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Bücher und Buchkapitel

Janik, K.; Stellmach, H.; Mittelberger, C.; Hause, B.; Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC) (Musetti, R. & Pagliari, L., eds.). Methods Mol. Biol. 1875, 321-331, (2019) ISBN: 978-1-4939-8837-2 DOI: 10.1007/978-1-4939-8837-2_24

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.
Bücher und Buchkapitel

Hause, B.; Yadav, H.; Creation of composite plants – transformation of Medicago truncatula roots (de Bruijn, F., ed.). 1179-1184, (2019) ISBN: 9781119409144 DOI: 10.1002/9781119409144.ch152

Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.
Publikation

Gelová, Z.; ten Hoopen, P.; Novák, O.; Motyka, V.; Pernisová, M.; Dabravolski, S.; Didi, V.; Tillack, I.; Oklešťková, J.; Strnad, M.; Hause, B.; Haruštiaková, D.; Conrad, U.; Janda, L.; Hejátko, J.; Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis J. Exp. Bot. 69, 441-454, (2018) DOI: 10.1093/jxb/erx426

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin
Publikation

Ferlian, O.; Biere, A.; Bonfante, P.; Buscot, F.; Eisenhauer, N.; Fernandez, I.; Hause, B.; Herrmann, S.; Krajinski-Barth, F.; Meier, I. C.; Pozo, M. J.; Rasmann, S.; Rillig, M. C.; Tarkka, M. T.; van Dam, N. M.; Wagg, C.; Martinez-Medina, A.; Growing Research Networks on Mycorrhizae for Mutual Benefits Trends Plant Sci. 23, 975-984, (2018) DOI: 10.1016/j.tplants.2018.08.008

Research on mycorrhizal interactions has traditionally developed into separate disciplines addressing different organizational levels. This separation has led to an incomplete understanding of mycorrhizal functioning. Integration of mycorrhiza research at different scales is needed to understand the mechanisms underlying the context dependency of mycorrhizal associations, and to use mycorrhizae for solving environmental issues. Here, we provide a road map for the integration of mycorrhiza research into a unique framework that spans genes to ecosystems. Using two key topics, we identify parallels in mycorrhiza research at different organizational levels. Based on two current projects, we show how scientific integration creates synergies, and discuss future directions. Only by overcoming disciplinary boundaries, we will achieve a more comprehensive understanding of the functioning of mycorrhizal associations.
Publikation

Fattorini, L.; Hause, B.; Gutierrez, L.; Veloccia, A.; Della Rovere, F.; Piacentini, D.; Falasca, G.; Altamura, M. M.; Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis BMC Plant Biol. 18, 182, (2018) DOI: 10.1186/s12870-018-1392-4

BackgroundAdventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored.ResultsMeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression.ConclusionsThe JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species.
Publikation

Farag, M. A.; Labib, R. M.; Noleto, C.; Porzel, A.; Wessjohann, L. A.; NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools LWT 90, 491-498, (2018) DOI: 10.1016/j.lwt.2017.12.069

Quantitative NMR metabolomics approach was developed to distinguish two cinnamon species (Ceylon Cinnamon, Cinnamomum verum and Chinese Cinnamon, Cinnamomum cassia) that are interchangeably used in food products. The results of the analyses of 10 bark accessions revealed for 9 key sensory metabolites, with (E)-cinnamaldehyde as the major form. Multivariate data analyses revealed for eugenol leading presence in C. verum versus fatty acid enrichment in C. cassia. This research provides the first NMR metabolites fingerprinting of the two major cinnamon resources. Compounds related to C. verum aroma and taste were identified and quantified that can be utilized as markers for the authentication of this valuable drug. Novel insight on metabolites mediating for C. verum antidiabetic effect is also presented.
Publikation

Schober, D.; Jacob, D.; Wilson, M.; Cruz, J. A.; Marcu, A.; Grant, J. R.; Moing, A.; Deborde, C.; de Figueiredo, L. F.; Haug, K.; Rocca-Serra, P.; Easton, J.; Ebbels, T. M. D.; Hao, J.; Ludwig, C.; Günther, U. L.; Rosato, A.; Klein, M. S.; Lewis, I. A.; Luchinat, C.; Jones, A. R.; Grauslys, A.; Larralde, M.; Yokochi, M.; Kobayashi, N.; Porzel, A.; Griffin, J. L.; Viant, M. R.; Wishart, D. S.; Steinbeck, C.; Salek, R. M.; Neumann, S.; nmrML: A Community Supported Open Data Standard for the Description, Storage, and Exchange of NMR Data Anal. Chem. 90, 649-656, (2018) DOI: 10.1021/acs.analchem.7b02795

