Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

Interspecific variation in the response of fish to anthropogenic noise

1. Elevated levels of anthropogenic noise, especially those observed through boating activity, can negatively impact fish species, but it remains unclear which species are most affected and which behavioural metrics are best used in assessing fish responses to underwater noise. The effects of boat sounds on freshwater species are of particular interest because freshwater environments are less studied than the marine realm despite comparably high levels of biodiversity.
2. In the current study, we examine the behavioural responses to boat noise in two freshwater species that differ in their hypothesised response to sound inputs: the spottail shiner (Notropis hudsonius), a species with known hearing specialisations, and the bluegill sunfish (Lepomis macrochirus), a species with more generalised hearing capabilities. Fish were presented with boat noise in a laboratory setting, and their swimming, escape and foraging behaviours were assessed to examine differential responses in relation to hypothesised hearing abilities.
3. Both species showed a decrease in general swimming behaviours but an increase in erratic movements in response to boat noise, indicative of stress responses for both species. Despite the similarities in response based on swimming behaviours however, only spottail shiners exhibited true escape responses to the onset of the noise stimulus, suggesting a more extreme reaction in the species with a more refined hearing ability.
4. Taken together, these results show that freshwater fish can respond to increased levels of anthropogenic noise, but that the severity of the response may differ based on auditory structures and therefore presumed hearing ability. The differences seen between behavioural metrics used (swimming vs. escape responses) also demonstrate how care must be taken in choosing a metric when developing exposure guidelines for underwater sound exposures, as different metrics could lead to differential impact assessments.

     

Photosynthesis of lichens from lichen-dominated communities in the alpine/nival belt of the Alps – II: Laboratory and field measurements of CO2 exchange and water relations

CO₂ exchange and water relations of selected lichen species were investigated in the field and also in the laboratory, at a height of 3106 m above sea level in the Austrian Alps, during the short snowless summer period from middle of July to the end of August. In the course of the field investigations, clear summer days were quite rare. Altogether 14 diurnal courses of CO₂ exchange were measured spanning a time of 255 h of measurements.The air temperatures measured close to the ground ranged between −0.7 and 17.1 °C and their daily fluctuation was lower than 10.7 °C. Fog was present for more than one-third of the measuring period and relative humidity (RH) exceeded 90% in almost half of the time. Temperature optimum of net photosynthesis (NP) of Xanthoria elegans and Brodoa atrofusca determined in the laboratory increased with increasing photosynthetic photon flux density (PPFD) from 1.5 to 11.3 °C and the maximal CO₂ uptake was found to be at 10 °C. In the field the lichens were metabolically active at air temperatures between −0.7 and 12.8 °C. The light compensation points (LCP) of both lichen species ranged in the laboratory between 50 and 200 μmol m⁻² s⁻¹ PPFD (0–20 °C) and in the field between 22 and 56 μmol m⁻² s⁻¹ PPFD (3–8 °C). At 30 °C the NP of X. elegans surpassed the LCP, whereas B. atrofusca remained below the LCP. NP in X. elegans did not reach light saturation at 1500 μmol m⁻² s⁻¹ PPFD. NP in B. atrofusca reached light saturation at low temperatures (−5 to +5 °C). At higher temperatures light saturation was almost detectable. On sunny days the lichens in the field were metabolically active only for 3 h during the early morning. In this time they reached the maximal values or values close to their maximal CO₂ uptake in situ. Under dry weather conditions the lichens dried out to a minimal water content (WC) of 5–12% which is below the moisture compensation point (MCP) of 34–25%. The optimal WC was between 90% and 120% dry weight (DW) in B. atrofusca and Umbilicaria cylindrica, in X. elegans between 140% and 180% DW. Species specific differences in water-holding capacity, desiccation intensity and in the compensation points of temperature, light and moisture are responsible for differences in metabolic activity. The lichens were active during less than half of the observation time. Total time of NP of X. elegans was 24% of the measuring period, for U. cylindrica 22% and for B. atrofusca 16%.     

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.