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Guber, Andrey

Publications and source records attributed to Guber, Andrey.

12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

2-D soil zymography: Accounting for the spatial variation of pH

Soil zymography is commonly used to quantify spatial distribution of hydrolytic enzyme activities on soil and plant root surfaces. It is recommended to adjust pH in zymography substrates and calibration solutions with respect to soil/root pH. However, pH values may vary greatly within a few mm of plant rhizosphere, potentially altering the distribution of pH in zymography membranes. Despite the fact that the effect of pH on the calibration of zymography membranes is generally known, its potential impact on zymography results is unaccounted for in processing zymography images and calculations of enzyme activity. In this study we assessed the effect of pH variations on the persistency of the methylumbelliferone (MUF) calibration. The studied pH values ranged from 4.5 to 7.5. The MUF calibration curves greatly deviated from that at a reference pH of 6.5, with a marked nonlinear increase of deviation with greater membrane brightness. We suggest that the problem can be partially alleviated by reducing the membrane incubation time. However, such deviations suggest the need for a more comprehensive resolution via mapping pH and using pH-specific calibrations to process zymography images. Here, we developed a MATLAB code to implement a pixel-based correction of enzyme activity for pH in processing time-lapse zymography images.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The soil pore structure encountered by roots affects plant‐derived carbon inputs and fate

Summary Plant roots are the main supplier of carbon (C) to the soil, the largest terrestrial C reservoir. Soil pore structure drives root growth, yet how it affects belowground C inputs remains a critical knowledge gap. By combining X‐ray computed tomography with 14 C plant labelling, we identified root–soil contact as a previously unrecognised influence on belowground plant C allocations and on the fate of plant‐derived C in the soil. Greater contact with the surrounding soil, when the growing root encounters a pore structure dominated by small (< 40 μm Ø) pores, results in strong rhizodeposition but in areas of high microbial activity. The root system of Rudbeckia hirta revealed high plasticity and thus maintained high root–soil contact. This led to greater C inputs across a wide range of soil pore structures. The root–soil contact Panicum virgatum , a promising bioenergy feedstock crop, was sensitive to the encountered structure. Pore structure built by a polyculture, for example, restored prairie, can be particularly effective in promoting lateral root growth and thus root–soil contact and associated C benefits. The findings suggest that the interaction of pore structure with roots is an important, previously unrecognised, stimulus of soil C gains.

Plant Sciences↗
DOE OSTI2023

Amino mapping: possibility to visualize amino-N compounds in the rhizosphere of Zea Mays L.

Abstract Understanding N uptake by plants, the N cycle, and their relationship to soil heterogeneity has generated a great deal of interest in the distribution of amino-N compounds in soil. Visualization of the spatial distribution of amino-N in soil can provide insights into the role of labile N in plant-microbial mechanisms of N acquisition and plant N uptake, but until now, it has remained technically challenging. Here, we describe a novel technique to visualize the amino-N distribution at the root-soil interface. The technique is based on time-lapse amino mapping (TLAM) using membranes saturated with the fluorogenic OPAME reagent (O-phthalaldehyde and β-mercaptoethanol). OPAME in the membrane reacts with organic compounds containing a NH 2 functional group at the membrane-soil interface, generating a fluorescent product visible under UV light and detectable by a digital camera. The TLAM amino-mapping technique was applied to visualize and quantify the concentration of amino-N compounds in the rhizosphere of maize (Zea MaysL.). A ten times greater amino-N concentration was detected in the rhizosphere compared to non-rhizosphere soil. The high content of amino-N was mainly associated with the root tips and was 3 times larger than the average amino-N content at seminal roots. The amino-N rhizosphere was 2 times broader around the root tips than around other parts of the roots. We concluded that TLAM is a promising approach for monitoring the fate of labile N in soils. However, the technique needs to be standardized for different soil types, plant species, and climate conditions to allow wider application.

Agriculture↗
DOE OSTI2022

Are enzymes transported in soils by water fluxes?

Transport of extracellular hydrolytic enzymes in soils has always been a subject of doubt. The considerations against its importance are that (i) enzymes benefit their producers the most when they remain in close proximity; and (ii) enzymes are large molecules with low mobility due to high affinity to fine soil particles and organic matter. However, soil mineral colloids (SMC), to which extracellular enzymes also have an affinity and which are known to facilitate transport of a broad variety of chemicals and microorganisms in soils, can serve as vehicles for enzyme transport as well. Since current literature lacks information on enzyme transport in soils, our goal was to determine whether enzymes are transported and, if so, whether they are transported in a free- or in a colloid-associated form. We conducted column transport experiments with four hydrolytic enzymes, namely, β-glucosidase, acid-phosphatase, cellobiohydrolase, and xylosidase, in soils with contrasting textures. The eluents containing enzymes were applied on top of soil columns, while enzyme activities, SMC, and electrical conductivity were measured in the effluents from the columns. Our results provided evidence of joint enzyme transport with soil colloids. The enzymes associated with the coarse SMC (1 μm < Ø) contributed 52–88% of the total enzyme activity in the effluents. The remaining enzyme activity was attributed to the enzymes associated with organic colloids, fine SMC (Ø < 1 μm) and free enzymes in solution. Furthermore, this study suggested a dual effect of ionic strength in the soil suspension on enzyme activity and their release from soils with soil colloids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Soil oxidoreductase zymography: Visualizing spatial distributions of peroxidase and phenol oxidase activities at the root-soil interface

