Soil Zymography Research Articles

Changes in enzyme activity, structure and growth strategies of the rhizosphere microbiome influenced by elevated temperature and CO2

The impacts of global warming and increased CO2 levels on soil processes and crop growth are concerning. Soil enzymes in the rhizosphere, produced mainly by microbes, play a vital role in nutrients mobilization for plants. Nevertheless, a comprehensive understanding of how microbial communities in the rhizosphere respond to increased temperatures and CO2 levels, particularly in relation to nutrient acquisition, is still lacking. Addressing this problem, we grew soybeans under elevated temperature (ET, +2 °C) and CO2 levels (eCO2, +300 ppm), both individually and in combination (eCO2 + eT), in rhizobox mesocosms. Enzyme activity and microbial communities in soybean rhizospheres were investigated using soil zymography. eCO2 increased enzyme activity by 2.5 % to 8.7 %, while eT expanded the hotspot area from 1.8 % to 3.3 %. The combined factors amplified both the hotspot area by 5.3 % to 10.1 % and enzyme activity by 35.4 % to 67.3 %. Compared to ambient conditions, rhizosphere communities under eCO2 were predominantly comprised of r-strategist keystone taxa, including Acidobacteria, Proteobacteria, and Ascomycota. On the contrary, eT induced a shift in the microbial community towards K-selected taxa, characterized by an increased relative abundance of Basidiomycota and Actinobacteria. Furthermore, the combination of eCO2 and eT led to an increase in the relative abundance of key bacterial species (Acidobacteria, Proteobacteria, and Actinobacteria) as well as fungi (Ascomycota and Basidiomycota). These findings indicate the potential significance of enzyme hotspots in modulating responses to climate change. Changes in enzyme activity and hotspot area could indicate the alteration in microbial growth strategies. The treatments exhibited distinct changes in the composition of microbial communities, in network organization, and in the proportion of species designated as r or K-strategists. Overall, these findings highlight the combined effects of global change factors on bacterial and fungal communities, providing insights into their growth strategies and nutrient mobilization under climate change scenarios.

Hyperaccumulator extracts promoting the phytoremediation of rare earth elements (REEs) by Phytolacca americana: Role of active microbial community in rhizosphere hotspots

The increased usage of rare earth elements (REEs) leads to the extensive exploitation of rare earth mines, and the REEs pollution in soil caused by the legacy mine tailings has brought great harm to environment and human health. Although Phytolacca americana can remove REEs from contaminated soil to some extent, there is still an urgent problem to improve its efficiency. Hyperaccumulator extract is a new organic material with potential in metal phytoextraction, but its role in REEs phytoremediation and the related underlying processes remain unclear. In this study, hyperaccumulator extracts from P. americana root (PR), stem (PS), leaf (PL) and EDTA were used to improve the phytoremediation efficiency of REEs with P. americana. Soil zymography was applied to assess the enzyme hotspots’ spatial distribution in the rhizosphere, and the hotspots' microbial communities were also identified. The results indicated that the application of hyperaccumulator extracts improved the biomass and REEs uptake of P. americana, and the highest REEs content in plant was observed in the treatment of PS, which increased 299% compared to that of the control. Hotspots area of β-glucosidase, leucine aminopeptidase and acid phosphatase were concentrated in the pant rhizosphere along the roots and increased 2.2, 5.3 and 2.2 times after PS application compared to unamended soils. The PS application increased the relative abundance of Proteobacteria, Cyanobacteria, Bacteroidota and Firmicutes phyla in rhizosphere. Soil fungi have a higher contribution on promoting REEs activation than that of bacteria. Available P and extractable REEs were leading predictors for the plant biomass and REEs concentrations. The co-occurrence network showed that the application of PS creates a more efficient and stable microbial network compared to other treatments. In conclusion, stem-derived hyperaccumulator extract is excellent in stimulating REEs phytoremediation with P. americana by improving hotspots microbial activities and form a healthy rhizosphere microenvironment.

