@phdthesis{Lu2024, author = {Lu, Jinping}, title = {The vacuolar TPC1 channel and its luminal calcium sensing site in the luminal pore entrance}, doi = {10.25972/OPUS-25135}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251353}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The slowly activating vacuolar SV/TPC1 channel is ubiquitously expressed in plants and provides a large cation conductance in the vacuolar membrane. Thereby, monovalent (K+, Na+) and in principle also divalent cations, such as Ca2+, can pass through the channel. The SV/TPC1 channel is activated upon membrane depolarization and cytosolic Ca2+ but inhibited by luminal calcium. With respect to the latter, two luminal Ca2+ binding sites (site 1 Asp240/Asp454/Glu528, site 2 Glu239/Asp240/Glu457) were identified to coordinate luminal Ca2+. In this work, the characteristics of the SV/TPC1 channels in terms of regulation and function were further elucidated, focusing on the TPC1s of Arabidopsis thaliana and Vicia faba. For electrophysiological analysis of the role of distinct pore residues for channel gating and luminal Ca2+ sensing, TPC1 channel variants were generated by site-directed mutagenesis and transiently expressed as eGFP/eYFP-fusion constructs in Arabidopsis thaliana mesophyll protoplasts of the TPC1 loss-of-function mutant attpc1-2. 1. As visualized by confocal fluorescence laser-scanning microscopy, all AtTPC1 (WT, E605A/Q, D606N, D607N, E605A/D606N, E605Q/D606N/D607N, E457N/E605A/D606N) and VfTPC1 channel variants (WT, N458E/A607E/ N608D) were correctly targeted to the vacuole membrane. 2. Patch-clamp studies revealed that removal of one of the negative charges at position Glu605 or Asp606 was already sufficient to promote voltage-dependent channel activation with higher voltage sensitivity. The combined neutralization of these residues (E605A/D606N), however, was required to additionally reduce the luminal Ca2+ sensitivity of the AtTPC1 channel, leading to hyperactive AtTPC1 channels. Thus, the residues Glu605/Asp606 are functionally coupled with the voltage sensor of AtTPC1 channel, thereby modulating channel gating, and form a novel luminal Ca2+ sensing site 3 in AtTPC1 at the luminal entrance of the ion transport pathway. 3. Interestingly, this novel luminal Ca2+ sensing site 3 (Glu605/Asp606) and Glu457 from the luminal Ca2+ sensing site 2 of the luminal Ca2+-sensitive AtTPC1 channel were neutralized by either asparagine or alanine in the TPC1 channel from Vicia faba and many other Fabaceae. Moreover, the VfTPC1 was validated to be a hyperactive TPC1 channel with higher tolerance to luminal Ca2+ loads which was in contrast to the AtTPC1 channel features. As a result, VfTPC1 but not AtTPC1 conferred the hyperexcitability of vacuoles. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the AtTPC1 triple mutant (E457N/E605A/D606N) gained VfTPC1-like characteristics. However, when VfTPC1 was mutated for the three AtTPC1-homologous polymorphic site residues, the VfTPC1 triple mutant (N458E/A607E/N608D) still sustained VfTPC1-WT-like features. These findings indicate that the hyperactivity of VfTPC1 is achieved in part by the loss of negatively charged amino acids at positions that - as part of the luminal Ca2+ sensing sites 2 and 3 - are homologous to AtTPC1-Glu457/Glu605/Asp606 and are likely stabilized by other unknown residues or domains. 4.The luminal polymorphic pore residues (Glu605/Asp606 in AtTPC1) apparently do not contribute to the unitary conductance of TPC1. Under symmetrical K+ conditions, a single channel conductance of about 80 pS was determined for AtTPC1 wild type and the AtTPC1 double mutant E605A/D606A. This is in line with the three-fold higher unitary conductance of VfTPC1 (232 pS), which harbors neutral luminal pore residues at the homologous sites to AtTPC1. In conclusion, by studying TPC1 channel from Arabidopsis thaliana and Vicia faba, the present thesis provides evidence that the natural TPC1 channel variants exhibit differences in voltage gating, luminal Ca2+ sensitivity and luminal Ca2+ binding sites.}, language = {en} } @phdthesis{Isasa2024, author = {Isasa, Emilie}, title = {Relationship between wood properties, drought-induced embolism and environmental preferences across temperate diffuse-porous broadleaved trees}, doi = {10.25972/OPUS-30356}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303562}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {In the scope of climate warming and the increase in frequency and intensity of severe heat waves in Central Europe, identification of temperate tree species that are suited to cope with these environmental changes is gaining increasing importance. A number of tree physiological characteristics are associated with drought-stress resistance and survival following severe heat, but recent studies have shown the importance of plant hydraulic and anatomical traits for predicting drought-induced tree mortality, such as vessel diameter, and their potential to predict species distribution in a changing climate. A compilation of large global datasets is required to determine traits related to drought-induced embolism and test whether embolism resistance can be determined solely by anatomical traits. However, most measurements of plant hydraulic traits are labour-intense and prone to measurement artefacts. A fast, accurate and widely applicable technique is necessary for estimating xylem embolism resistance (e.g., water potential at 50\% loss of conductivity, P50), in order to improve forecasts of future forest changes. These traits and their combination must have evolved following the selective pressure of the environmental conditions in which each species occurs. Describing these environmental-trait relationships can be useful to assess potential responses to environmental change and mitigation strategies for tree species, as future warmer temperatures may be compounded by drier conditions.}, subject = {Pflanzen{\"o}kologie}, language = {en} } @phdthesis{Kopic2024, author = {Kopic, Eva}, title = {On the physiological role of post-translational regulation of the \(Arabidopsis\) guard cell outward rectifying potassium channel GORK}, doi = {10.25972/OPUS-34880}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-348806}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Das streng regulierte Gleichgewicht zwischen CO2-Aufnahme und Transpiration ist f{\"u}r Pflanzen essentiell und h{\"a}ngt von kontrollierten Turgor{\"a}nderungen ab, die durch die Aktivit{\"a}t verschiedener Anionen- und Kationenkan{\"a}le verursacht werden. Diese Kan{\"a}le sind Teil von Signalkaskaden, die z. B. durch Phytohormone wie ABA (Abscisins{\"a}ure) und JA (Jasmonat) ausgel{\"o}st werden, die beide bei Trockenstress in den Schließzellen wirken. Dar{\"u}ber hinaus ist bekannt, dass JA an der Reaktion der Pflanze auf Pathogenbefall oder Verwundung beteiligt ist. GORK (guard cell outward rectifying K+ channel) ist der einzige bekannte, ausw{\"a}rts gleichrichtende K+-Kanal in Schließzellen und somit f{\"u}r den K+-Efflux beim Schließen der Stomata verantwortlich. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass GORK ein wesentlicher Bestandteil des JA-induzierten Stomatschlusses ist. Dies gilt f{\"u}r beide Ausl{\"o}ser, sowohl die Blattverwundung als auch die direkte Anwendung von JA. Patch-Clamp-Experimente an Protoplasten von Schließzellen untermauerten dieses Ergebnis, indem sie GORK-K+-Ausw{\"a}rtsstr{\"o}me als direktes Ziel von JA-Signalen entlarvten. Da bekannt ist, dass zytosolische Ca2+-Signale sowohl bei ABA- als auch bei JA-Signalen eine Rolle spielen, wurde die Interaktion von GORK mit Ca2+-abh{\"a}ngigen Kinasen untersucht. Eine antagonistische Regulation von GORK durch CIPK5-CBL1/9-Komplexe und ABI2 konnte durch DEVC (double electrode voltage clamp) sowie Protein-Protein-Interaktions-Experimente identifiziert und durch in-vitro Kinase-Assays untermauert werden. Patch-Clamp-Aufzeichnungen an Protoplasten von Schließzellen der cipk5-2 Funktions-Verlust-Mutante zeigten die Bedeutung von CIPK5 f{\"u}r den JA-induzierten Stomaschluss via Aktivierung von GORK. Die Interaktion verschiedener CDPKs (Ca2+-abh{\"a}ngige Proteinkinasen) mit GORK wurde ebenfalls untersucht. Neben der Ca2+-Signal{\"u}bertragung ist auch die Produktion von ROS (reaktive Sauerstoffspezies) f{\"u}r die ABA- und MeJA-Signal{\"u}bertragung von Bedeutung. In DEVC-Experimenten konnte ein reversibler Effekt von ROS auf die GORK-Kanalaktivit{\"a}t nachgewiesen werden, was ein Teil der Erkl{\"a}rung f{\"u}r diese ROS-Effekte bei ABA- und MeJA-Signalen sein k{\"o}nnte.}, subject = {Spalt{\"o}ffnung}, language = {en} } @phdthesis{Huang2023, author = {Huang, Shouguang}, title = {Role of ABA-induced Ca\(^{2+}\) signals, and the Ca\(^{2+}\)-controlled protein kinase CIPK23, in regulation of stomatal movements}, doi = {10.25972/OPUS-20473}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204737}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Stomata are pores in the leaf surface, formed by pairs of guard cells. The guard cells modulate the aperture of stomata, to balance uptake of CO2 and loss of water vapor to the atmosphere. During drought, the phytohormone abscisic acid (ABA) provokes stomatal closure, via a signaling chain with both Ca2+-dependent and Ca2+-independent branches. Both branches are likely to activate SLAC1-type (Slow Anion Channel Associated 1) anion channels that are essential for initiating the closure of stomata. However, the importance of the Ca2+-dependent signaling branch is still debated, as the core ABA signaling pathway only possesses Ca2+-independent components. Therefore, the aim of this thesis was to address the role of the Ca2+-dependent branch in the ABA signaling pathway of guard cells. In the first part of the thesis, the relation between ABA-induced Ca2+ signals and stomatal closure was studied, with guard cells that express the genetically encoded Ca2+-indicator R-GECO1-mTurquoise. Ejection of ABA into the guard cell wall rapidly induced stomatal closure, however, only in ¾ of the guard cells ABA evoked a cytosolic Ca2+ signal. A small subset of stomata (¼ of the experiments) closed without Ca2+ signals, showing that the Ca2+ signals are not essential for ABA-induced stomatal closure. However, stomata in which ABA evoked Ca2+ signals closed faster as those in which no Ca2+ signals were detected. Apparently, ABA-induced Ca2+ signals enhance the velocity of stomatal closure. In addition to ABA, hyperpolarizing voltage pulses could also trigger Ca2+ signals in wild type guard cells, which in turn activated S-type anion channels. However, these voltage pulses failed to elicit S-type anion currents in the slac1/slah3 guard cells, suggesting that SLAC1 and SLAH3 contribute to Ca2+-activated conductance. Taken together, our data indicate that ABA-induced Ca2+ signals enhance the activity of S-type anion channels, which accelerates stomatal closure. The second part of the thesis deals with the signaling pathway downstream of the Ca2+ signals. Two types of Ca2+-dependent protein kinase modules (CPKs and CBL/CIPKs) have been implicated in guard cells. We focused on the protein kinase CIPK23 (CBL-Interacting Protein Kinase 23), which is activated by the Ca2+-dependent protein CBL1 or 9 (Calcineurin B-Like protein 1 or 9) via interacting with the NAF domain of CIPK23. The CBL1/9-CIPK23 complex has been shown to affect stomatal movements, but the underlying molecular mechanisms remain largely unknown. We addressed this topic by using an estrogen-induced expression system, which specifically enhances the expression of wild type CIPK23, a phosphomimic CIPK23T190D and a kinase dead CIPK23K60N in guard cells. Our data show that guard cells expressing CIPK23T190D promoted stomatal opening, while CIPK23K60N enhanced ABA-induced stomatal closure, suggesting that CIPK23 is a negative regulator of stomatal closure. Electrophysiological measurements revealed that the inward K+ channel currents were similar in guard cells that expressed CIPK23, CIPK23T190D or CIPK23K60N, indicating that CIPK23-mediated inward K+ channel AKT1 does not contribute to stomatal movements. Expression of CIPK23K60N, or loss of CIPK23 in guard cells enhanced S-type anion activity, while the active CIPK23T190D inhibited the activity of these anion channels. These results are in line with the detected changes in stomatal movements and thus indicate that CIPK23 regulates stomatal movements by inhibiting S-type anion channels. CIPK23 thus serves as a brake to control anion channel activity. Overall, our findings demonstrate that CIPK23-mediated stomatal movements do not depend on CIPK23-AKT1 module, instead, it is achieved by regulating S-type anion channels SLAC1 and SLAH3. In sum, the data presented in this thesis give new insights into the Ca2+-dependent branch of ABA signaling, which may help to put forward new strategies to breed plants with enhanced drought stress tolerance, and in turn boost agricultural productivity in the future.}, language = {en} } @phdthesis{Zhou2023, author = {Zhou, Yang}, title = {The Exploitation of Opsin-based Optogenetic Tools for Application in Higher Plants}, doi = {10.25972/OPUS-23696}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236960}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The discovery, heterologous expression, and characterization of channelrhodopsin-2 (ChR2) - a light-sensitive cation channel found in the green alga Chlamydomonas reinhardtii - led to the success of optogenetics as a powerful technology, first in neuroscience. ChR2 was employed to induce action potentials by blue light in genetically modified nerve cells. In optogenetics, exogenous photoreceptors are expressed in cells to manipulate cellular activity. These photoreceptors were in the beginning mainly microbial opsins. During nearly two decades, many microbial opsins and their mutants were explored for their application in neuroscience. Until now, however, the application of optogenetics to plant studies is limited to very few reports. Several optogenetic strategies for plant research were demonstrated, in which most attempts are based on non-opsin optogenetic tools. Opsins need retinal (vitamin A) as a cofactor to generate the functional protein, the rhodopsin. As most animals have eyes that contain animal rhodopsins, they also have the enzyme - a 15, 15'-Dioxygenase - for retinal production from food-supplied provitamin A (beta-carotene). However, higher plants lack a similar enzyme, making it difficult to express functional rhodopsins successfully in plants. But plant chloroplasts contain plenty of beta-carotene. I introduced a gene, coding for a 15, 15'-Dioxygenase with a chloroplast target peptide, to tobacco plants. This enzyme converts a molecule of β-carotene into two of all-trans-retinal. After expressing this enzyme in plants, the concentration of all-trans-retinal was increased greatly. The increased retinal concentration led to increased expression of several microbial opsins, tested in model higher plants. Unfortunately, most opsins were observed intracellularly and not in the plasma membrane. To improve their localization in the plasma membrane, some reported signal peptides were fused to the N- or C-terminal end of opsins. Finally, I helped to identify three microbial opsins -- GtACR1 (a light-gated anion channel), ChR2 (a light-gated cation channel), PPR (a light-gated proton pump) which express and work well in the plasma membrane of plants. The transgene plants were grown under red light to prevent activation of the expressed opsins. Upon illumination with blue or green light, the activation of these opsins then induced the expected change of the membrane potential, dramatically changing the phenotype of plants with activated rhodopsins. This study is the first which shows the potential of microbial opsins for optogenetic research in higher plants, using the ubq10 promoter for ubiquitous expression. I expect this to be just the beginning, as many different opsins and tissue-specific promoters for selective expression now can be tested for their usefulness. It is further to be expected that the here established method will help investigators to exploit more optogenetic tools and explore the secrets, kept in the plant kingdom.