Decomposition of organic material in the rhizosphere – the most dynamic microbial habitat in soil – involves arrays of oxidoreductase and hydrolytic enzymes. Spatial distributions of various hydrolytic activities in soil have already been explored by zymographic techniques. However, the distribution of oxidative activity in the rhizosphere remains to be studied. Thus, we extended a Time-Lapse Zymography technique, using Amplex Red® reagent, to visualize and quantify distributions of phenol oxidase and peroxidase activities in the rhizosphere of Zea mays L. growing in a Haplic Phaeozem and the non-rhizospheric soil. The gross oxidative activity was greatest at the root surfaces, and fell to background soil levels 1.26 and 0.73 mm from seminal (>1 mm diameter) and lateral (<0.5 mm diameter) roots, respectively. The rhizosphere extent relative to the root radius was 59% broader around lateral than around seminal roots. The greatest activities, up to 30 nmol cm –2 min –1 , were peroxidase-dominated and closely associated with roots. Here, the results confirm the utility of the approach for studying spatio-temporal distributions of oxidative activities in soil. However, actual activity of oxidoreductases in the field will be strongly controlled by fluctuating environmental conditions such as soil aeration and the gradient of reactive oxygen species, which need to be considered especially in anoxic soils.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Pore architecture and particulate organic matter in soils under monoculture switchgrass and restored prairie in contrasting topography

Bioenergy cropping systems can substantially contribute to climate change mitigation. However, limited information is available on how they affect soil characteristics, including pores and particulate organic matter (POM), both essential components of the soil C cycle. The objective of this study was to determine effects of bioenergy systems and field topography on soil pore characteristics, POM, and POM decomposition under new plant growth. We collected intact soil cores from two systems: monoculture switchgrass (Panicum virgatum L.) and native prairie, at two contrasting topographical positions (depressions and slopes), planting half of the cores with switchgrass. Pore and POM characteristics were obtained using X-ray computed micro-tomography (μCT) (18.2 µm resolution) before and after new switchgrass growth. Diverse prairie vegetation led to higher soil C than switchgrass, with concomitantly higher volumes of 30–90 μm radius pores and greater solid-pore interface. Yet, that effect was present only in the coarse-textured soils on slopes and coincided with higher root biomass of prairie vegetation. Surprisingly, new switchgrass growth did not intensify decomposition of POM, but even somewhat decreased it in monoculture switchgrass as compared to non-planted controls. Our results suggest that topography can play a substantial role in regulating factors driving C sequestration in bioenergy systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Time-lapse approach to correct deficiencies of 2D soil zymography

Membrane zymography is commonly used in rhizosphere ecology to map enzyme activities in intact soil samples and plant roots. The method consists of incubating a membrane saturated with an enzyme-specific fluorogenic substrate on the soil/root surface followed by measurements of fluorescence intensity of the product in the membrane. The traditional zymography is based on assumptions that fluorescence on membrane images is linearly increasing with time during zymography and an increase in product content is numerically equal to enzyme activity in soil below the membrane. These assumptions are unlikely to hold in experimental settings. In this work we introduce a new zymography technique, time-lapse zymography (TLZ); the approach that eliminates the need for assumptions of the traditional zymography and provides more realistic estimates of enzymatic activities. We assessed the new technique in a series of laboratory and modeling experiments, including quantification of the fluorescent product diffusion (e.g. MUF: 4-methylumbelliferone) from plant roots, enzyme activity measurements on the roots, and HYDRUS-2D & HP2 software calibration with obtained data. The calibrated model was used to analyze the processes governing spatial and temporal dynamics of MUF contents in the membrane. The results indicated that the enzyme diffusion within the membrane-soil system was negligible, and measured zymograms were adequately reproduced solely by accounting for substrate and product diffusions and for catalytic enzyme reaction described by the Michaelis-Menten equation. TLZ enabled identifying and using linear parts on MUF time series and accounting for MUF losses in each zymogram pixel, considerably improving the accuracy as compared to the traditional method. Results demonstrated that enzymatic activity from only a thin soil layer (~0.2 mm) is adequately represented in zymograms.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Dynamics of N 2 O in vicinity of plant residues: a microsensor approach

