Selective feeding by the aquatic oligochaete Tubifex tubifex (Tubificidae,Clitellata)

The particle size distribution of faecal pellets produced by the tubificid worm Tubifex tubifex in laboratory culture, was measured with a Coulter® Multisizer. The faecal material from worms cultured in a range of sediments was composed of particles with a mean diameter of less than 63 m, and only a few isolated larger particles were found by microscopic analysis. This suggests that this species actively selects the silt-clay fraction, avoiding larger sand particles. A more detailed analysis of faeces revealed that about 75%, by volume, was composed of particles with a mean diameter < 25m, and the mode was < 10m. T. tubifex fed selectively on the organic rich particles of the sediment, and this feeding was independent of particle size. Measurement of the organic content of faeces (measured as % loss on ignition) showed that they had a consistently higher organic content than the sediment, considered as whole sediments or the <63 m sieved fraction. On the basis of these results, we hypothesise that this species exhibits two levels of selectivity in its feeding behaviour. Thus selection is primarily based on particle size, avoiding the ingestion of sand particles and also, on the preferential selection of particles associated with organic material, within the fine (silt-clay) fraction of the sediment.     

A comparison of soil quality in adjacent cultivated,forest and native grassland soils

Changes in soil quality after 45 years of continuous production of corn (Zea mays L.) by the conventional tillage method (C) compared with adjacent poplar forest (F) and native grassland (G) sites were examined. The investigated parameters were: total and humified organic C, total N, light fraction content and composition, water-soluble organic C (WSOC), water-soluble carbohydrates (WSC), phenolic substances, biomass C, cumulative CO₂-C (soil respiration) (C _m), enzyme activities (alkaline phosphatase, protease, -glucosidase, urease, catalase and dehydrogenase). Empirical indexes of soil quality were also calculated: biomass C/organic C, specific respiration of biomass C (qCO₂), death rate quotient (qD), metabolic potential (MP), biological index of fertility (BIF), enzyme activity number (EAN) and hydrolysing coefficient (HC). Results indicate that long-term corn production at an intensive level caused a marked decline in all examined parameters. Between the undisturbed systems, native grassland showed higher values of soil quality parameters than forest site. The indexes most responsive to management practices that may provide indications of the effects of soil cultivation, as well as of the differently undisturbed ecosystems were: organic C, WSC, C _m, protease, -glucosidase, urease and HC. Soil enzyme activities were well related with, and not more sensitive than organic carbon.     

Purification,and characterization of a β-mannanase of Trichoderma reesei C-30

Trichoderma reesei was studied for its ability to produce -mannanase activity on a variety of carbon sources. The highest -mannanase activity was produced on cellulose, whereas -mannan-containing carbon sources (such as kojac powder or locust bean gum) gave lower enzyme titres. The enzyme responsible for the major -mannanolytic activity from T. reesei was purified to physical homogeneity by preparative chromatofocusing and anion exchange fast protein liquid chromatography. This -mannanase is a glycoprotein, with a molecular mass of 46 (±2) kDa and an isoelectric point of 5.2. It has an optimal pH at 5.0 and broad pH stability (2.5–7.0). It is stable for 60 min at 55° C, and has an optimal temperature for activity at 75° C. During incubation with locust bean gum, the enzyme releases mainly tri- and disaccharides. Correspondence to: C. P. Kubicek     

Double-stranded regions in denatured DNA from mouse cells

Approximately 2% of the DNA of the mouse genome reassociates at infinitely low C ₀ t values, 10^-7 to 10^-6 moles 1^-1 s. The melting profile of the reassociation product, which is resistant to nuclease S₁ digestion, has been characterized by hydroxyapatite column chromatography. The properties of these nuclease resistant sequences suggest that they exist as DNA-hairpins and that they originate from reverted base sequences within the genome.     

