Toxic metal contamination effects mediated by hotspot intensity of soil enzymes and microbial community structure.

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.

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 ß-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.

Contrasting distribution of enzyme activities in the rhizosphere of European beech and Norway spruce.

Recent policies and silvicultural management call for forest regeneration that involve the selection of tree species able to cope with low soil nutrient availability in forest ecosystems. Understanding the impact of different tree species on the rhizosphere processes (e.g., enzyme activities) involved in nutrient mobilisation is critical in selecting suitable species to adapt forests to environmental change. Here, we visualised and investigated the rhizosphere distribution of enzyme activities (cellobiohydrolase, leucine-aminopeptidase, and acid phosphomonoesterase) using zymography. We related the distribution of enzyme activities to the seedling root morphological traits of European beech (Fagus sylvatica) and Norway spruce (Picea abies), the two most cultivated temperate tree species that employ contrasting strategies in soil nutrient acquisition. We found that spruce showed a higher morphological heterogeneity along the roots than beech, resulting in a more robust relationship between rhizoplane-associated enzyme activities and the longitudinal distance from the root apex. The rhizoplane enzyme activities decreased in spruce and increased in beech with the distance from the root apex over a power-law equation. Spruce revealed broader rhizosphere extents of all three enzymes, but only acid phosphomonoesterase activity was higher compared with beech. This latter result was determined by a larger root system found in beech compared with spruce that enhanced cellobiohydrolase and leucine-aminopeptidase activities. The root hair zone and hair lengths were significant variables determining the distribution of enzyme activities in the rhizosphere. Our findings indicate that spruce has a more substantial influence on rhizosphere enzyme production and diffusion than beech, enabling spruce to better mobilise nutrients from organic sources in heterogeneous forest soils.

Visualization and quantification of carbon "rusty sink" by rice root iron plaque: Mechanisms, functions, and global implications.

Paddies contain 78% higher organic carbon (C) stocks than adjacent upland soils, and iron (Fe) plaque formation on rice roots is one of the mechanisms that traps C. The process sequence, extent and global relevance of this C stabilization mechanism under oxic/anoxic conditions remains unclear. We quantified and localized the contribution of Fe plaque to organic matter stabilization in a microoxic area (rice rhizosphere) and evaluated roles of this C trap for global C sequestration in paddy soils. Visualization and localization of pH by imaging with planar optodes, enzyme activities by zymography, and root exudation by 14 C imaging, as well as upscale modeling enabled linkage of three groups of rhizosphere processes that are responsible for C stabilization from the micro- (root) to the macro- (ecosystem) levels. The 14 C activity in soil (reflecting stabilization of rhizodeposits) with Fe2+ addition was 1.4-1.5 times higher than that in the control and phosphate addition soils. Perfect co-localization of the hotspots of ß-glucosidase activity (by zymography) with root exudation (14 C) showed that labile C and high enzyme activities were localized within Fe plaques. Fe2+ addition to soil and its microbial oxidation to Fe3+ by radial oxygen release from rice roots increased Fe plaque (Fe3+ ) formation by 1.7-2.5 times. The C amounts trapped by Fe plaque increased by 1.1 times after Fe2+ addition. Therefore, Fe plaque formed from amorphous and complex Fe (oxyhydr)oxides on the root surface act as a "rusty sink" for organic matter. Considering the area of coverage of paddy soils globally, upscaling by model revealed the radial oxygen loss from roots and bacterial Fe oxidation may trap up to 130 Mg C in Fe plaques per rice season. This represents an important annual surplus of new and stable C to the existing C pool under long-term rice cropping.

Differential responses of fungal and bacterial necromass accumulation in soil to nitrogen deposition in relation to deposition rate.

Influenced by nitrogen (N) deposition, changes in soil organic carbon (SOC) sequestration in terrestrial ecosystems could provide strong feedback to climate change. Mounting evidence showed that microbial necromass contributes substantially to SOC sequestration; however, how N deposition influences microbial necromass accumulation in soils remains elusive. We investigated the impacts of N deposition on soil microbial necromass, assessed by amino sugars, at seven forest sites along a north-south transect in eastern China. We found that the responses of fungal and bacterial necromass accumulation to N deposition depended on the deposition rate, with high N deposition (>50 kg N ha-1 yr-1) stimulating fungal necromass accumulation from 29.1 % to 35.2 %, while low N deposition damaging the accumulation of bacterial necromass in soil by 12.1 %. On the whole, N deposition benefitted the dominance of fungal over bacterial necromass, with their ratio being significantly greater at high-N level. The accumulation of microbial necromass was primarily governed by soil properties, including nutrients stoichiometry, clay content and pH, while the composition of microbial necromass was conjointly affected by soil properties and microbial community structure. The latitudinal distribution of microbial necromass contributions to SOC pool was not altered by N deposition, and was firmly controlled by the climatic and edaphic factors. Collectively, our results reveal the impacts of N deposition on microbial necromass accumulation in soil and the geographical pattern across forest ecosystems in eastern China, providing implications for our accurate predictions of global change impacts on SOC sequestration.

