Glomalin-related soil protein contains non-mycorrhizal-related heat-stable proteins, lipids and humic materials

Glomalin is reportedly a stable and persistent protein produced in copious quantities by mycorrhizal fungi and may be an important pool of organic N in soil. Glomalin-related soil protein (GRSP), however, is only operationally defined by its extraction method, and has been only poorly characterized at best. The goal of this study was to characterize the molecular structures within GRSP. Synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy and pyrolysis field-ionization mass spectrometry (Py-FIMS) revealed that GRSP contains a consortium of proteins along with many impurities. Employing proteomic techniques, we found that glomalin itself may be a thioredoxin-containing chaperone; however, no homologies with proteins or DNA of mycorrhizal origin were detected. Proteomics techniques further revealed that this fraction contains large amounts of soil-related heat-stable proteins and proteins of non-mycorrhizal origin. Results of this research show that the current extraction procedure that defines GRSP yields a mixture of compounds and thereby overestimates glomalin stocks when quantified using the Bradford assay. The chemical nature of glomalin has yet to be conclusively determined; it is unlikely that the chemical structure of glomalin can be elucidated from the mixture extracted as GRSP. Instead, an investigation into the specific biochemistry of immunoreactive assays currently used to define GRSP, followed by proteomic characterization of monoxenic mycorrhizal cultures may be required to advance our understanding of the chemical nature and agronomic significance of GRSP in soils.     

Role of allophanes in the accumulation of glomalin‐related soil protein in tropical soils (Martinique,French West Indies)

Thermo‐stable, operationally defined soil protein, known as glomalin, may make an important contribution to carbon storage in soils. The term glomalin is used because this putative protein, or group of proteins, was originally thought to be produced only by Glomus fungi. There is currently little information on the glomalin‐related soil protein (GRSP) content of tropical soils, particularly allophanic soils that are known to have different carbon dynamics to temperate climate soils. We have measured the Bradford‐reactive GRSP content of soils sampled from forests and grasslands on the tropical island of Martinique and compared the observations with soil composition. Two operationally defined fractions of GRSP were measured, namely easily‐extractable and total GRSP. The contents of GRSP in moist soils were in the range of 2–36 g kg^?1, accounting for about 8% of soil organic carbon, and were greater in topsoils than in corresponding subsoils. Both the GRSP contents and the fraction of soil organic carbon attributed to GRSP were greater than those reported for temperate climate soils. Both total and easily extractable GRSP contents were positively correlated to soil organic carbon content. The fraction of soil organic carbon that could be attributed to soil protein decreased with increasing allophane content for allophanic soils. No other trends of GRSP content with soil properties or land use were found. GRSP extraction was decreased about seven‐fold by air‐drying of soils, confirming the irreversible change in the soil microstructure of allophanic soils. Total and easily extractable GRSP were correlated and we conclude that both are good probes of thermo‐stable soil protein content for these soils. No attempt was made to verify the fungal origin of the protein detected.     

Chemical characteristics of glomalin-related soil protein (GRSP) extracted from soils of varying organic matter content

Glomalin is described in the literature as a N-linked glycoprotein and the putative gene product of arbuscular mycorrhizal fungi (AMF). Since the link between glomalin and various protein fractions in soil is not yet clearly defined, glomalin-related soil protein (GRSP) more appropriately describes glomalin's existence in natural organic matter (NOM). The objective of this study was to examine the chemical characteristics of GRSP present in several mineral and organic soils of varying organic carbon content. GRSP was isolated using high temperature sodium citrate extraction followed by either trichloroacetic acid (TCA) or hydrochloric acid (HCl) precipitation. GRSP was characterized by quantitative solid-state ¹³C DPMAS NMR, infrared (IR) spectroscopy, elemental analysis, and the Bradford assay for protein content. GRSP accounted for 25% and 52% of total C in the mineral soils and organic soil, respectively. Molar C/N and H/C ratios reveal that GRSP has less nitrogen than bovine serum albumin (BSA), and that GRSP extracted from the Pahokee peat soil possessed a more unsaturated, and thus aromatic character relative to the mineral soil GRSP, respectively. GRSP's high aromatic (42-49%) and carboxyl (24-30%) carbon contents and low aliphatic (4-11%) and carbohydrate-type carbon contents (4-16%) suggests that GRSP does not resemble a typical glycoprotein. In fact, the NMR spectra of GRSP closely resemble that of humic acid. GRSP extracted from mineral and organic soils possessed the same NMR fingerprint regardless of the precipitation method used (i.e., either TCA or HCl). It is likely that the current GRSP extraction methods, because of their similarity to the method used to extract humic acid, are coextracting both materials.     

