The effect of organic carbon content on soil compression characteristics.

We investigated the effect of soil organic carbon (SOC) on the consolidation behaviour of soil from two long term field experiments at Rothamsted; the Broadbalk Wheat Experiment and Hoosfield Spring Barley. These experiments are located on soil with similar particle size distributions, and include treatments with SOC contents ranging from approximately 1-3.5 g/100 g. Soils taken from plots with contrasting SOC contents were compressed and deformed in a triaxial cell and the normal consolidation and critical state lines were determined. We found that the compression index was independent of SOC, but the void ratio at any given effective stress was highly correlated with organic carbon content. By comparison with uniaxial compression data, the apparent influence of SOC on the compression index is likely to be due to its effect on soil hydraulic properties rather than any intrinsic effects of strength. The plastic limit test appears to be a useful and simple test to allow direct comparison of soil physical behaviour and expected soil density.

A comparison between water uptake and root length density in winter wheat: effects of root density and rhizosphere properties.

BACKGROUND AND AIMS: We aim to quantify the variation in root distribution in a set of 35 experimental wheat lines. We also compared the effect of variation in hydraulic properties of the rhizosphere on water uptake by roots. METHODS: We measured the root length density and soil drying in 35 wheat lines in a field experiment. A 3D numerical model was used to predict soil drying profiles with the different root length distributions and compared with measured soil drying. The model was used to test different scenarios of the hydraulic properties of the rhizosphere. RESULTS: We showed that wheat lines with no detectable differences in root length density can induce soil drying profiles with statistically significant differences. Our data confirmed that a root length density of at least 1 cm/cm3 is needed to drain all the available water in soil. In surface layers where the root length density was far greater than 1 cm/cm3 water uptake was independent of rooting density due to competition for water. However, in deeper layers where root length density was less than 1 cm/cm3, water uptake by roots was proportional to root density. CONCLUSION: In a set of wheat lines with no detectable differences in the root length density we found significant differences in water uptake. This may be because small differences in root density at depth can result in larger differences in water uptake or that the hydraulic properties of the rhizosphere can greatly affect water uptake.

Multiple abiotic stress, nitrate availability and the growth of wheat.

In the field, wheat experiences a combination of physical and nutrient stresses. There has been a tendency to study root impedance and water stress in separation and less is known about how they might interact. In this study, we investigated the effect of root impedance on the growth of three wheat varieties (Cadenza, Xi19 and Battalion) at different levels of nitrate availability, from 0-20 mM nitrate, in sand culture. This model system allows soil strength to be increased while maintaining adequate water availability. In a separate pot experiment, we grew the same wheat varieties in a loamy sand where soil was allowed to dry sufficiently to both reduce water potential and increase root impedance. This pot experiment also had a range of nitrate availabilities 0-20 mM nitrate. Once the seedlings were established we limited water supply to apply a matric potential of approximately -200 kPa to the roots. Soil drying increased the penetrometer resistance from approximately 300 kPa to more than 1 MPa. There were differences between the two experimental systems; growth was smaller in the soil-based experiment compared to the sand culture. However, the effects of the experimental treatment, root impedance or water withholding, relative to the control were comparable. Our data confirmed that leaf elongation in Cadenza (carrying the tall Rht allele) was the most sensitive to root impedance. Leaf stunting occurred irrespective of nitrate availability. Leaf elongation in the Xi19 and Battalion (carrying the semi-dwarf Rht allele) was less sensitive to root impedance and drought than Candenza. We suggest that the critical stress in a pot experiment where the soil was allowed to dry to approximately -200 kPa was root impedance and not water availability.

The effect of microbial activity on soil water diffusivity.

In this study, we explored the effects of microbial activity on the evaporation of water from cores of a sandy soil under laboratory conditions. We applied treatments to stimulate microbial activity by adding different amounts of synthetic analogue root exudates. For comparison, we used soil samples without synthetic root exudates as control and samples treated with mercuric chloride to suppress microbial activity. Our results suggest that increasing microbial activity reduces the rate of evaporation from soil. Estimated diffusivities in soil with the largest amounts of added root exudates were one third of those estimated in samples where microbial activity was suppressed by adding mercuric chloride. We discuss the effect of our results with respect to water uptake by roots. HIGHLIGHTS: We explored effects of microbial activity on the evaporation of water from cores of a sandy soil.We found the effect of microbial activity on water release characteristic was small.Increasing microbial activity reduced evaporation from soil, while microbial suppression increased it.Effect of microbial activity on root water uptake was estimated to be equivalent to a change in soil structure.

The emergent rhizosphere: imaging the development of the porous architecture at the root-soil interface.