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. We developed a MATLAB code to implement a pixel-based correction of enzyme activity for pH in processing time-lapse zymography images.

High-resolution chemical imaging to understand Cd activation in rice rhizosphere of karstic soils

Cadmium (Cd) activation, especially at a high spatial resolution, in paddy soils with a high geogenic Cd background is yet to be understood. To investigate the temporal and spatial patterns of Cd activation in rice rhizosphere, pot and rhizotron experiments were conducted using four paddy soils with high geogenic Cd (0.11–3.70 mg kg−1) from Guangxi, southwestern China. The pot experiment results showed that porewater Cd concentrations initially decreased and then increased over the complete rice growth period, reaching its lowest value during the late-tillering and early-filling stages. Besides, correlation analysis identified organic matter and root manganese (Mn) content as the main factors affecting rice Cd uptake, with Mn having a negative effect and organic matter having a positive effect. Sub-millimeter two-dimensional chemical imaging revealed that the distribution of labile Cd in the rhizosphere (by diffusive gradients in thin-films, or DGT) was influenced by the root system and soil properties, such as pH (by planar optode) and acid phosphatase activity (by soil zymography). Soil acid phosphatase activity increased under Cd stress. The overall pH at rice rhizosphere decreased. Moreover, a close relationship was found between the spatial distributions of soil labile Mn and Cd at the rhizosphere, with higher Mn being associated with lower Cd lability. This study highlights Mn as a key element in regulating rice Cd uptake and enlightens future Mn-based strategies for addressing Cd pollution in rice paddy soils, especially in karst areas with high geochemical background.

Multi-imaging platform for rhizosphere studies: Phosphorus and oxygen fluxes

Rhizosphere is a soil volume of high spatio-temporal heterogeneity and intensive plant-soil-microbial interactions, for which visualization and process quantification is of highest scientific and applied relevance, but still very challenging. A novel methodology for quick assessment of two-dimensional distribution of available phosphorus (P) in rhizosphere was suggested, tested, and development up to the application platform. Available P was firstly trapped by an in-situ diffusive gradients in thin-films (DGT) sampler with precipitated zirconia as the binding gel, and subsequently, the loaded gel was analyzed with an optimized colorimetric imaging densitometry (CID). The imaging platform was established linking: i) DGT, ii) planar optode, and iii) soil zymography techniques to simultaneously determine available P, oxygen, and acid phosphatase in rhizosphere at sub-millimeter spatial scales. The DGT identified available P level in rice rhizosphere were spatially overlapping to the localized redox hotspots and phosphatase activity. The spatial relationship between available P and acid phosphatase activity was dependent on root development. The root radial oxygen loss (ROL) remained active during the experimental observations (2–3 days), while a flux of available P of 10 pg cm−2 s−1 was visualized within 2–3 mm of roots, confirming the correlative response of rice roots to oxygen secretion and P uptake. Summarizing, the established imaging platform is suitable to capture spatial heterogeneity and temporal dynamics of root activities, nutrient bioavailability, ROL and enzyme activities in rhizosphere.

Will root-associated fungi affect enzyme activity in the root-detritusphere?

Root-associated fungal groups play a key role in plant productivity and belowground processes. However, how root-associated fungi will affect rhizosphere processes after the death of root still remain unknown. We applied soil zymography to visualize the spatio-temporal distributions of enzyme activities involved in root decomposition processes of European Spruce (Picea abies) between rhizosphere (living root) and detritusphere (7, 14, and 28 days after shoot cutting), and evaluated the effects of different root associated functional fungal groups (ectomycorrhizal fungi: EcMF; non-mycorrhizal rhizosphere fungi: NMRF; a mixture of EcMF and NMRF: Mix) on microbial growth rate and enzyme functions. In the root-detritusphere, the amount of available substrate released by dead root was increased, which stimulated microbial growth and enzyme activity. This was supported by larger hotspots area and broader extension of C-, N-, and P-enzyme activity (1.3–12.8 times) compared to rhizosphere (at day 0 after shoot cutting). This further shifted microorganisms from K-strategies in the rhizosphere to r-strategies in the detritusphere. Since NMRF benefited from the newly available resources as well as from the decreased competition with EcMF after root dead, enzyme activities were higher under a mixture of EcMF and NMRF than that under bare root or individual fungi alone. Furthermore, plants inoculated with EcMF increased the secretion of enzymes after shooting cutting, which may be explained by that the C and nutrients remaining in the root support the growth of EcMF to searching for new nutrients in the short-term. Overall, turnover of fungal biomass in detritusphere and soil represents an important process in the environment.

