Maize (Zea mays L.) root exudation profiles change in quality and quantity during plant development - A field study.

Deciphering root exudate composition of soil-grown plants is considered a crucial step to better understand plant-soil-microbe interactions affecting plant growth performance. In this study, two genotypes of Zea mays L. (WT, rth3) differing in root hair elongation were grown in the field in two substrates (sand, loam) in custom-made, perforated columns inserted into the field plots. Root exudates were collected at different plant developmental stages (BBCH 14, 19, 59, 83) using a soil-hydroponic-hybrid exudation sampling approach. Exudates were characterized by LC-MS based non-targeted metabolomics, as well as by photometric assays targeting total dissolved organic carbon, soluble carbohydrates, proteins, amino acids, and phenolics. Results showed that plant developmental stage was the main driver shaping both the composition and quantity of exuded compounds. Carbon (C) exudation per plant increased with increasing biomass production over time, while C exudation rate per cm² root surface area h-1 decreased with plant maturity. Furthermore, exudation rates were higher in the substrate with lower nutrient mobility (i.e., loam). Surprisingly, we observed higher exudation rates in the root hairless rth3 mutant compared to the root hair-forming WT sibling, though exudate metabolite composition remained similar. Our results highlight the impact of plant developmental stage on the plant-soil-microbe interplay.

Correlative Imaging of the Rhizosphere─A Multimethod Workflow for Targeted Mapping of Chemical Gradients.

Examining in situ processes in the soil rhizosphere requires spatial information on physical and chemical properties under undisturbed conditions. We developed a correlative imaging workflow for targeted sampling of roots in their three-dimensional (3D) context and assessed the imprint of roots on chemical properties of the root-soil contact zone at micrometer to millimeter scale. Maize (Zea mays) was grown in 15N-labeled soil columns and pulse-labeled with 13CO2 to visualize the spatial distribution of carbon inputs and nitrogen uptake together with the redistribution of other elements. Soil columns were scanned by X-ray computed tomography (X-ray CT) at low resolution (45 µm) to enable image-guided subsampling of specific root segments. Resin-embedded subsamples were then analyzed by X-ray CT at high resolution (10 µm) for their 3D structure and chemical gradients around roots using micro-X-ray fluorescence spectroscopy (µXRF), nanoscale secondary ion mass spectrometry (NanoSIMS), and laser-ablation isotope ratio mass spectrometry (LA-IRMS). Concentration gradients, particularly of calcium and sulfur, with different spatial extents could be identified by µXRF. NanoSIMS and LA-IRMS detected the release of 13C into soil up to a distance of 100 µm from the root surface, whereas 15N accumulated preferentially in the root cells. We conclude that combining targeted sampling of the soil-root system and correlative microscopy opens new avenues for unraveling rhizosphere processes in situ.

Plant Age and Soil Texture Rather Than the Presence of Root Hairs Cause Differences in Maize Resource Allocation and Root Gene Expression in the Field.

Understanding the biological roles of root hairs is key to projecting their contributions to plant growth and to assess their relevance for plant breeding. The objective of this study was to assess the importance of root hairs for maize nutrition, carbon allocation and root gene expression in a field experiment. Applying wild type and root hairless rth3 maize grown on loam and sand, we examined the period of growth including 4-leaf, 9-leaf and tassel emergence stages, accompanied with a low precipitation rate. rth3 maize had lower shoot growth and lower total amounts of mineral nutrients than wild type, but the concentrations of mineral elements, root gene expression, or carbon allocation were largely unchanged. For these parameters, growth stage accounted for the main differences, followed by substrate. Substrate-related changes were pronounced during tassel emergence, where the concentrations of several elements in leaves as well as cell wall formation-related root gene expression and C allocation decreased. In conclusion, the presence of root hairs stimulated maize shoot growth and total nutrient uptake, but other parameters were more impacted by growth stage and soil texture. Further research should relate root hair functioning to the observed losses in maize productivity and growth efficiency.

Direct Imaging of Plant Metabolites in the Rhizosphere Using Laser Desorption Ionization Ultra-High Resolution Mass Spectrometry.

