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Quantifying the influence of pollen aging on the adhesive
properties of Hypochaeris radicata pollen
(2022)
Simple Summary Pollination is the transfer of pollen from a plant's male part (anther) to the corresponding female part (stigma). It is a fundamental biological process that ensures plant reproduction. Most studies investigate pollination from a biological perspective, but the underlying physical processes are poorly understood. Many plants rely on insects to transport pollen and the forces with which pollen adhere to insects and floral surfaces are fundamental for successful pollination.
We quantified pollen adhesion by measuring the forces necessary to detach Hypochaeris radicata (catsear, a common insect-pollinated plant) pollen from glass and studied for the first time how the adhesion forces change with pollen aging.
Our results show that newly formed adhesion bonds between H. radicata pollen and glass are stronger for fresh pollen than for old ones. On the other hand, when H. radicata pollen age in contact with glass, the adhesion between pollen and glass strengthens over time. These effects are probably caused by the viscous liquid covering most pollen (pollenkitt) changing its viscoelastic properties as it dries.
Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transferred from anther to insect and from insect to stigma. The influence of the properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer have been poorly studied.
Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen aging by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt-the viscous, lipid liquid on the surface of most pollen grains-influences the forces necessary to detach a pollen from hydrophilic surfaces.
Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time.
This study shows for the first time the pollen aging effect on the pollination mechanism.
Volatile organic compounds (VOCs) are involved in microbial interspecies communication and in the mode of action of various antagonistic interactions. They are important for balancing host-microbe interactions and provide the basis for developing biological control strategies to control plant pathogens.
We studied the interactions between the bacterial antagonist Serratia plymuthica HRO-C48 and three fungal plant pathogens Rhizoctonia solani, Leptosphaeria maculans and Verticillium longisporum. Significant differences in fungal growth inhibition by the Serratia-emitted VOCs in pairwise dual culture assays and changes in the transcriptome of the bacterium and in the volatilomes of both interacting partners were observed. Even though the rate of fungal growth inhibition by Serratia was variable, the confrontation of the bacterium with the VOCs of all three fungi changed the levels of expression of the genes involved in stress response, biofilm formation, and the production of antimicrobial VOCs. Pairwise interacting microorganisms switched between defense (downregulation of gene expression) and attack (upregulation of gene expression and metabolism followed by growth inhibition of the interacting partner) modes, subject to the combinations of microorganisms that were interacting.
In the attack mode HRO-C48 significantly inhibited the growth of R. solani while simultaneously boosting its own metabolism; by contrast, its metabolism was downregulated when HRO-C48 went into a defense mode that was induced by the L. maculans and V. longisporum VOCs. L. maculans growth was slightly reduced by the one bacterial VOC methyl acetate that induced a strong downregulation of expression of genes involved in almost all metabolic functions in S. plymuthica.
Similarly, the interaction between S. plymuthica and V. longisporum resulted in an insignificant growth reduction of the fungus and repressed the rate of bacterial metabolism on the transcriptional level, accompanied by an intense volatile dialogue. Overall, our results indicate that VOCs substantially contribute to the highly break species-specific interactions between pathogens and their natural antagonists and thus deserving of increased consideration for pathogen control.
Ciliates represent a crucial link between phytoplankton and bacteria and mesozooplankton in pelagic food webs, but little is known about the processes influencing the dynamics of individual species.
Using long-term, high-frequency observations, we compared the diversity and the temporal variability in biomass and species composition of the ciliate community in large, deep, mesotrophic Lake Constance to that of the phytoplankton and rotifer communities in the same lake.
Furthermore, we used boosted regression trees to evaluate possible environmental predictors (temperature, three prey groups, four predator/competitor groups) influencing ciliate net growth.
The biomass of all ciliate species showed a common, recurrent seasonal pattern, often with peaks in spring and summer.
The ciliate community was more diverse than the rotifer community, exhibited highly synchronous dynamics and its species were regularly encountered during the season. The top-down control by copepods likely contributes to the ciliates' synchronized decline prior to the clear-water phase when food concentration is still high.