Behavior of Bradford-reactive substances is consistent with predictions for glomalin

There is considerable controversy concerning detection in soils of the protein, glomalin, which is produced by arbuscular mycorrhizal fungi. Glomalin was originally defined as a substance that cross reacts with a monoclonal antibody formed against a substance in the cell walls of an arbuscular mycorrhizal fungus. Thus, one can use an immunological approach to detect glomalin. However, it was recently shown that other proteins cross react with the antibody. The other, more common, approach involves assay of soil protein using the Bradford reaction. This approach assumes that the Bradford assay is specific to protein, and that the assayed protein is largely glomalin, either because other proteins are in low concentration, or because the extraction process eliminates the possibility of their detection. These assumptions, however, have been called into question recently. One way to test whether the Bradford assay can be useful in quantifying glomalin is to determine whether the concentrations of Bradford-reactive substances are consistent with predictions for glomalin. For example, if recently produced glomalin is more labile than older glomalin, the concentrations of the two fractions should not be highly correlated. Moreover, when a contrast is established between mycorrhizal and nonmycorrhizal vegetation, recently produced glomalin should soon occur in higher concentration in soils supporting mycorrhizal vegetation. Older glomalin should not be found in higher concentrations in the soils of mycorrhizal vegetation until some time later. We tested these predictions by employing the Bradford assay during the course of a three-year field experiment in which canola (nonmycorrhizal) and soy (mycorrhizal) were grown in separate plots in year 1, both of which were followed by maize (mycorrhizal) in years 2 and 3. The correlation between the concentrations of fraction 1 Bradford-reactive substances (also known as easily extractable glomalin and frequently assumed to be recently produced) and fraction 2 (the more difficult-to-extract fraction and frequently assumed to be older glomalin), was very poor. In year 1, the concentration of fraction 1 was significantly greater in soy plots than in canola plots. Finally, fraction 2 was only significantly higher in the former soy plots than in former canola plots in years 2 and 3. These data support the hypothesis that the Bradford assay was useful in detecting glomalin in this case.     

Glomalin extraction and measurement

We investigated extraction from soil of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, and we examined its measurement. The most commonly used protocols for extracting glomalin require autoclaving of soil in citrate solution, followed by centrifugation to separate the supernatant, and then measurement by either Bradford protein assay or enzyme-linked immunosorbent assay (ELISA). We found that lengthening the time of autoclaving increased easily extractable glomalin extraction. Delay of centrifugation after autoclaving, however, diminished Bradford-reactive substances in the supernatant, suggesting that extracted substances might be reversibly immobilized on soil particles. Surprisingly, increasing the volume of extraction solution did not accelerate extraction of “total glomalin”, but instead, substantially increased the amount extracted. Multiple autoclave cycles nevertheless denature glomalin, which may not be as heat-resistant as thought. Repeated 1-h autoclaving of supernatant diminished both its Bradford-reactive substances (7.3% h^?1) and immunoreactive protein (22% during the first hour and 9.5% h^?1 of the remainder thereafter), although a large initial volume of extractant could reduce the loss of immunoreactive protein. Proteins and polyphenols that survive the extraction process are measured non-specifically by the Bradford assay. When we added other glycoproteins to dry soils, we recovered a maximum 34% bovine serum albumin and 22% bovine mucin, primarily in the first two, 1-h extraction cycles. These added proteins may adhere to soil organic matter and thereby be protected from denaturation. In addressing the endpoint of glomalin extraction, we found that the Michaelis–Menten equation closely fits cumulative glomalin per extraction cycle such that its asymptote provides an objective estimate of total extractable glomalin for a given set of extraction conditions. Additionally, the equation provides a curvature parameter that reflects the soil-specific efficiency of an extraction protocol. Although the soils that we investigated with 7.6% or more soil organic matter had the most asymptotic total glomalin, they were extracted the least efficiently.     