The interplay of rhizosphere components such as root exudates, microbes, and minerals results in small-scale gradients of organic molecules in the soil around roots. The current methods for the direct chemical imaging of plant metabolites in the rhizosphere often lack molecular information or require labeling with fluorescent tags or isotopes. Here, we present a novel workflow using laser desorption ionization (LDI) combined with mass spectrometric imaging (MSI) to directly analyze plant metabolites in a complex soil matrix. Undisturbed samples of the roots and the surrounding soil of Zea mays L. plants from either field- or laboratory-scale experiments were embedded and cryosectioned to 100 µm thin sections. The target metabolites were detected with a spatial resolution of 25 µm in the root and the surrounding soil based on accurate masses using ultra-high mass resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS). Using this workflow, we could determine the rhizosphere gradients of a dihexose (e.g., sucrose) and other plant metabolites (e.g., coumaric acid, vanillic acid). The molecular gradients for the dihexose showed a high abundance of this metabolite in the root and a strong depletion of the signal intensity within 150 µm from the root surface. Analyzing several sections from the same undisturbed soil sample allowed us to follow molecular gradients along the root axis. Benefiting from the ultra-high mass resolution, isotopologues of the dihexose could be readily resolved to enable the detection of stable isotope labels on the compound level. Overall, the direct molecular imaging via LDI-FT-ICR-MS allows for the first time a non-targeted or targeted analysis of plant metabolites in undisturbed soil samples, paving the way to study the turnover of root-derived organic carbon in the rhizosphere with high chemical and spatial resolution.

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.

An improved method for the segmentation of roots from X-ray computed tomography 3D images: Rootine v.2.

BACKGROUND: X-ray computed tomography is acknowledged as a powerful tool for the study of root system architecture of plants growing in soil. In this paper, we improved the original root segmentation algorithm "Rootine" and present its succeeding version "Rootine v.2". In addition to gray value information, Rootine algorithms are based on shape detection of cylindrical roots. Both algorithms are macros for the ImageJ software and are made freely available to the public. New features in Rootine v.2 are (i) a pot wall detection and removal step to avoid segmentation artefacts for roots growing along the pot wall, (ii) a calculation of the root average gray value based on a histogram analysis, (iii) an automatic calculation of thresholds for hysteresis thresholding of the tubeness image to reduce the number of parameters and (iv) a false negatives recovery based on shape criteria to increase root recovery. We compare the segmentation results of Rootine v.1 and Rootine v.2 with the results of root washing and subsequent analysis with WinRhizo. We use a benchmark dataset of maize roots (Zea mays L. cv. B73) grown in repacked soil for two scenarios with differing soil heterogeneity and image quality. RESULTS: We demonstrate that Rootine v.2 outperforms its preceding version in terms of root recovery and enables to match better the root diameter distribution data obtained with root washing. Despite a longer processing time, Rootine v.2 comprises less user-defined parameters and shows an overall greater usability. CONCLUSION: The proposed method facilitates higher root detection accuracy than its predecessor and has the potential for improving high-throughput root phenotyping procedures based on X-ray computed tomography data analysis.

Assembly Patterns of the Rhizosphere Microbiome Along the Longitudinal Root Axis of Maize (Zea mays L.).

It is by now well proven that different plant species within their specific root systems select for distinct subsets of microbiota from bulk soil - their individual rhizosphere microbiomes. In maize, root growth advances several centimeters each day, with the locations, quality and quantity of rhizodeposition changing. We investigated the assembly of communities of prokaryotes (archaea and bacteria) and their protistan predators (Cercozoa, Rhizaria) along the longitudinal root axis of maize (Zea mays L.). We grew maize plants in an agricultural loamy soil and sampled rhizosphere soil at distinct locations along maize roots. We applied high-throughput sequencing, followed by diversity and network analyses in order to track changes in relative abundances, diversity and co-occurrence of rhizosphere microbiota along the root axis. Apart from a reduction of operational taxonomic unit (OTU) richness and a strong shift in community composition between bulk soil and root tips, patterns of microbial community assembly along maize-roots were more complex than expected. High variation in beta diversity at root tips and the root hair zone indicated substantial randomness of community assembly. Root hair zone communities were characterized by massive co-occurrence of microbial taxa, likely fueled by abundant resource supply from rhizodeposition. Further up the root where lateral roots emerged processes of community assembly appeared to be more deterministic (e.g., through competition and predation). This shift toward significance of deterministic processes was revealed by low variability of beta diversity, changes in network topology, and the appearance of regular phylogenetic co-occurrence patterns in bipartite networks between prokaryotes and their potential protistan predators. Such patterns were strongest in regions with fully developed laterals, suggesting that a consistent rhizosphere microbiome finally assembled. For the targeted improvement of microbiome function, such knowledge on the processes of microbiome assembly on roots and its temporal and spatial variability is crucially important.

