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The soil volume affected by roots – the rhizosphere – is one of the most important microbial hotspots determining the processes, dynamics and cycling of carbon (C), nutrients and water in terrestrial ecosystems. Rhizosphere visualization is necessary to understand, localize and quantify the ongoing processes and functions, but quantitative conclusions are very uncertain because of: 1) the continuum of the parameters between the root surface and root-free soil, i.e., there are no sharp borders, 2) differences in the distributions of various parameters (C, nutrients, pH, enzyme and microbial activities, gases, water etc.) across and along roots, 3) temporal changes of the parameters and processes with root growth as well as with water and C flows. In situ techniques: planar optodes, zymography, sensitive gels, 14C and neutron imaging as well as destructive approaches (thin layer slicing) have been used to analyze the rhizosphere extent and the gradients of various physico-chemical and biological characteristics: pH, CO 2 , O 2 , redox potential, enzyme activities, content of water, nutrients and excess elements, and organic compounds. A literature analysis allows the conclusion that: i) the rhizosphere extent for most of the parameters assessed by non-destructive visualization techniques is 0.5–4 mm but exceeds 4 mm for gases, nitrate, water and redox potential. ii) The rhizosphere extent of nutrients (N, P) is decoupled from the extent of the corresponding enzyme activities. iii) The imbalance between element flows to and uptake by roots may lead to accumulation of excess elements and formation of root carapaces (e.g. CaCO 3 rhizoliths, Fe plaque) ranging up to a few cm. iv) All destructive approaches show a much (3–5 times) larger rhizosphere extent compared to visualization techniques. These conclusions are crucial for a mechanistic understanding of rhizosphere properties and functioning, estimation of the nutrient stocks available to roots, and for rhizosphere modelling considering root growth and architecture. Overall, roots function as ecosystem engineers and build their environment, serving as the main factors shaping rhizosphere extent. Sharp gradients are formed within a few days for nutrients and enzymes, but more time is necessary for the establishment of specific microbial communities. Despite the very strong dynamics of most parameters, their stationarity is reached within a few days because the release of C and enzymes or nutrient uptake are very quickly compensated by utilization by surrounding microorganisms or/and sorption and diffusion processes. We conclude that despite the dynamic nature of each property, the rhizosphere gradients, their extent and shape are quasi-stationary because of the opposite directions of their formation processes. Image 1 • The rhizosphere size and shape were summarized based on gradients of various physical, chemical and biological properties. • The rhizosphere extent for most of the parameters assessed by non-destructive visualization techniques is 0.5–4 mm. • Sharp gradients are formed within few days for nutrients and enzymes; longer periods are necessary for microbial communities. • The rhizosphere gradients are dynamic but quasi-stationary because of the opposite directions of their forming processes. • The stationarity of most parameters is reached within a few days. [ABSTRACT FROM AUTHOR]

The input of labile organics by plant roots stimulates microbial activity and therefore facilitates biochemical process rates in the rhizosphere compared to bulk soil, forming microbial hotspots. However, the extent to which the functional properties of soil microorganisms are different in the hotspots formed in soils with contrasting fertility remains unclear. We identified the hotspots related to different levels of Zea mays L. root architecture by zymography of leucine aminopeptidase in two soils with contrasting fertility. The hotspots localized by tiny wet-needle approach around first- and second-order roots were compared for parameters of microbial growth and enzyme kinetics. The pattern of hotspot distribution was more dispersed and the hotspot area was one order of magnitude smaller around first-versus second-order roots. The specific microbial growth rate (μ m) and biomass of active microorganisms were soil-specific, with no difference between the hotspots and bulk soil in the fertile soil. In contrast, in the soil poor in organic matter and nutrients, 1.2-fold higher μ m and greater growing biomass were found in the hotspots versus bulk soil. Lower enzyme affinity (1.3–2.2 times higher K m value) of β-glucosidase and leucine aminopeptidase to the substrate was detected in the hotspots versus bulk soil, whereas only β-glucosidase showed higher potential enzyme activity (V max) in the hotspots, being 1.7–2.1 times greater than that in bulk soil. Notably, the activity of C-acquiring enzyme, β-glucosidase positively correlated with the biomass of actively growing microorganisms. The fertile soil, on the whole, showed greater V max and catalytic efficiency (V max / K m) and an approximately 2.5 times shorter substrate turnover time as compared to the poor soil. Therefore, we conclude that i) the differences in microbial growth strategy between rhizosphere hotspots and bulk soil were dependent on soil fertility; ii) affinity of hydrolytic enzyme systems to substrate was mainly modulated by plant, whereas potential enzymatic activity was driven by both plant and soil quality. • The hotspots around roots were successfully localized by tiny wet-needle approach. • Stimulation on microbial growth in the hotspots was soil-specific. • Roots modulated enzyme affinity to substrate. • The fertile soil showed greater catalytic efficiency than the poor soil. [ABSTRACT FROM AUTHOR]