Decomposition and the contribution of glomalin-related soil protein (GRSP) in heavy metal sequestration: Field experiment

Glomalin is a metal-sorbing glycoprotein excreted by arbuscular mycorrhizal fungi (AMF). One method of estimating glomalin in soils is as glomalin-related soil protein (GRSP). In this study the role of GRSP in sequestering Pb and Cd was investigated in an in situ field experiment. The effect of metal sequestration on the subsequent decomposition of GRSP was also investigated. GRSP was determined using the Bradford method as total glomalin-related soil protein (T-GRSP) and as easily extractable glomalin-related soil protein (EE-GRSP). After 140 days, GRSP bound Pb accounted for 0.21–1.78% of the total Pb, and GRSP bound Cd accounted for 0.38–0.98% of the total Cd content in the soil. However when compared on a soil organic matter (SOM) basis, only 4% of the Pb or Cd was bound to the GRSP fraction of the SOM compared with 40–54% of the Pb or Cd bound to the humin and fulvic acids in the SOM fraction. In soils contaminated with the highest levels of Pb and Cd, the T-GRSP (EE-GRSP) decomposition after 140 days was reduced by 8.0 (6.6)% and 7.0 (7.5)%, respectively, when compared with the controls. In the high Pb or Cd treatment groups we found that the fraction of metal bound to GRSP increased even though the total GRSP content declined over time. The mass ratio between Pb and GRSP-carbon changed from 2.3 to 271.4 mg (100 g)⁻¹ in all Pb levels soil, while with the high-Cd treatment group the mass ratio between Cd and GRSP-carbon (0.36 mg (100 g)⁻¹) was higher than the mass ratio seen with Cd-bound humic acid fractions. Our in situ field study shows that while GRSP does bind Pb and Cd, in the soils we investigated, the levels are insignificant compared to soil organic matter such as humic and fulvic acids.     

Glomalin‐related soil protein in French temperate forest soils: interference in the Bradford assay caused by co‐extracted humic substances

Thermostable soil protein, known as glomalin, is an important component of soil carbon stocks. Thought to originate from endomycorrhizal fungi, Glomales, this operationally‐defined fraction of soil organic matter contains proteins of diverse origin as well as non‐protein material, including humic substances. Accumulation results from the balance between production/release and subsequent degradation. Quantification of the protein is subject to uncertainty because of the co‐extraction of other components that interfere with the Bradford assay. We studied 10 topsoils from French temperate forests, taken from the national forest monitoring network (Renecofor). Two fractions were extracted, easily extractable (EE) at neutral pH and total extractable (T) at pH 8. Protein was quantified with the colorimetric Bradford method, either by direct calibration using bovine serum albumin (BSA) or by extrapolation of the standard addition plot of BSA. Solubilized organic matter was characterized by using absorbance at 465 and 665 nm and by three‐dimensional fluorescence excitation‐emission spectroscopy. Neither soil properties nor forest cover influenced glomalin‐related soil protein (GRSP) content. Direct assay gave the GRSP_EE to be about 1 g kg^?1 soil, and GRSP_T in the range 3–10 g kg^?1, accounting for about 2% of soil organic carbon and about 15% of soil nitrogen. Standard addition plots indicated a two to sixfold under‐estimation of protein in total extracts, caused by negative interference with the Bradford assay. The GRSP_EE was correlated significantly with both estimates of GRSP_T. Under‐estimation of GRSP_T by direct assay was not related to the E4:E6 ratio but was correlated significantly with the intensity of absorbance at either 460 or 660 nm and with one of the fluorescence peaks. We conclude that GRSP_EE is not necessarily more recent than GRSP_T and that both fractions may be probes of protein content, but that absolute contents may be under‐estimated because of co‐extracted humic substances.     

Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) produce a protein, glomalin, quantified operationally in soils as glomalin-related soil protein (GRSP). GRSP concentrations in soil can range as high as several mg g⁻¹ soil, and GRSP is highly positively correlated with aggregate water stability. Given that AMF are obligate biotrophs (i.e. depending on host cells for their C supply), it is difficult to explain why apparently large amounts of glomalin would be produced and secreted actively into the soil, since the carbon could not be directly recaptured by the mycelium (and benefits to the AMF via increased soil structure would be diffuse and indirect). This apparent contradiction could be resolved by learning more about the pathway of delivery of glomalin into soil; namely, does this occur via secretion, or is glomalin tightly bound in the fungal walls and only released after hyphae are being degraded by the soil microbial community? In order to address this question, we grew the AMF Glomus intraradices in in vitro cultures and studied the release of glomalin from the mycelium and the accumulation of glomalin in the culture medium. Numerous protein-solubilizing treatments to release glomalin from the fungal mycelium were unsuccessful (including detergents, acid, base, solvents, and chaotropic agents), and the degree of harshness required to release the compound (autoclaving, enzymatic digestion) is consistent with the hypothesis that glomalin is tightly bound in hyphal and spore walls. Further, about 80% of glomalin (by weight) produced by the fungus was contained in hyphae and spores compared to that released into the culture medium, strongly suggesting that glomalin arrives mainly in soil via release from hyphae, and not primarily through secretion. These results point research on functions of glomalin and GRSP in a new direction, focusing on the contributions this protein makes to the living mycelium, rather than its role once it is released into the soil.     