The high temporal autocorrelation of the ciliate biomasses together with the inter-annual recurrent seasonal patterns and the low explanatory power of the environmental predictors suggest that the dynamics of individual ciliate species are strictly controlled, yet it remains difficult to determine the responsible factors.
Current evidence suggests that migratory animals extract map information from the geomagnetic field for true navigation. The sensory basis underlying this feat is elusive, but presumably involves magnetic particles.
A common experimental manipulation procedure consists of pre-treating animals with a magnetic pulse, with the aim of re-magnetising particles to alter the internal representation of the external field prior to a navigation task.
Although pulsing provoked deflected bearings in caged songbirds, analogous studies with free-flying songbirds yielded inconsistent results.
Here, we pulsed European robins (Erithacus rubecula) at an offshore stopover site during spring migration and monitored their free-flight behaviour with a regional-scale network of radio-receiving stations.
We found no pulse effect on departure probability, nocturnal departure timing departure direction or consistency of flight direction.
This suggests either no use of the geomagnetic map by our birds, or that magnetic pulses do not affect the sensory system underlying geomagnetic map detection.
Nature conservation is currently shaping many terrestrial ecosystems in Africa.
This is particularly evident in Sub-Saharan Africa (SSA), where conservation is intended to recover wildlife populations, with special focus on elephants.
Rising numbers of elephants induce woody biomass losses but increase soil organic carbon (SOC) stocks from decaying wood and dung.
We hypothesized that these increases under wildlife conservation in SSA go along with rising contents of plant residues in SOC, traceable by the molecular markers lignin and n-alkanes.
In contrast, agricultural intensification would reduce them due to lower C input and faster SOC turnover through tillage.
To test this, we analyzed lignin by the CuO oxidation method and n-alkanes by fast pressurized solvent extraction in topsoils (0-10 cm) of Arenosols and corresponding plant samples (trees, grasses and crops).
Sampling sites followed conservation gradients with low, medium and high elephant densities and intensification gradients with rangeland and cropland in the woodland savanna of the Namibian Zambezi Region.
Patterns of lignin-derived phenols were retained in the soil, whereas n-alkanes showed shifts in chain lengths. n-Alkanes also showed no clear increase or decrease under conservation or intensification, respectively.
Differently, lignin-derived phenols showed lower values under intensification than under conservation. Confirming our hypothesis, rising SOC contents with rising elephant densities (from 4.4 at low to 5.7 g kg(-1) SOC at high elephant densities) went along with an increasing accumulation of lignin-derived phenols (24.4-34.8 g kg(-1) VSCOC).
This increase is associated with the input of woody debris to the soil, as indicated by V-units and carbon isotopes, modulated by clay and woody biomass.
We conclude, that increasing input of woody residues into soil by browsing behaviour of elephants is an important mechanism for controlling SOC supply in the context of wildlife conservation and is traceable with lignin-derived phenols, but not with n-alkanes.
Rhizophagus irregularis is one of the most extensively studied arbuscular mycorrhizal fungi (AMF) that forms symbioses with and improves the performance of many crops. Lack of transformation protocol for R. irregularis renders it challenging to investigate molecular mechanisms that shape the physiology and interactions of this AMF with plants.
Here, we used all published genomics, transcriptomics, and metabolomics resources to gain insights into the metabolic functionalities of R. irregularis by reconstructing its high-quality genome-scale metabolic network that considers enzyme constraints. Extensive validation tests with the enzyme-constrained metabolic model demonstrated that it can be used to (i) accurately predict increased growth of R. irregularis on myristate with minimal medium; (ii) integrate enzyme abundances and carbon source concentrations that yield growth predictions with high and significant Spearman correlation (rS = 0.74) to measured hyphal dry weight; and (iii) simulate growth rate increases with tighter association of this AMF with the host plant across three fungal structures.
Based on the validated model and system-level analyses that integrate data from transcriptomics studies, we predicted that differences in flux distributions between intraradical mycelium and arbuscles are linked to changes in amino acid and cofactor biosynthesis.