Root exposure to apple replant disease soil triggers local defense response and rhizoplane microbiome dysbiosis.

A soil column split-root experiment was designed to investigate the ability of apple replant disease (ARD)-causing agents to spread in soil. 'M26' apple rootstocks grew into a top layer of Control soil, followed by a barrier-free split-soil layer (Control soil/ARD soil). We observed a severely reduced root growth, concomitant with enhanced gene expression of phytoalexin biosynthetic genes and phytoalexin content in roots from ARD soil, indicating a pronounced local plant defense response. Amplicon sequencing (bacteria, archaea, fungi) revealed local shifts in diversity and composition of microorganisms in the rhizoplane of roots from ARD soil. An enrichment of operational taxonomic units affiliated to potential ARD fungal pathogens (Ilyonectria and Nectria sp.) and bacteria frequently associated with ARD (Streptomyces, Variovorax) was noted. In conclusion, our integrated study supports the idea of ARD being local and not spreading into surrounding soil, as only the roots in ARD soil were affected in terms of growth, phytoalexin biosynthetic gene expression, phytoalexin production and altered microbiome structure. This study further reinforces the microbiological nature of ARD, being likely triggered by a disturbed soil microbiome enriched with low mobility of the ARD-causing agents that induce a strong plant defense and rhizoplane microbiome dysbiosis, concurring with root damage.

Soil Texture, Sampling Depth and Root Hairs Shape the Structure of ACC Deaminase Bacterial Community Composition in Maize Rhizosphere.

Preservation of the phytostimulatory functions of plant growth-promoting bacteria relies on the adaptation of their community to the rhizosphere environment. Here, an amplicon sequencing approach was implemented to specifically target microorganisms with 1-aminocyclopropane-1-carboxylate deaminase activity, carrying the acdS gene. We stated the hypothesis that the relative phylogenetic distribution of acdS carrying microorganisms is affected by the presence or absence of root hairs, soil type, and depth. To this end, a standardized soil column experiment was conducted with maize wild type and root hair defective rth3 mutant in the substrates loam and sand, and harvest was implemented from three depths. Most acdS sequences (99%) were affiliated to Actinobacteria and Proteobacteria, and the strongest influence on the relative abundances of sequences were exerted by the substrate. Variovorax, Acidovorax, and Ralstonia sequences dominated in loam, whereas Streptomyces and Agromyces were more abundant in sand. Soil depth caused strong variations in acdS sequence distribution, with differential levels in the relative abundances of acdS sequences affiliated to Tetrasphaera, Amycolatopsis, and Streptomyces in loam, but Burkholderia, Paraburkholderia, and Variovorax in sand. Maize genotype influenced the distribution of acdS sequences mainly in loam and only in the uppermost depth. Variovorax acdS sequences were more abundant in WT, but Streptomyces, Microbacterium, and Modestobacter in rth3 rhizosphere. Substrate and soil depth were strong and plant genotype a further significant single and interacting drivers of acdS carrying microbial community composition in the rhizosphere of maize. This suggests that maize rhizosphere acdS carrying bacterial community establishes according to the environmental constraints, and that root hairs possess a minor but significant impact on acdS carrying bacterial populations.

Exploring microbial determinants of apple replant disease (ARD): a microhabitat approach under split-root design.

Apple replant disease (ARD) occurs worldwide in apple orchards and nurseries and leads to a severe growth and productivity decline. Despite research on the topic, its causality remains unclear. In a split-root experiment, we grew ARD-susceptible 'M26' apple rootstocks in different substrate combinations (+ARD: ARD soil; -ARD: gamma-irradiated ARD soil; and Control: soil with no apple history). We investigated the microbial community composition by 16S rRNA gene amplicon sequencing (bacteria and archaea) along the soil-root continuum (bulk soil, rhizosphere and rhizoplane). Significant differences in microbial community composition and structure were found between +ARD and -ARD or +ARD and Control along the soil-root continuum, even for plants exposed simultaneously to two different substrates (-ARD/+ARD and Control/+ARD). The substrates in the respective split-root compartment defined the assembly of root-associated microbial communities, being hardly influenced by the type of substrate in the respective neighbor compartment. Root-associated representatives from Actinobacteria were the most dynamic taxa in response to the treatments, suggesting a pivotal role in ARD. Altogether, we evidenced an altered state of the microbial community in the +ARD soil, displaying altered alpha- and beta-diversity, which in turn will also impact the normal development of apple rhizosphere and rhizoplane microbiota (dysbiosis), concurring with symptom appearance.