Nutrients in the rhizosphere: A meta-analysis of content, availability, and influencing factors.

Nutrient deficiency in most terrestrial ecosystems constrains global primary productivity. Rhizosphere nutrient availability directly regulates plant growth and is influenced by many factors, including soil properties, plant characteristics and climate. A quantitatively comprehensive understanding of the role of these factors in modulating rhizosphere nutrient availability remains largely unknown. We reviewed 123 studies to assess nutrient availability in the rhizosphere compared to bulk soil depending on various factors. The increase in microbial nitrogen (N) content and N-cycling related enzyme activities in the rhizosphere led to a 10% increase in available N relative to bulk soil. The available phosphorus (P) in the rhizosphere decreased by 12% with a corresponding increase in phosphatase activities, indicating extreme demand and competition between plants and microorganisms for P. Greater organic carbon (C) content around taproots (+17%) confirmed their stronger ability to store more organic compounds than the fibrous roots. This corresponds to higher bacterial and fungal contents and slightly higher available nutrients in the rhizosphere of taproots. The maximal rhizosphere nutrient accumulation was common for low-fertile soils, which is confirmed by the negative correlation between most soil chemical properties and the effect sizes of available nutrients. Increases in rhizosphere bacterial and fungal population densities (205-254%) were much higher than microbial biomass increases (indicated as microbial C: +19%). Consequently, despite the higher microbial population densities in the rhizosphere, the biomass of individual microbial cells decreased, pointing on their younger age and faster turnover. This meta-analysis shows that, contrary to the common view, most nutrients are more available in the rhizosphere than in bulk soil because of higher microbial activities around roots.

Bridging Microbial Functional Traits With Localized Process Rates at Soil Interfaces.

In this review, we introduce microbially-mediated soil processes, players, their functional traits, and their links to processes at biogeochemical interfaces [e.g., rhizosphere, detritusphere, (bio)-pores, and aggregate surfaces]. A conceptual view emphasizes the central role of the rhizosphere in interactions with other biogeochemical interfaces, considering biotic and abiotic dynamic drivers. We discuss the applicability of three groups of traits based on microbial physiology, activity state, and genomic functional traits to reflect microbial growth in soil. The sensitivity and credibility of modern molecular approaches to estimate microbial-specific growth rates require further development. A link between functional traits determined by physiological (e.g., respiration, biomarkers) and genomic (e.g., genome size, number of ribosomal gene copies per genome, expression of catabolic versus biosynthetic genes) approaches is strongly affected by environmental conditions such as carbon, nutrient availability, and ecosystem type. Therefore, we address the role of soil physico-chemical conditions and trophic interactions as drivers of microbially-mediated soil processes at relevant scales for process localization. The strengths and weaknesses of current approaches (destructive, non-destructive, and predictive) for assessing process localization and the corresponding estimates of process rates are linked to the challenges for modeling microbially-mediated processes in heterogeneous soil microhabitats. Finally, we introduce a conceptual self-regulatory mechanism based on the flexible structure of active microbial communities. Microbial taxa best suited to each successional stage of substrate decomposition become dominant and alter the community structure. The rates of decomposition of organic compounds, therefore, are dependent on the functional traits of dominant taxa and microbial strategies, which are selected and driven by the local environment.

Spatiotemporal Dynamics of Maize (Zea mays L.) Root Growth and Its Potential Consequences for the Assembly of the Rhizosphere Microbiota.

Numerous studies have shown that plants selectively recruit microbes from the soil to establish a complex, yet stable and quite predictable microbial community on their roots - their "microbiome." Microbiome assembly is considered as a key process in the self-organization of root systems. A fundamental question for understanding plant-microbe relationships is where a predictable microbiome is formed along the root axis and through which microbial dynamics the stable formation of a microbiome is challenged. Using maize as a model species for which numerous data on dynamic root traits are available, this mini-review aims to give an integrative overview on the dynamic nature of root growth and its consequences for microbiome assembly based on theoretical considerations from microbial community ecology.