Therefore, our results demonstrated that the enzyme-constrained metabolic model can be employed to pinpoint mechanisms driving developmental and physiological responses of R. irregularis to different environmental cues.
In conclusion, this model can serve as a template for other AMF and paves the way to identify metabolic engineering strategies to modulate fungal metabolic traits that directly affect plant performance.
IMPORTANCE Mounting evidence points to the benefits of the symbiotic interactions between the arbuscular mycorrhiza fungus Rhizophagus irregularis and crops; however, the molecular mechanisms underlying the physiological responses of this fungus to different host plants and environments remain largely unknown.
We present a manually curated, enzyme-constrained, genome-scale metabolic model of R. irregularis that can accurately predict experimentally observed phenotypes.
We show that this high-quality model provides an entry point into better understanding the metabolic and physiological responses of this fungus to changing environments due to the availability of different nutrients.
The model can be used to design metabolic engineering strategies to tailor R. irregularis metabolism toward improving the performance of host plants.
Cell division and the resulting changes to the cell organization affect the shape and functionality of all tissues.
Thus, understanding the determinants of the tissue-wide changes imposed by cell division is a key question in developmental biology.
Here, we use a network representation of live cell imaging data from shoot apical meristems (SAMs) in Arabidopsis thaliana to predict cell division events and their consequences at the tissue level.
We show that a support vector machine classifier based on the SAM network properties is predictive of cell division events, with test accuracy of 76%, which matches that based on cell size alone.
Furthermore, we demonstrate that the combination of topological and biological properties, including cell size, perimeter, distance and shared cell wall between cells, can further boost the prediction accuracy of resulting changes in topology triggered by cell division.
Using our classifiers, we demonstrate the importance of microtubule-mediated cell-to-cell growth coordination in influencing tissue-level topology.
Together, the results from our network-based analysis demonstrate a feedback mechanism between tissue topology and cell division in A. thaliana SAMs.
In plants, the trehalose biosynthetic pathway plays key roles in the regulation of carbon allocation and stress adaptation.
Engineering of the pathway holds great promise to increase the stress resilience of crop plants.
The synthesis of trehalose proceeds by a two-step pathway in which a trehalose-phosphate synthase (TPS) uses UDP-glucose and glucose-6-phosphate to produce trehalose-6 phosphate (T6P) that is subsequently dephosphorylated by trehalose-6 phosphate phosphatase (TPP).
While plants usually do not accumulate high amounts of trehalose, their genome encodes large families of putative trehalose biosynthesis genes, with many members lacking obvious enzymatic activity.
Thus, the function of putative trehalose biosynthetic proteins in plants is only vaguely understood. To gain a deeper insight into the role of trehalose biosynthetic proteins in crops, we assessed the enzymatic activity of the TPS/TPP family from tomato (Solanum lycopersicum L.) and investigated their expression pattern in different tissues as well as in response to temperature shifts. From the 10 TPS isoforms tested, only the 2 proteins belonging to class I showed enzymatic activity, while all 5 TPP isoforms investigated were catalytically active.
Most of the TPS/TPP family members showed the highest expression in mature leaves, and promoter-reporter gene studies suggest that the two class I TPS genes have largely overlapping expression patterns within the vasculature, with only subtle differences in expression in fruits and flowers.
The majority of tomato TPS/TPP genes were induced by heat stress, and individual family members also responded to cold.
This suggests that trehalose biosynthetic pathway genes could play an important role during temperature stress adaptation.
In summary, our study represents a further step toward the exploitation of the TPS and TPP gene families for the improvement of tomato stress resistance.
Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened.
In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and -capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS).Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones.
Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha -agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and -capture system offers a switchable scFv display.
Efficient plasmid clearance upon removal of selective pres-sure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (fac-tor in the range of-21-49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone.
Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of-2-3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.
Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors.
These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated.
GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others.
All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers.
Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent.
This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses.
Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer.
This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.