• Warming facilitates the consumption of labile organics because of the faster microbial growth. • Microbial respiration and enzyme activities acclimates to 8-year warming. • The increased labile organics weakens the memory effect of soil microbial functions. • Microbial memory to warming is strongly dependent on microbial physiological state. The feedback between soil carbon (C) and climate change has the potential to decrease over time, which is mainly due to the thermal acclimation of microbial decomposition of soil organic matter (SOM). The adaption and stabilization mechanisms of microbial functions in response to long-term warming in agroecosystems, however, remain intensive debate. Therefore, we explored the controls of thermal acclimation based on an 8-year field warming (ambient, +1.6 °C, +3.2 °C) in agroecosystem and a short-term incubation at five constant temperature levels (from 5 to 25 °C with 5 °C intervals) under microbial steady-state (without glucose, non-activated microbial community) and activate microbial growth induced by sufficient glucose addition. At the steady-state, field warming (+3.2 °C) increased the specific growth rate whilst decreased the portion of growing microbial biomass as compared to ambient soils. This indicated that 8-year field warming facilitated the consumption of labile organics due to faster microbial growth. Consequently, microbial growth, activities of β-glucosidase and leucine aminopeptidase, as well as microbial respiration acclimated to field warming (+1.6 °C, +3.2 °C) over 8-year, responded up to 40% weaker to further short-term temperature increase (above 15 °C). Also, the temperature sensitivity of enzyme activity decreased with field warming magnitude under higher incubation temperatures, which further indicated enzyme acclimation. Under activation mode, however, the higher enzyme activities suggested that the increased labile C remarkably relieved the substrate limitation of microbial growth and activated dormant microorganisms in field warmed soils, and weaken the thermal acclimation of soil microbial functions. We thus found experimental evidence that the transition between microbial physiological states (i.e., dormant vs. active) owing to variations in C availability is the most plausible explanation for the alterations in temperature sensitivity of enzyme activities and microbial respiration. The compensating effect of the increased labile substrate under climate warming on the thermal acclimation of soil microbe-driven CO 2 may thus be larger than that currently predicted, with important consequences for atmospheric CO 2 concentrations. [ABSTRACT FROM AUTHOR]

Metal contamination from mine waste is a widespread threat to soil health. Understanding of the effects of toxic metals from mine waste on the spatial patterning of rhizosphere enzymes and the rhizosphere microbiome remains elusive. Using zymography and high-throughput sequencing, we conducted a mesocosm experiment with mine-contaminated soil, to compare the effects of different concentrations of toxic metals on exoenzyme kinetics, microbial communities, and maize growth. The negative effects of toxic metals exerted their effects largely on enzymatic hotspots in the rhizosphere zone, affecting both resistance and the area of hotspots. This study thus revealed the key importance of such hotspots in overall changes in soil enzymatic activity under metal toxicity. Statistical and functional guild analysis suggested that these enzymatic changes and associated microbial community changes were involved in the inhibition of maize growth. Keystone species of bacteria displayed negative correlations with toxic metals and positive correlations with the activity of enzymatic hotspots, suggesting a potential role. This study contributes to an emerging paradigm, that changes both in the activity of soil enzymes and soil biota – whether due to substrate addition or in this case toxicity – are largely confined to enzymatic hotspot areas. [Display omitted] • The interaction between enzyme activities and mine soil was analyzed through zymography. • Mine soil inhabited the growth of maize. • Mine soil decreased the area and resistance of enzyme hotspots. • The enzyme hotspots are highly related to bacterial keystone species and co-occurrence characteristics [ABSTRACT FROM AUTHOR]