The rhizosphere is the zone of soil influenced by a plant root and is critical for plant health and nutrient acquisition. All below ground resources must pass through this dynamic zone prior to their capture by plant roots. However, researching the undisturbed rhizosphere has proved very challenging. Here we compare the temporal changes to the intact rhizosphere pore structure during the emergence of a developing root system in different soils. High resolution X-ray Computed Tomography (CT) was used to quantify the impact of root development on soil structural change, at scales relevant to individual micro-pores and aggregates (µm). A comparison of micro-scale structural evolution in homogenously packed soils highlighted the impacts of a penetrating root system in changing the surrounding porous architecture and morphology. Results indicate the structural zone of influence of a root can be more localised than previously reported (µm scale rather than mm scale). With time, growing roots significantly alter the soil physical environment in their immediate vicinity through reducing root-soil contact and crucially increasing porosity at the root-soil interface and not the converse as has often been postulated. This 'rhizosphere pore structure' and its impact on associated dynamics are discussed.

Root growth in field-grown winter wheat: Some effects of soil conditions, season and genotype.

This work compared root length distributions of different winter wheat genotypes with soil physical measurements, in attempting to explain the relationship between root length density and soil depth. Field experiments were set up to compare the growth of various wheat lines, including near isogenic lines (Rht-B1a Tall NIL and Rht-B1c Dwarf NIL) and wheat lines grown commercially (cv. Battalion, Hystar Hybrid, Istabraq, and Robigus). Experiments occurred in two successive years under rain fed conditions. Soil water content, temperature and penetrometer resistance profiles were measured, and soil cores taken to estimate vertical profiles of pore distribution, and root number with the core-break method and by root washing. Root length distributions differed substantially between years. Wetter soil in 2014/2015 was associated with shallower roots. Although there was no genotypic effect in 2014/2015, in 2013/2014 the dwarf wheat had the most roots at depth. In the shallower layers, some wheat lines, especially Battalion, seemed better at penetrating non-structured soil. The increase in penetrometer resistance with depth was a putative explanation for the rapid decrease in root length density with depth. Differences between the two years in root profiles were greater than those due to genotype, suggesting that comparisons of different genotypic effects need to take account of different soil conditions and seasonal differences. We also demonstrate that high yields are not necessarily linked to resource acquisition, which did not seem to be limiting in the low yielding dwarf NIL.

Effect of microbial activity on penetrometer resistance and elastic modulus of soil at different temperatures.

We explore the effect of microbial activity stimulated by root exudates on the penetrometer resistance of soil and its elastic modulus. This is important because it is a measure of the mechanical strength of soil and it correlates closely with the rate of elongation of roots. A sandy soil was incubated with a synthetic root exudate at different temperatures, for different lengths of time and with selective suppression of either fungi or bacteria. The shape of the temperature response of penetrometer resistance in soil incubated with synthetic exudate was typical of a poikilothermic temperature response. Both penetrometer resistance and small strain shear modulus had maximum values between 25 and 30°C. At temperatures of 20°C and less, there was little effect of incubation with synthetic root exudate on the small strain shear modulus, although penetrometer resistance did increase with temperature over this range (4-20°C). This suggests that in this temperature range the increase in penetrometer resistance was related to a greater resistance to plastic deformation. At higher temperatures (> 25°C) penetrometer resistance decreased. Analysis of the DNA sequence data showed that at 25°C the number of Streptomyces (Gram-positive bacteria) increased, but selective suppression of either fungi or bacteria suggested that fungi have the greater role with respect to penetrometer resistance. HIGHLIGHTS: Effect of microbial activity stimulated by synthetic root exudates on the mechanical properties.We compared penetrometer measurements and estimates of elastic modulus with microbial community.Penetrometer resistance of soil showed a poikilothermic temperature response.Penetrometer resistance might be affected more by fungi than bacteria.

Methods to estimate changes in soil water for phenotyping root activity in the field.

BACKGROUND AND AIMS: There is an urgent need to develop new high throughput approaches to phenotype roots in the field. Excavating roots to make direct measurements is labour intensive. An alternative to excavation is to measure soil drying profiles and to infer root activity. METHODS: We grew 23 lines of wheat in 2013, 2014 and 2015. In each year we estimated soil water profiles with electrical resistance tomography (ERT), electromagnetic inductance (EMI), penetrometer measurements and measurements of soil water content. We determined the relationships between the measured variable and soil water content and matric potential. RESULTS: We found that ERT and penetrometer measurements were closely related to soil matric potential and produced the best discrimination between wheat lines. We found genotypic differences in depth of water uptake in soil water profiles and in the extent of surface drying. CONCLUSIONS: Penetrometer measurements can provide a reliable approach to comparing soil drying profiles by different wheat lines, and genotypic rankings are repeatable across years. EMI, which is more sensitive to soil water content than matric potential, and is less effective in drier soils than the penetrometer or ERT, nevertheless can be used to rapidly screen large populations for differences in root activity.

Kinetics of competitive binding with application to thrombin complexes.

The kinetics of competitive binding is treated analytically, allowing the rate constants to be determined accurately from simple experiments. The method is especially suited to situations where traditional approximations and numerical integration fail, e.g., when the dissociation constants are small or when the concentration of one receptor cannot be measured accurately. The method is applied to the competitive binding of hirudin to thrombin and anhydrothrombin and found to be accurate to a few parts in ten million. The fitted rate constants show that anhydrothrombin binds hirudin more weakly than thrombin, with a 2.6-fold increase in its dissociation constant. The small relative difference in binding free energy (0.6 kcal/mol indicates that anhydrothrombin is structurally similar to thrombin.

