Measurement of field scale N2O emission fluxes from a wheat crop using micrometeorological techniques

Measurements of N₂O emission fluxes from a 3 ha field of winter wheat were measured using eddy covariance and relaxed eddy accumulation continuously over 10 days during April 1994. The measurements averaged fluxes over approximately 10⁵ m² of the field, which was fertilised with NH₄NO₃ at a rate of 43 kg N ha^-1 at the beginning of the measurements. The emission fluxes became detectable after the first heavy rainfall, which occured 4 days after fertiliser application. Emissions of N₂O increased rapidly during the day following the rain to a maximum of 280 ng N m^-2s^-1 and declined over the following week. During the period of significant emission fluxes, a clear diurnal cycle in N₂O emission was observed, with the daytime maximum coinciding with the soil temperature maximum at 12 cm depth. The temperature dependence of the N₂O emission was equivalent to an activation energy for N₂O production of 108 kJ mol^-1. The N₂O fluxes measured using relaxed eddy accumulation, averaged over 30 to 270 min, were in agreement with those of the eddy covariance system within 60%. The total emission of N₂O over the period of continuous measurement (10 days) was equivalent to about 10 kg N₂O-N, or 0.77% of the N fertiliser applied.     

The global nitrogen cycle in the twenty-first century

Global nitrogen fixation contributes 413 Tg of reactive nitrogen (N_r) to terrestrial and marine ecosystems annually of which anthropogenic activities are responsible for half, 210 Tg N. The majority of the transformations of anthropogenic N_r are on land (240 Tg N yr⁻¹) within soils and vegetation where reduced N_r contributes most of the input through the use of fertilizer nitrogen in agriculture. Leakages from the use of fertilizer N_r contribute to nitrate (NO₃⁻) in drainage waters from agricultural land and emissions of trace N_r compounds to the atmosphere. Emissions, mainly of ammonia (NH₃) from land together with combustion related emissions of nitrogen oxides (NO_x), contribute 100 Tg N yr⁻¹ to the atmosphere, which are transported between countries and processed within the atmosphere, generating secondary pollutants, including ozone and other photochemical oxidants and aerosols, especially ammonium nitrate (NH₄NO₃) and ammonium sulfate (NH₄)₂SO₄. Leaching and riverine transport of NO₃ contribute 40–70 Tg N yr⁻¹ to coastal waters and the open ocean, which together with the 30 Tg input to oceans from atmospheric deposition combine with marine biological nitrogen fixation (140 Tg N yr⁻¹) to double the ocean processing of N_r. Some of the marine N_r is buried in sediments, the remainder being denitrified back to the atmosphere as N₂ or N₂O. The marine processing is of a similar magnitude to that in terrestrial soils and vegetation, but has a larger fraction of natural origin. The lifetime of N_r in the atmosphere, with the exception of N₂O, is only a few weeks, while in terrestrial ecosystems, with the exception of peatlands (where it can be 10²–10³ years), the lifetime is a few decades. In the ocean, the lifetime of N_r is less well known but seems to be longer than in terrestrial ecosystems and may represent an important long-term source of N₂O that will respond very slowly to control measures on the sources of N_r from which it is produced.     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

The effector enzyme regulates the duration of G protein signaling in vertebrate photoreceptors by increasing the affinity between transducin and RGS protein

The photoreceptor-specific G protein transducin acts as a molecular switch, stimulating the activity of its downstream effector in its GTP-bound form and inactivating the effector upon GTP hydrolysis. This activity makes the rate of transducin GTPase an essential factor in determining the duration of photoresponse in vertebrate rods and cones. In photoreceptors, the slow intrinsic rate of transducin GTPase is accelerated by the complex of the ninth member of the regulators of G protein signaling family with the long splice variant of type 5 G protein beta subunit (RGS9.Gbeta5L). However, physiologically rapid GTPase is observed only when transducin forms a complex with its effector, the gamma subunit of cGMP phosphodiesterase (PDEgamma). In this study, we addressed the mechanism by which PDEgamma regulates the rate of transducin GTPase. We found that RGS9.Gbeta5L alone has a significant ability to activate transducin GTPase, but its affinity for transducin is low. PDEgamma acts by enhancing the affinity between activated transducin and RGS9.Gbeta5L by more than 15-fold, which is evident both from kinetic measurements of transducin GTPase rate and from protein binding assays with immobilized transducin. Furthermore, our data indicate that a single RGS9.Gbeta5L molecule is capable of accelerating the GTPase activity of approximately 100 transducin molecules/s. This rate is faster than the rates reported previously for any RGS protein and is sufficient for timely photoreceptor recovery in both rod and cone photoreceptors.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Identification of candidate genes and mutations in QTL regions for immune responses in chicken