Abstract Roots increase microbial activities depending on exudate composition, especially on the ratios of sugars, carboxylic and amino acids, and thus structure enzyme activities in the rhizosphere. We introduce a new approach combining soil zymography and simulated exudates released from Rhizon® samplers to stimulate microbial activities but avoid the direct release of enzymes by living roots. This enabled visualizing, localizing and analyzing the effects of simulated root exudates on activity of five microbial enzymes involved in carbon (C) (β-glucosidase, cellobiohydrolase), nitrogen (N) (leucine aminopeptidase), phosphorus (P) (phosphatase) and sulfur (S) (sulfatase) cycles. We tested the hypotheses that 1) artificial exudates stimulate microorganisms for enzyme production and form spatial gradients around roots, and 2) the extent of microbial enzyme activities in the rhizosphere is component-specific. In line with these hypotheses, the activities of P-, N- and S-related enzymes were higher near the artificial root and gradually decreased as a function of distance from the root. The pattern for C-cycle enzymes was uniform and independent of the exudate composition. Among all components, alanine increased the rhizosphere extent much stronger than other substances, while methionine had no effect on the spatial distribution of enzyme activities. V max of all enzymes increased with alanine addition, but decreased after adding citrate. The ratios of enzyme activities demonstrated that rhizosphere microorganisms release more leucine aminopeptidase than other enzymes to meet their N demand. Glucose increased the K m of cellobiohydrolase and β-glucosidase, while alanine had the greatest effect on the K m of leucine aminopeptidase and sulfatase. Phosphatase is the enzyme most sensitive to the composition of root exudates; consequently, any factor influencing root exudate composition can strongly affect the P cycle. We conclude that the rhizosphere extent of microbial-derived enzyme activities is component- and enzyme-specific and that this extent depends on the substrate stoichiometry and microbial nutrient demand. Graphical abstract Image 1 Highlights • We introduce a new approach to avoid direct release of enzymes by roots. • Distributions of microbial enzyme activities were enzyme- and component-specific. • Alanine increased the rhizosphere extent stronger than other substances. • Phosphatase had the most sensitive enzyme system to exudate composition. [ABSTRACT FROM AUTHOR]

Plants stimulate microbial enzyme production in the rhizosphere, regulating soil organic matter decomposition and nutrient cycling. The availability of labile organic compounds (i.e. exudates) and water is the main prerequisite for such microbial activity and enzyme production, thus shaping the rhizosphere. Root morphology (i.e., root hairs) and exudate composition define the spatial distribution of properties and functions in the rhizosphere. However, the role of root architecture and exudate composition in this spatial self-organization of the rhizosphere remains unknown. We coupled three in situ imaging approaches: 14C imaging to localize exudates, soil zymography to analyze enzyme activity distribution, and neutron radiography for water fluxes to trace the spatial structure of the rhizosphere of three maize genotypes (wild-type, mutant with defective root-hair prolongation rth3 , and mutant with reduced benzoxazinoid content in root exudates bx1). The co-localization of these three soil images revealed the pivotal role of both optimal water content (neutron radiography) and root exudation (14C imaging) for β-glucosidase production by the rhizosphere microbiome and its hydrolytic activity (zymography). Root hairs increased the exudate release and enlarged the spatial extent of increased β - glucosidase activity around the root axis by 35%, leading to a two-fold faster decomposition of 14C exudates compared to the mutant with defective root hairs. In contrast, benzoxazinoids suppressed β - glucosidase activity by 30%, reflecting decreased microbial activity, whereas their absence broadened the rhizosphere. Overall, root hairs in wild-type maize increased microbial activity (i.e. β - glucosidase production), whereas the benzoxazinoids in root exudates suppressed microorganisms. [Display omitted] • Zymography, 14C imaging, and neutron radiography were coupled to localize rhizosphere processes. • Exudates are released at root tip in wild-type maize, but along the whole root in rth3 and bx1 mutants. • Root hairs enlarged the spatial extent of increased β - glucosidase activity around the root by 35%. • Benzoxazinoids (secondary metabolites) suppressed β - glucosidase activity in the rhizosphere by 30%. • Hotspot co-localization revealed that exudates and water increased β - glucosidase activity. [ABSTRACT FROM AUTHOR]