Is glomalin an appropriate indicator of forest soil reactive nitrogen status?

In this paper we address total glomalin‐related soil protein (T‐GRSP) as a possible indicator of differences in forest soils related to reactive nitrogen and forest composition. We focused especially on the relationship between T‐GRSP (g kg⁻¹), soil organic carbon (SOC), and reactive nitrogen (N_r) availability among different categories of temperate forests and different horizons. Our study included 105 sampling sites divided into 5 categories, which vary in elevation and tree species composition (coniferous, deciduous, mixed). We detected significantly higher T‐GRSP and SOC in the F+H horizon under conifers. We assume that this observation might be attributed to suppression of decomposition of T‐GRSP and SOC by nature of coniferous litter. The lack of significant differences in T‐GRSP/SOC among the categories and the positive correlations between T‐GRSP and SOC in most of the categories confirmed the strong relationship of T‐GRSP with SOC. We found a significantly higher content of T‐GRSP in the F+H horizon for all studied forest categories. However, the contribution of T‐GRSP to SOC is significantly higher in the A horizon, which might be caused by stabilization of glomalin by mineral fraction, including clay minerals or by the belowground origin of glomalin. We found the increase of SOC with increasing N_r in the A horizon for most categories of forest. T‐GRSP follows this trend in the case of deciduous forests (decid), mixed forest (mixed), and mountain forests (mount). On the other hand, we detected a decrease of T‐GRSP with increasing N_r in the F+H horizon of coniferous forests (conif). Moreover the T‐GRSP/SOC decreases with the increase of N_r in the A horizon of conif, mixed and mount, which points to the higher sensitivity of forest with prevalence of coniferous trees. Our observations have confirmed an ecosystem‐specific relationship between T‐GRSP, SOC and N_r. We concluded that T‐GRSP in combination with T‐GRSP/SOC has the potential to reveal qualitative changes in soil organic matter (SOM) connected with increasing N_r.     

Soil structural stability and erosion rates influenced by agricultural management practices in a semi‐arid Mediterranean agro‐ecosystem

Unsuitable agricultural practices can cause loss in soil quality and erodibility to thus increase or trigger desertification under Mediterranean conditions. A field experiment was performed at the El Teularet‐Sierra de Enguera Experimental Station (eastern Spain) to assess the influence during a 5‐yr period of different agricultural practices on physical and chemical indicators of soil quality (total and water‐soluble carbohydrates, glomalin‐related soil proteins (GRSP), total organic carbon, aggregate stability (AS), vegetation cover and soil erosion). The management practices included residual herbicide use, ploughing, ploughing + oats, addition of oat straw mulch and a control (land abandonment). Adjacent soil under natural vegetation was used as a reference for local, high‐quality soil and as a control for comparison with the agricultural soils under different management practices. Oat straw mulching led to higher levels of water‐soluble carbohydrates, GRSP and AS and lower soil erosion rates, resulting in values similar to those in the soil under native vegetation. The lowest levels of carbohydrates and GRSP were for the plots that were treated with herbicide or were ploughed. The maintenance of and increases in stable aggregates promoted by the different agricultural management practices over the years were attributed to increases in labile organic fractions such as carbohydrates and to the GRSP content. The results demonstrate that land abandonment (control plot) or the use of a cover (plants or straw) contributes to increases in soil quality and reduces the risk of erosion. The research also shows that sustainable agricultural management allows soil to recover and that the use of straw mulching is the most effective management strategy.     