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.
Publikation

Reisberg, M.; Arnold, N.; Porzel, A.; Neubert, R. H.; Dräger, B.; Malusides, novel glucosylceramides isolated from apple pomace (Malus domestica) Z. Naturforsch. C 73, 33-39, (2018) DOI: 10.1515/znc-2017-0059

Three new glucosylceramides (GluCers) named malusides I–III (1–3) were isolated from apple (cultivars of Malus domestica) pomace (fruit material remaining after juice extraction). An unusual oxo/hydroxy group pattern within the sphingadienine (d18:2) type sphingoid base was observed. All compounds contained the same α-hydroxylated fatty acid (h16:0) and a β-D-glucose moiety. Their structures were assigned on the basis of one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopic analyses and mass spectrometry (MS) measurements.
Publikation

Rasheed, D. M.; Porzel, A.; Frolov, A.; El Seedi, H. R.; Wessjohann, L. A.; Farag, M. A.; Comparative analysis of Hibiscus sabdariffa (roselle) hot and cold extracts in respect to their potential for α-glucosidase inhibition Food Chem. 250, 236-244, (2018) DOI: 10.1016/j.foodchem.2018.01.020

Roselle (Hibiscus sabdariffa) is a functional food with potential health benefits, consumed either as hot or cold beverage. To ensure quality control of its various products, accurate measurement of active metabolites is warranted. Herein, we propose a combination of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and nuclear magnetic resonance (NMR) analytical platforms for the untargeted characterization of metabolites in two roselle cultivars, Aswan and Sudan-1. The analyses revealed 33 metabolites, including sugars, flavonoids, anthocyanins, phenolic and aliphatic organic acids. Their relative contents in cultivars were assessed via principle component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS). Impact of the different extraction methods (decoction, infusion and maceration) was compared by quantitative 1H NMR spectroscopy, revealing cold maceration to be optimal for preserving anthocyanins, whereas infusion was more suited for recovering organic acids. The metabolite pattern revealed by the different extraction methods was found in good correlation for their ability to inhibit α-glucosidase enzyme.
Publikation

Moreno, M.; Zacarias, A.; Porzel, A.; Velasquez, L.; Gonzalez, G.; Alegría-Arcos, M.; Gonzalez-Nilo, F.; Gross, E. K. U.; IR and NMR spectroscopic correlation of enterobactin by DFT Spectrochim. Acta A 198, 264-277, (2018) DOI: 10.1016/j.saa.2018.02.060

Emerging and re-emerging epidemic diseases pose an ongoing threat to global health. Currently, Enterobactin and Enterobactin derivatives have gained interest, owing to their potential application in the pharmaceutical field. As it is known [J. Am. Chem. Soc (1979) 101, 20, 6097–6104], Enterobactin (H6EB) is an efficient iron carrier synthesized and secreted by many microbial species. In order to facilitate the elucidation of enterobactin and its analogues, here we propose the creation of a H6EB standard set using Density Functional Theory Infrared (IR) and NMR spectra. We used two exchange-correlation (xc) functionals (PBE including long-range corrections LC-PBE and mPW1), 2 basis sets (QZVP and 6-31G(d)) and 2 grids (fine and ultrafine) for most of the H6EB structures dependent of dihedral angles. The results show a significant difference between the OH and NH bands, while the CO amide and O(CO) IR bands are often found on top of each other. The NMR DFT calculations show a strong dependence on the xc functional, basis set, and grid used for the H6EB structure. Calculated 1H and 13C NMR spectra enable the effect of the solvent to be understood in the context of the experimental measurements. The good agreement between the experimental and the calculated spectra using LC-PBE/QZVP and ultrafine grid suggest the possibility of the systems reported here to be considered as a standard set. The dependence of electrostatic potential and frontier orbitals with the catecholamide dihedral angles of H6EB is described. The matrix-assisted laser desorption/ionization time of the flight mass spectrometry (MALDI-TOF MS) of H6EB is also reported of manner to enrich the knowledge about its reactivity.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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