Root hairs proliferation and warming strongly influence exudate release, enzyme activities and microbial substrate utilization. However, how the presence of root hairs regulates those processes in the rhizosphere under elevated temperature is poorly known. To clarify these interactions, a wild type maize (with root hairs) and its hairless mutant were grown for 3 weeks at 20 and 30 °C, respectively. We combined zymography (localize hotspots of β-glucosidase) with substrate-induced respiration and microcalorimetry to monitor exudate effects on enzyme kinetics, microbial growth and heat production in the rhizosphere hotspots in response to warming. Root hairs effects were more pronounced at the elevated temperature: i) β-glucosidase activity of the wild type at 30 °C was 21% higher than that of the hairless maize; ii) temperature shifted the microbial growth strategy, whereas root hairs promoted the fraction of growing microbial biomass; iii) K m and the activation energy for β-glucosidase under the hairless mutant was lower than that under wild maize. These results suggest that microorganisms inhabiting hotspots of the wild type synthesized more enzymes to fulfill their higher energy and nutrient demands than those of the hairless mutant. In contrast, at higher temperature the hairless maize produced an enzyme pool with higher efficiencies rather than higher enzyme production, enabling metabolic needs to be met at lower cost. We therefore conclude that root hairs play an important role in regulating enzyme systems and microbial growth to adapt to climate warming. Image 1 • Zymography is combined with microcalorimetry and substrate-induced respiration. • Temperature causes a shift in the microbial growth strategy. • Root hairs (i.e. C input) increases the active portion of microbial biomass. • Enzyme structure–function and thermal–substrate tradeoffs occur in hotspots under warming. • C input changes the enzyme system and substrate use efficiency under warming. [ABSTRACT FROM AUTHOR]

The mobilization of soil nutrients bound in organic matter is largely mediated by enzymes derived from plants, soil microorganisms and animal residues. Land-use change alters important soil characteristics that may affect the activities of soil enzymes. However, mechanistic understanding of how land use and management practices influence the catalytic properties of enzymes in top- and subsoil are still scarce, especially in African ecosystems. We linked catalytic properties i.e. substrate affinity constant (K m) and maximum reaction rate (V max), determined by Michaelis–Menten kinetics, to a set of environmental and microbial variables in the soils of a land-use sequence (6 ecosystems) ranging from natural forests to agricultural fields at Mt. Kilimanjaro. The sensitivity of K m and V max of four extracellular hydrolytical enzymes, β-galactosidase, cellobiohydrolase, phosphatase and chitinase to changing environmental conditions were tested by fluorogenic substrates in topsoils and subsoils. The β-galactosidase activity increased with increasing soil depth. Other extracellular enzyme (cellobiohydrolase, phosphatase and chitinase) activities decreased with depth. The affinity of enzymes to substrates was higher in soils of natural compared to agricultural ecosystems: i.e. higher under forests than under cropland. The activity of β-galactosidase, cellobiohydrolase and chitinase enzyme were highest in lower mountain forest and grassland (less disturbed ecosystems). This indicated that changes in land use and management practice not only affects enzyme activity but also controls enzyme kinetics (K m and K a) thus pointing towards the expression of different enzyme systems. Therefore, we concluded that anthropogenic activities result in alteration of C and nutrient cycling by affecting microbial activities and enzymes catalytic properties. • Anthropogenic activities at Mt. Kilimanjaro affects nutrient cycles, microbial activities and enzymes catalytic propertie. • Land-use intensification not only affects enzyme activity but also controls enzyme systems. • The impact of land use on enzyme activities decreases with soil depth. • Elevation affects enzyme systems due to changing temperature and a possible shift in microbial communities. [ABSTRACT FROM AUTHOR]