Plant invasion of native grassland on serpentine soils has no major effects upon selected physical and biological properties

Plant invasions alter soil microbial community composition; this study examined whether invasion-induced changes in the soil microbial community were reflected in soil aggregation, an ecosystem property strongly influenced by microorganisms. Soil aggregation is regulated by many biological factors including roots, arbuscular mycorrhizal fungal hyphae, and microbially-derived carbon compounds. We measured root biomass, fungal-derived glomalin-related soil protein (GRSP), and aggregate mean weight diameter in serpentine soils dominated by an invasive plant (Aegilops triuncialis (goatgrass) or Centaurea solstitialis (yellow starthistle)), or by native plants (Lasthenia californica and Plantago erecta, or Hemizonia congesta). Root biomass tended to increase in invaded soils. GRSP concentrations were lower in goatgrass-dominated soils than native soils. In contrast, starthistle dominated soil contained a higher amount of one fraction of GRSP, easily extractable immunoreactive soil protein (EE-IRSP) and a lower amount of another GRSP fraction, easily extractible Bradford reactive soil protein (EE-BRSP). Soil aggregation increased with goatgrass invasion, but did not increase with starthistle invasion. In highly aggregated serpentine soils, small increases in soil aggregation accompanying plant invasion were not related to changes in GRSP and likely have limited ecological significance.     

Minimal direct contribution of arbuscular mycorrhizal fungi to DOC leaching in grassland through losses of glomalin-related soil protein

Arbuscular mycorrhizal fungi (AMF) have multiple influences on ecosystem C cycling, but most research has focused on ecosystem C gains. We explore here the possibility of direct contributions of AMF to ecosystem C losses, namely via leaching of glomalin-related soil protein (GRSP). We tested the hypothesis that GRSP, an operationally defined SOM pool to which AMF contribute (especially as evidenced with monoclonal antibody MAb32B11-based detection), is mobile in soils and can be lost in leachate. For two New Zealand soils, we showed that only insignificant amounts of GRSP were lost: a maximum of 0.03% of MAb32B11-immunoreactive GRSP present in soils was lost during the week-long experiment, representing a minute fraction of total leachate dissolved organic carbon (0.06%). Our data showed that this pathway of C loss may be relatively unimportant in many soils. However, other indirect contributions of AMF to soil C losses remain yet to be explored.     

南京典型利用方式土壤中球囊霉素含量及剖面分布特征

采用Brad-ford染色法研究了南京市5种典型利用方式土壤不同土层中(0~10、10~20、20~40 cm)球囊霉素的含量。结果表明:土壤中总球囊霉素含量为1.96~3.12 mg/g,占土壤有机碳的12.5%~29.0%,所占比例随土壤有机碳含量的增加而降低。林地和草地土壤中球囊霉素和有机碳的含量均高于3种耕作土壤(水稻田、茶园土和菜园土)。随着土层深度(0~40 cm)的增加,5种不同利用方式土壤中总球囊霉素和有机碳的含量均减小;与其他土层相比,0~10 cm土层总球囊霉素和有机碳含量均最大。耕作土壤中易提取球囊霉素更易于向总球囊霉素转化。发现5种土地利用方式下土壤中总球囊霉素含量与土壤有机碳含量极显著正相关,与土壤p H显著负相关;易提取球囊霉素与土壤有机碳含量极显著负相关。总球囊霉素和易提取球囊霉素可作为评价土壤丛枝菌根真菌活性和土壤质量的重要指标。     

球囊霉素及其在土壤生态系统中的作用

球囊霉素(Glomalin)是一种在土壤中大量存在的、由丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)产生的具有良好热稳性的特殊糖蛋白.球囊霉素因其在促进土壤团聚体形成,保持团聚体稳定性,增加土壤有机碳库,提高植物抗逆能力以及降低重金属在土壤中的毒性等方面的作用备受人们关注.目前由于提...     

Molecular quantification of slug density in the soil using monoclonal antibodies

Molecular ecology techniques are increasingly used to study invertebrate foodwebs and trophic interactions in the field. However, the study of subterranean foodwebs is currently constrained by the difficult, laborious and often expensive methods that need to be employed to simply measure invertebrate population densities accurately. Here we describe and field-test a novel monoclonal antibody-based system for tracking slug populations. Proteins were extracted from soil blocks using sodium chloride and a new slug-specific monoclonal antibody was developed, capable of detecting the proteins liberated by the salt. Detection sensitivity and limits, using enzyme-linked immunosorbent assays (ELISA), were measured for a range of soils and the system proved to be effective, whether the soil was heavy clay or sandy. The sensitivity of the assay varied between soils and needed to be calibrated by ELISA. There was a linear relationship between slug biomass in any given soil and slug proteins detected by ELISA. A field experiment was performed comparing ELISA with the most accurate conventional approach. The latter involved taking blocks of soil from the field and flooding them gradually over 10 days to drive slugs to the surface, where they were collected and weighed. Parallel blocks of soil were taken 1 m away and subjected to the salt extraction/ELISA approach. Results using the two systems proved to be very similar, but ELISA produced results more rapidly. The many advantages of using ELISA to measure slug density are discussed.     