Dynamics of localised nitrogen supply and relevance for root growth of Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') in soil.

Root growth responds to local differences in N-form and concentration. This is known for artificial systems and assumed to be valid in soil. The purpose of this study is to challenge this assumption for soil mesocosms locally supplied with urea with and without nitrification inhibitor. Soil column experiments with Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') were performed to investigate soil solution chemistry and root growth response of these two species with contrasting root architectures to the different N-supply simultaneously. Root growth was analysed over time and separately for the fertiliser layer and the areas above and below with X-ray CT (via region growing) and WinRHIZO. Additionally, NO3- and NH4+ in soil and soil solution were analysed. In Vicia faba, no pronounced differences were observed, although CT analysis indicated different root soil exploration for high NH4+. In Hordeum vulgare, high NO3- inhibited lateral root growth while high NH4+ stimulated the formation of first order laterals. The growth response to locally distributed N-forms in soil is species specific and less pronounced than in artificial systems. The combination of soil solution studies and non-invasive imaging of root growth can substantially improve the mechanistic understanding of root responses to different N-forms in soil.

Compatibility of X-ray computed tomography with plant gene expression, rhizosphere bacterial communities and enzyme activities.

Non-invasive X-ray computed tomography (XRCT) is increasingly used in rhizosphere research to visualize development of soil-root interfaces in situ. However, exposing living systems to X-rays can potentially impact their processes and metabolites. In order to evaluate these effects, we assessed the responses of rhizosphere processes 1 and 24 h after a low X-ray exposure (0.81 Gy). Changes in root gene expression patterns occurred 1 h after exposure with down-regulation of cell wall-, lipid metabolism-, and cell stress-related genes, but no differences remained after 24 h. At either time point, XRCT did not affect either root antioxidative enzyme activities or the composition of the rhizosphere bacterial microbiome and microbial growth parameters. The potential activities of leucine aminopeptidase and phosphomonoesterase were lower at 1 h, but did not differ from the control 24 h after exposure. A time delay of 24 h after a low X-ray exposure (0.81 Gy) was sufficient to reverse any effects on the observed rhizosphere systems. Our data suggest that before implementing novel experimental designs involving XRCT, a study on its impact on the investigated processes should be conducted.

Roots compact the surrounding soil depending on the structures they encounter.

Contradictory evidence exists regarding whether and to which extend roots change soil structure in their vicinity. Here we attempt to reconcile disparate views allowing for the two-way interaction between soil structure and root traits, i.e. changes in soil structure due to plants and changes in root growth due to soil structure. Porosity gradients extending from the root/biopore surface into the bulk soil were investigated with X-ray µCT for undisturbed soil samples from a field chronosequence as well as for a laboratory experiment with Zea mays growing into three different bulk densities. An image analysis protocol was developed, which enabled a fast analysis of the large sample pool (n > 300) at a resolution of 19 µm. Lab experiment showed that growing roots only compact the surrounding soil if macroporosity is low and dominated by isolated pores. When roots can grow into a highly connected macropore system showing high connectivity the rhizosphere is more porous compared to the bulk soil. A compaction around roots/biopores in the field chronosequence was only observed in combination with high root/biopore length densities. We conclude that roots compact the rhizosphere only if the initial soil structure does not offer a sufficient volume of well-connected macropores.

Apple Replant Disease: Causes and Mitigation Strategies.

After replanting apple (Malus domestica Borkh.) on the same site severe growth suppressions, and a decline in yield and fruit quality are observed in all apple producing areas worldwide. The causes of this complex phenomenon, called apple replant disease (ARD), are only poorly understood up to now which is in part due to inconsistencies in terms and methodologies. Therefore we suggest the following definition for ARD: ARD describes a harmfully disturbed physiological and morphological reaction of apple plants to soils that faced alterations in their (micro-) biome due to the previous apple cultures. The underlying interactions likely have multiple causes that extend beyond common analytical tools in microbial ecology. They are influenced by soil properties, faunal vectors, and trophic cascades, with genotype-specific effects on plant secondary metabolism, particularly phytoalexin biosynthesis. Yet, emerging tools allow to unravel the soil and rhizosphere (micro-) biome, to characterize alterations of habitat quality, and to decipher the plant reactions. Thereby, deep insights into the reactions taking place at the root rhizosphere interface will be gained. Counteractions are suggested, taking into account that culture management should emphasize on improving soil microbial and faunal diversity as well as habitat quality rather than focus on soil disinfection.