Contribution of Biogas Slurry-Derived Colloids to Plant P Uptake and Phosphatase Activities: Spatiotemporal Response.

The bioavailability for varied-size phosphorus (P)-binding colloids (Pcoll) especially from external P sources in soil terrestrial ecosystems remains unclear. This study evaluated the differential contribution of various-sized biogas slurry (BS)-derived colloids to plant available P uptake in the rhizosphere and the corresponding patterns of phosphatase response. Keeping the same content of total P input (15 mg kg-1), we applied different size-fractioned BS-derived colloids including nanosized colloids (NCs, 1-20 nm), fine-sized colloids (FCs, 20-220 nm), and medium-sized colloids (MCs, 220-450 nm) respectively to conduct a 45-day rice (Oryza sativa L.) rhizotron experiment. During the whole cultivation period, the dynamics of chemical characteristics and P fractions in each experimental rhizosphere soil solution were analyzed. The spatial and temporal dynamics examination of P-transforming enzymes (acid phosphatases) in the rice rhizosphere was visualized by a soil zymography technique after 5, 25, and 45 days of rice transplantation. The results indicated that the acid phosphatase activities and its hot spot areas were significantly 1) correlated with the relative bioavailability of colloidal P (RBAcoll), 2) increased with the colloid-free (truly dissolved P) and BS-derived NC addition, and 3) affected by the plant growth stage. With the nanosized BS colloid addition, the RBAcoll and plant biomass were respectively found to be the highest (64% and 1.22 g plant-1), in which the acid phosphatase-catalyzed hydrolysis of organic Pcoll played an important role. All of the above suggested that nanosized BS-derived colloids are an effective alternative to conventional phosphorus fertilizer for promoting plant P uptake and P bioavailability.

Contrasting mechanisms of nutrient mobilization in rhizosphere hotspots driven by straw and biochar amendment

Straw return strategies are widely used green management practices that can alter soil organic matter transformation and dynamics through changes in microbial community structure and functions. How the exogenous input of organic materials of contrasting qualities affects the composition of dominant taxa, growth, and microbial functional properties related to nutrient acquisition in space remains unclear. In this study, we investigated the hotsopts and kinetics of C- and N-acquiring hydrolases, microbial growth, and bacterial community structure in maize rhizosphere hotspots after the addition of straw and straw-derived biochar using soil zymography, substrate-induced respiration and high-throughput sequencing. Compared with no amendment and maize straw-derived biochar, straw addition increased the growing biomass and microbial specific growth rate by 1.2–1.6 and 1.7–2.0-fold, respectively, indicating the relative dominance of fast-growing r-strategists. This corresponds to an increased relative abundance of the keystone taxa Firmicutes and their gene copies encoding β-1,4-glucosidase (BG) and β-N-acetylglucosaminidase (NAG). The potential activity and affinity (Vmax and Km) of BG increased 2.2 and 1.8 times, respectively, and those of NAG increased 4.0 and 2.0 times, respectively. In contrast, the relative abundance of Actinobacteria belonging to K-strategists increased in the biochar-amended soil. This resulted in slower growth and retarded enzymatic activity than the straw return treatment. Biochar enhanced the root biomass by 31% and increased the rhizosphere hotspot extents of BG and NAG by 26% and 47%, respectively. The highest robustness and modularity of the co-occurrence network indicated a more stable network with biochar input. In summary, the addition of straw accelerated rhizosphere nutrient cycling by triggering microbial growth, especially fast-growth r-strategists (Firmicutes), and synthesizing a large number of enzymes. In contrast, the addition of biochar increased rhizosphere nutrient mobilization by expanding the extent of rhizosphere hotspots to mobilize nutrients from a larger soil volume. This suggests that there are different strategies for nutrient mobilization in the rhizosphere with contrasting exogenous C addition.