Re-examining the glomalin-purity of glomalin-related soil protein fractions through immunochemical,lectin-affinity and soil labelling experiments

Due to analytical similarities with the mycorrhizal glycoprotein glomalin, ubiquitous citrate and heat-extractable soil protein fractions have been assumed to be predominantly glomalin-stabilised within soil. Often termed glomalin-related soil protein (GRSP), little however is actually known of the “glomalin-purity” of these soil fractions. We undertook western and lectin blots and crossed immuno/lectin affinity electrophoresis (CIE/CLAE) analysis of “easily extractible” GRSP fractions, as well as liquid chromatography-tandem mass spectrometry (LC–MS/MS) of “total” GRSP fractions. To further test whether soil saprobes contribute to GRSP production, we amended soil with ¹⁴C-sucrose and examined whether ¹⁴C could be traced in the GRSP pool over a 500-day incubation period.While only four of six bands on SDS–PAGE profiles of easily extracted GRSP reacted with anti-glomalin MAb32B11 and the lectin Con A under our blotting conditions, CIE/CLAE indicated the presence of a single protein moiety in the easily extractible GRSP pool. LC–MS/MS analysis of total GRSP pooled from various soils also showed that although traces of protein tentatively assignable to soil bacteria were present in GRSP, their concentrations were low. Additionally, specific activity of total GRSP in ¹⁴C-labelled soil was relatively depleted compared to the bulk soil and soil microbial biomass. This suggests that little GRSP of heterotrophic origin was laid down over the incubation period, although the potential presence of a pre-existing ¹⁴C-free GRSP background, as well as of low microbial dynamics in the absence of any further substrate inputs to the soil warrant caution with this inference.     

Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method

 The Bradford protein quantification assay is based on an absorbance shift in Coomassie brilliant blue G-250 (CBB). Samples extracted for glomalin, a protein produced by arbuscular mycorrhizal (AM) fungi, are quantified using the Bradford assay. CBB is known to react with polyphenolic substances, and co-extraction of glomalin and humic substances is known to occur. The effects of increasing concentrations polyphenolic compounds were measured. The addition of any amount of polyphenolic compounds increased the Bradford reactive fraction (BRF) of soil extract. Caution is required when interpreting BRF data, as comparison of BRF data from different studies or different field sites is problematic. The BRF may represent recalcitrant organic material in soil, though its relationship to AM fungi remains unclear.     

Arbuscular mycorrhizal fungi contribute to C and N enrichment of soil organic matter in forest soils

Increasing evidence suggests that accretion of microbial turnover products is an important driver for isotopic carbon (C) and nitrogen (N) enrichment of soil organic matter (SOM). However, the exact contribution of arbuscular mycorrhizal fungi (AMF) to soil isotopic patterns remains unknown. In this study, we compared ¹³C and ¹⁵N patterns of glomalin-related soil protein (GRSP), which includes a main fraction derived from AMF, litter, and bulk soil in four temperate rainforests. GRSP was an abundant C and N pool in these forest soils, showing significant ¹³C and ¹⁵N enrichment relative to litter and bulk soil. Hence, cumulative accumulation of recalcitrant AMF turnover products in the soil profile likely contributes to ¹³C and ¹⁵N enrichment in forest soils. Further research on the relationship between GRSP and AMF should clarify the exact extent of this process.     

Revision of two colorimetric methods to quantify glomalin-related compounds in soils subjected to different managements

The aim of this study was to analyze two colorimetric methods used to determine easily extracted glomalin-related soil proteins (EE-GRSP). The historically and most commonly used method for measurement of EE-GRSP as total protein has been the Bradford assay. After some troubles/inconsistencies with this method, we carefully analyzed the Bradford assay, measuring a dilution series of the EE-GRSP fraction and analyzing the time stability of the product. In addition, we did similar analysis of another colorimetric method that quantifies total protein, the bicinchoninic acid (BCA) assay. Unexpectedly, we found that the EE-GRSP concentration values determined by Bradford assay were dependent and variable with the dilution level of the soil extract; moreover, the Bradford assay shows a great instability with the time when soil samples were analyzed but not when protein solution as bovine serum albumin (BSA) was used as control. On the contrary, the BCA assay was independent of the dilution levels of the soil extract and showed stability in the time either for soil samples or BSA protein quantification. These results were consistent and independent on the different type of soils corresponding to different locations and with different textures.