Filamentous Marine Fungi as Producers of <Emphasis Type="Italic">O</Emphasis>-Glycosylhydrolases: β-1,3-Glucanase from <Emphasis Type="Italic">Chaetomium indicum</Emphasis>

Ninety fungal strains (42 species) isolated from marine habitats were studied for their ability to produce extracellular enzymes. Cultural filtrates of these strains were shown to contain a series of glycosidases (-glucosidases, N-acetyl--glucosaminidases, -galactosidases -mannosidases) and glucanases (1,3--glucanases, amylases) which varied with habitat. The level of activity depended on the species of fungi. Several promising strains capable of producing both individual enzymes and a set of enzymes for splitting carbohydrate-containing compound have been isolated. Optimal conditions for growth of Chaetomium indicum and for biosynthesis of -1,3-glucanase were determined. -1,3-Glucanase was isolated using ion-exchange chromatography, ultrafiltration, and gel filtration. The presence of 2 enzyme forms was shown; both forms were exo--1,3-glucanases.     

Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii

The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55°C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS–polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55–70°C and pH 5–10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially -cyclodextrin. With a longer incubation period, the -cyclodextrin to -cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5–20.0%) and glucans containing branching points (-1,6 glycosidic linkages) shifted the product pattern to: -cyclodextin > -cyclodextrin > -cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl₂ shifted the apparent melting point of the enzyme from 60°C to 69°C.Dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 80th birthday.     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

Nitrogen deposition effects on soil organic matter chemistry are linked to variation in enzymes,ecosystems and size fractions

Recent research has dramatically advanced our understanding of soil organic matter chemistry and the role of N in some organic matter transformations, but the effects of N deposition on soil C dynamics remain difficult to anticipate. We examined soil organic matter chemistry and enzyme kinetics in three size fractions (>250 μm, 63–250 μm, and <63 μm) following 6 years of simulated atmospheric N deposition in two ecosystems with contrasting litter biochemistry (sugar maple, Acer saccharum—basswood, Tilia americana and black oak, Quercus velutina—white oak, Q. alba). Ambient and simulated (80-kg NO₃ ⁻–N ha⁻¹ year⁻¹) atmospheric N deposition were studied in three replicate stands in each ecosystem. We found striking, ecosystem-specific effects of N deposition on soil organic matter chemistry using pyrolysis gas chromatography/mass spectrometry. First, furfural, the dominant pyrolysis product of polysaccharides, was significantly decreased by simulated N deposition in the sugar maple–basswood ecosystem (15.9 vs. 5.0%) but was increased by N deposition in the black oak–white oak ecosystem (8.8 vs. 24.0%). Second, simulated atmospheric N deposition increased the ratio of total lignin derivatives to total polysaccharides in the >250 μm fraction of the sugar maple–basswood ecosystem from 0.9 to 3.3 but there were no changes in other size classes or in the black oak–white oak ecosystem. Third, simulated N deposition increased the ratio of lignin derivatives to N-bearing compounds in the 63–250 and >250 μm fractions in both ecosystems but not in the <63 μm fraction. Relationships between enzyme kinetics and organic matter chemistry were strongest in the particulate fractions (>63 μm) where there were multiple correlations between oxidative enzyme activities and concentrations of lignin derivatives and between glycanolytic enzyme activities and concentrations of carbohydrates. Within silt-clay fractions (<63 μm), these enzyme-substrate correlations were attenuated by interactions with particle surfaces. Our results demonstrate that variation in enzyme activity resulting from atmospheric N deposition is directly linked to changes in soil organic matter chemistry, particularly those that occur within coarse soil size fractions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distance-constraint approach to protein folding. I. Statistical analysis of protein conformations in terms of distances between residues