}, language = {en} } @phdthesis{Kunz2023, author = {Kunz, Marcel}, title = {Diffusion kinetics of organic compounds and water in plant cuticular model wax under the influence of diffusing barrier-modifying adjuvants}, doi = {10.25972/OPUS-27487}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-274874}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {To reach their target site, systemic pesticides must enter the plant from a spray droplet applied in the field. The uptake of an active ingredient (AI) takes place via the barrier-forming cuticular membrane, which is the outermost layer of the plant, separating it from the surrounding environment. Formulations are usually used which, in addition to the AI, also contain stabilizers and adjuvants. Adjuvants can either have surface-active properties or they act directly as barrier-modifying agents. The latter are grouped in the class of accelerating adjuvants, whereby individual variants may also have surface-active properties. The uptake of a pesticide from a spray droplet depends essentially on its permeability through the cuticular barrier. Permeability defines a combined parameter, which is the product of AI mobility and AI solubility within the cuticle. In recent decades, several tools have been developed that allowed the determination of individual parameters of organic compound penetration across the cuticular membrane. Nevertheless, earlier studies showed that mainly cuticular waxes are the barrier-determining component of the cuticular membrane and additionally, it was shown that mainly the very-long-chain aliphatic compounds (VLCAs) are responsible for establishing an effective barrier. However, the barrier-determining role of the individual VLCAs, being classified according to their respective functional groups, is still unknown. Therefore, the following objectives were pursued and achieved in this work: (1) A new ATR-FTIR-based approach was developed to measure the temperature-dependent real-time diffusion kinetics of organic models for active ingredients (AIs) in paraffin wax, exclusively consisting of very-long chain alkanes. (2) The developed ATR-FTIR approach was applied to determine the diffusion kinetics of self-accelerating adjuvants in cuticular model waxes of different VLCA composition. At the same time, wax-specific changes were recorded in the respective IR spectra, which provided information about the respective wax modification. (3) The ATR-FTIR method was used to characterize the diffusion kinetics, as well as to determine the wax-specific sorption capacities for an AI-modeling organic compound and water in cuticular model waxes after adjuvant treatment. Regarding the individual chemical compositions and structures, conclusions were drawn about the adjuvant-specific modes of action (MoA). In the first chapter, the ATR-FTIR based approach to determine organic compound diffusion kinetics in paraffin wax was successfully established. The diffusion kinetics of the AI modelling organic compounds heptyl parabene (HPB) and 4-cyanophenol (CNP) were recorded, comprising different lipophilicities and molecular volumes typical for AIs used in pesticide formulations. Derived diffusion coefficients ranged within 10-15 m2 s-1, thus being thoroughly higher than those obtained from previous experiments using an approach solely investigating desorption kinetics in reconstituted cuticular waxes. An ln-linear dependence between the diffusion coefficients and the applied diffusion temperature was demonstrated for the first time in cuticular model wax, from which activation energies were derived. The determined activation energies were 66.2 ± 7.4 kJ mol-1 and 56.4 ± 9.8 kJ mol-1, being in the expected range of already well-founded activation energies required for organic compound diffusion across cuticular membranes, which again confirmed the significant contribution of waxes to the cuticular barrier. Deviations from the assumed Fickian diffusion were attributed to co-occurring water diffusion and apparatus-specific properties. In the second and third chapter, mainly the diffusion kinetics of accelerating adjuvants in the cuticular model waxes candelilla wax and carnauba wax were investigated, and simultaneously recorded changes in the wax-specific portion of the IR spectrum were interpreted as indications of plasticization. For this purpose, the oil derivative methyl oleate, as well as the organophosphate ester TEHP and three non-ionic monodisperse alcohol ethoxylates (AEs) C12E2, C12E4 and C12E6 were selected. Strong dependence of diffusion on the respective principal components of the mainly aliphatic waxes was demonstrated. The diffusion kinetics of the investigated adjuvants were faster in the n-alkane dominated candelilla wax than in the alkyl ester dominated carnauba wax. Furthermore, the equilibrium absorptions, indicating equilibrium concentrations, were also higher in candelilla wax than in carnauba wax. It was concluded that alkyl ester dominated waxes feature higher resistance to diffusion of accelerating adjuvants than alkane dominated waxes with shorter average chain lengths due to their structural integrity. This was also found either concerning candelilla/policosanol (n-alcohol) or candelilla/rice bran wax (alkyl-esters) blends: with increasing alcohol concentration, the barrier function was decreased, whereas it was increased with increasing alkyl ester concentration. However, due to the high variability of the individual diffusion curves, only a trend could be assumed here, but significant differences were not shown. The variability itself was described in terms of fluctuating crystalline arrangements and partial phase separation of the respective wax mixtures, which had inevitable effects on the adjuvant diffusion. However, diffusion kinetics also strongly depended on the studied adjuvants. Significantly slower methyl oleate diffusion accompanied by a less pronounced reduction in orthorhombic crystallinity was found in carnauba wax than in candelilla wax, whereas TEHP diffusion was significantly less dependent on the respective wax structure and therefore induced considerable plasticization in both waxes. Of particular interest was the AE diffusion into both waxes. Differences in diffusion kinetics were also found here between candelilla blends and carnauba wax. However, these depended equally on the degree of ethoxylation of the respective AEs. The lipophilic C12E2 showed approximately Fickian diffusion kinetics in both waxes, accompanied by a drastic reduction in orthorhombic crystallinity, especially in candelilla wax, whereas the more hydrophilic C12E6 showed significantly retarded diffusion kinetics associated with a smaller effect on orthorhombic crystallinity. The individual diffusion kinetics of the investigated adjuvants sometimes showed drastic deviations from the Fickian diffusion model, indicating a self-accelerating effect. Hence, adjuvant diffusion kinetics were accompanied by a distinct initial lag phase, indicating a critical concentration in the wax necessary for effective penetration, leading to sigmoidal rather than to exponential diffusion kinetics. The last chapter dealt with the adjuvant-affected diffusion of the AI modelling CNP in candelilla and carnauba wax. Using ATR-FTIR, diffusion kinetics were recorded after adjuvant treatment, all of which were fully explicable based on the Fickian model, with high diffusion coefficients ranging from 10-14 to 10-13 m2 s-1. It is obvious that the diffusion coefficients presented in this work consistently demonstrated plasticization induced accelerated CNP mobilities. Furthermore, CNP equilibrium concentrations were derived, from which partition- and permeability coefficients could be determined. Significant differences between diffusion coefficients (mobility) and partition coefficients (solubility) were found on the one hand depending on the respective waxes, and on the other hand depending on treatment with respective adjuvants. Mobility was higher in candelilla wax than in carnauba wax only after methyl oleate treatment. Treatment with TEHP and AEs resulted in higher CNP mobility in the more polar alkyl ester dominated carnauba wax. The partition coefficients, on the other hand, were significantly lower after methyl oleate treatment in both candelilla and carnauba wax as followed by TEHP or AE treatment. Models were designed for the CNP penetration mode considering the respective adjuvants in both investigated waxes. Co-penetrating water, which is the main ingredient of spray formulations applied in the field, was likely the reason for the drastic differences in adjuvant efficacy. Especially the investigated AEs favored an enormous water uptake in both waxes with increasing ethoxylation level. Surprisingly, this effect was also found for the lipophilic TEHP in both waxes. This led to the assumption that the AI permeability is not exclusively determined by adjuvant induced plasticization, but also depends on a "secondary plasticization", induced by adjuvant-attracted co-penetrating water, consequently leading to swelling and drastic destabilization of the crystalline wax structure. The successful establishment of the presented ATR-FTIR method represents a milestone for the study of adjuvant and AI diffusion kinetics in cuticular waxes. In particular, the simultaneously detectable wax modification and, moreover, the determinable water uptake form a perfect basis to establish the ATR-FTIR system as a universal screening tool for wax-adjuvants-AI-water interaction in crop protection science.}, subject = {Pflanzen}, language = {en} } @article{SteinerZacharyBaueretal.2023, author = {Steiner, Thomas and Zachary, Marie and Bauer, Susanne and M{\"u}ller, Martin J. and Krischke, Markus and Radziej, Sandra and Klepsch, Maximilian and Huettel, Bruno and Eisenreich, Wolfgang and Rudel, Thomas and Beier, Dagmar}, title = {Central Role of Sibling Small RNAs NgncR_162 and NgncR_163 in Main Metabolic Pathways of Neisseria gonorrhoeae}, series = {mBio}, volume = {14}, journal = {mBio}, doi = {10.1128/mbio.03093-22}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313323}, year = {2023}, abstract = {Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.}, language = {en} } @article{DeğirmenciRogeFerreiraVukosavljevicetal.2023, author = {Değirmenci, Laura and Rog{\´e} Ferreira, Fabio Luiz and Vukosavljevic, Adrian and Heindl, Cornelia and Keller, Alexander and Geiger, Dietmar and Scheiner, Ricarda}, title = {Sugar perception in honeybees}, series = {Frontiers in Physiology}, volume = {13}, journal = {Frontiers in Physiology}, issn = {1664-042X}, doi = {10.3389/fphys.2022.1089669}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302284}, year = {2023}, abstract = {Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments have demonstrated collective responses of the whole Gr-set. We here disentangle the contributions of all three honeybee sugar receptors (AmGr1-3), combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. We show an expanded sugar spectrum of the AmGr1 receptor. Mutants lacking AmGr1 have a reduced response to sucrose and glucose but not to fructose. AmGr2 solely acts as co-receptor of AmGr1 but not of AmGr3, as we show by electrophysiology and using bimolecular fluorescence complementation. Our results show for the first time that AmGr2 is indeed a functional receptor on its own. Intriguingly, AmGr2 mutants still display a wildtype-like sugar taste. AmGr3 is a specific fructose receptor and is not modulated by a co-receptor. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. Our comprehensive study on the functions of AmGr1, AmGr2 and AmGr3 in honeybees is the first to combine investigations on sugar perception at the receptor level and simultaneously in vivo. We show that honeybees rely on two gustatory receptors to sense all relevant sugars.}, language = {en} } @phdthesis{Lambour2023, author = {Lambour, Benjamin}, title = {Regulation of sphingolipid long-chain bases during cell death reactions and abiotic stress in \(Arabidopsis\) \(thaliana\)}, doi = {10.25972/OPUS-32591}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-325916}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Sphingobasen (LCBs) sind die Bausteine der Biosynthese von Sphingolipiden. Sie werden als Strukturelemente der pflanzlichen Zellmembran definiert und spielen eine wichtige Rolle f{\"u}r das Schicksal der Zellen. Komplexe Ceramide machen einen wesentlichen Teil der gesamten Sphingolipide aus, die einen großen Teil der eukaryotischen Membranen bilden. Gleichzeitig sind LCBs bekannte Signalmolek{\"u}le f{\"u}r zellul{\"a}re Prozesse in Eukaryonten und sind an Signal{\"u}bertragungswegen in Pflanzen beteiligt. Es hat sich gezeigt, dass hohe LCB-Konzentrationen mit der Induktion des programmierten Zelltods sowie mit dem durch Pathogene ausgel{\"o}sten Zelltod in Verbindung stehen. Mehrere Studien haben die regulierende Funktion der Sphingobasen beim programmierten Zelltod (PCD) in Pflanzen best{\"a}tigt: (i) Spontaner PCD und ver{\"a}nderte Zelltodreaktionen, die durch mutierte verwandte Gene des Sphingobasen-Stoffwechsels verursacht werden. (ii) Zelltodbedingungen erh{\"o}hen den Gehalt an LCBs. (iii) PCD aufgrund eines gest{\"o}rten Sphingolipid-Stoffwechsels, der durch von nekrotrophen Krankheitserregern produzierte Toxine wie Fumonisin B1 (FB1) hervorgerufen wird. Um den Zelltod zu verhindern und die Zelltodreaktion zu kontrollieren, kann daher die Regulierung des Gehalts an freien LCBs entscheidend sein. Die Ergebnisse der vorliegenden Studie stellten das Verst{\"a}ndnis der Sphingobasen und Sphingolipidspiegel w{\"a}hrend der PCD in Frage. Wir lieferten eine detaillierte Analyse der Sphingolipidspiegel, die Zusammenh{\"a}nge zwischen bestimmten Sphingolipidarten und dem Zelltod aufzeigte. Dar{\"u}ber hinaus erm{\"o}glichte uns die Untersuchung der Sphingolipid-Biosynthese ein Verst{\"a}ndnis des Fluxes nach Akkumulation hoher LCB-Konzentrationen. Weitere Analysen von Abbauprodukten oder Sphingolipid-Mutantenlinien w{\"a}ren jedoch erforderlich, um vollst{\"a}ndig zu verstehen, wie die Pflanze mit hohen Mengen an Sphingobasen umgeht.}, subject = {Ackerschmalwand}, language = {en} } @phdthesis{Jaślan2023, author = {Jaślan, Justyna Joanna}, title = {R-type currents in \(Arabidopsis\) guard cells: properties and molecular nature}, doi = {10.25972/OPUS-18883}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188836}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {In contrast to the well described molecular basis for S-type anion currents, the genes underlying R-type anion currents were unknown until 2010. Meyer S. and colleagues (2010) showed that, localized in the guard cell plasma membrane, AtALMT12 is an R-type anion channel involved in stomatal closure. However, knocking out AtALMT12 did not fully shut down R-type currents; the almt12 loss-of-function mutant has residual R-type-like currents indicating that ALMT12 is not the only gene encoding Arabidopsis thaliana R-type channels (Meyer S. et al., 2010). This PhD thesis is focussed on understanding the properties, regulation and molecular nature of the R-type channels in Arabidopsis thaliana plants. To fulfil these aims, the patch clamp technique was used to characterize electrical features of R-type currents in various conditions such as the presence/absence of ATP, variation in cytosolic calcium concentration or the presence of cytosolic chloride. Electrophysiological study revealed many similarities between the features of Arabidopsis thaliana R-type currents (Col0) and residual R-type currents (the almt12 loss-of-function mutant). Strong voltage dependency, channel activity in the same voltage range, position of maximal recorded current and blockage by cytosolic ATP all pointed to a shared phylogenetic origin of the channels underlying these R-type currents. Expression patterns of the ALMT family members for Col0 and the almt12 mutant revealed ALMT13 and AMT14 as potential candidates of the R-type channels. Electrical characterization of Col0, almt12 and the two double loss-of-function mutants (almt12/almt13 and almt12/almt14) strongly suggest that ALMT13 mediates the calcium-dependent R-type current component that is directly regulated by cytosolic calcium. Additionally, similarly to ALMT12, ALMT14 could participate as a calcium-independent R-type anion channel. Differences in response to the cytosolic calcium concentration between ALMT12, ALMT13 and ALMT14 suggest their possible involvement in different signalling pathways leading to stomatal closure. Moreover, a study performed for the two Arabidopsis thaliana ecotypes Col0 and WS showed drastically increased ALMT13 expression for WS, which is related to R-type current properties. The WS ecotype has calcium-dependent R-type current behaviour, while it is calcium-independent in Col0. Furthermore, this plant line showed lower peak current densities compared to Col0 and almt mutants. These facts strongly suggest interaction between ALMT12 and ALMT13, with ALMT13 as a repressor of the ALMT12. Acquired patch clamp data revealed sulphate-dependent increases in ALMT13 current. This could be caused by changes in absolute open probability and/or permeability for sulphate and possibly chloride and links ALMT13 with sulphate-mediated stomatal closure under drought stress. It was then confirmed that ATP affects R-type currents. In contrast to Vicia faba, ATP was identified as a negative regulator of the Arabidopsis thaliana R-type anion channels. The effect of ATP is ambiguous but there is a high probability that it is a result of direct block and phosphorylation. However, the phosphorylation site and place of ATP binding needs further investigation. The story of the ALMT family, as examined in this thesis, sheds light on the complexity of the stomatal closure process.}, language = {en} } @phdthesis{Fei2023, author = {Fei, Lin}, title = {Optogenetic regulation of osmolarity and water flux}, doi = {10.25972/OPUS-32309}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323092}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Optogenetics is a powerful technique that utilizes light to precisely regulate physiological activities of neurons and other cell types. Specifically, light-sensitive ion channels, pumps or enzymes are expressed in cells to enable their regulation by illumination, thus allowing for precise control of biochemical signaling pathways. The first part of my study involved the construction, optimization, and characterization of two optogenetic tools, KCR1 and NCR1. Elena Govorunova et al. discovered a lightgated potassium channel, KCR1, in the protozoan Hyphochytrium catenoides. Traditional potassium ion channels are classified as either ligand-gated or voltage-gated and possess conserved pore-forming domains and K+ -selective filters. However, KCR1 is unique in that it does not contain the signature sequence of previously known K+ channels and is a channelrhodopsin. We synthesized the KCR1 plasmid according to the published sequence and expressed it in Xenopus oocytes. Due to the original KCR1 current being too small, I optimized it into KCR1 2.0 to improve its performance by fusing LR (signal peptide LucyRho, enhances expression) at the N-terminal and T (trafficking signal peptide) and E (ER export signal peptide) at the C-terminal. Additionally, I investigated the light sensitivity, action spectrum, and kinetics of KCR1 2.0 in Xenopus oocytes. The potassium permeability of KCR1 2.0, PK/Pna  24, makes KCR1 2.0 a powerful hyperpolarizing tool that can be used to inhibit neuronal firing in animals. Inspired by KCR1, we used the KCR1 sequence as a template for gene sequence alignment with the sequences in H. catenoides. We found that NCR1 and KCR1 have similar gene sequences. NCR1 was characterized by us as a light-gated sodium channel. This NCR1 was also characterized and published by Govorunova et al. very recently, with the name HcCCR. Due to the original NCR1 current being too small, I optimized it into NCR1 2.0 to improve its performance by fusing LR at the N-terminal and T and E at the C-terminal, which significantly improved the expression level and greatly increased the current amplitude of NCR1. Full-length NCR1 2.0 contains 432 amino acids. To test whether the number of amino acids changes the characteristics of NCR1 2.0, we designed NCR1 2.0 (330), NCR1 2.0 (283), and NCR1 2.0 (273) by retaining the number of amino acids at 330, 280, and 273 in NCR1 2.0, respectively. As the number of amino acids decreased, the current in NCR1 2.0 increased. I also investigated the light sensitivity, action spectrum, and kinetics of NCR1 2.0 (273) in the Xenopus Abstract 2 oocytes. We performed four point mutations at amino acid positions 133 and 116 of NCR1 2.0 and analyzed the reversal potentials of the mutants. The mutations were as follows: NCR1 2.0 (273 D116H), NCR1 2.0 (273 D116E), NCR1 2.0 (283 V133H), and NCR1 2.0 (283 D116Q). The second part of this study focuses on light-induced water transport using optogenetic tools. We explored the use of optogenetic tools to regulate water flow by changing the osmolarity in oocytes. Water flux through AQP1 is driven by the osmotic gradient that results from concentration differences of small molecules or ions. Therefore, we seek to regulate ion concentrations, using optogenetic tools to regulate the flux of water noninvasively. To achieve this, I applied the light-gated cation channels XXM 2.0 and NCR1 2.0 to regulate the concentration of Na+ , while K + channel KCR1 2.0 was used to regulate K + concentration. As Na+ flows into the Xenopus oocytes, the membrane potential of the oocytes becomes positive, and Clcan influx through the light-gated anion channel GtACR1. By combining these optogenetic tools to regulate NaCl or KCl concentrations, I can change the osmolarity inside the oocytes, thus regulating the flux of water. I co-expressed AQP1 with optogenetic tools in the oocytes to accelerate water flux. Overall, I designed three combinations (1: AQP1, XXM 2.0 and GtACR1. 