Genetic approaches for breeding heat stress tolerance in faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

Vicia faba L. (faba bean) is an important legume and is cultivated essentially as a cool-season crop. Changes in sowing dates and lack of precipitation expose faba bean crop to drought and heat stresses. The gradual rise in global temperatures owing to climate change is likely to exacerbate the detrimental effects of hot and dry climatic conditions on faba bean cultivation. High temperature stress is particularly damaging to faba bean during the flowering period, when the viability of pollen is critical for successful reproduction. Recent studies have shown that maintenance of protein homeostasis through synthesis of heat shock proteins plays a key role in the heat response of plants. To date, there has been no significant work linking the heat response of faba bean to the repertoire of its heat shock proteins. While quantitative trait loci have been identified for resistance against biotic stresses in faba bean, there is no parallel success with abiotic stresses in this species. Programs aiming at genetic improvement of the heat/drought resistance of this crop by both conventional breeding and molecular breeding methods are hampered because of the large and majorly ill-analyzed genome of faba bean plants. Likewise, molecular and biotechnology-related tools are poorly developed for faba bean; as a result, the fruits of transgenic research developed with model plant species are not reaching this crop. While specifically discussing the prospects for the genetic improvement of faba bean against heat and drought stresses, we highlight the areas of research which need to be strengthened on faba bean.     

A New Route to Glucose Sensing Based on Surface Plasmon Resonance Using Polyindole

In the present study, we report the first polyindole-modified metal (Au) as a glucose sensor utilizing surface plasmon resonance (SPR) technique. Polyindole (PIn) was deposited by spin coating to modify the surface of the gold disk. Sensor surface was prepared by immobilizing glucose oxidase on the polyindole-modified gold disk. Different concentrations of glucose were taken to analyze the sensor response. A change in refractive index of the film was observed due to the chemical reactions of glucose with glucose oxidase. The response of the sensor is fast and highly sensitive to low concentrations of glucose and the sensitivity increases in the range of 0.075–0.5 μM. The use of Au/polyindole (PIn/Au) substrates for SPR-based study of bio-molecular sensing has been demonstrated.     

Hydrogen Peroxide Stimulates Activity and Alters Behavior in Drosophila melanogaster

Circadian rhythms in animals are regulated at the level of individual cells and by systemic signaling to coordinate the activities of multiple tissues. The circadian pacemakers have several physiological outputs, including daily locomotor rhythms. Several redox-active compounds have been found to function in regulation of circadian rhythms in cells, however, how particular compounds might be involved in regulating specific animal behaviors remains largely unknown. Here the effects of hydrogen peroxide on Drosophila movement were analyzed using a recently developed three-dimensional real-time multiple fly tracking assay. Both hydrogen peroxide feeding and direct injection of hydrogen peroxide caused increased adult fly locomotor activity. Continuous treatment with hydrogen peroxide also suppressed daily locomotor rhythms. Conditional over-expression of the hydrogen peroxide-producing enzyme superoxide dismutase (SOD) also increased fly activity and altered the patterns of locomotor activity across days and weeks. The real-time fly tracking system allowed for detailed analysis of the effects of these manipulations on behavior. For example, both hydrogen peroxide feeding and SOD over-expression increased all fly motion parameters, however, hydrogen peroxide feeding caused relatively more erratic movement, whereas SOD over-expression produced relatively faster-moving flies. Taken together, the data demonstrate that hydrogen peroxide has dramatic effects on fly movement and daily locomotor rhythms, and implicate hydrogen peroxide in the normal control of these processes.     

Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.     

Quantifying Intracellular Particle Flows by DIC Object Tracking

Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.     

Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.     

Protein insertion and patterning of PEG bearing langmuir monolayers

Protein adsorption to multicomponent lipid monolayers is presented as a means of inducing protein-specific binding pockets or imprints in membranes. Adsorption of the acidic protein ferritin to Langmuir monolayers of cationic dioctadecyldimethylammonium bromide (DOMA), nonionic methyl stearate (SME), and poly(ethylene glycol) (PEG) bearing phospholipids is investigated as a model system. The number, size, and distribution of protein binding pockets (domains of SME and DOMA in a PEG matrix) are defined by controlling the molar ratios, miscibility, and lateral mobility of the lipids. Protein patterning of binary SME:DOMA monolayers is limited by protein-protein interactions that hinder desorption to regenerate the imprint site. The incorporation of PEG bearing phospholipids as a third lipid component provides a successful approach to prevent protein surface aggregation during imprinting. Atomic force microscopy reveals a user-defined distribution of protein molecules where protein-protein interactions on the monolayer are eliminated, thus facilitating protein desorption and regeneration of the protein binding pockets.     

Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.     

Regulation of FasL/Fas in human trophoblasts: possible implications for chorioamnionitis

Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor alpha-induced and interferon gamma-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.