Plant residues decomposing within the soil matrix are known to serve as hotspots of N 2 O production. However, the lack of technical tools for microscale in-situ N 2 O measurements limits understanding of hotspot functioning. Our aim was to assess performance of microsensor technology for evaluating the temporal patterns of N 2 O production in immediate vicinity to decomposing plant residues. We incorporated intact switchgrass leaves and roots into soil matrix and monitored O 2 depletion and N 2 O production using electrochemical microsensors along with N 2 O emission from the soil. We also measured residue’s water absorption and b-glucosidase activity on the surface of the residue - the characteristics related to microenvironmental conditions and biological activity near the residue. N 2 O production in the vicinity of switchgrass residues began within 0–12 h after the wetting, reached peak at ~0.6 day and decreased by day 2. N 2 O was higher near leaf than near root residues due to greater leaf N contents and water absorption by the leaves. However, N 2 O production near the roots started sooner than near the leaves, in part due to high initial enzyme levels on root surfaces. Electrochemical microsensor is a useful tool for in-situ micro-scale N 2 O monitoring in immediate vicinity of soil incorporated plant residues. Monitoring provided valuable information on N 2 O production near leaves and roots, its temporal dynamic, and the factors affecting it. The N 2 O production from residues measured by microsensors was consistent with the N 2 O emission from the whole soil, demonstrating the validity of the microsensors for N 2 O hotspot studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

How “hot” are hotspots: Statistically localizing the high-activity areas on soil and rhizosphere images

The topic of microbial hotspots in soil requires not only visualizing their spatial distribution and biochemical analyses, but also statistical approaches to identify these hotspots and separate them from the surrounding activities (background). We hypothesized that each hotspot type (e.g. enzyme activities in the rhizosphere, root exudation, localization of herbicide accumulation) is a result of local process driven by biotic and/or abiotic factors, and the process rates in the hotspots are much faster than those in the soil background. We further hypothesized that the background and hotspot activities in soil belong to different statistical distributions. Consequently, hotspot determination should be based on statistical separation of activities significantly higher than the background. We analyzed for the statistical distributions of grey values on three groups of published images: 1) 14 C images of carbon input by roots into the rhizosphere, 2) 14 C glyphosate accumulation in the plant, and 3) zymogram of leucine aminopeptidase activity in rooted soil. The two Gaussian distributions were fit (the first representing the background, the second the hotspots) to the distribution of grey values in the images, the parameters (means and standard deviations, SD) of the fitted distributions were calculated, and the background was removed. Thus, we identified hotspots as areas outside of the Mean+2SD image intensity (corresponding to the upper ~ 2.5% of activity, being over 97.5% of background values) and finally, visualized images of solely hotspot locations. Finally, these results were compared with previously used decisions on hotspot intensity thresholding (i.e. Top-25% and 17 standard thresholding approaches in ImageJ) and discussed the advantages of the Mean+2SD as well as Mean+3SD approaches. These advantages include: i) simple unification of the thresholding approach for several imaging methods with various principles of activity distribution, ii) identification of hotspots with various activity levels, iii) analysis of “time-specific” hotspots in temporal sequences of images. Compared with 17 standard thresholding methods, we concluded that objectively elucidating and separating the hotspots should be based on statistical distribution analysis, e.g. using the Mean+2SD or Mean+3SD approaches. Furthermore, this simple Mean+2SD approach delivered suitable results for three groups of images and so, helps to understand the processes responsible for the highest activities and elucidate hotspots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Contribution of decomposing plant roots to N 2 O emissions by water absorption

The “sponge effect”, or water absorption by incorporated plant leaf residues, was recently identified as one of the mechanisms that drives activity in microbial hotspots. We explored the presence of the sponge effect in plant root residues, and its role in root decomposition and associated N 2 O and CO 2 emissions. Young soybean (Glycine max) plants were grown in microcosms with two soil materials dominated by (i) large (>30 μm Ø) and (ii) small (<10 μm Ø) pores. After termination, the microcosms with the decomposing roots were incubated at 50% and 75% water-filled pore space (WFPS) soil moisture levels. Root decomposition, water absorption by the decomposing roots, and water redistribution were quantified using X-ray computed micro-tomography (μCT), including dual-energy scanning. The results demonstrated occurrence of the sponge effect in young, in-situ grown soybean roots and sharp gradients in the distribution of the added liquid within ~150 µm distance from the decomposing roots. At 50% WFPS the large pore soil emitted 185% more N 2 O than the small pore soil; and, during the first 5 days of incubation, more N 2 O than the large pore soil at 75% WFPS. This finding indicates that the decomposing roots acted as hotspots of N 2 O production, potentially due to sponge effect and associated anoxic conditions. This study suggests that the interactions between pore characteristics and soil moisture can play a significant role in defining the contribution of detritusphere, specifically, decomposing young roots, to soil biogeochemical processes, including microbial activity and denitrification dynamics.

54 ENVIRONMENTAL SCIENCES↗