Root growth, function and rhizosphere microbiome analyses show local rather than systemic effects in apple plant response to replant disease soil.

Apple replant disease (ARD) is the phenomenon of soil decline occurring after repeated planting of apple trees at the same site. This study aimed to elucidate whether ARD is systemic, i.e. whether the contact of parts of the root system with ARD soil causes the whole plant to show poor shoot and root growth. A split-root experiment was conducted with seedlings of 'M26', offering the same plant for its root system the choice between the substrates ARD soil (+ARD), γ-sterilized ARD soil (-ARD) or soil from a grass parcel (Control) with the following combinations: +ARD/+ARD, -ARD/-ARD; +ARD/-ARD; +ARD/Control. Root growth was analysed throughout the 34-day growing period. Samples from bulk, rhizosphere and rhizoplane soil were collected separately for each compartment, and analysed by fingerprints of 16S rRNA gene or ITS fragments amplified from total community (TC) DNA. The response of the plant to +ARD was not systemic as root growth in -ARD compartment was always superior to root growth in +ARD soil. Crosswise 15N-labelling of the N-fertilizer applied to the split-root compartments showed that nitrate-N uptake efficiency was higher for roots in -ARD soil compared to those in +ARD. Bacterial and fungal community composition in the rhizoplane and rhizosphere of the same plants differed significantly between the compartments containing +ARD/-ARD or +ARD/Control. The strongest differences between the bacterial fingerprints were observed in the rhizoplane and rhizosphere. Bacterial genera with increased abundance in response to ARD were mainly Streptomyces but also Sphingobium, Novosphingobium, Rhizobium, Lysobacter and Variovorax. The strongest differences between the fungal fingerprints were observed in bulk soil. Our data showed that the response of the apple plant to ARD soil is local and not systemic.

Quantification of Root Growth Patterns From the Soil Perspective via Root Distance Models.

The rhizosphere, the fraction of soil altered by plant roots, is a dynamic domain that rapidly changes during plant growth. Traditional approaches to quantify root growth patterns are very limited in estimating this transient extent of the rhizosphere. In this paper we advocate the analysis of root growth patterns from the soil perspective. This change of perspective addresses more directly how certain root system architectures facilitate the exploration of soil. For the first time, we propose a parsimonious root distance model with only four parameters which is able to describe root growth patterns throughout all stages in the first 3 weeks of growth of Vicia faba measured with X-ray computed tomography. From these models, which are fitted to the frequency distribution of root distances in soil, it is possible to estimate the rhizosphere volume, i.e., the volume fraction of soil explored by roots, and adapt it to specific interaction distances for water uptake, rhizodeposition, etc. Through 3D time-lapse imaging and image registration it is possible to estimate root age dependent rhizosphere volumes, i.e., volumes specific for certain root age classes. These root distance models are a useful abstraction of complex root growth patterns that provide complementary information on root system architecture unaddressed by traditional root system analysis, which is helpful to constrain dynamic root growth models to achieve more realistic results.

How much is too much?-Influence of X-ray dose on root growth of faba bean (Vicia faba) and barley (Hordeum vulgare).

X-ray CT is a powerful technology to study root growth in soil in-situ. Root systems can be studied in its true 3D geometry over time. Hence, the same plant can be scanned multiple times during development. A downside is the potential of X-rays to interfere with biological processes and therefore plant growth. The aim of this study is to evaluate the influence of cumulative X-ray dose on Vicia faba and Hordeum vulgare during a growth period of 17 days. One control treatment without X-ray scanning was compared to two treatments being scanned every two and four days, respectively. Scanned treatments received a maximum cumulative dose of less than 8 Gy. Plant species differed in their susceptibility to X-ray dose. For Vicia faba, mean total root length was reduced significantly. Leave growth was reduced as well. Number and length of second order laterals was reduced significantly, as well as length of first order laterals. Hordeum vulgare showed no negative impact of X-ray dose on any of the root parameters. Large differences between the two species investigated were detected in respect to susceptibility to X-ray dose. Results indicate that for X-ray CT studies involving temporal resolution a control treatment without scanning is required.