Effects of temperature and glycerides on the enhancement of Agkistrodon piscivorus piscivorus phospholipase A2 activity by lysolecithin and palmitic acid.

The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature-dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer.

Preparation and characterization of anhydrothrombin.

Anhydrothrombin, a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehydroalanine by promoting the beta-elimination of phenylmethylsulfonic acid from phenylmethylsulfonyl fluoride-inactivated thrombin under conditions in which the enzyme is unfolded. After the elimination reaction was quenched, the resulting anhydrothrombin was folded by diluting the denaturant, Gdn.HCl, to nondenaturing concentrations. Anhydrothrombin was purified by PAB affinity chromatography. Both native thrombin and anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by reverse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptide, as shown by amino acid analysis. This anhydrothrombin peptide was found to be 18.7 +/- 1.6 lower in mass units than the native peptide by electrospray mass spectrometry, in accord with the elimination of a water molecule. The anhydrothrombin preparation was monomeric, as determined by sedimentation equilibrium. Anhydrothrombin was used in a competitive titration of the complex of native thrombin with the leech saliva protein hirudin, a potent thrombin inhibitor, as measured by the recovery of thrombin amidolytic activity. This demonstrated that anhydrothrombin is capable of nativelike binding interactions with macromolecular ligands.

Tetracyanoquinodimethane mediated glucose sensor based on a self-assembling alkanethiol/phospholipid bilayer.

An amperometric tetracyanoquinodimethane (TCNQ) mediated biosensor for glucose is described, based on a self-assembling alkanethiol/phospholipid bilayer laid down onto a gold surface. Gold was sputter deposited onto chromium coated silicon wafers to a thickness of 200 nm. A monolayer of alkanethiol was allowed to self-assemble from an ethanolic solution of dodecanethiol onto a freshly cleaned gold electrode in an overnight incubation. The monolayer was characterized by ellipsometric, impedance and cyclic voltammetry measurements (capacitance = 1.60 +/- 0.06 microF/cm2, and thickness 1.34 +/- 0.15 nm). A mixture of phospholipid liposomes containing free amino groups was placed on the monolayer and allowed to incubate overnight. The self-assembly of a phospholipid monolayer and allowed to incubate overnight. The onto the alkanethiol monolayer, resulted in the formation of a bilayer. The formation of bilayer was again characterized by impedance and cyclic voltammetry measurements (capacitance = 0.98 +/- 0.09 microF/cm2, and thickness = 1.85 +/- 0.22 nm). TCNQ has been incorporated into the liposomes before the formation of the bilayer. Glucose oxidase was cross-linked with the amino-groups of the phospholipids using bis [2-(sulphosuccinimiidooxicarbonyloxy) ethyl] sulphone. TCNQ which was incorporated in the bilayer acted as an efficient mediator to regenerate glucose oxidase. Cyclic voltammetry of the modified electrode and a response curve for the glucose sensor are reported.

Thrombin hydrolysis of an N-terminal peptide from fibrinogen Lille: kinetic and NMR studies.

Fibrinogen Lille, a congenital dysfibrinogenemia, has been reported to arise from a mutation from Asp to Asn at position 7 of the A alpha chain of human fibrinogen, thereby reducing the thrombin-catalyzed rate of hydrolysis of the Arg(16)-Gly(17) peptide bond of this chain. Synthetic peptides of relevant portions of the wild-type and mutant A alpha chains were prepared, and the thrombin-catalyzed rates of hydrolysis of their Arg(16)-Gly(17) peptide bonds were determined. In addition, transferred NOE measurements were made to deduce their conformations, when complexed to bovine thrombin. The kinetics data showed little difference in the hydrolysis rates between the wild-type and mutant peptides, and the NMR data indicate no difference in the bound conformation of these two peptides. Therefore, electrostatic (or salt-bridge) interactions between Asp(7) and thrombin do not influence the bound conformations of these peptides. Asp(7) may interact with a remote residue of fibrinogen, not present in these synthetic peptides, or there may be additional mutations beyond A alpha (1-20) which have not been detected in fibrinogen Lille. Alternatively, when thrombin binds to fibrinogen at its secondary binding site, its primary (active) site may display different reactivities toward wild-type fibrinogen and fibrinogen Lille.

Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site.

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).

Enzymatic coupled assay procedures that employ high-performance liquid chromatography: the synthesis of orotidylate from ribose.

High-performance liquid chromatographic assay procedures have been designed to monitor the catalytic activities of ribokinase and phosphoribosyl alpha-1-pyrophosphate (PRibPP) synthetase. These methods are only of qualitative value, when crude protein extracts are to be examined, because of the presence of myokinase. However, the product of the PRibPP synthetase reaction, can be detected quantitatively even in crude protein extracts through the addition of two enzymes (orotate phosphoribosyltransferase and inorganic pyrophosphatase) that catalyze the conversion of PRibPP into a spectroscopically detectable nucleotide product (orotidylate).