Plant flavones enrich rhizosphere Oxalobacteraceae to improve maize performance under nitrogen deprivation.

Beneficial interactions between plant roots and rhizosphere microorganisms are pivotal for plant fitness. Nevertheless, the molecular mechanisms controlling the feedback between root architecture and microbial community structure remain elusive in maize. Here, we demonstrate that transcriptomic gradients along the longitudinal root axis associate with specific shifts in rhizosphere microbial diversity. Moreover, we have established that root-derived flavones predominantly promote the enrichment of bacteria of the taxa Oxalobacteraceae in the rhizosphere, which in turn promote maize growth and nitrogen acquisition. Genetic experiments demonstrate that LRT1-mediated lateral root development coordinates the interactions of the root system with flavone-dependent Oxalobacteraceae under nitrogen deprivation. In summary, these experiments reveal the genetic basis of the reciprocal interactions between root architecture and the composition and diversity of specific microbial taxa in the rhizosphere resulting in improved plant performance. These findings may open new avenues towards the breeding of high-yielding and nutrient-efficient crops by exploiting their interaction with beneficial soil microorganisms.

Impact of manure on soil biochemical properties: A global synthesis.

Manure application mitigates land degradation and improves soil fertility. Despite many individual studies on manure effects, a comprehensive overview of its consequences for a broad range of soil properties is lacking. Through a meta-analysis of 521 observations spanning the experiments from days after pulse addition up to 113 years with continues manure input, we quantified and generalized the average responses of soil biochemical properties depending on climate factors, management, soil, and manure characteristics. Large increase of pools with fast turnover (microbial carbon (C) and nitrogen (N): +88% and +84%, respectively) compared to stable organic matter pools (+27% for organic C, and +33% for total N) reflects acceleration of C and N cycles and soil fertility improvement. Activities of enzymes acquiring C-, energy-, N-, phosphorus- and sulfur were 1.3-3.3 times larger than those in soil without manure for all study durations included. Soil C/N ratio remained unaffected, indicating the stability of coupled C and N cycles. Microbial C/N ratio decreased, indicating a shift towards bacterial domination, general increase of C and N availability and acceleration of element cycling. Composted manure or manure without mineral fertilizers induced the greatest increase compared to non-composted manure or manure with mineral fertilizers, respectively, in most biochemical properties. The optimal manure application rate for adjusting proper soil pH was 25 Mg ha-1 year-1. Among manure types, swine manure caused the greatest increase of N-cycle-related properties: microbial N (+230%), urease (+258%) and N-acetyl-ß-D-glucosaminidase (+138%) activities. Manure application strategies should avoid P and N losses and pollution via runoff, leaching or gaseous emissions due to fast mineralization and priming of soil organic matter. In conclusion, manure application favors C accumulation and accelerates nutrient cycling by providing available organic substances and nutrients and thus increasing enzyme activities.

Combined biochar and nitrogen application stimulates enzyme activity and root plasticity.

Biochar (BC) and nitrogen (N) fertilizers are frequently applied to improve soil properties and increase crop productivity. Nonetheless, our mechanistic understanding of plant-soil interactions under single or combined application of BC and N remains incomplete. For the first time, we applied a split-root system to evaluate how BC or N contributes to the changes in soil enzyme activities, N and phosphorus (P) cycling as well as root plasticity. Left and right parts of rhizoboxes were filled with silty-clay loamy soil amended with BC (15 g kg-1 soil, from wheat straw, 300 °C), N (0.05 g KNO3-N kg-1 soil) or a control (no amendments), resulting in the following combinations: BC/Control, N/Control, BC/N. Soil enzyme activities, available N and P, root morphology and plant biomass were analyzed after plant harvest. Plant biomass (shoot + root) ranged from 0.56 g pot-1 (BC/Control) to 0.91 g pot-1(BC/N). The decreased soil bulk density and increased P availability in the BC compartment (BC/Control and BC/N) stimulated root length by 1.4-1.8 times - an effect that was independent of N availability in the same rhizobox. Biochar stimulated activities of ß-glucosidase and leucine aminopeptidase (by 33-39%) compared to N due to the coupling of C, N and P cycles in BC/N treated soil. Nitrogen fertilization also increased ß-glucosidase activity compared to the unfertilized control, whereas root elongation remained unaffected. Thus, the combined application of BC/N had more efficient benefits for plant growth than BC or N alone. This is linked with i) the stimulation of enzyme activities at the BC locations to reduce N limitation for both microorganisms and plants, and ii) an increase of fine root production to improve N uptake efficiency. Thus, combined BC/N application is potentially especially sustainable to overcome nutrient limitation as well as to maintain crop productivity because it accelerates root-microbial interactions.