A statistical analysis of protein conformations in terms of the distance between residues, represented by their C atoms, is presented. We consider four factors that contribute to the determination of the distanced _i,i+k between a given pair ofith and(i+k)th residues in the native conformation of a globular protein: (1) the distancek along the chain, (2) the size of the protein, (3) the conformational states of theith to(i+k)th residues, and (4) the amino acid types of the and(i+k)th residues. In order to account for the dependence on the distancek along the chain, the statistics are taken for three ranges, viz., short, medium, and long ranges (k8; 9k20; andk21; respectively). In the statistics of short-range distances, a mean distanceD _k and its standard deviationS _k are calculated for each value ofk, with and without taking into account the conformational states of all residues fromi toi+k (factors 1 and 3). As an Appendix, the relations for converting from the distances between residues into other conformational parameters are discussed. In the statistics of long-range distances, a reduced distanced* _ij (the actual distance divided by the radius of gyration) is used to scale the data so that they become independent of protein size, and then a mean reduced distanceD _l (a, a) and its standard deviation _l (a, a) are calculated for each amino acid pair (a, a) (factors 2 and 4). The effect of the neighboring residues along the chain on the value of the distanced* _ij is explored by a linear regression analysis between the actual reduced distanced* _ij and the mean value over theD _l for all possible pairs of residues in the two segments of the (i–2)th to the (i+2)th and the (j–2)th to the (j+2)th residues. The effect is assessed in terms of the tangentA _l (a, a) of the calculated regression line for each amino acid pair (a, a). In the statistics of medium-range distances, only factors 1 and 4 are considered, to simplify the analysis. The scaled distanced _i,i+k =(d _i,i+k -D _k )/S _k is used to eliminate the dependence onk, the distance along the chain. The propertiesD _m (a, a), _m (a, a) andA _m (a, a) corresponding toD _l (a, a), _l (a, a), andA _l (a, a), and also calculated for each amino acid pair (a, a). The results are interpreted as follows: the smaller values ofD _l (a, a) andD _m (a, a) indicate a preference of the pair (a, a) for a contact (e.g., pairs between hydrophobic amino acids, and pairs of Cys with aromatic amino acids), and the larger values of these quantities indicate a preference for distant mutual location (e.g., pairs between strong hydrophilic amino acids); the smaller values of _l (a, a) and _m (a, a) indicate a strong preference for either contact or noncontact (e.g., pairs between hydrophobic amino acids, and pairs between strong hydrophobic and hydrophilic amino acids, respectively), and the larger values of these quantities indicate the ambivalent/neutral nature of the preference for contact and noncontact (e.g., pairs containing Ser or Thr); the smaller values ofA _l (a, a) andA _m (a, a) indicate that the distance of an (a, a) pair is determined independently of the amino acid character of the neighboring residues along the chain (e.g., some pairs of Cys or Met with other amino acids) and the larger values of these quantities indicare that such amino acid character contributes strongly to the determination of the distance (e.g., pairs containing Ser or Thr, and pairs between amino acids with small side chains). The difference between the statistics for the long- and medium-range distances is also discussed; the former reflect the difference between the hydrophobic and hydrophilic character of the residues, but the latter cannot be easily interpretable only in terms of hydrophobicity and hydrophilicity. The data analyzed here are used in the optimization of an object function to compute protein conformation in a subsequent paper.     

Effects of hyperphenylalaninemia in the fetal stage on the postnatal development of fetal rat brain

The effect of exposure at different prenatal stages to maternal hyperphenylalaninemia (HyPhe) on the somatic and neurological development of fetuses in rats was studied, with special respect to the change of relevant enzyme activities in the brain. While evident somatic damage was found only in the fetuses exposed to maternal HyPhe at a last stage of gestation, distinct mental retardation seemingly due to some irreversible damage to the brain was observed in all the treated fetuses regardless of the timing of exposure, and a significantly reduced activity of 2, 3-cyclic nucleotide 3-phosphohydrolase (CNPase), a marker enzyme of myelin, was confirmed in the mantle region of the brain.Dedicated to Professor Yasuzo Tsukoda.     

cDNA Sequence, Protein Structure, and Evolution of the Single Hemocyanin from Aplysia californica, an Opisthobranch Gastropod

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5UTR of 77 bp, a 3UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (M_r ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (M_r ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373±47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343±43 million years ago.[Reviewing Editor: Dr. Axel Meyer]The sequence reported in this paper has been deposited in the GenBank database under accession number AJ556169.     

