefeldin A (BFA)-sensitive compartments, indicating that it participates in conventional secretion. Conversely, treatment with the immunogenic peptide flg22 or the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), induced Exo70B2 transport into the vacuole where it colocalized with autophagic markers AUTOPHAGY-RELATED PROTEIN 8 (ATG8) and NEIGHBOR OF BRCA1 GENE 1 (NBR1). According with its role in immunity, we discovered that Exo70B2 interacts with and is phosphorylated by the MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3). Mimicking phosphorylation inhibited Exo70B2 localization at sites of active secretion. By contrast, lines expressing phosphonull variants displayed higher Effector-Triggered Immunity and were hypersensitive to BTH, conditions known to induce the secretory pathway. Our results suggest a molecular mechanism by which phosphorylation of Exo70B2 regulates interaction with the plasma membrane, and couples the secretory pathway with cellular signalling.

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.

Background Metabolomics is the comprehensive study of a multitude of small molecules to gain insight into an organism’s metabolism. The research field is dynamic and expanding with applications across biomedical, biotechnological and many other applied biological domains. Its computationally-intensive nature has driven requirements for open data formats, data repositories and data analysis tools. However, the rapid progress has resulted in a mosaic of independent – and sometimes incompatible – analysis methods that are difficult to connect into a useful and complete data analysis solution.Findings The PhenoMeNal (Phenome and Metabolome aNalysis) e-infrastructure provides a complete, workflow-oriented, interoperable metabolomics data analysis solution for a modern infrastructure-as-a-service (IaaS) cloud platform. PhenoMeNal seamlessly integrates a wide array of existing open source tools which are tested and packaged as Docker containers through the project’s continuous integration process and deployed based on a kubernetes orchestration framework. It also provides a number of standardized, automated and published analysis workflows in the user interfaces Galaxy, Jupyter, Luigi and Pachyderm.Conclusions PhenoMeNal constitutes a keystone solution in cloud infrastructures available for metabolomics. It provides scientists with a ready-to-use, workflow-driven, reproducible and shareable data analysis platform harmonizing the software installation and configuration through user-friendly web interfaces. The deployed cloud environments can be dynamically scaled to enable large-scale analyses which are interfaced through standard data formats, versioned, and have been tested for reproducibility and interoperability. The flexible implementation of PhenoMeNal allows easy adaptation of the infrastructure to other application areas and ‘omics research domains.

Drought is one of the major stress factors affecting growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants responses to water deficit by multiple physiological and metabolic adaptations at the molecular, cellular and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponic or agar culture. These experimental setups give access to different aspects of plant response to drought, like decrease of tissue water potential, reduction of stomata conductance and photosynthesis efficiency, accumulation of low-molecular weight solutes (metabolic adjustment) and drought protective proteins. Till now, this pattern of markers was successfully extended to the methods of enzyme chemistry, molecular biology and omics techniques. Thus, conventional tests can be efficiently complemented by determination of phytohormone and reactive oxygen species (ROS) contents, activities of antioxidant enzymes, as well as comprehensive profiling of transcriptome, proteome and metabolome.