Comparable effects of manure and its biochar on reducing soil Cr bioavailability and narrowing the rhizosphere extent of enzyme activities.

Chromium (Cr) contamination is especially hazardous to soil biota. Application of manure and biochar has been frequently proposed to remediate Cr-contaminated soil. However, the understanding of mechanisms behind manure and biochar impacts on soil enzyme activities requires advanced visualization technologies. For the first time, we compared manure and its biochar influence on the spatial distribution of ß-glucosidase, N-acetyl-glucosaminidase and phosphomonoesterase activities in Cr-contaminated soil using direct zymography. Maize was planted for 45 days in (a) soil mixed with manure, (b) soil mixed with manure-derived biochar and (c) soil without any addition. Soil pH decreased over 45 days, inducing an increase in acid soluble Cr. The concomitant decrease in ß-glucosidase and N-acetyl-glucosaminidase activities explained the narrowing rhizosphere extent of enzyme activities by 13-44%, indicating that increased Cr bioavailability decreases microbial activities. A larger maize performance index and the greatest plant shoot/root ratio after biochar application suggested enhanced maize growth (p < 0.05). In contrast, manure induced the narrowest extent of ß-glucosidase and phosphomonoesterase activities due to the addition of labile organic compounds and nutrients following its application. Our study emphasizes the importance of pH on Cr bioavailability and enzyme activities and demonstrates that biochar application is more ideally suited for remediating Cr-contaminated soil.

Visualization of Enzyme Activities in Earthworm Biopores by In Situ Soil Zymography.

Earthworms produce biopores with strongly increased microbial and enzyme activities and consequently they form microbial hotspots in soil. In extremely dynamic microhabitats and hotspots such as earthworm biopores, the in situ enzyme activities are a footprint of process rates and complex biotic interactions. The effect of earthworms on enzyme activities inside biopores, relative to earthworm-free soil, can be visualized by in situ soil zymography. Here, we describe the details of the approach and discuss its advantages and limitations. Direct zymography provides high spatial resolution for quantitative images of enzyme activities in biopores.

Nonlinear temperature sensitivity of enzyme kinetics explains canceling effect-a case study on loamy haplic Luvisol.

The temperature sensitivity of enzymes responsible for organic matter decomposition in soil is crucial for predicting the effects of global warming on the carbon cycle and sequestration. We tested the hypothesis that differences in temperature sensitivity of enzyme kinetic parameters V max and K m will lead to a canceling effect: strong reduction of temperature response of catalytic reactions. Short-term temperature response of V max and K m of three hydrolytic enzymes responsible for decomposition of cellulose (ß-glucosidase, cellobiohydrolase) and hemicelluloses (xylanase) were analyzed in situ from 0 to 40°C. The apparent activation energy varied between enzymes from 20.7 to 35.2 kJ mol(-1) corresponding to the Q 10 values of the enzyme activities of 1.4-1.9 (with V max - Q 10 1.0-2.5 and K m - Q 10 0.94-2.3). Temperature response of all tested enzymes fitted well to the Arrhenius equation. Despite that, the fitting of Arrhenius model revealed the non-linear increase of two cellulolytic enzymes activities with two distinct thresholds at 10-15°C and 25-30°C, which were less pronounced for xylanase. The nonlinearity between 10 and 15°C was explained by 30-80% increase in V max . At 25-30°C, however, the abrupt decrease of enzyme-substrate affinity was responsible for non-linear increase of enzyme activities. Our study is the first demonstrating nonlinear response of V max and K m to temperature causing canceling effect, which was most strongly pronounced at low substrate concentrations and at temperatures above 15°C. Under cold climate, however, the regulation of hydrolytic activity by canceling in response to warming is negligible because canceling was never observed below 10°C. The canceling, therefore, can be considered as natural mechanism reducing the effects of global warming on decomposition of soil organics at moderate temperatures. The non-linearity of enzyme responses to warming and the respective thresholds should therefore be investigated for other enzymes, and incorporated into Earth system models to improve the predictions at regional and global levels.