A knowledge based system for diagnosing microbial activities during a fermentation process

A knowledge based system, LAexpert, was developed to diagnose microbial activities during a fermentation process on the basis of specific rates determined on-line. The LAexpert is a supervisor for a process control system and assists operators in fault diagnosis. The LAexpert was implemented using a fuzzy expert system shell based on the object oriented programming tool Smalltalk V/Mac running in a Macintosh II computer. The shell can handle uncertainties both in the measurements and knowledge by fuzzy reasoning.List of Symbols X g/l biomass dry weight (g/l) - S g/l substrate concentration (g/l) - P g/l product concentration (g/l) - _c, _c, _c 1/h specific rates calculated from on-line measured data of X, S and P (1/h) - _d, _d, _d 1/h specific rates read from database of BIOACS (1/h)     

Simultaneous synthesis of enantiomerically pure (R)-1-phenylethanol and (R)-alpha-methylbenzylamine from racemic alpha-methylbenzylamine using omega-transaminase/alcohol dehydrogenase/glucose dehydrogenase coupling reaction

A simultaneous synthesis of (R)-1-phenylethanol and (R)--methylbenzylamine from racemic -methylbenzyl- amine was achievied using an -transaminase, alcohol dehydrogenase, and glucose dehydrogenase in a coupled reaction. Racemic -methylbenzylamine (100 mM) was converted to 49 mM (R)-1-phenylethanol (> 99% ee) and 48 mM (R)--methylbenzylamine (> 98% ee) in 18 h at 37 °C. This method was also used to overcome product inhibition of -TA by the ketone product in the kinetic resolution of racemic amines at high concentration.     

Isolation,characterization, and numerical taxonomy of Simonsiella strains from the oral cavities of cats,dogs, sheep,and humans

Forty-nine strains of the gliding prokaryote Simonsiella were isolated from the oral cavities of cats (8), dogs (19), sheep (4), and humans (18) in Southern California by a direct isolation procedure using a complex serum-enriched medium. The numerical taxonomic analysis (unweighted pair-group method using arithmetic averages) of 57 differential traits for each strain was based on standard bacteriological diagnostic tests and included the molar guanine-pluscytosine contents of the DNA and the relative percentages of fatty acid contents reported earlier. The resulting phenogram clustered the strains of Simonsiella into groups that correlated with sources of origin. The study included the neotype strain of Simonsiella crassa (ATCC 27504, ICPB 3651, NCTC 10283) of Australian sheep origin. The strains isolated from dogs, sheep, and humans form clusters of organisms that appear to have become adapted to live in and possibly to have evolved with their respective hosts. In our judgment, these source-of-origin clusters represent different ecospecies.List of Abbreviations ATCC American Type Culture Collection - BSTSY bovine serum-tryptic soy-yeast extract - DNA deoxyribonucleic acid - ECL equivalent chain length of carbon atoms - ICPB International Collection of Phytopathogenic Bacteria - NCTC National Collection of Type Cultures To whom offprint requests should be sent.     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

Identification of a positive regulatory protein in Escherichia coli: the product of the cysB gene

Summary From the specialized transducing bacteriophage cysB, recombinant phages cysB242 and cysB257 have been obtained, each of which carries an amber mutation in the cysB cistron. A comparison of polyacrylamide gel electrophoretic profiles of labelled extracts from uv-irradiated bacteria that had been infected with cysB ⁺ or with cysB-amber phages, led to the identification of a 39,000-dalton polypeptide as the product of the cysB gene. The native protein was purified to near radiochemical purity and was found to be an oligomer with an isoelectric point close to pH 7.     

Physicochemical and biological factors affecting atmospheric methane oxidation in gray forest soils

The decline of methane oxidizing activities in gray forest soil upon its conversion into arable land was shown to be caused by major changes in biotic and physicochemical properties of soil. Using the method of immune serums, methane-oxidizing bacteria were detected in both forest and agricultural soils, but their populations differed significantly in both abundance and composition. In the forest soil, the number of methanotrophs was an order of magnitude higher than in arable soil, amounting to 3.5 × 10⁸ and 0.24 × 10⁸ cells/g soil, respectively. All methane-oxidizing bacteria identified in the forest soil belonged to the genus Methylocystis, and 94% of these were represented by a single species, M. parvus. The arable soil was dominated by type I methanotrophs (Methylobacter and Methylomonas, 67.6%), occurring along with bacteria of the genus Methylocystis. In addition, arable soil is characterized by a low content of microbial biomass, lower porosity and water resistance of soil aggregates, and the predominance of nitrogen mineralization processes over those of nitrogen immobilization. These factors can also contribute to lower rates of methane oxidation in arable soil as compared to forest soil.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 255–260.Original Russian Text Copyright © 2005 by Kravchenko, Semenov, Kuznetsova, Bykova, Dulov, Pardini, Gispert, Boeckx, Cleemput, Galchenko.     