2: AQP1, NCR1 2.0 and GtACR1. 3: AQP1, KCR1 2.0 and GtACR1) to regulate the flow of water in oocytes. The shrinking or swelling of the oocytes can only be achieved when AQP1, light-gated cation channels (XXM 2.0/NCR1 2.0/KCR1 2.0), and light-gated anion channels (GtACR1) are expressed together. The illumination after expression of either or both alone does not result in changes in oocyte morphology. In sum, I demonstrated a novel strategy to manipulate water movement into and out of Xenopus oocytes, non-invasively through illumination. These findings provide a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.}, subject = {Osmolarit{\"a}t}, language = {en} } @phdthesis{YuStrzelczyk2023, author = {Yu-Strzelczyk, Jing}, title = {Generation and Characterization of novel proteins for light-activated hyperpolarization of cell membranes}, doi = {10.25972/OPUS-26675}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266752}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The light-gated cation channel Channelrhodopsin-2 was discovered and characterized in 2003. Already in 2005/2006 five independent groups demonstrated that heterologous expression of Channelrhodopsin-2 is a highly useful and simply applicable method for depolarizing and thereby activating nerve cells. The application of Channelrhodopsin-2 revolutionized neuroscience research and the method was then called optogenetics. In recent years more and more light-sensitive proteins were successfully introduced as "optogenetic tools", not only in neuroscience. Optogenetic tools for neuronal excitation are well developed with many different cation-conducting wildtype and mutated channelrhodopsins, whereas for inhibition of neurons in the beginning (2007) only hyperpolarizing ion pumps were available. The later discovered light-activated anion channels (anion channelrhodopsins) can be useful hyperpolarizers, but only at low cytoplasmic anion concentration. For this thesis, I optimized CsR, a proton-pumping rhodopsin from Coccomyxa subellipsoidea, which naturally shows a robust expression in Xenopus laevis oocytes and plant leaves. I improved the expression and therefore the photocurrent of CsR about two-fold by N-terminal modification to the improved version CsR2.0, without altering the proton pump function and the action spectrum. A light pulse hyperpolarised the mesophyll cells of CsR2.0-expressing transgenic tobacco plants (N. tabacum) by up to 20 mV from the resting membrane potential of -150 to -200 mV. The robust heterologous expression makes CsR2.0 a promising optogenetic tool for hyperpolarization in other organisms as well. A single R83H point-mutation converted CsR2.0 into a light-activated (passive) proton channel with a reversal potential close to the Nernst potential for intra-/extra-cellular H+ concentration. This light-gated proton channel is expected to become a further useful optogenetic tool, e.g. for analysis of pH-regulation in cells or the intercellular space. Ion pumps as optogenetic tools require high expression levels and high light intensity for efficient pump currents, whereas long-term illumination may cause unwanted heating effects. Although anion channelrhodopsins are effective hyperpolarizing tools in some cases, their effect on neuronal activity is dependent on the cytoplasmic chloride concentration which can vary among neurons. In nerve cells, increased conductance for potassium terminates the action potential and K+ conductance underlies the resting membrane potential in excitable cells. Therefore, several groups attempted to synthesize artificial light-gated potassium channels but 2 all of these published innovations showed serious drawbacks, ranging from poor expression over lacking reversibility to poor temporal precision. A highly potassium selective light-sensitive silencer of action potentials is needed. To achieve this, I engineered a light-activated potassium channel by the genetic fusion of a photoactivated adenylyl cyclase, bPAC, and a cAMP-gated potassium channel, SthK. Illumination activates bPAC to produce cAMP and the elevated cAMP level opens SthK. The slow diffusion and degradation of cAMP makes this construct a very light-sensitive, long-lasting inhibitor. I have successfully developed four variants with EC50 to cAMP ranging from 7 over 10, 21, to 29 μM. Together with the original fusion construct (EC50 to cAMP is 3 μm), there are five different light- (or cAMP-) sensitive potassium channels for researchersto choose, depending on their cell type and light intensity needs.}, subject = {Proteine}, language = {en} } @phdthesis{Li2023, author = {Li, Kunkun}, title = {Dissecting the interconnection of Ca\(^{2+}\) and pH signaling in plants with a novel biosensor for dual imaging}, doi = {10.25972/OPUS-24973}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249736}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Calcium ion (Ca2+) and protons (H+) are both regarded as second messengers, participating in plant growth and stress mechanisms. However, H+ signals in plant physiology are less well investigated compared to Ca2+ signals. If interconnections between these two second messengers exist remains to be uncovered because appropriate imaging tools to monitor Ca2+ and H+ simultaneously in the same cell as well as accurate bioinformatics analysis remain to be developed. To overcome this problem and unravel the role and possible interconnection of Ca2+ and H+ in plants, a new biosensor named CapHensor was developed and optimized to visualize intracellular Ca2+ and H+ changes simultaneously and ratiometrically in the same cell. The CapHensor consisted of an optimized green fluorescent pH sensor (PRpHluorin) and an established red fluorescent Ca2+ sensor (R-GECO1) that were combined in one construct via a P2A sequence. A P2A self-cleavage site between the two sensors allowed to express equal amounts but spatially separated sensors, which enabled artifact-free and ratiometric imaging of cellular Ca2+ and pH side-by-side. The function of the CapHensor was verified in pollen tubes, since they possess standing Ca2+ and pH gradients. We found better imaging quality and the signal-to-noise ratio to be enhanced in live-cell imaging when two R-GECO1 proteins were fused in tandem within the CapHensor construct. To guarantee exclusive subcellular localization and avoid mixed signals from different compartments, Nuclear Export Sequence (NES) and Nuclear Localization Sequence (NLS) were used to target PRpHluorin and R-GECO1 to distinct compartments. After optimization and verification its function, CapHensor was successfully expressed in different cell types to investigate the role of Ca2+ and H+ signals to control polar growth of pollen tube, stomatal movement or leaf defense signaling. Results obtained in the past indicated both Ca2+ gradients and pH gradients in pollen tubes play roles in polar growth. However, the role and temporal relationship between the growth process and changes in Ca2+ and pH have not been conclusively resolved. Using CapHensor, I found cytosolic acidification at the tip could promote and alkalization to suppress growth velocity in N. tabacum pollen tubes, indicating that cytosolic H+ concentrations ([H+]cyt) play an important role in regulation pollen tubes growth despite the accompanied changes in cytosolic Ca2+ concentrations ([Ca2+]cyt). Moreover, growth correlated much better with the tip [H+]cyt regime than with the course of the tip [Ca2+]cyt regime. However, surprisingly, tip-focused [Ca2+]cyt andII [H+]cyt oscillations both lagged behind growth oscillations approximately 33 s and 18 s, respectively, asking for a re-evaluation of the role that tip [Ca2+]cyt may play in pollen tube growth. Live-cell CapHensor imaging combined with electrophysiology uncovered that oscillatory membrane depolarization correlated better with tip [H+]cyt oscillations than with tip [Ca2+]cyt oscillations, indicative for a prominent role of [H+]cyt to also control electrogenic membrane transport. Using CapHensor, reading out cellular movement at the same time enabled to provide a precise temporal and spatial resolution of ion signaling events, pointing out a prominent role of [H+]cyt in pollen tube tip growth. For leaf cells, a special CapHensor construct design had to be developed, containing additional NES localization sequences to avoid overlapping of fluorescense signals from the nucleus and the cytosol. Once this was achieved, the role of Ca2+ and pH changes in guard cells, another typical single-cell system was investigated. Cytosolic pH changes have been described in stomatal movement, but the physiological role of pH and the interaction with changing Ca2+ signals were still unexplored. Combining CapHensor with the here developed technique to monitor stomatal movement in parallel, the role of Ca2+ and H+ in stomatal movement was studied in detail and novel aspects were identified. The phytohormone ABA and the bacterial elicitor flagellin (flg22) are typical abiotic and biotic stresses, respectively, to trigger stomatal closure. What kind of Ca2+ and H+ signals by ABA and flg22 are set-off in guard cells and what their temporal relationship and role for stomatal movement is were unknown. Similar [Ca2+]cyt increases were observed upon ABA and flg22 triggered stomatal closure, but [H+]cyt dynamics differed fundamentally. ABA triggered pronounced cytosolic alkalization preceded the [Ca2+]cyt responses significantly by 57 s while stomata started to close ca. 205 s after phytohormone application. With flg22, stomatal closure was accompanied only with a mild cytosolic alkalization but the [Ca2+]cyt response was much more pronounced compared to the ABA effects. Where the cytosolic alkalization originates from was unclear but the vacuole was speculated to contribute in the past. In this thesis, vacuolar pH changes were visualized by the dye BCECF over time, basically displaying exactly the opposite course of the concentration shift in the vacuole than observed in the cytosol. This is indicative for the vacuolar pH dynamics to be coupled strongly to the cytosolic pH changes. In stomatal closure signalling, reactive oxygen species (ROS) were proposed to play a major role, however, only very high concentration of H2O2 (> 200 µM), which resulted in the loss of membrane integrity, induced stomatal closure. Unexpectedly, physiological concentrations of ROS led to cytosolic acidificationIII which was associated with stomatal opening, but not stomatal closure. To study the role of [H+]cyt to steer stomatal movement in detail, extracellular and intracellular pH variations were evoked in N. tabacum guard cells and their behaviour was followed. The results demonstrated cytosolic acidification stimulated stomatal opening while cytosolic alkalization triggered stomatal closure accompanied by [Ca2+]cyt elevations. This demonstrated pH regulation to be an important aspect in stomatal movement and to feed-back on the Ca2+-dynamics. It was remarkable that cytosolic alkalization but not [Ca2+]cyt increase seemed to play a crucial role in stomatal closure, because more pronounced cytosolic alkalization, evoked stronger stomatal closure despite similar [Ca2+]cyt increases. Increases in [Ca2+]cyt, which are discussed as an early stomatal closure signal in the past, could not trigger stomatal closure alone in my experiments, even when extremely strong [Ca2+]cyt signals were triggered. Regarding the interaction between the two second messengers, [Ca2+]cyt and [H+]cyt were negatively correlated most of the times, which was different from pollen tubes showing positive correlation of [Ca2+]cyt and [H+]cyt regimes. [Ca2+]cyt elevations were always associated with a cytosolic alkalization and this relationship could be blocked by the presence of vanadate, a plasma membrane H+-pump blocker, indicating plasma membrane H+-ATPases to contribute to the negative correlation of [Ca2+]cyt and [H+]cyt. To compare with guard cells, cytosolic and nuclear versions of CapHensor were expressed in N. benthamiana mesophyll cells, a multicellular system I investigated. Mesophyll cell responses to the same stimuli as tested in guard cells demonstrated that ABA and H2O2 did not induce any [Ca2+]cyt and [H+]cyt changes while flg22 induced an increase in [Ca2+]cyt and [H+]cyt, which is different from the response in guard cells. I could thus unequivocally demonstrate that guard cells and mesophyll cells do respond differently with [Ca2+]cyt and [H+]cyt changes to the same stimuli, a concept that has been proposed before, but never demonstrated in such detail for plants. Spontaneous Ca2+ oscillations have been observed for a long time in guard cells, but the function or cause is still poorly understood. Two populations of oscillatory guard cells were identified according to their [Ca2+]cyt and [H+]cyt phase relationship in my study. In approximately half of the oscillatory cells, [H+]cyt oscillations preceded [Ca2+]cyt oscillations whereas [Ca2+]cyt was the leading signal in the other half of the guard cells population. Strikingly, natural [H+]cyt oscillations were dampened by ABA but not by flg22. This effect could be well explained by dampening of vacuolar H+ oscillations in the presence of ABA, but not through flg22. Vacuolar pH contributes to spontaneous [H+]cyt oscillations and ABA but not flg22 can block the interdependence of naturalIV [Ca2+]cyt and [H+]cyt signals. To study the role of [Ca2+]cyt oscillations in stomatal movement, solutions containing high and low KCl concentrations were applied aiming to trigger [Ca2+]cyt oscillations. The triggering of [Ca2+]cyt oscillations by this method was established two decades ago leading to the dogma that [Ca2+]cyt increases are the crucial signal for stomatal closure. However, I found stomatal movement by this method was mainly due to osmotic effects rather than [Ca2+]cyt increases. Fortunately, through this methodology, I found a strong correlation between cytosolic pH and the transport of potassium across the plasma membrane and vacuole existed. The plasma membrane H+-ATPases and H+-coupled K+ transporters were identified as the cause of [H+]cyt changes, both very important aspects in stomata physiology that were not visualized experimentally before. Na+ transport is also important for stomatal regulation and leaves generally since salt can be transported from the root to the shoot. Unlike well-described Ca2+- dependent mechanisms in roots, how leaves process salt stress is not at all understood. I applied salt on protoplasts from leaves, mesophyll cells and guard cells and combined live-cell imaging with Vm recordings to understand the transport and signaling for leaf cells to cope with salt stress. In both, mesophyll and guard cells, NaCl did not trigger Ca2+-signals as described for roots but rather triggered Ca2+ peaks when washing salt out. However, membrane depolarization and pronounced alkalinization were very reliably triggered by NaCl, which could presumably act as a signal for detoxification of high salt concentrations. In line with this, I found the vacuolar cation/H+ antiporter NHX1 to play a role in sodium transport, [H+]cyt homeostasis and the control of membrane potential. Overexpression of AtNHX1 enabled to diminish [H+]cyt changes and resulted in a smaller depolarization responses druing NaCl stress. My results thus demonstrated in contrast to roots, leaf cells do not use Ca2+-dependent signalling cascades to deal with salt stress. I could show Na+ and K+ induced [H+]cyt and Vm responses and Cl- transport to only have a minor impact. Summing all my results up briefly, I uncovered pH signals to play important roles to control pollen tube growth, stomatal movement and leaf detoxification upon salt. My results strongly suggested pH changes might be a more important signal than previously thought to steer diverse processes in plants. Using CapHensor in combination with electrophysiology and bioinformatics tools, I discovered distinct interconnections between [Ca2+]cyt and [H+]cyt in different cell types and distinct [Ca2+]cyt and [H+]cyt signals are initiated through diverse stimuli and environmental cues. The CapHensor will be very useful in the future to further investigate the coordinated role of Ca2+ and pH changes in controlling plant physiology.