Top-Down Effect of Arthropod Predator Chinese Mitten Crab on Freshwater Nutrient Cycling.

Aquatic litter decomposition is highly dependent on contributions and interactions at different trophic levels. The invasion of alien aquatic organisms like the channeled apple snail (Pomacea canaliculata) might lead to changes in the decomposition process through new species interactions in the invaded wetland. However, it is not clear how aquatic macroinvertebrate predators like the Chinese mitten crab (Eriocheir sinensis) will affect the nutrient cycle in freshwater ecosystems in the face of new benthic invasion. We used the litter bag method to explore the top-down effect of crabs on the freshwater nutrient cycle with the help of soil zymography (a technology previously used in terrestrial ecosystems). The results showed significant feeding effects of crabs and snails on lotus leaf litter and cotton strips. Crabs significantly inhibited the intake of lotus litter and cotton strips and the ability to transform the environment of snails by predation. Crabs promoted the decomposition of various litter substrates by affecting the microbial community structure in the sediment. These results suggest that arthropod predators increase the complexity of detrital food webs through direct and indirect interactions, and consequently have an important impact on the material cycle and stability of freshwater ecosystems. This top-down effect makes macrobenthos play a key role in the biological control and engineering construction of freshwater ecosystems.

Bio-converted organic wastes shape microbiota in maize rhizosphere: Localization and identification in enzyme hotspots

Organic fertilizers increase soil fertility, microbial diversity and heterogeneity of microbial activity hotspots. Black soldier fly (Hermetia illucens L.) frass (BSFF) derived from organic waste composts has been extensively marketed as organic fertilizer, but its effects on rhizosphere microbiota remain unknown. We compared the effects of BSFF from three organic wastes (straw, manure, and kitchen waste) on maize growth in acidic soils. Sufficient P, an optimal soil pH, and a satisfactory soil C/N ratio, as provided by the straw-derived frass, increased the maize growth more than the other frass types. Maize growth increased due to nutrient mobilization by active rhizosphere microbiota, which was localized using in situ soil zymography and identified through DNA amplicon sequencing and quantitative PCR. Specifically, the area of hotspots of acid phosphatase activity along the maize roots increased for 2.8 times after straw-derived frass application compared to unamended soils. Microbial diversity raised within these enzyme activity hotspots and was accompanied by the increased abundances of plant growth-promoting microbial taxa like Luteibacter, Chrysosporium, and Cladorrhinum. Microorganisms formed efficient and mutualistic networks within the rhizosphere hotspots induced by straw-derived frass to accelerate organic P mineralization, as inferred by random forest analysis and the quantitative of phoC and phoD genes. Concluding, the straw-derived frass benefits maize growth on acidic soils through abiotic (soil physico-chemical properties) and biotic (active rhizosphere microbiota) stimuli.

Soil Enzyme Activity Response to Substrate and Nutrient Additions on Undisturbed Forest Subsoil Samples