There are two categories of immune responses – innate and adaptive immunity – both having polygenic backgrounds and a significant environmental component. In our study, adaptive immunity was represented by the specific antibody response toward keyhole limpet hemocyanin (KLH); innate immunity was represented by natural antibodies toward lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Defining genetic bases of immune responses leads from defining quantitative trait loci (QTL) toward a single mutation responsible for variation in the phenotypic trait. The goal of the reported study was to define candidate genes and mutations for the immune traits of interest in chicken by performing an association study of SNPs located in candidate genes defined in QTL regions. Candidate genes and SNPs in QTL regions were selected in silico. SNP association was based on a custom SNP panel, GoldenGate genotyping assay (Illumina) and two statistical models: random mixed model and CAR score. The most significant SNP for immune response toward KLH was located in the JMJD6 gene located on GGA18. Four SNPs in candidate genes FOXJ1 (GGA18), EPHB1 (GGA9), PTGER4 (GGAZ) and PRKCB (GGA14) showed association with natural antibodies for LPS. A single SNP in ITGB4 (GGA18) was associated with natural antibodies for LTA. All associated SNPs mentioned above showed additive effects.     

Methane emissions from sheep pasture,measured with an open‐path eddy covariance system

Methane (CH₄) is an important greenhouse gas, contributing 0.4–0.5 W m^?2 to global warming. Methane emissions originate from several sources, including wetlands, rice paddies, termites and ruminating animals. Previous measurements of methane flux from farm animals have been carried out on animals in unnatural conditions, in laboratory chambers or fitted with cumbersome masks. This study introduces eddy covariance measurements of CH₄, using the newly developed LI‐COR LI‐7700 open‐path methane analyser, to measure field‐scale fluxes from sheep grazing freely on pasture. Under summer conditions, fluxes of methane in the morning averaged 30 nmol m^?2 s^?1, whereas those in the afternoon were above 100 nmol m^?2 s^?1, and were roughly two orders of magnitude larger than the small methane emissions from the soil. Methane emissions showed no clear relationship with air temperature or photosynthetically active radiation, but some diurnal pattern was apparent, probably linked to sheep grazing behaviour and metabolism. Over the measurement period (days 60–277, year 2010), cumulative methane fluxes were 0.34 mol CH₄ m^?2, equating to 134.3 g CO₂ equivalents m^?2. By comparison, a carbon dioxide (CO₂) sink of 819 g CO₂ equivalents m^?2 was measured over the same period, but it is likely that much of this would be released back to the atmosphere during the winter or as off‐site losses (through microbial and animal respiration). By dividing methane fluxes by the number of sheep in the field each day, we calculated CH₄ emissions per head of livestock as 7.4 kg CH₄ sheep^?1 yr^?1, close to the published IPCC emission factor of 8 kg CH₄ sheep^?1 yr^?1.     

Structural Requirements of the Photoreceptor Phosphodiesterase ��-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its γ-subunit (Pγ), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pγ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pγ78–87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pγ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pγ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63–70 and/or the intact α2 helix of Pγ stabilize binding of C-terminal Pγ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pγ molecule and tested for activation by transducin, we found that the C-terminal region (Pγ63–87) by itself could not be displaced but that transducin could relieve inhibition of certain Pγ truncation mutants. Our results are consistent with two distinct mechanisms of Pγ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pγ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pγ to effectively relieve its inhibitory constraint.     

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.