}, subject = {Pflanzen}, language = {en} } @phdthesis{SchliermanngebStratmann2023, author = {Schliermann [geb. Stratmann], Anna Theresa}, title = {The Role of FGF Receptor 2 in GDF5 mediated Signal Transduction}, doi = {10.25972/OPUS-19288}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192889}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Bone morphogenetic proteins (BMPs) are involved in various aspects of cell-cell communication in complex life forms. They act as morphogens, help differentiate different cell types from different progenitor cells in development, and are involved in many instances of intercellular communication, from forming a body axis to healing bone fractures, from sugar metabolism to angiogenesis. If the same protein or protein family carries out many functions, there is a demand to regulate and fine-tune their biological activities, and BMPs are highly regulated to generate cell- and context-dependent outcomes. Not all such instances can be explained yet. Growth/differentiation factor (GDF)5 (or BMP14) synergizes with BMP2 on chondrogenic ATDC5 cells, but antagonizes BMP2 on myoblastic C2C12 cells. Known regulators of BMP2/GDF5 signal transduction failed to explain this context-dependent difference, so a microarray was performed to identify new, cell-specific regulatory components. One identified candidate, the fibroblast growth factor receptor (FGFR)2, was analyzed as a potential new co-receptor to BMP ligands such as GDF5: It was shown that FGFR2 directly binds BMP2, GDF5, and other BMP ligands in vitro, and FGFR2 was able to positively influence BMP2/GDF5-mediated signaling outcome in cell-based assays. This effect was independent of FGFR2s kinase activity, and independent of the downstream mediators SMAD1/5/8, p42/p44, Akt, and p38. The elevated colocalization of BMP receptor type IA and FGFR2 in the presence of BMP2 or GDF5 suggests a signaling complex containing both receptors, akin to other known co-receptors of BMP ligands such as repulsive guidance molecules. This unexpected direct interaction between FGF receptor and BMP ligands potentially opens a new category of BMP signal transduction regulation, as FGFR2 is the second receptor tyrosine kinase to be identified as BMP co-receptor, and more may follow. The integration of cell surface interactions between members of the FGF and BMP family especially may widen the knowledge of such cellular communication mechanisms which involve both growth factor families, including morphogen gradients and osteogenesis, and may in consequence help to improve treatment options in osteochodnral diseases.}, subject = {Molekularbiologie}, language = {en} } @article{SexauerBhasinSchoenetal.2023, author = {Sexauer, Moritz and Bhasin, Hemal and Sch{\"o}n, Maria and Roitsch, Elena and Wall, Caroline and Herzog, Ulrike and Markmann, Katharina}, title = {A micro RNA mediates shoot control of root branching}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-43738-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357472}, year = {2023}, abstract = {Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots.}, language = {en} } @article{AmatobiOzbekUnalSchaebleretal.2023, author = {Amatobi, Kelechi M. and Ozbek-Unal, Ayten Gizem and Sch{\"a}bler, Stefan and Deppisch, Peter and Helfrich-F{\"o}rster, Charlotte and Mueller, Martin J. and Wegener, Christian and Fekete, Agnes}, title = {The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition}, series = {Journal of Lipid Research}, volume = {64}, journal = {Journal of Lipid Research}, number = {10}, doi = {10.1016/j.jlr.2023.100417}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349961}, pages = {100417}, year = {2023}, abstract = {Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.}, language = {en} } @article{LuDreyerDickinsonetal.2023, author = {Lu, Jinping and Dreyer, Ingo and Dickinson, Miles Sasha and Panzer, Sabine and Jaślan, Dawid and Navarro-Retamal, Carlos and Geiger, Dietmar and Terpitz, Ulrich and Becker, Dirk and Stroud, Robert M. and Marten, Irene and Hedrich, Rainer}, title = {Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole two pore channels}, series = {eLife}, volume = {12}, journal = {eLife}, doi = {10.7554/eLife.86384}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350264}, year = {2023}, abstract = {To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.}, language = {en} } @article{ThomasFiebigKuhnetal.2023, author = {Thomas, Sarah and Fiebig, Juliane E. and Kuhn, Eva-Maria and Mayer, Dominik S. and Filbeck, Sebastian and Schmitz, Werner and Krischke, Markus and Gropp, Roswitha and Mueller, Thomas D.}, title = {Design of glycoengineered IL-4 antagonists employing chemical and biosynthetic glycosylation}, series = {ACS Omega}, volume = {8}, journal = {ACS Omega}, number = {28}, issn = {2470-1343}, doi = {10.1021/acsomega.3c00726}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350278}, pages = {24841-24852}, year = {2023}, abstract = {Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.}, language = {en} } @article{FaistAnkenbrandSickeletal.2023, author = {Faist, Hanna and Ankenbrand, Markus J. and Sickel, Wiebke and Hentschel, Ute and Keller, Alexander and Deeken, Rosalia}, title = {Opportunistic bacteria of grapevine crown galls are equipped with the genomic repertoire for opine utilization}, series = {Genome Biology and Evolution}, volume = {15}, journal = {Genome Biology and Evolution}, number = {12}, doi = {10.1093/gbe/evad228}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350172}, year = {2023}, abstract = {Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall-specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.}, language = {en} } @phdthesis{Muralidhara2022, author = {Muralidhara, Prathibha}, title = {Perturbations in plant energy homeostasis alter lateral root plasticity via SnRK1-bZIP63-ARF19 signalling}, doi = {10.25972/OPUS-20563}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205636}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Photosynthetic plants have a remarkable ability to modify their metabolism and development according to ever changing environmental conditions. The root system displays continuous growth of the primary root and formation of lateral roots enabling efficient water and nutrient uptake and anchorage of the plant in soil. With regard to lateral roots, development is post-embryonic, originating from the pericycle of the primary root. Coordinated activity of several molecular signalling pathways controlled by the hormone auxin is important throughout all stages of lateral root development.At first, two adjacent Xylem Pole Pericycle (XPP) cells are activated and the nuclei of these cells migrate towards a common cell wall.This is followed by XPP cells acquiring volume thus swelling up.The XPP cells then undergo anticlinal cell division, followed by a series of periclinal and anticlinal divisions,leading to lateral root primordia.These break through the radial cell layers and emerge out the primary root. Although root system plasticity is well-described in response to environmental cues such as ion nutrition in the soil, little is known on how root development is shaped according to the endogenous energy status of the plant.In this study, we were able to connect limited perturbations in photosynthetic energy supply to lateral root development.We established two experimental systems - treatment with low light and unexpected darkness which led to short-term energy imbalance in the plant.These short perturbations administered, showed an increase in the emerged lateral root density and decrease in root hexose availability and activation of the low energy marker gene ASN1 (ASPARAGINE SYNTHETASE 1).Although not demonstrated, presumably, these disturbances in the plant energy homeo-stasis activates SnRK1 (SNF1 RELATED KINASE 1),an evolutionary conserved kinase mediat-ing metabolic and transcriptional responses towards low energy conditions. In A. thaliana, two catalytic α-subunits of this kinase (SnRK1.α1 and SnRK1.α2) are functionally active and form ternary complexes with the regulatory β- and γ- subunits. Whereas unexpected darkness results in an increase in emerged lateral root density, the snrk1.α1 loss-of-function mutant displayed decrease in emerged lateral root density. As this effect is not that pronounced in the snrk1.α2 loss-of-function mutant, the α1 catalytic subunit is important for the observed lateral root phenotype under short-term energy perturbations. Moreover, root expression patterns of SnRK1.α1:GFP supports a role of this catalytic subunit in lateral root development. Furthermore, the lateral root response during short-term perturbations requires the SnRK1 downstream transcriptional regulator bZIP63 (BASIC LEU-CINE ZIPPER 63), as demonstrated here by a loss-of-function approach. Phenotypic studies showed that in comparison to wild-type, bzip63 mutants displayed decreased lateral root density upon low-light and unexpected darkness conditions. Previous work has demonstrat-ed that SnRK1 directly phosphorylates bZIP63 at three serine residues. Alanine-exchange mutants of the SnRK1 dependent bZIP63 phosphorylation sites behave similarly to bzip63 loss-of-function mutants and do not display increased lateral root density upon short-term unexpected darkness. This data strongly supports an impact of SnRK1-bZIP63 signalling in mediating the observed lateral root density phenotype. Plants expressing a bZIP63:YFP fu-sion protein showed specific localization patterns in primary root and in all developmental stages of the lateral root. bzip63 loss-of-function mutant lines displayed reduced early stage lateral root initiation events under unexpected darkness as demonstrated by Differen-tial Interference Contrast microscopy (DIC) and the use of a GATA23 reporter line. This data supports a role of bZIP63 in early lateral root initiation. Next, by employing Chromatin Immunoprecitation (ChIP) sequencing, we were able to iden-tify global binding targets of bZIP63, including the auxin-regulated transcription factor (TF) ARF19 (AUXIN RESPONSE FACTOR 19), a well-described central regulator of lateral root development. Additional ChIP experiments confirmed direct binding of bZIP63 to an ARF19 promoter region harboring a G-Box cis-element, a well-established bZIP63 binding site. We also observed that short-term energy perturbation upon unexpected darkness induced tran-scription of ARF19, which was impaired in the bzip63 loss-of-function mutant. These results propose that bZIP63 mediates lateral root development under short-term energy perturba-tion via ARF19. In conclusion, this study provides a novel mechanistic link between energy homeostasis and plant development. By employing reverse genetics, confocal imaging and high-throughput sequencing strategies, we were able to propose a SnRK1-bZIP63-ARF19 signalling module in integrating energy signalling into lateral root developmental programs.}, subject = {Arabidopsis thaliana}, language = {en} } @phdthesis{Yang2022, author = {Yang, Shang}, title = {Characterization and engineering of photoreceptors with improved properties for optogenetic application}, doi = {10.25972/OPUS-20527}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205273}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Optogenetics became successful in neuroscience with Channelrhodopsin-2 (ChR2), a light-gated cation channel from the green alga Chlamydomonas reinhardtii, as an easy applicable tool. The success of ChR2 inspired the development of various photosensory proteins as powerful actuators for optogenetic manipulation of biological activity. However, the current optogenetic toolbox is still not perfect and further improvements are desirable. In my thesis, I engineered and characterized several different optogenetic tools with new features. (i) Although ChR2 is the most often used optogenetic actuator, its single-channel conductance and its Ca2+ permeability are relatively low. ChR2 variants with increased Ca2+ conductance were described recently but a further increase seemed possible. In addition, the H+ conductance of ChR2 may lead to cellular acidification and unintended pH-related side effects upon prolonged illumination. Through rational design, I developed several improved ChR2 variants with larger photocurrent, higher cation selectivity, and lower H+ conductance. (ii) The light-activated inward chloride pump NpHR is a widely used optogenetic tool for neural silencing. However, pronounced inactivation upon long time illumination constrains its application for long-lasting neural inhibition. I found that the deprotonation of the Schiff base underlies the inactivation of NpHR. Through systematically exploring optimized illumination schemes, I found illumination with blue light alone could profoundly increase the temporal stability of the NpHR-mediated photocurrent. A combination of green and violet light eliminates the inactivation effect, similar to blue light, but leading to a higher photocurrent and therefore better light-induced inhibition. (iii) Photoactivated adenylyl cyclases (PACs) were shown to be useful for light-manipulation of cellular cAMP levels. I developed a convenient in-vitro assay for soluble PACs that allows their reliable characterization. Comparison of different PACs revealed that bPAC from Beggiatoa is the best optogenetic tool for cAMP manipulation, due to its high efficiency and small size. However, a residual activity of bPAC in the dark is unwanted and the cytosolic localization prevents subcellular precise cAMP manipulation. I therefore introduced point mutations into bPAC to reduce its dark activity. Interestingly, I found that membrane targeting of bPAC with different linkers can remarkably alter its activity, in addition to its localization. Taken together, a set of PACs with different activity and subcellular localization were engineered for selection based on the intended usage. The membrane-bound PM-bPAC 2.0 with reduced dark activity is well-tolerated by hippocampal neurons and reliably evokes a transient photocurrent, when co-expression with a CNG channel. (iv) Bidirectional manipulation of cell activity with light of different wavelengths is of great importance in dissecting neural networks in the brain. Selection of optimal tool pairs is the first and most important step for dual-color optogenetics. Through N- and C-terminal modifications, an improved ChR variant (i.e. vf-Chrimson 2.0) was engineered and selected as the red light-controlled actuator for excitation. Detailed comparison of three two-component potassium channels, composed of bPAC and the cAMP-activated potassium channel SthK, revealed the superior properties of SthK-bP. Combining vf-Chrimson 2.0 and improved SthK-bP "SthK(TV418)-bP" could reliably induce depolarization by red light and hyperpolarization by blue light. A residual tiny crosstalk between vf-Chrimson 2.0 and SthK(TV418)-bP, when applying blue light, can be minimized to a negligible level by applying light pulses or simply lowering the blue light intensity.}, language = {en} } @phdthesis{Staiger2022, author = {Staiger, Simona}, title = {Chemical and physical nature of the barrier against active ingredient penetration into leaves: effects of adjuvants on the cuticular diffusion barrier}, doi = {10.25972/OPUS-19937}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199375}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Agrochemicals like systemic active ingredients (AI) need to penetrate the outermost barrier of the plant, known as the plant cuticle, to reach its right target site. Therefore, adjuvants are added to provide precise and efficient biodelivery by i.a. modifying the cuticular barrier and increasing the AI diffusion. This modification process is depicted as plasticization of the cuticular wax which mainly consists of very long-chain aliphatic (VLCA) and cyclic compounds. Plasticization of cuticular waxes is pictured as an increase of amorphous domains and/or a decrease of crystalline fractions, but comprehensive, experimental proof is lacking to date. Hence, the objective of this thesis was to i) elucidate the permeation barrier of the plant cuticle to AIs in terms of the different wax fractions and ii) holistically investigate the modification of this barrier using selected oil and surface active adjuvants, an aliphatic leaf wax and an artificial model wax. Therefore, the oil adjuvant methyl oleate (MeO) and other oil derivatives like methyl linolenate (MeLin), methyl stearate (MeSt) and oleic acid (OA) were selected. Three monodisperse, non-ionic alcohol ethoxylates with increasing ethylene oxide monomer (EO) number (C10E2, C10E5, C10E8) were chosen as representatives of the group of surface active agents (surfactants). Both adjuvant classes are commonly used as formulation aids for agrochemicals which are known for its penetration enhancing effect. The aliphatic leaf wax of Schefflera elegantissima was selected, as well as a model wax comprising the four most abundant cuticular wax compounds of this species. Permeation, transpiration and penetration studies were conducted using enzymatically isolated cuticles of Prunus laurocerasus and Garcinia xanthochymus. Cuticular permeability to the three organic solutes theobromine, caffeine and azoxystrobin differing in lipophilicity was measured using a steady-state two-chamber system separated by the isolated leaf cuticles of the evergreen species P. laurocerasus and G. xanthochymus. Treating the isolated cuticles with methanol selectively removed the cyclic fraction, and membrane permeability to the organic compounds was not altered. In contrast, fully dewaxing the membranes using chloroform resulted in a statistically significant increase in permeance for all compounds and species, except caffeine with cuticles of G. xanthochymus due to a matrix-specific influence on the semi-hydrophilic compound. Crystalline regions may reduce the accessibility to the lipophilic pathway across the waxes and also block hydrophilic domains in the cuticle. Knowing that the aliphatic wax fraction builds the cuticular diffusion barrier, the influence of the adjuvants on the phase behaviour of an aliphatic cuticular wax as well as the influence on the cuticular penetration of AIs were investigated. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were selected to investigate the phase behaviour and thus possible plasticization of pure Schefflera elegantissima leaf wax, its artificial model wax comprising the four most abundant compounds (n-nonacosane, n-hentriacontane, 1-triacontanol and 1-dotriacontanol) and wax adjuvant mixtures. DSC thermograms showed a shift of the melting ranges to lower temperatures and decreased absolute values of the total enthalpy of transition (EOT) for all adjuvant leaf wax blends at 50 \% (w/w) adjuvant proportion. The highest decrease was found for C10E2 followed by MeO > OA and C10E8 > MeLin > MeSt. The aliphatic crystallinity determined by FTIR yielded declined values for the leaf and the artificial wax with 50 \% MeO. All other adjuvant leaf wax blends did not show a significant decrease of crystallinity. As it is assumed that the cuticular wax is formed by crystalline domains which consist of aliphatic hydrocarbon chains and an amorphous fraction comprising aliphatic chain ends and functional groups, the plasticizers are depicted as wax disruptors influencing amorphization and/or crystallization. The adjuvants can increase crystalline domains using the aliphatic tail whereas their more hydrophilic head is embedded in the amorphous wax fraction. DSC and FTIR showed similar trends using the leaf wax and the model wax in combination with the adjuvants. In general, cuticular transpiration increased after adding the pure adjuvants to the surface of isolated cuticles or leaf envelopes. As waxes build the cuticular permeation barrier not only to AIs but also to water, the adjuvant wax interaction might affect the cuticular barrier properties leading to increased transpiration. Direct evidence for increased AI penetration with the adjuvants was given using isolated cuticles of P. laurocerasus in combination with the non-steady-state setup simulation of foliar penetration (SOFP) and caffeine at relative humidity levels (RH) of 30, 50 and 80 \%. The increase in caffeine penetration was much more pronounced using C10E5 and C10E8 than MeO but always independent of RH. Only C10E2 exhibited an increased penetration enhancing effect positively related to RH. The role of the molecular structure of adjuvants in terms of humectant and plasticizer properties are discussed. Hence, the current work shows for the first time that the cuticular permeation barrier is associated with the VLCAs rather than the cyclic fraction and that adjuvants structurally influence this barrier resulting in penetration enhancing effects. Additionally, this work demonstrates that an artificial model wax is feasible to mimic the wax adjuvant interaction in conformity with a leaf wax, making it feasible for in-vitro experiments on a larger scale (e.g. screenings). This provides valuable knowledge about the cuticular barrier modification to enhance AI penetration which is a crucial factor concerning the optimization of AI formulations in agrochemistry.}, subject = {Adjuvans}, language = {en} } @article{LuebbeLamarqueDelzonetal.2022, author = {L{\"u}bbe, Torben and Lamarque, Laurent J. and Delzon, Sylvain and Torres Ruiz, Jos{\´e} M. and Burlett, R{\´e}gis and Leuschner, Christoph and Schuldt, Bernhard}, title = {High variation in hydraulic efficiency but not xylem safety between roots and branches in four temperate broad-leaved tree species}, series = {Functional Ecology}, volume = {36}, journal = {Functional Ecology}, number = {3}, issn = {0269-8463}, doi = {10.1111/1365-2435.13975}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318587}, pages = {699 -- 712}, year = {2022}, abstract = {Xylem hydraulic safety and efficiency are key traits determining tree fitness in a warmer and drier world. While numerous plant hydraulic studies have focused on branches, our understanding of root hydraulic functioning remains limited, although roots control water uptake, influence stomatal regulation and have commonly been considered as the most vulnerable organ along the hydraulic pathway. We investigated 11 traits related to xylem safety and efficiency along the hydraulic pathway in four temperate broad-leaved tree species. Continuous vessel tapering from coarse roots to stems and branches caused considerable reduction in hydraulic efficiency. Wood density was always lowest in roots, but did not decline linearly along the flow path. In contrast, xylem embolism resistance (P50) did not differ significantly between roots and branches, except for one species. The limited variation in xylem safety between organs did not adequately reflect the corresponding reductions in vessel diameter (by ~70\%) and hydraulic efficiency (by ~85\%). Although we did not observe any trade-off between xylem safety and specific conductivity, vessel diameter, vessel lumen fraction and wood density were related to embolism resistance, both across and partly within organs. We conclude that coarse roots are not highly vulnerable to xylem embolism as commonly believed, indicating that hydraulic failure during soil drying might be restricted to fine roots.}, language = {en} } @article{SchilcherHilsmannAnkenbrandetal.2022, author = {Schilcher, Felix and Hilsmann, Lioba and Ankenbrand, Markus J. and Krischke, Markus and Mueller, Martin J. and Steffan-Dewenter, Ingolf and Scheiner, Ricarda}, title = {Honeybees are buffered against undernourishment during larval stages}, series = {Frontiers in Insect Science}, volume = {2}, journal = {Frontiers in Insect Science}, issn = {2673-8600}, doi = {10.3389/finsc.2022.951317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304646}, year = {2022}, abstract = {The negative impact of juvenile undernourishment on adult behavior has been well reported for vertebrates, but relatively little is known about invertebrates. In honeybees, nutrition has long been known to affect task performance and timing of behavioral transitions. Whether and how a dietary restriction during larval development affects the task performance of adult honeybees is largely unknown. We raised honeybees in-vitro, varying the amount of a standardized diet (150 µl, 160 µl, 180 µl in total). Emerging adults were marked and inserted into established colonies. Behavioral performance of nurse bees and foragers was investigated and physiological factors known to be involved in the regulation of social organization were quantified. Surprisingly, adult honeybees raised under different feeding regimes did not differ in any of the behaviors observed. No differences were observed in physiological parameters apart from weight. Honeybees were lighter when undernourished (150 µl), while they were heavier under the overfed treatment (180 µl) compared to the control group raised under a normal diet (160 µl). These data suggest that dietary restrictions during larval development do not affect task performance or physiology in this social insect despite producing clear effects on adult weight. We speculate that possible effects of larval undernourishment might be compensated during the early period of adult life.}, language = {en} } @techreport{MuellerSchererLorenzenAmmeretal.2022, author = {M{\"u}ller, J{\"o}rg and Scherer-Lorenzen, Michael and Ammer, Christian and Eisenhauer, Nico and Seidel, Dominik and Schuldt, Bernhard and Biedermann, Peter and Schmitt, Thomas and K{\"u}nzer, Claudia and Wegmann, Martin and Cesarz, Simone and Peters, Marcell and Feldhaar, Heike and Steffan-Dewenter, Ingolf and Claßen, Alice and B{\"a}ssler, Claus and von Oheimb, Goddert and Fichtner, Andreas and Thorn, Simon and Weisser, Wolfgang}, title = {BETA-FOR: Erh{\"o}hung der strukturellen Diversit{\"a}t zwischen Waldbest{\"a}nden zur Erh{\"o}hung der Multidiversit{\"a}t und Multifunktionalit{\"a}t in Produktionsw{\"a}ldern. Antragstext f{\"u}r die DFG Forschungsgruppe FOR 5375}, doi = {10.25972/OPUS-29084}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290849}, pages = {210}, year = {2022}, abstract = {Der in j{\"u}ngster Zeit beobachtete kontinuierliche Verlust der β-Diversit{\"a}t in {\"O}kosystemen deutet auf homogene Gemeinschaften auf Landschaftsebene hin, was haupts{\"a}chlich auf die steigende Landnutzungsintensit{\"a}t zur{\"u}ckgef{\"u}hrt wird. Biologische Vielfalt ist mit zahlreichen Funktionen und der Stabilit{\"a}t von {\"O}kosystemen verkn{\"u}pft. Es ist daher zu erwarten, dass eine abnehmende β-Diversit{\"a}t auch die Multifunktionalit{\"a}t verringert. Wir kombinieren hier Fachwissen aus der Forstwissenschaft, der {\"O}kologie, der Fernerkundung, der chemischen {\"O}kologie und der Statistik in einem gemeinschaftlichen und experimentellen β-Diversit{\"a}tsdesign, um einerseits die Auswirkungen der Homogenisierung zu bewerten und andererseits Konzepte zu entwickeln, um negative Auswirkungen durch Homogenisierung in W{\"a}ldern r{\"u}ckg{\"a}ngig zu machen. Konkret werden wir uns mit der Frage besch{\"a}ftigen, ob die Verbesserung der strukturellen β-Komplexit{\"a}t (ESBC) in W{\"a}ldern durch Waldbau oder nat{\"u}rliche St{\"o}rungen die Biodiversit{\"a}t und Multifunktionalit{\"a}t in ehemals homogenen Produktionsw{\"a}ldern erh{\"o}hen kann. Unser Ansatz wird m{\"o}gliche Mechanismen hinter den beobachteten Homogenisierungs-Diversit{\"a}ts-Beziehungen identifizieren und zeigen, wie sich diese auf die Multifunktionalit{\"a}t auswirken. An elf Standorten in ganz Deutschland haben wir dazu zwei Waldbest{\"a}nde als zwei kleine "Waldlandschaften" ausgew{\"a}hlt. In einem dieser beiden Best{\"a}nde haben wir ESBC (Enhancement of Structural Beta Complexity)-Behandlungen durchgef{\"u}hrt. Im zweiten, dem Kontrollbestand, werden wir die gleich Anzahl 50x50m Parzellen ohne ESBC einrichten. Auf allen Parzellen werden wir 18 taxonomische Artengruppen aller trophischer Ebenen und 21 {\"O}kosystemfunktionen, einschließlich der wichtigsten Funktionen in W{\"a}ldern der gem{\"a}ßigten Zonen, messen. Der statistische Rahmen wird eine umfassende Analyse der Biodiversit{\"a}t erm{\"o}glichen, indem verschiedenen Aspekte (taxonomische, funktionelle und phylogenetische Vielfalt) auf verschiedenen Skalenebenen (α-, β-, γ-Diversit{\"a}t) quantifiziert werden. Um die Gesamtdiversit{\"a}t zu kombinieren, werden wir das Konzept der Multidiversit{\"a}t auf die 18 Taxa anwenden. Wir werden neue Ans{\"a}tze zur Quantifizierung und Aufteilung der Multifunktionalit{\"a}t auf α- und β-Skalen verwenden und entwickeln. Durch die experimentelle Beschreibung des Zusammenhangs zwischen β-Diversit{\"a}t und Multifunktionalit{\"a}t in einer Reallandschaft wird unsere Forschung einen neuen Weg einschlagen. Dar{\"u}ber hinaus werden wir dazu beitragen, verbesserte Leitlinien f{\"u}r waldbauliche Konzepte und f{\"u}r das Management nat{\"u}rlicher St{\"o}rungen zu entwickeln, um Homogenisierungseffekte der Vergangenheit umzukehren.}, subject = {Wald{\"o}kosystem}, language = {en} } @article{ScherzerHuangIosipetal.2022, author = {Scherzer, S{\"o}nke and Huang, Shouguang and Iosip, Anda and Kreuzer, Ines and Yokawa, Ken and Al-Rasheid, Khaled A. S. and Heckmann, Manfred and Hedrich, Rainer}, title = {Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap}, series = {Scientific reports}, volume = {12}, journal = {Scientific reports}, doi = {10.1038/s41598-022-06915-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300411}, year = {2022}, abstract = {Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap's prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.}, language = {en} } @article{KumarWaitePaligietal.2022, author = {Kumar, Manish and Waite, Pierre-Andr{\´e} and Paligi, Sharath Shyamappa and Schuldt, Bernhard}, title = {Influence of juvenile growth on xylem safety and efficiency in three temperate tree species}, series = {Forests}, volume = {13}, journal = {Forests}, number = {6}, issn = {1999-4907}, doi = {10.3390/f13060909}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278889}, year = {2022}, abstract = {The evolution of the internal water transport system was a prerequisite for high plant productivity. In times of climate change, understanding the dependency of juvenile growth on xylem hydraulic physiology is therefore of high importance. Here, we explored various wood anatomical, hydraulic, and leaf morphological traits related to hydraulic safety and efficiency in three temperate broadleaved tree species (Acer pseudoplatanus, Betula pendula, and Sorbus aucuparia). We took advantage of a severe natural heat wave that resulted in different climatic growing conditions for even-aged plants from the same seed source growing inside a greenhouse and outside. Inside the greenhouse, the daily maximum vapour pressure deficit was on average 36\% higher than outside during the growing seasons. Because of the higher atmospheric moisture stress, the biomass production differed up to 5.6-fold between both groups. Except for one species, a high productivity was associated with a high hydraulic efficiency caused by large xylem vessels and a large, supported leaf area. Although no safety-efficiency trade-off was observed, productivity was significantly related to P\(_{50}\) in two of the tree species but without revealing any clear pattern. A considerable plasticity in given traits was observed between both groups, with safety-related traits being more static while efficiency-related traits revealed a higher intra-specific plasticity. This was associated with other wood anatomical and leaf morphological adjustments. We confirm that a high hydraulic efficiency seems to be a prerequisite for a high biomass production, while our controversial results on the growth-xylem safety relationship confirm that safety-efficiency traits are decoupled and that their relationship with juvenile growth and water regime is species-specific.}, language = {en} } @phdthesis{vonRueden2022, author = {von R{\"u}den, Martin Frederik}, title = {The Venus flytrap - Role of oxylipins in trap performance of Dionaea muscipula}, doi = {10.25972/OPUS-27385}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-273854}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {A part of the plant kingdom consists of a variety of carnivorous plants. Some trap their prey using sticky leaves, others have pitfall traps where prey cannot escape once it has fallen inside. A rare trap type is the snap-trap: it appears only twice in the plant kingdom, in the genera Aldrovanda and Dionaea. Even Charles Darwin himself described Dionaea muscipula, the Venus flytrap, with the following words "This plant, commonly called Venus' fly-trap, from the rapidity and force of its movements, is one of the most wonderful in the world". For a long time now, the mechanisms of Dionaea's prey recognition, capture and utilization are of interest for scientists and have been studied intensively. Dionaea presents itself with traps wide-open, ready to catch insects upon contact. For this, the insect has to touch the trigger hairs of the opened trap twice within about 20-30 seconds. Once the prey is trapped, the trap lobes close tight, forming a hermetically sealed "green stomach". Until lately, there was only limited knowledge about the molecular and hormonal mechanisms which lead to prey capture and excretion of digestive fluids. It is known that the digestion process is very water-consuming; therefore, the interplay of digestion-inducing and digestion inhibiting substances was to be analyzed in this work, to elucidate the fine-tuning of the digestive pathway. Special attention was given to the impact of phytohormones on mRNA transcript levels of digestion-related proteins after various stimuli as well as their effect on Dionaea's physiological responses. Jasmonic acid (JA) and its isoleucine-conjugated form, JA-Ile, are an important signal in the jasmonate pathway. In the majority of non-carnivorous plants, jasmonates are critical for the defense against herbivory and pathogens. In Dionaea, this defense mechanism has been restructured towards offensive prey catching. One question in this work was how the frequency of trigger hair bendings is related to the formation of jasmonates and the induction of the digestion process. Upon contact of a prey with the trigger hairs in the inside of the trap, the trap closes and jasmonates are produced biosynthetically. JA-Ile interacts with the COI1- receptor, thereby activating the digestion pathway which leads to the secretion of digestive fluid and production of transporters needed to take up prey-derived nutrients. In this work it could be shown that the number of trigger hair bendings is positively correlated with the level and duration of transcriptional induction of several digestive enzymes/hydrolases. Abscisic acid (ABA) acts, along with many other functions, as the plant "drought stress hormone". It is synthesized either by roots as the primary sensor for water shortage or by guard cells in the leaves. ABA affects a network of several thousand genes whose regulation prepares the plant for drought and initiates protective measurements. It was known from previous work that the application of ABA for 48 hours increased the required amount of trigger hair bendings to achieve trap closure. As the digestion process is very water-intensive, the question arose how exactly the interplay between the jasmonate- and the ABA-pathway is organized, and if ABA could stop the running digestion process once it had been activated. In the present work it could be shown that the application of ABA on intact traps prior to mechanically stimulating the trigger hairs (mechanostimulation) already significantly reduced the transcription of digestive enzymes for an incubation time as short as 4 h, showing that already short-term exposure to ABA counteracts the effects of jasmonates when it comes to initiating the digestion process, but does not inhibit trap closure. Incubation for 24 and 48 hours with 100 μM active ABA had no effect on trap reopening, only very high levels of 200 μM of active ABA inhibited trap reopening but also led to tissue necrosis. As the application of ABA could reduce the transcription of digestive hydrolases, it is likely that Dionaea can stop the digestion process, if corresponding external stimuli are received. Another factor, which only emerged later, was the effect of the wounding-induced systemic jasmonate burst. As efficient as ABA was in inhibiting marker hydrolase expression after mechanostimulation in intact plants, the application of ABA on truncated traps was not able to inhibit mechanostimulation-induced marker hydrolase expression. One reason might be that the ABA-signal is perceived in the roots, and therefore truncated traps were not able to react to it. Another reason might be that the wounding desensitized the tissue for the ABAsignal. Further research is required at this point. Inhibitors of the jasmonate pathway were also used to assess their effect on the regulation of Dionaea´s hunting cycle. Coronatine-O-methyloxime proved to be a potent inhibitor of mechanostimulation-induced expression of digestive enzymes, thus confirming the key regulatory role of jasmonates for Dionaea´s prey consumption mechanism. In a parallel project, the generation of in vitro cultures from sterilized seeds and single plant parts proved successful, which may be important for stock-keeping of future transgenic lines. Protoplasts were generated from leaf blade tissue and transiently transformed, expressing the reporter protein YFP after 24 h of incubation. In the future this might be the starting point for the generation of transgenic lines or the functional testing of DNA constructs.