Previous studies have found that C turnover is bound to hotspots of microbial activity. The objective of this study was to analyze the effects of pure energy substrate (glucose), nutrient (mineral N or P) and combined substrate and nutrient (glucose + N, glucose + P, sterile DOC, artificial root exudate extract) additions to enzyme activity inside and outside hotspots as a proxy for microbial C turnover in a subsoil. By means of different substrate and nutrient additions, we tested how the limitations of our site were distributed on a small scale and depth-dependently to contribute to an increase in knowledge of subsoil mechanistics. The study site is a sandy Dystric Cambisol under an over 100-year-old beech forest stand in Lower Saxony, Germany. Forty-eight undisturbed soil samples from two depth increments (15–27 cm and 80–92 cm) of three profiles were sprayed homogeneously with easily available C, N and P sources to investigate the impacts of substrates and nutrients on three enzyme activities (acid phosphatase, β-glucosidase and N-acetylglucosaminidase) by using the soil zymography approach. Comparisons of upper and lower subsoils showed significantly fewer and smaller hotspots in the lower subsoil but with a high degree of spatial variation in comparison to the upper subsoil. Different patterns of enzyme distribution between upper and lower subsoil suggest microbial communities with a lower diversity are found in deeper soil regions of the site. Both substrate and nutrient additions stimulated enzyme activities significantly more outside the initial hotspots than within. Because of this, we conclude that microorganisms in the initial hotspots are less limited than in the surrounding bulk soil. Changes in enzyme activities owing to both substrate and nutrient addition were stronger in the lower subsoil than in the upper subsoil, showing differences in limitations and possible changes in microbial community structure with increasing depth. The results of our study emphasize the need to consider spatial factors in microbial turnover processes, especially in lower subsoil regions where stronger substrate and nutrient limitations occur.

Zymography: developing of the enzyme soil activity visualization method

The enzymes produced by the soil biota are a key link in the regulation of biochemical processes. The soil enzyme activity can be visualized with zymography, a method based on using fluorescent substrates and obtaining two-dimensional images (zymograms). A variant of a zymographic measuring system has been proposed. Characteristics of lighting, photographic equipment and shooting modes, reagents preparation and calibration are presented. Preparing and analyzing soil samples of different texture (sand and clay loam) and processing the study results have been described. The ways of introducing the substrate are considered in this study, namely pipetting, short-time dipping, and saturation. An analysis of the kinetics of incubation of samples was carried out. The possibilities and disadvantages of the method were also considered and options for solving possible methodological problems during the analysis were proposed. The zymography is a promising method that allows comparing data with the results of other methods. The use of neural network technologies makes it possible to obtain the volumetric distribution of soil enzymes with high reliability. The soil zymography requires qualitative preparatory work and extreme accuracy during the analysis. It is necessary to ensure maximum contact between the substrate and the soil, as this is one of the key factors determining the quality of the results. The most optimal way to introduce the substrate is to saturate the membranes with substrate solution for 60 minutes. At this stage of the development of the method, it is not possible to establish a universal sample incubation time, since this depends on characteristics of both the studied soils and the experiment conditions. Also, it is necessary to document the conditions in detail for discussion the study results.

Phosphorus acquisition strategies of wheat are related to biochar types added in cadmium-contaminated soil: Evidence from soil zymography and root morphology

Biochar application for the remediation of cadmium (Cd)-contaminated soils may result in a relative deficiency of phosphorus (P) due to the disruption of soil nutrient balance. However, the P acquisition strategies of plants in such situation are still unclear. In this study, analyses on soil zymography and root morphology were combined for the first time to investigate the effects of pristine and P-modified biochars from apple tree branches on the P acquisition strategies of wheat under Cd stress. The results show that the application of pristine biochar exacerbated the soil's relative P deficiency. Wheat was forced to improve foraging for P by forming longer and thinner roots (average diameter 0.284 mm) as well as releasing more phosphatase to promote P mobilization in the soil. Moreover, bioavailable Cd affected the P acquisition strategies of wheat through stimulating the release of phosphatase from roots. The P-modified biochar maintained high levels of Olsen-P (>100 mg kg−1) in the soil over time by slow release, avoiding the creation of relative P deficiency in the soil; and increased the average root diameter (0.338 mm) and growth performance index, which promoted shoot growth (length and biomass). Furthermore, the P-modified biochar reduced DTPA-extracted Cd concentration in soils by 79.8 % (pristine biochar by 26.9 %), and decreased the Cd translocation factor from root to shoot as well as Cd concentration in the shoots. Therefore, P-modified biochar has a great potential to regulate the soil element balance (carbon, nitrogen, and P), promote wheat growth, and remediate the Cd-contaminated soil.