The potential of Miscanthus to sequester carbon in soils: comparing field measurements in Carlow,Ireland to model predictions

Growing bioenergy crops such as Miscanthus has the potential to mitigate atmospheric carbon dioxide emissions by the replacement of fossil fuels and by storing carbon (C) in the soil due to land use change. Here we compare direct measurements of soil organic C fractions made in Carlow (Ireland) to model predictions made by RothC and a cohort model. Our results show that when Miscanthus is grown on land previously under arable agriculture, the soil organic C will increase to a level above that of native pasture, as Miscanthus organic material is shown to have a slow decomposition rate. In addition we demonstrate that for measured organic C, fractions of different lability are similar to the C pools used in RothC. Using the model predictions from RothC and Miscanthus yields from MISCANFOR, we predict that in Ireland, changing the land use from arable to Miscanthus plantations has the potential to store between 2 and 3 Mg C ha^?1 y^?1 depending on the crop yield and the initial soil organic C level.     

Volume estimation of biological objects by systematic sections

The absolute volume of biological objects is often estimated stereologically from an exhaustive set of systematic sections. The usual volume estimator is the sum of the section contents times the distance between sections. For systematic sectioning with a random start, it has been recently shown that is unbiased when m, the ratio between projected object length and section distance, is an integer number (Cruz-Orive 1985). As this quantity is no integer in the real world, we have explored the properties of in the general and realistic situation m . The unbiasedness of under appropriate sampling conditions is demonstrated for the arbitrary compact set in 3 dimensions by a rigorous proof. Exploration of further properties of for the general triaxial ellipsoid leads to a new class of non-elementary real functions with common formal structure which we denote as np-functions. The relative mean square error (CE ²) of in ellipsoids is an oscillating differentiable np-function, which reduces to the known result CE ²= 1/(5m ⁴) for integer m. As a biological example the absolute volumes of 10 left cardiac ventricles and their internal cavities were estimated from systematic sections. Monte Carlo simulation of replicated systematic sectioning is shown to be improved by using m instead of m . In agreement with the geometric model of ellipsoids with some added shape irregularities, mean empirical CE was proportional to m ^–1.36 and m^–1.73 in the cardiac ventricle and its cavity. The considerable variance reduction by systematic sectioning is shown to be a geometric realization of the principle of antithetic variates.     

Warming and grazing increase mineralization of organic P in an alpine meadow ecosystem of Qinghai-Tibet Plateau, China

Background and aims

Little is known about the soil phosphorus (P) biogeochemical cycling in response to combined warming and grazing, especially in the alpine meadow ecosystem of the Qinghai-Tibet Plateau. Here, we used a free-air temperature enhancement system in a controlled warming-grazing experiment to test the hypothesis that combined warming and grazing would significantly accelerate mineralization of soil organic P.

Methods

A two factorial design of warming (1.2–1.7°C temperature increase) and moderate grazing was utilized. A fractionation method was applied to investigate the sizes of different soil inorganic and organic P fractions.

Results

Results showed that both warming and grazing significantly decreased the quantity of organic P extracted by first NaOH (N(I)Po), as well as the total extractable organic P (TPo) at the 0–10?cm depth. Warming also decreased the total P of soil at 0–10?cm. The combined warming and grazing treatment (WG) led to the reduction of major soil organic P fractions (N(I)Po, TPo) by 40–48% and 28–32%, respectively compared with other treatments at 0–10?cm. The activities of acid and alkaline phosphomonoesterase (AcPME and AlPME) were both enhanced by warming and grazing, and their interaction. Decreased concentrations of soil N(I)Po and TPo were accompanied by increased AcPME activity (P?<?0.01) and soil temperature (P?<?0.05), indicating the enhanced mineralization of organic P under rising temperature. Meanwhile, leaf biomass P of two major species (Potentilla anserine and Gentiana straminea) within these plots were significantly enhanced by either grazing or warming.

Conclusions

The microbial mineralization of soil organic P could be strongly increased under combined warming and grazing conditions as driven by increasing plant demand for P and enhanced microbial activities.