}, subject = {Venusfliegenfalle}, language = {en} } @article{deSouzaRiedererLeide2022, author = {de Souza, Aline Xavier and Riederer, Markus and Leide, Jana}, title = {Multifunctional contribution of the inflated fruiting calyx: implication for cuticular barrier profiles of the solanaceous genera Physalis, Alkekengi, and Nicandra}, series = {Frontiers in Plant Science}, volume = {13}, journal = {Frontiers in Plant Science}, issn = {1664-462X}, doi = {10.3389/fpls.2022.888930}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280251}, year = {2022}, abstract = {Pivotal barrier properties of the hydrophobic plant cuticle covering aerial plant surfaces depend on its physicochemical composition. Among plant species and organs, compounds of this boundary layer between the plant interior and the environment vary considerably but cuticle-related studies comparing different organs from the same plant species are still scarce. Thus, this study focused on the cuticle profiles of Physalis peruviana, Physalis ixocarpa, Alkekengi officinarum, and Nicandra physalodes species. Inflated fruiting calyces enveloping fruits make Physalis, Alkekengi, and Nicandra highly recognizable genera among the Solanoideae subfamily. Although the inflation of fruiting calyces is well discussed in the literature still little is known about their post-floral functionalities. Cuticular composition, surface structure, and barrier function were examined and compared in fully expanded amphistomatous leaves, ripe astomatous fruits, and fully inflated hypostomatous fruiting calyces. Species- and organ-specific abundances of non-glandular and glandular trichomes revealed high structural diversity, covering not only abaxial and adaxial leaf surfaces but also fruiting calyx surfaces, whereas fruits were glabrous. Cuticular waxes, which limit non-stomatal transpiration, ranged from <1 μg cm\(^{-2}\) on P. peruviana fruiting calyces and N. physalodes fruits to 22 μg cm\(^{-2}\) on P. peruviana fruits. Very-long-chain aliphatic compounds, notably n-alkanes, iso-, and anteiso-branched alkanes, alkanols, alkanoic acids, and alkyl esters, dominated the cuticular wax coverages (≥86\%). Diversity of cuticular wax patterns rose from leaves to fruiting calyces and peaked in fruits. The polymeric cutin matrix providing the structural framework for cuticular waxes was determined to range from 81 μg cm\(^{-2}\) for N. physalodes to 571 μg cm\(^{-2}\) for A. officinarum fruits. Cuticular transpiration barriers were highly efficient, with water permeabilities being ≤5 × 10\(^{-5}\) m s\(^{-1}\). Only the cuticular water permeability of N. physalodes fruits was 10 × 10\(^{-5}\) m s\(^{-1}\) leading to their early desiccation and fruits that easily split, whereas P. peruviana, P. ixocarpa, and A. officinarum bore fleshy fruits for extended periods after maturation. Regarding the functional significance, fruiting calyces establish a physicochemical shield that reduces water loss and enables fruit maturation within a protective microclimate, and promotes different seed dispersal strategies among plant species investigated.}, language = {en} } @article{LambourGlenzForneretal.2022, author = {Lambour, Benjamin and Glenz, Ren{\´e} and Forner, Carmen and Krischke, Markus and Mueller, Martin J. and Fekete, Agnes and Waller, Frank}, title = {Sphingolipid long-chain base phosphate degradation can be a rate-limiting step in long-chain base homeostasis}, series = {Frontiers in Plant Science}, volume = {13}, journal = {Frontiers in Plant Science}, issn = {1664-462X}, doi = {10.3389/fpls.2022.911073}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-277679}, year = {2022}, abstract = {Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels.}, language = {en} } @article{SiverinoFahmyGarciaMumcuogluetal.2022, author = {Siverino, Claudia and Fahmy-Garcia, Shorouk and Mumcuoglu, Didem and Oberwinkler, Heike and Muehlemann, Markus and Mueller, Thomas and Farrell, Eric and van Osch, Gerjo J. V. M. and Nickel, Joachim}, title = {Site-directed immobilization of an engineered bone morphogenetic protein 2 (BMP2) variant to collagen-based microspheres induces bone formation in vivo}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {7}, issn = {1422-0067}, doi = {10.3390/ijms23073928}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284572}, year = {2022}, abstract = {For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.}, language = {en} } @article{WeithmannLinkBanzragchetal.2022, author = {Weithmann, Greta and Link, Roman M. and Banzragch, Bat-Enerel and W{\"u}rzberg, Laura and Leuschner, Christoph and Schuldt, Bernhard}, title = {Soil water availability and branch age explain variability in xylem safety of European beech in Central Europe}, series = {Oecologia}, volume = {198}, journal = {Oecologia}, number = {3}, doi = {10.1007/s00442-022-05124-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324228}, pages = {629-644}, year = {2022}, abstract = {Xylem embolism resistance has been identified as a key trait with a causal relation to drought-induced tree mortality, but not much is known about its intra-specific trait variability (ITV) in dependence on environmental variation. We measured xylem safety and efficiency in 300 European beech (Fagus sylvatica L.) trees across 30 sites in Central Europe, covering a precipitation reduction from 886 to 522 mm year-1. A broad range of variables that might affect embolism resistance in mature trees, including climatic and soil water availability, competition, and branch age, were examined. The average P50 value varied by up to 1 MPa between sites. Neither climatic aridity nor structural variables had a significant influence on P50. However, P50 was less negative for trees with a higher soil water storage capacity, and positively related to branch age, while specific conductivity (Ks) was not significantly associated with either of these variables. The greatest part of the ITV for xylem safety and efficiency was attributed to random variability within populations. We conclude that the influence of site water availability on P50 and Ks is low in European beech, and that the high degree of within-population variability for P50, partly due to variation in branch age, hampers the identification of a clear environmental signal.}, subject = {Bodenwasser}, language = {en} } @phdthesis{vonMeyer2021, author = {von Meyer, Katharina}, title = {Molecular characterization of defensin-like proteins in the fertilization process of \(Nicotiana\) \(tabacum\)}, doi = {10.25972/OPUS-19214}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192141}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Flowering plants or angiosperms have developed a fertilization mechanism that involves a female egg and central cell, as well as two male sperm cells. A male gametophyte carries the two non-mobile sperm cells, as they need to be delivered to the female gametophyte, the embryo sac. This transport is initiated by a pollen grain that is transmitted onto the stigma of the angiosperm flower. Here it hydrates, germinates, and forms a pollen tube, which navigates through the female plant tissue towards the ovary. The pollen tube grows into an ovule through the funiculus and into one of the two synergid cells. There, growth arrests and the pollen tube bursts, releasing the two sperm cells. One of the sperm cells fuses with the egg cell, giving rise to the embryo, the other one fuses with the central cell, developing into the endosperm, which nourishes the embryo during its development. After a successful fertilization, each ovule develops into a seed and a fruit is formed. This usually consists of several fertilized ovules. The directional growth of the pollen tube through the maternal tissues towards the ovule, as well as sperm cell release, requires a complex communication between the male and the female gametophyte to achieve reproductive success. Over the last years many studies have been performed, contributing to the understanding of cell-cell communication events between the two gametophytes, nevertheless still many aspects remain to be elucidated. This work focused on two topics: i.) Analysis of biological processes affected by pollination and fertilization in the Nicotiana tabacum flower and identification of cysteine rich proteins (CRPs) expressed via isolating and sequencing RNA from the tissue and analyzing the resulting data. ii.) Identification of the defensin-like protein (DEFL) responsible for pollen tube attraction towards the ovule in tobacco. First, tissue samples of pollen tubes and mature ovules were taken at different stages of the fertilization process (unpollinated ovules, after pollination, and after fertilization of the flower). RNA was then isolated and a transcriptome was created. The resulting reads were assembled and transcriptome data analysis was performed. Results showed that pollen tubes and mature ovules differ severely from each other, only sharing about 23 \% of the transcripts, indicating that different biological processes are dominant in the two gametophytes. A MapMan analysis revealed that in the pollen tube the most relevant biological processes are related to the cell wall, signaling, and transport, which supports the fact that the pollen tube grows fast to reach the ovule. On the other hand, in the ovule the values of highest significance were obtained for processes related to protein synthesis and regulation. Upon comparing the transcripts in the ovule before and after pollination, as well as after fertilization, it showed that pollination of the flower causes a bigger alteration in the ovule on the transcriptomic level compared to the step from pollination to fertilization. A total of 953 CRPs were identified in Nicotiana tabacum, including 116 DEFLs. Among those, the peptide responsible for pollen tube attraction towards the ovule should be found. Based on in-silico analysis four candidate peptides were chosen for further analysis, two of which had increased expression levels upon pollination and fertilization and the other two displayed an opposite expression. Quantitative real time PCR experiments were performed for the candidates, confirming the in-silico data in vivo. The candidate transcripts were then expressed in a cell free system and applied to pollen tubes in order to test their effect on the growing cells. Positive controls were used, where pollen tubes grew towards freshly dissected ovules. The four candidates did not provoke a pollen tube attraction towards the peptide, leaving open the chance to work on the 112 remaining DEFLs in the future.}, subject = {Samenpflanzen}, language = {en} } @phdthesis{Thomas2021, author = {Thomas, Sarah Katharina}, title = {Design of novel IL-4 antagonists employing site-specific chemical and biosynthetic glycosylation}, doi = {10.25972/OPUS-17517}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175172}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases. For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R. This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist. For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability. The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab.}, subject = {Glykosylierung}, language = {en} } @phdthesis{BergmannBueno2021, author = {Bergmann Bueno, Amauri}, title = {Ecophysiological adaptations of cuticular water permeability of plants to hot arid biomes}, doi = {10.25972/OPUS-16783}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167832}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Arid environments cover almost one-third of the land over the world. Plant life in hot arid regions is prone to the water shortage and associated high temperatures. Drought-stressed plants close the stomata to reduce water loss. Under such conditions, the remaining water loss exclusively happens across the plant cuticle. The cuticular water permeability equals the minimum and inevitable water loss from the epidermal cells to the atmosphere under maximally stomatal closure. Thus, low cuticular water permeability is primordial for plant survival and viability under limited water source. The assumption that non-succulent xerophytes retard water loss due to the secretion of a heavier cuticle is often found in the literature. Intuitively, this seems to be plausible, but few studies have been conducted to evaluate the cuticular permeability of xerophilous plants. In chapter one, we investigated whether the cuticular permeability of Quercus coccifera L. grown in the aridest Mediterranean-subtype climate is indeed lower than that of individuals grown under temperate climate conditions. Also, the cuticular wax chemical compositions of plants grown in both habitats were qualitatively and quantitatively analysed by gas-chromatography. In few words, our findings showed that although the cuticular wax deposition increased in plants under Mediterranean climate, the cuticular permeability remained unaltered, regardless of habitat. The associated high temperatures in arid regions can drastically increase the cuticular water permeability. Thereby, the thermal stability of the cuticular transpirational barrier is decisive for safeguarding non-succulent xerophytes against desiccation. The successful adaptation of plants to hot deserts might be based on finding different solutions to cope with water and heat stresses. Water-saver plants close the stomata before the leaf water potential drastically changes in order to prevent damage, whereas water-spender plants reduce the leaf water potential by opening the stomata, which allow them to extract water from the deep soil to compensate the high water loss by stomatal transpiration. In chapter two, we compare the thermal stability of the cuticular transpiration barrier of the desert water-saver Phoenix dactylifera L. and the water-spender Citrullus colocynthis (L.) Schrad. In short, the temperature-dependent increase of the cuticular permeability of P. dactylifera was linear over the whole temperature range (25-50°C), while that of C. colocynthis was biphasic with a steep increase at temperatures ≥ 40°C. This drastic increase of cuticular permeability indicates a thermally induced breakdown of the C. colocynthis cuticular transpiration barrier, which does not occur in P. dactylifera. We further discussed how the specific chemical composition of the cutin and cuticular waxes might contribute to the pronounced thermal resistance of the P. dactylifera cuticular transpiration barrier. A multitude of morpho and physiological modifications, including photosynthetic thermal tolerance and traits related to water balance, led to the successful plant colonisation of hot arid regions over the globe. High evaporative demand and elevated temperatures very often go along together, thereby constraining the plant life in arid environments. In chapter 3, we surveyed cuticular permeability, leaf thermal tolerance, and cuticular wax chemical composition of 14 non-succulent plant species native from some of the hottest and driest biomes in South-America, Europe, and Asia. Our findings showed that xerophilous flowering plants present high variability for cuticular permeability and leaf thermal tolerance, but both physiological features could not be associated with the species original habitat. We also provide substantial evidence that non-succulent xerophytes with more efficient cuticular transpirational barrier have higher leaf thermal tolerance, which might indicate a potential coevolution of these features in hot arid biomes. We further discussed the efficiency of the cuticular transpiration barrier in function to the cuticular wax chemical composition in the general discussion section.}, subject = {Plant cuticle}, language = {en} } @phdthesis{ScheideNoeth2021, author = {Scheide-N{\"o}th, Jan-Philipp}, title = {Activation of the Interleukin-5 receptor and its inhibition by cyclic peptides}, doi = {10.25972/OPUS-18250}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182504}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The cytokine interleukin-5 (IL-5) is part of the TH2-mediated immune response. As a key regulator of eosinophilic granulocytes (eosinophils), IL-5 controls multiple aspects of eosinophil life. Eosinophils play a pathogenic role in the onset and progression of atopic diseases as well as hypereosinophilic syndrome (HES). Here, cytotoxic proteins and pro-inflammatory mediators stored in intracellular vesicles termed granula are released upon activation thereby causing local inflammation to fight the pathogen. However, if such inflammation persists, tissue damage and organ failure can occur. Due to the close relationship between eosinophils and IL-5 this cytokine has become a major pharmaceutical target for the treatment of atopic diseases or HES. As observed with other cytokines, IL-5 signals by assembling a heterodimeric receptor complex at the cell surface in a stepwise mechanism. In the first step IL-5 binds to its receptor IL-5Rα (CD125). This membrane-located complex then recruits the so-called common beta chain βc (CD131) into a ternary ligand receptor complex, which leads to activation of intracellular signaling cascades. Based on this mechanism various strategies targeting either IL-5 or IL-5Rα have been developed allowing to specifically abrogate IL-5 signaling. In addition to the classical approach of employing neutralizing antibodies against IL 5/IL-5Rα or antagonistic IL-5 variants, two groups comprising small 18 to 30mer peptides have been discovered, that bind to and block IL-5Rα from binding its activating ligand IL-5. Structure-function studies have provided detailed insights into the architecture and interaction of IL-5IL-5Rα and βc. However, structural information for the ternary IL-5 complex as well as IL-5 inhibiting peptides is still lacking. In this thesis three areas were investigated. Firstly, to obtain insights into the second receptor activation step, i.e. formation of the ternary ligand-receptor complex IL-5•IL-5Rα•βc, a high-yield production for the extracellular domain of βc was established to facilitate structure determination of the ternary ligand receptor assembly by either X-ray crystallography or cryo-electron microscopy. In a second project structure analysis of the ectodomain of IL-5Rα in its unbound conformation was attempted. Data on IL-5Rα in its ligand-free state would provide important information as to whether the wrench-like shaped ectodomain of IL-5Rα adopts a fixed preformed conformation or whether it is flexible to adapt to its ligand binding partner upon interaction. While crystallization of free IL-5Rα failed, as the crystals obtained did not diffract X rays to high resolution, functional analysis strongly points towards a selection fit binding mechanism for IL-5Rα instead of a rigid and fixed IL-5Rα structure. Hence IL-5 possibly binds to a partially open architecture, which then closes to the known wrench-like architecture. The latter is then stabilized by interactions within the D1-D2 interface resulting in the tight binding of IL-5. In a third project X-ray structure analysis of a complex of the IL-5 inhibitory peptide AF17121 bound to the ectodomain of IL-5Rα was performed. This novel structure shows how the small cyclic 18mer peptide tightly binds into the wrench-like cleft formed by domains D1 and D2 of IL-5Rα. Due to the partial overlap of its binding site at IL-5Rα with the epitope for IL-5 binding, the peptide blocks IL-5 from access to key residues for binding explaining how the small peptide can effectively compete with the rather large ligand IL-5. While AF17121 and IL-5 seemingly bind to the same site at IL-5Rα, functional studies however showed that recognition and binding of both ligands differ. With the structure for the peptide-receptor complex at hand, peptide design and engineering could be performed to generate AF17121 analogies with enhanced receptor affinity. Several promising positions in the peptide AF17121 could be identified, which could improve inhibition capacity and might serve as a starting point for AF17121-based peptidomimetics that can yield either superior peptide based IL-5 antagonists or small-molecule-based pharmacophores for future therapies of atopic diseases or the hypereosinophilic syndrome.