Soy and mustard effectively mobilize phosphorus from inorganic and organic sources

We aimed to investigate phosphorus (P) mobilization by different plant species from organic and inorganic sources in relation to different P mobilization mechanisms. Knowledge about P mobilization is important for producing crops on P sources other than phosphate rock-derived fertilizers. We conducted a greenhouse experiment with four plant species (maize, soy, lupin, mustard) and three P sources (FePO4, phytate, struvite). We determined pH and phosphomonoesterase activity in the rhizosphere using pH imaging and soil zymography. At harvest, root exudates were analyzed for phosphomonoesterase activity, pH, organic acids, and dissolved organic carbon (DOC). Plants were analyzed for biomass, root length, and P content. Struvite was more plant-available than phytate and FePO4 as indicated by higher plant P contents. Soy had the highest biomass and P content, irrespective of P source. Soy exuded up to 12.5 times more organic acids and up to 4.2 times more DOC than the other plant species. Lupin had a 122.9 times higher phosphomonoesterase activity than the other plant species with phytate. The pH in the exudate solution of mustard was on average 0.8 pH units higher than of the other plant species. P uptake by mustard and soy seemed to have also benefited from large root lengths. Taken together, our study indicates that soy has a particularly high potential to mobilize P from struvite and phytate, while mustard has a high potential to mobilize P from FePO4. Therefore, soy and mustard seem to be good options for agricultural production that relies less on phosphate rock-derived fertilizers.

The spatial distribution of rhizosphere microbial activities under drought: water availability is more important than root-hair-controlled exudation.

Root hairs and soil water content are crucial in controlling the release and diffusion of root exudates and shaping profiles of biochemical properties in the rhizosphere. But whether root hairs can offset the negative impacts of drought on microbial activity remains unknown. Soil zymography, 14 C imaging and neutron radiography were combined to identify how root hairs and soil moisture affect rhizosphere biochemical properties. To achieve this, we cultivated two maize genotypes (wild-type and root-hair-defective rth3 mutant) under ambient and drought conditions. Root hairs and optimal soil moisture increased hotspot area, rhizosphere extent and kinetic parameters (Vmax and Km ) of β-glucosidase activities. Drought enlarged the rhizosphere extent of root exudates and water content. Colocalization analysis showed that enzymatic hotspots were more colocalized with root exudate hotspots under optimal moisture, whereas they showed higher dependency on water hotspots when soil water and carbon were scarce. We conclude that root hairs are essential in adapting rhizosphere properties under drought to maintain plant nutrition when a continuous mass flow of water transporting nutrients to the root is interrupted. In the rhizosphere, soil water was more important than root exudates for hydrolytic enzyme activities under water and carbon colimitation.

Keep oxygen in check: An improved in-situ zymography approach for mapping anoxic hydrolytic enzyme activities in a paddy soil

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O2) on in-situ microbial hotspots and enzyme activities in anoxic ecosystems remain unclear. To fill this knowledge gap, we applied soil zymography to localize hotspots and activities of phosphomonoesterase (PME), β-glucosidase (BG), and leucine aminopeptidase (LAP) in three compartments of rice-planted rhizoboxes (top bulk, rooted, and bottom bulk paddy soil) under oxic (+O2) and anoxic (O2) conditions. Short-term (35 min) aeration decreased PME activity by 13–49 %, BG by 4–52 %, and LAP by 12–61 % as compared with O2 in three soil compartments. The percentage of hotspot area was higher by 3–110 % for PME, by 10–60 % for BG, and by 12–158 % for LAP under +O2 vs. O2 conditions depending on a rice growth stage. Irrespective of the aeration conditions, the rhizosphere extent of rice plants for three enzymes was generally greater under higher moisture conditions and at earlier growth stage. Higher O2 sensitivity for the tested enzymes at bottom bulk soil versus other compartments suggested that short-term aeration during conventional zymography may lead to underestimation of nutrient mobilization in subsoil compared to top bulk soil. The intolerance of anaerobic microorganisms against the toxicity of O2 in the cells and the shift of microbial metabolic pathways may explain such a short-term suppression by O2. Our findings, therefore, show that anoxic conditions and soil moisture should be kept during zymography and probably other in-situ soil imaging methods when studying anoxic systems.