}, subject = {Interleukin 5}, language = {en} } @article{KarimiFreundWageretal.2021, author = {Karimi, Sohail M. and Freund, Matthias and Wager, Brittney M. and Knoblauch, Michael and Fromm, J{\"o}rg and M. Mueller, Heike and Ache, Peter and Krischke, Markus and Mueller, Martin J. and M{\"u}ller, Tobias and Dittrich, Marcus and Geilfus, Christoph-Martin and Alfaran, Ahmed H. and Hedrich, Rainer and Deeken, Rosalia}, title = {Under salt stress guard cells rewire ion transport and abscisic acid signaling}, series = {New Phytologist}, volume = {231}, journal = {New Phytologist}, number = {3}, doi = {10.1111/nph.17376}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259635}, pages = {1040-1055}, year = {2021}, abstract = {Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.}, language = {en} } @phdthesis{Kumari2021, author = {Kumari, Khushbu}, title = {The role of lipid transfer proteins (LTPs) during the fertilization process in Arabidopsis thaliana}, doi = {10.25972/OPUS-19961}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199613}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Double fertilization is a defining characteristic of flowering plants (angiosperms). As the sperm cells of higher plants are non-motile, they need to be transported to the female gametophyte via the growing pollen tube. The pollen-tube journey through the female tissues represents a highly complex process. To provide for successful reproduction it demands intricate communication between the cells of the two haploid gametophytes - the polar growing pollen tube (carrying the two non-motile sperm cells) and the ovule (hosting the egg cell/synergid cells). The polar growth of the pollen tube towards the female gamete is guided by different signaling molecules, including sugars, amino acids and peptides. Some of these belong to the family of lipid transfer proteins (LTPs), which are secreted cysteine-rich peptides. Depending on the plant species several lines of evidence have also suggested potential roles for LTPs during pollen germination or pollen-tube guidance. Although Arabidopsis thaliana has 49 annotated genes for LTPs, several of which are involved in plant immunity and cell-to-cell communication, the role of most members of this family during fertilization is unknown. The aim of this project was therefore to systematically identify LTPs which play a role in the fertilization process in A. thaliana, particularly during pollen tube guidance. To identify candidate proteins, the expression profile of LTPs in reproductive tissue was investigated. This was accomplished by in-silico bioinformatic analysis using different expression databases. Following confirmion of these results by qRT-PCR analysis, seven Type-I nsLTPs (LTP1, LTP2, LTP3, LTP4, LTP5, LTP6 and LTP12) were found to be exclusively expressed in pistils. Except for LTP12, all other pistil expressed LTPs were transcriptionally induced upon pollination. Using reporter-based transcriptional and translational fusions the temporal and spatial expression patterns together with protein localizations for LTP2, 3, 4, 5, 6, and 12 were determined in planta. Stable transgenic plants carrying PromLTP::GUS constructs of the six different LTP candidates showed that most of LTPs were expressed in the stigma/stylar region and were induced upon pollination. With respect to protein localization on the cellular level, they split into two categories: LTP2, LTP5 and LTP6 were localized in the cell wall, while LTP3, LTP4 and LTP12 were specifically targeted to the plasma membrane. For the functional characterization of the candidate LTPs, several T-DNA insertion mutant plant lines were investigated for phenotypes affecting the fertilization process. Pollen development and quality as well as their in-vitro germination rate did not differ between the different single ltp mutant lines and wildtype plants. Moreover, in-vivo cross pollination experiments revealed that tube growth and fertilization rate of the mutant plants were similar to wildtype plants. Altogether, no discernible phenotype was evident in other floral and vegetative parts between different single ltp mutant lines and wildtype plants. As there was no distinguishable phenotype observed for single ltp-ko plants, double knock out plants of the two highly homologous genes LTP2 (expressed in the female stigma, style and transmitting tract) and LTP5 (expressed in the stigma, style, pollen pollen-tube and transmitting tract) were generated using the EPCCRISPR-Cas9 genome editing technique. Two ltp2ltp5 mutant transgenic-lines (\#P31-P2 and \#P31-P3) with frameshift mutations in both the genes could be established. Further experiments showed, that the CRISPR/Cas9-mediated knock-out of LTP2/LTP5 resulted in significantly reduced fertilization success. Cell biological analyses revealed that the ltp2ltp5 double mutant was impaired in pollen tube guidance towards the ovules and that this phenotype correlated with aberrant callose depositions in the micropylar region during ovule development. Detailed analysis of in-vivo pollen-tube growth and reciprocal cross pollination assay suggested that, the severely compromised fertility was not caused by any defect in development of the pollen grains, but was due to the abnormal callose deposition in the embryo sac primarily concentrated at the synergid cell near the micropylar end. Aberrant callose deposition in ltp2ltp5 ovules pose a complete blockage for the growing pollen tube to change its polarity to enter the funiculus indicating funicular and micropylar defects in pollen tube guidance causing fertilization failure. Our finding suggests that female gametophyte expressed LTP2 and LTP5 play a crucial role in mediating pollen tube guidance process and ultimately having an effect on the fertilization success. In line with the existence of a N-terminal signal peptide, secreted LTPs might represent a well-suited mobile signal carrier in the plant's extracellular matrix. Previous reports suggested that, LTPs could act as chemoattractant peptide, imparting competence to the growing pollen tube, but the molecular mechanism is still obscure. The results obtained in this thesis further provide strong evidence, that LTP2/5 together regulate callose homeostasis and testable models are discussed. Future work is now required to elucidate the detailed molecular link between these LTPs and their potential interacting partners or receptors expressed in pollen and synergid cells, which should provide deeper insight into their functional role as regulatory molecules in the pollen tube guidance mechanism.}, subject = {Fertilization in angiosperm}, language = {en} } @phdthesis{Seufert2021, author = {Seufert, Pascal}, title = {Chemical and physical structure of the barrier against water transpiration of leaves: Contribution of different wax compounds}, doi = {10.25972/OPUS-20896}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208963}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The cuticle is constituted of the biopolymer cutin and intra- and epicuticular waxes. In some cases, it has epicuticular wax crystals, protruding from the epicuticular wax film. One of the most important tasks is protection against desiccation. Many investigations were conducted to find the transport limiting component of the cuticle. It is evidentially confirmed that the waxes form this barrier. These waxes are multifactorial blends made of very-long-chain aliphatic (VLCA) compounds and triterpenoids (TRP). The VLCAs were proposed to constitute the transpiration barrier to water. However, experimental confirmation was lacking so far. The present study focuses on the development of a method to selectively extract TRPs from the cuticle and the impact of the removal on the transpiration barrier. The plants deployed in this study exhibited several features. They had no epicuticular crystals on their surfaces, were astomatous, had a rather durable and possibly isolatable cuticle. A broad range of wax compositions was covered from plants with no TRP content and low wax load like Hedera helix and Zamioculcas zamiifolia to plants with high TRP content and high wax load like Nerium oleander. The selective extraction was conducted using a sequence of solvents. TRPs were extracted almost exhaustively from CMs with the first MeOH extract. Only a minor amount of shorter chained VLCAs was obtained. The remaining waxes, consisting mostly of VLCAs and some remnant TRPs, were removed with the following TCM extract. After the extractions, the water permeance of native cuticular membranes (CM), MeOH extracted (M) and dewaxed cuticular discs (MX) was investigated gravimetrically. Compared to the water permeance of CMs, Ms showed no or only a small increase in water conductance. MXs, however, always showed strongly increased values. The knowledge about the wax compounds constituting the transport-limiting properties is vital for different projects. For various issues, it would be favourable to have a standardized wax mixture as an initial point of research. It could be used to develop screening procedures to investigate the impact of adjuvants on cuticular waxes or the influence of wax constituents on the properties of cuticular waxes. This work concentrated on the development of an artificial wax mixture, which mimics the physical properties of a plant leaf wax sufficiently. As target wax, the leaf wax of Schefflera elegantissima was chosen. The wax of this plant species consisted almost exclusively of VLCAs, had a rather simple composition regarding compound classes and chain length distribution and CMs could be isolated. Artificial binary, ternary and quaternary waxes corresponding to the conditions within the plant wax were investigated using differential scanning calorimetry (DSC), X-ray diffraction (XRD) techniques and Fourier-transform infrared (FTIR) spectroscopy. Phase diagrams were mapped out for a series of binary, ternary and quaternary wax mixtures. FTIR experiments were conducted using, ternary and a quaternary artificial wax blends. The blends were chosen to represent the conditions within the wax of the adaxial CM plant wax. The FTIR experiments exhibited an increasing resemblance of the artificial wax to the plant wax (adaxial CM wax) with an increasing number of compounds in the artificial wax. The same trend was found for DSC thermograms. Thermograms of ternary and quaternary blends exhibited more overlapping peaks and occurred in a temperature range more similar to the range of the whole leaf plant wax. The XRD spectrum at room temperature showed good conformity with the quaternary blend. The current work illustrates a method for selective extraction of TRPs from isolated CMs. It gives direct experimental proof of the association of the water permeance barrier with the VLCA rather than to the TRPs. Furthermore, the possibility to mimic cuticular waxes using commercially available wax compounds is investigated. The results show promising feasibility for its viability, enabling it to perform as a standardized initial point for further research (e.g. to examine the influence of different constituents on waxes), revealing valuable knowledge about the structure and the chemistry-function relationship of cuticular waxes.}, subject = {Kutikula}, language = {en} } @article{SchilcherHilsmannRauscheretal.2021, author = {Schilcher, Felix and Hilsmann, Lioba and Rauscher, Lisa and Değirmenci, Laura and Krischke, Markus and Krischke, Beate and Ankenbrand, Markus and Rutschmann, Benjamin and Mueller, Martin J. and Steffan-Dewenter, Ingolf and Scheiner, Ricarda}, title = {In vitro rearing changes social task performance and physiology in honeybees}, series = {Insects}, volume = {13}, journal = {Insects}, number = {1}, issn = {2075-4450}, doi = {10.3390/insects13010004}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252305}, year = {2021}, abstract = {In vitro rearing of honeybee larvae is an established method that enables exact control and monitoring of developmental factors and allows controlled application of pesticides or pathogens. However, only a few studies have investigated how the rearing method itself affects the behavior of the resulting adult honeybees. We raised honeybees in vitro according to a standardized protocol: marking the emerging honeybees individually and inserting them into established colonies. Subsequently, we investigated the behavioral performance of nurse bees and foragers and quantified the physiological factors underlying the social organization. Adult honeybees raised in vitro differed from naturally reared honeybees in their probability of performing social tasks. Further, in vitro-reared bees foraged for a shorter duration in their life and performed fewer foraging trips. Nursing behavior appeared to be unaffected by rearing condition. Weight was also unaffected by rearing condition. Interestingly, juvenile hormone titers, which normally increase strongly around the time when a honeybee becomes a forager, were significantly lower in three- and four-week-old in vitro bees. The effects of the rearing environment on individual sucrose responsiveness and lipid levels were rather minor. These data suggest that larval rearing conditions can affect the task performance and physiology of adult bees despite equal weight, pointing to an important role of the colony environment for these factors. Our observations of behavior and metabolic pathways offer important novel insight into how the rearing environment affects adult honeybees.}, language = {en} } @article{RoellRameshaLinketal.2021, author = {R{\"o}ll, Alexander and Ramesha, Mundre N. and Link, Roman M. and Hertel, Dietrich and Schuldt, Bernhard and Patil, Shekhargouda L. and H{\"o}lscher, Dirk}, title = {Water availability controls the biomass increment of Melia dubia in South India}, series = {Forests}, volume = {12}, journal = {Forests}, number = {12}, issn = {1999-4907}, doi = {10.3390/f12121675}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250150}, year = {2021}, abstract = {Farmland tree cultivation is considered an important option for enhancing wood production. In South India, the native leaf-deciduous tree species Melia dubia is popular for short-rotation plantations. Across a rainfall gradient from 420 to 2170 mm year\(^{-1}\), we studied 186 farmland woodlots between one and nine years in age. The objectives were to identify the main factors controlling aboveground biomass (AGB) and growth rates. A power-law growth model predicts an average stand-level AGB of 93.8 Mg ha\(^{-1}\) for nine-year-old woodlots. The resulting average annual AGB increment over the length of the rotation cycle is 10.4 Mg ha\(^{-1}\) year\(^{-1}\), which falls within the range reported for other tropical tree plantations. When expressing the parameters of the growth model as functions of management, climate and soil variables, it explains 65\% of the variance in AGB. The results indicate that water availability is the main driver of the growth of M. dubia. Compared to the effects of water availability, the effects of soil nutrients are 26\% to 60\% smaller. We conclude that because of its high biomass accumulation rates in farm forestry, M. dubia is a promising candidate for short-rotation plantations in South India and beyond.}, language = {en} } @article{TangYangNageletal.2021, author = {Tang, Ruijing and Yang, Shang and Nagel, Georg and Gao, Shiqiang}, title = {mem-iLID, a fast and economic protein purification method}, series = {Bioscience Reports}, volume = {41}, journal = {Bioscience Reports}, number = {7}, doi = {10.1042/BSR20210800}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261420}, year = {2021}, abstract = {Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.