Plant Species Interactions in the Rhizosphere Increase Maize N and P Acquisition and Maize Yields in Intercropping

The aim of the study was to examine interspecific plant interactions that contribute to plant nitrogen (N) and phosphorus (P) acquisition and are likely the reason for overyielding in intercropping. We conducted a field and a rhizobox experiment with the same soil. Maize (Zea mays L.) was grown alone or intercropped with the companions faba bean (Vicia faba L.), soy (Glycine max (L.) Merr.), blue lupin (Lupinus angustifolius L.), or white mustard (Sinapis alba L.). We determined the isotopic N signature (δ15N) of maize as well as soil parameters (pH, phosphatase activity, nitrate) in the field experiment. We analyzed phosphatase activities and rhizosphere pH by soil zymography and pH imaging in the rhizobox experiment. Maize N and P contents were larger in intercropping than monocropping, especially with soy and lupin in the field, indicating intercropping advantages for maize N and P acquisition. Intercropping with legumes decreased maize δ15N in the field, suggesting that 11–20% of maize aboveground biomass N was transferred from legumes to maize. Soil zymography revealed high phosphatase activities in the rhizosphere of lupin and faba bean. pH imaging showed a rhizosphere alkalinization by mustard, and a rhizosphere acidification by faba bean. These changes in the companions’ rhizosphere likely mobilized P and were also beneficial for maize in intercropping. Taken together, our study provides evidence that the companions’ ability to mobilize N and P in the rhizosphere promotes increases in maize nutrient contents and causes maize overyielding in intercropping and thus can contribute to fertilizer savings.

Reduction in root active zones under drought stress controls spatial distribution and catalytic efficiency of enzyme activities in rhizosphere of wheat

Adaptation of plants and microbes to drought stress affects various biochemical processes such as enzyme activities in the rhizosphere. Nonetheless, it is largely unknown how this adaptation changes the spatial distribution of enzyme activities and catalytic properties of enzymes in the rhizosphere. Also, it is very important to know about the spatial pattern of enzyme activities in seminal and lateral roots regions to clarify how the interaction of drought and root types affects rhizosphere extent and enzyme activities. To fill these knowledge gaps, we grew wheat plants under drought (30% WHC, three weeks) and optimum (70% WHC) conditions and then coupled soil zymography with enzyme kinetics for studying β-glucosidase (GLU), acid phosphatase (ACP), and leucine aminopeptidase (LAP). The results showed that drought considerably decreased the hotspot percentage of GLU and ACP activities by 63% and 67% in comparison with optimum moisture. In the regions of the seminal root, the rhizosphere extent of GLU and LAP, normalized rhizosphere extent, and total activity of GLU, ACP, and LAP were higher in optimum than in drought treatment. For the lateral root regions, no difference in the rhizosphere extent and total enzyme activities was found between drought and optimum moisture. Besides, we found higher normalized total enzyme activity in lateral roots regions than in seminal roots regions. Drought elevated the maximum enzymatic reaction velocity (Vmax) and Michaelis–Menten constant (Km) of GLU whereas dropped the Vmax and Km (non-significant) of ACP and LAP in comparison with optimum moisture. The catalytic efficiency of GLU and ACP were 37% and 27% higher in optimum than in drought treatment. Our results revealed that effects of drought on rhizosphere extent and total enzyme activities in lateral roots regions were weaker than in seminal roots regions. Drought decreased microbial hotspots area, rhizosphere expansion of enzyme activities in seminal roots regions, and catalytic efficiency of enzyme systems. In conclusion, drought decreases root biomass and root exudations in the rhizosphere, leading to narrower microbial hotspots area and rhizosphere extent and thus to less enzyme catalytic efficiency.

Mutualistic interaction between arbuscular mycorrhiza fungi and soybean roots enhances drought resistant through regulating glucose exudation and rhizosphere expansion

Glucose is one of the low molecular weight components of root exudates to mediate the cross-talk between plants and microbes, but less is known about their contribution to drought resistance of plants and root-associated microbiome. To fill this knowledge gap, we optimized the visualization of glucose exudation and coupled it with another in situ tool – soil zymography ‒ as well as destructive analysis of enzyme kinetics (β-glucosidase; acid phosphomonoesterase) and microbial biomass. This helped identify how microbial functionality ‒ affected by drought and P limitation ‒ will show more resistance in the hotspots of soybean rhizosphere (grown in the rhizoboxes for 10 weeks) associated with arbuscular mycorrhizal fungi (AMF) symbiosis than those without AMF. Drought reduced glucose exudation, mainly allocated to root tips, and narrowed the rhizosphere enzymatic hotspot by three times. However, AMF inoculation enhanced glucose exudation compared to non-mycorrhizal plants and enlarged enzymatic hotspot area by 53% under drought condition. Despite the 50% reduction in β-glucosidase and acid phosphomonoesterase activities owing to water deficit, AMF symbiont triggered up to 36% enzyme activities in correlation with the non-mycorrhizal ones. Therefore, the drought resistance of these two enzymes was enhanced by up to 63% in mycorrhizal plants. The biomass of microbial phosphorus increased by 45% under drought AMF-conditioned plants. We conclude that the cooperation between soybean and AMF induced the formation of favorable microsites around the root, specifically in overlapping localities between rhizosphere and mycorrhizosphere, characterized by enhanced glucose release, increasing rhizosphere expansion, high enzyme activities and shortened substrate turnover time. This, in turn, contributed to the stronger resistance of microbial functions (e.g., enzyme expression) to drought stress in the rhizosphere hotspots. Thus, in response to AMF inoculation and consequent high glucose availability, rhizosphere microorganisms increased P mining rate in those hotspots remaining active despite water scarcity.

Spatiotemporal characteristics of enzymatic hotspots in subtropical forests: In situ evidence from 2D zymography images

Soil enzymes play a central role in organic matter decomposition and nutrient cycling in forest ecosystems. However, the spatiotemporal characteristics of in situ enzyme activities in heterogeneous soil and their drivers are still poorly understood. We applied soil zymography in the field to visualize the seasonal dynamics of enzyme activity along soil profiles (28 cm depth) in Quercus variabilis (oak) and Pinus massoniana (pine) pure and mixed forests in southern China. Temporally, the enzymatic hotspot areas were largest in summer (21.9% ± 7.1%), smallest in autumn (6.1% ± 4.9%), and medium-sized in spring (13.4% ± 5.9%) and winter (12.5% ± 7.7%) in all forest soils. The seasonal characteristics were attributed to changes in soil temperature and moisture. Spatially, the enzymatic hotspot areas were the largest at 0–10 cm (6.7% ± 3.8%), followed by 10–20 cm (5.1% ± 3.9%) and 20–28 cm (2.6% ± 2.3%) in all forest soils. In addition, the enzymatic hotspot areas of mixed forest (6.5–33.3%) were larger than those of pure oak (1.3–28.5%) and pine (0.2–22.8%) forests. This positive effect of mixed forest on enzymatic hotspot areas was mainly attributed to the higher microbial biomass and total soil faunal abundance than those in pure forests, especially under dry and cold climate conditions. These results suggest that the negative effects of drought and cold on enzymatic hotspot areas could be mitigated in the mixed forest. This study provided the first in situ evidence of the seasonal dynamics of enzymatic hotspots in forest soil profiles, which are of paramount importance for understanding nutrient cycling and ecosystem functioning.