}, language = {en} } @phdthesis{Duan2021, author = {Duan, Xiaodong}, title = {Development of new channelrhodopsin versions with enhanced plasma membrane targeting and high calcium/sodium conductance}, doi = {10.25972/OPUS-18839}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188397}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The technique to manipulate cells or living animals by illumination after gene transfer of light-sensitive proteins is called optogenetics. Successful optogenetics started with the use of the light-gated cation channel channelrhodopsin-2 (ChR2). After early demonstrations of the power of ChR2, further light-sensitive ion channels and ion pumps were recruited to the optogenetic toolbox. Furthermore, mutations and chimera of ChR2 improved its versatility. However, there is still a need for improved optogenetic tools, e.g. with higher permeability for calcium or better expression in the plasma membrane. In this thesis, my work focuses on the design of highly functional channelrhodopsins with enhanced Na+ and Ca2+ conductance. First, I tested different N-terminal signal peptides to improve the plasma membrane targeting of Channelrhodopsins. We found that a N-terminal peptide, named LR, could improve the plasma membrane targeting of many rhodopsins. Modification with LR contributed to three to ten-fold larger photocurrents (than that of the original version) of multiple channelrhodopsins, like ChR2 from C. reinhardtii (CrChR2), PsChR, Chrimson, CheRiff, CeChR, ACRs, and the light-activated pump rhodopsins KR2, Jaw, HR. Second, by introducing point mutation, I could further improve the light sensitivity and photocurrent of different channelrhodopsins. For instance, ChR2-XXM 2.0, ChR2-XXL 2.0 and PsChR D139H 2.0 exhibited hundred times larger photocurrents than wild type ChR2 and they show high light sensitivity. Also, the Ca2+ permeable channelrhodopsins PsCatCh 2.0f and PsCatCh 2.0e show very large photocurrents and fast kinetics. In addition, I also characterized a novel bi-stable CeChR (from the acidophilic green alga Chlamydomonas eustigma) with a much longer closing time. Third, I analysed the ion selectivity of different ChRs, which provides a basis for rational selection of channelrhodopsins for different experimental purposes. I demonstrate that ChR2, Chronos, Chrimson, CheRiff and CeChR are highly proton conductive, compared with wild type PsChR. Interestingly, Chronos has the lowest potassium conductance among these channelrhodopsins. Furthermore, I found that mutation of an aspartate in TM4 of ChR2 (D156) and PsChR (D139) to histidine obviously increased both the sodium and calcium permeability while proton conductance was reduced. PsChR D139H 2.0 has the largest sodium conductance of any published channelrhodopsin variants. Additionally, I generated PsCatCh 2.0e which exhibits a ten-fold larger calcium current than the previously reported Ca2+ transporting CrChR2 mutant CatCh. In summary, my research work 1.) described strategies for improving plasma membrane trafficking efficiency of opsins; 2.) yielded channelrhodopsins with fast kinetics or high light sensitivity; 3.) provided optogenetic tools with improved calcium and sodium conductance. We could also improve the performance of channelrhodopsins with distinct action spectra, which will facilitate two-color neural excitation, both in-vitro and in-vivo.}, subject = {Optogenetik}, language = {en} } @phdthesis{LindenbergverhSchubert2021, author = {Lindenberg [verh. Schubert], Annekathrin}, title = {Timing of sensory preferences in \(Camponotus\) Ants}, doi = {10.25972/OPUS-16094}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160948}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Ants belong to the most successful insects living on our planet earth. One criterion of their tremendous success is the division of labor among workers that can be related to age (age¬- or temporal polyethism) and/ or body size (size-related polymorphism). Young ants care for the queen and brood in the nest interior and switch to foraging tasks in the outside environment with ongoing age. This highly flexible interior-exterior transition probably allows the ant workers to properly match the colony needs and is one of the most impressive behaviors a single worker undergoes during its life. As environmental stimuli are changing with this transition, workers are required to perform a new behavioral repertoire. This requires significant adaptions in sensory and higher¬-order integration centers in the brain, like the mushroom bodies. Furthermore, foragers need proper time measuring mechanisms to cope with daily environmental changes and to adapt their own mode of life. Therefore, they possess a functional endogenous clock that generates rhythms with a period length of approximately 24 hours. The species-rich genus of Camponotus ants constitute a rewarding model to study how behavioral duties of division of labor were performed and modulated within the colony and how synaptic plasticity in the brain is processed, as they can divide their labor to both, age and body size, simultaneously. In my PhD thesis, I started to investigate the behavioral repertoire (like foraging and locomotor activity) of two sympatric Camponotus species, C. mus and C. rufipes workers under natural and under controlled conditions. Furthermore, I focused on the division of labor in C. rufipes workers and started to examine structural and ultrastructural changes of neuronal architectures in the brain that are accompanied by the interior-exterior transition of C. rufipes ants. In the first part of my thesis, I started to analyze the temporal organization of task allocation throughout the life of single C. rufipes workers. Constant video-tracking of individually labeled workers for up to 11 weeks, revealed an age-related division of labor of interior and exterior workers. After emergence, young individuals are tended to by older ones within the first 48 hours of their lives before they themselves start nurturing larvae and pupae. Around 52\% switch to foraging duties at an age of 14-20 days. The workers that switched to foraging tasks are mainly media-sized workers and seem to be more specialized than nurses. Variations in proportion and the age of switching workers between and within different subcolonies indicate how highly flexible and plastic the age-related division of labor occurs in this ant species. Most of the observed workers were engaged in foraging tasks exclusively during nighttime. As the experiments were conducted in the laboratory, they are completely lacking environmental stimuli of the ants´ natural habitat. I therefore asked in a second study, how workers of the two closely related Camponotus species, C. rufipes and C. mus, adapt their daily activity patterns (foraging and locomotor activity) under natural (in Uruguay, South America) and controlled (in the laboratory) conditions to changing thermal conditions. Monitoring the foraging activity of both Camponotus species in a field experiment revealed, that C. mus workers are exclusively diurnal, whereas C. rufipes foragers are predominantly nocturnal. However, some nests showed an elevated daytime activity, which could be an adaption to seasonally cold night temperatures. To further investigate the impact of temperature and light on the differing foraging activity patterns in the field, workers of both Camponotus species were artificially exposed to different thermal regimes in the laboratory, simulating local winter and summer conditions. Here again, C. mus workers display solely diurnal locomotor activity, whereas workers of C. rufipes shifted their locomotor activity from diurnal under thermal winter conditions to nocturnal under thermal summer conditions. Hence, the combination of both, field work and laboratory studies, shows that daily activity is mostly shaped by thermal conditions and that temperature cycles are not just limiting foraging activity but can be used as zeitgeber to schedule the outside activities of the nests. Once an individual worker switches from indoor duties to exterior foraging tasks, it is confronted with an entirely new set of sensory information. To cope with changes of the environmental conditions and to facilitate the behavioral switch, workers need a highly flexible and plastic neuronal system. Hence, my thesis further focuses on the underlying neuronal adaptations of the visual system, including the optic lobes as the primary visual neuropil and the mushroom bodies as secondary visual brain neuropil, that are accompanied with the behavioral switch from nursing to foraging. The optic lobes as well as the mushroom bodies of light-deprived workers show an `experience-independent´ volume increase during the first two weeks of adulthood. An additional light exposure for 4 days induces an `experience-dependent´ decrease of synaptic complexes in the mushroom body collar, followed by an increase after extended light exposure for 14 days. I therefore conclude, that the plasticity of the central visual system represents important components for the optimal timing of the interior-exterior transitions and flexibility of the age-related division of labor. These remarkable structural changes of synaptic complexes suggest an active involvement of the mushroom body neuropil in the lifetime plasticity that promotes the interior-exterior transition of Camponotus rufipes ants. Beside these investigations of neuronal plasticity of synaptic complexes in the mushroom bodies on a structural level, I further started to examine mushroom body synaptic structures at the ultrastructural level. Until recently, the detection of synaptic components in projection neuron axonal boutons were below resolution using classical Transmission Electron Microscopy. Therefore, I started to implement Electron Tomography to increase the synaptic resolution to understand architectural changes in neuronal plasticity process. By acquiring double tilt series and consecutive computation of the acquired tilt information, I am now able to resolve individual clear-core and dense-core vesicles within the projection neuron cytoplasm of C. rufipes ants. I additionally was able to reveal single postsynaptic Kenyon cell dendritic spines (~62) that surround one individual projection neuron bouton. With this, I could reveal first insights into the complex neuronal architecture of single projection neuron boutons in the olfactory mushroom body lip region. The high resolution images of synaptic architectures at the ultrastructural level, received with Electron Tomography would promote the understanding of architectural changes in neuronal plasticity. In my PhD thesis, I demonstrate that the temporal organization within Camponotus colonies involves the perfect timing of different tasks. Temperature seems to be the most scheduling abiotic factors of foraging and locomotor activity. The ants do not only need to adapt their behavioral repertoire in accordance to the interior-exterior switch, also the parts in the peripheral and central that process visual information need to adapt to the new sensory environment.}, subject = {Rossameise}, language = {en} } @phdthesis{Tang2021, author = {Tang, Ruijing}, title = {Optogenetic Methods to Regulate Water Transport and Purify Proteins}, doi = {10.25972/OPUS-23173}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231736}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Water transport through the water channels, aquaporins (AQPs), is involved in epithelial fluid secretion and absorption, cell migration, brain edema, adipocyte metabolism, and other physiological or pathological functions. Modulation of AQP function has therapeutic potential in edema, cancer, obesity, brain injury, glaucoma, etc. The function of AQPs is in response to the osmotic gradient that is formed by the concentration differences of ions or small molecules. In terms of brain edema, it is a pathophysiological condition, resulting from dysfunction of the plasma membrane that causes a disorder of intracellular ion homeostasis and thus increases intracellular fluid content. Optogenetics can be used to regulate ion transport easily by light with temporal and spatial precision. Therefore, if we control the cell ion influx, boosting the water transport through AQPs, this will help to investigate the pathological mechanisms in e.g. brain edema. To this end, I investigated the possibility for optogenetic manipulating water transport in Xenopus oocytes. The main ions in Xenopus oocyte cytoplasm are ~10 mM Na+, ~50 mM Cl- and ~100 mM K+, similar to the mammalian cell physiological condition. Three light-gated channels, ChR2-XXM 2.0 (light-gated cation channel), GtACR1 (light-gated anion channel) and SthK-bPAC (light-gated potassium channel), were used in my study to regulate ion transport by light and thus manipulate the osmotic gradient and water transport. To increase water flow, I also used coexpression of AQP1. When expressing ChR2-XXM 2.0 and GtACR1 together, mainly Na+ influx was triggered by ChR2-XXM2.0 under blue light illumination, which then made the membrane potential more positive and facilitated Cl- influx by GtACR1. Due to this inward movement of Na+ and Cl-, the osmotic gradient was formed to trigger water influx through AQP1. Large amounts of water uptake can speedily increase the oocyte volume until membrane rupture. Next, when co-expressing GtACR1 and SthK-bPAC, water efflux will be triggered with blue light because of the light-gated KCl efflux and then oocyte shrinking could be observed. I also developed an optogenetic protein purification method based on a light-induced protein interactive system. Currently, the most common protein purification method is based on affinity chromatography, which requires different chromatography columns and harsh conditions, such as acidic pH 4.5 - 6 and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. The change in conditions could influence the activity of target proteins. So, an easy and flexible protein purification method based on the photo-induced protein interactive system iLID was designed, which regulates protein binding with light in mild conditions and does not require a change of solution composition. For expression in E. coli, the blue light-sensitive part of iLID, the LOV2 domain, was fused with a membrane anchor and expressed in the plasma membrane, and the other binding partner, SspB, was fused with the protein of interest (POI), expressed in the cytosol. The plasma membrane fraction and the soluble cytosolic fraction of E. coli can be easily separated by centrifugation. The SspB-POI can be then captured to the membrane fraction by light stimulation and released to clean buffer in the dark after washing. This method does not require any specific column and functions in mild conditions, which are very flexible at scale and will facilitate extensive protein engineering and purification of proteins, sensitive to changed buffer conditions.}, language = {en} } @article{ZhouDingDuanetal.2021, author = {Zhou, Yang and Ding, Meiqi and Duan, Xiaodong and Konrad, Kai R. and Nagel, Georg and Gao, Shiqiang}, title = {Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics}, series = {Membranes}, volume = {11}, journal = {Membranes}, number = {4}, issn = {2077-0375}, doi = {10.3390/membranes11040287}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236617}, year = {2021}, abstract = {Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.}, language = {en} } @article{TianNagelGao2021, author = {Tian, Yuehui and Nagel, Georg and Gao, Shiqiang}, title = {An engineered membrane-bound guanylyl cyclase with light-switchable activity}, series = {BMC Biology}, volume = {19}, journal = {BMC Biology}, number = {1}, doi = {10.1186/s12915-021-00978-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259181}, pages = {54}, year = {2021}, abstract = {Background Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized. Results Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm\(^2\) of 473 nm (~ 18 J/m\(^2\)). Conclusions This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.}, language = {en} } @article{LiPradaDaminelietal.2021, author = {Li, Kunkun and Prada, Juan and Damineli, Daniel S. C. and Liese, Anja and Romeis, Tina and Dandekar, Thomas and Feij{\´o}, Jos{\´e} A. and Hedrich, Rainer and Konrad, Kai Robert}, title = {An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca\(^{2+}\) and H\(^{+}\) reveals new insights into ion signaling in plants}, series = {New Phytologist}, volume = {230}, journal = {New Phytologist}, number = {6}, doi = {10.1111/nph.17202}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-239847}, pages = {2292 -- 2310}, year = {2021}, abstract = {Whereas the role of calcium ions (Ca\(^{2+}\)) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca\(^{2+}\) and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca\(^{2+}\)- and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca\(^{2+}\)-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca\(^{2+}\). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+-increases and cytosolic acidification by c. 2 min, suggesting a Ca\(^{2+}\)/pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca\(^{2+}\)- and pH-responses. We propose close interrelation in Ca\(^{2+}\)- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.}, language = {en} } @article{NuhkatBroscheStoezleFeixetal.2021, author = {Nuhkat, Maris and Brosch{\´e}, Mikael and Stoezle-Feix, Sonja and Dietrich, Petra and Hedrich, Rainer and Roelfsema, M. Rob G. and Kollist, Hannes}, title = {Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response}, series = {New Phytologist}, volume = {232}, journal = {New Phytologist}, number = {4}, doi = {10.1111/nph.17711}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259646}, pages = {1692-1702}, year = {2021}, abstract = {Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O\(_{3}\) triggered a drop in whole-plant CO\(_{2}\) uptake. Within this early phase, O\(_{3}\) pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O\(_{3}\) induced cell death, systemic Ca\(^{2+}\) signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca\(^{2+}\)) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.}, language = {en} } @article{DindasDreyerHuangetal.2021, author = {Dindas, Julian and Dreyer, Ingo and Huang, Shouguang and Hedrich, Rainer and Roelfsema, M. Rob G.}, title = {A voltage-dependent Ca\(^{2+}\) homeostat operates in the plant vacuolar membrane}, series = {New Phytologist}, volume = {230}, journal = {New Phytologist}, number = {4}, doi = {10.1111/nph.17272}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259627}, pages = {1449-1460}, year = {2021}, abstract = {Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca\(^{2+}\) signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca\(^{2+}\) and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca\(^{2+}\) concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H\(^{+}\)/Ca\(^{2+}\) exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca\(^{2+}\) homeostat could contribute to calcium signalling when coupled to a recently discovered K\(^{+}\) channel-dependent module for electrical excitability of the vacuolar membrane.}, language = {en} }