The breakthrough paradox: How focusing on one form of innovation jeopardizes the advancement of science: How focusing on one form of innovation jeopardizes the advancement of science.

Research needs a balance of risk-taking in "breakthrough projects" and gradual progress. For building a sustainable knowledge base, it is indispensable to provide support for both.

The effect of root hairs on exudate composition: a comparative non-targeted metabolomics approach.

Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20-79 features, 13-31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula.

Total synthesis of [13 C2 ]-labeled phytosiderophores of the mugineic and avenic acid families.

We, herein, report the synthesis of 13 C2 -labeled natural products from the mugineic acid and avenic acid family. These phytosiderophores ("plant iron carriers") are built up from non-proteinogenic amino acids and play a key role in micronutrient uptake in gramineous plants. In this work, two central building blocks are prepared from labeled starting materials (13 C2 -bromoacetic acid, 13 C2 -glycine) and further employed in our recently reported divergent, branched synthetic strategy delivering eight isotopically labeled phytosiderophores. The required labeled building blocks (13 C2 -l-allylglycine and a related hydroxylated derivative) were prepared via enantioselective phase-transfer catalysis and enantio- and diastereoselective aldol condensation with a chiral auxiliary, respectively, both potentially valuable themselves for other synthetic routes toward labeled (natural) products.

A Unified Approach to Phytosiderophore Natural Products.

This work reports on the concise total synthesis of eight natural products of the mugineic acid and avenic acid families (phytosiderophores). An innovative "east-to-west" assembly of the trimeric products resulted in a high degree of divergence enabling the formation of the final products in just 10 or 11 steps each with a minimum of overall synthetic effort. Chiral pool starting materials (l-malic acid, threonines) were employed for the outer building blocks while the middle building blocks were accessed by diastereo- and enantioselective methods. A highlight of this work consists in the straightforward preparation of epimeric hydroxyazetidine amino acids, useful building blocks on their own, enabling the first synthesis of 3''-hydroxymugineic acid and 3''-hydroxy-2'-deoxymugineic acid.

Arsenic redox transformations and cycling in the rhizosphere of Pteris vittata and Pteris quadriaurita.

Pteris vittata (PV) and Pteris quadriaurita (PQ) are reported to hyperaccumulate arsenic (As) when grown in Asrich soil. Yet, little is known about the impact of their unique As accumulation mechanisms on As transformations and cycling at the soil-root interface. Using a combined approach of two-dimensional (2D), sub-mm scale solute imaging of arsenite (AsIII), arsenate (AsV), phosphorus (P), manganese (Mn), iron (Fe) and oxygen (O2), we found localized patterns of AsIII/AsV redox transformations in the PV rhizosphere (AsIII/AsV ratio of 0.57) compared to bulk soil (AsIII/AsV ratio of ≤0.04). Our data indicate that the high As root uptake, translocation and accumulation from the As-rich experimental soil (2080 mg kg-1) to PV fronds (6986 mg kg-1) induced As detoxification via AsV reduction and AsIII root efflux, leading to AsIII accumulation and re-oxidation to AsV in the rhizosphere porewater. This As cycling mechanism is linked to the reduction of O2 and MnIII/IV (oxyhydr)oxides resulting in decreased O2 levels and increased Mn solubilization along roots. Compared to PV, we found 4-fold lower As translocation to PQ fronds (1611 mg kg-1), 2-fold lower AsV depletion in the PQ rhizosphere, and no AsIII efflux from PQ roots, suggesting that PQ efficiently controls As uptake to avoid toxic As levels in roots. Analysis of root exudates obtained from soil-grown PV showed that As acquisition by PV roots was not associated with phytic acid release. Our study demonstrates that two closely-related As-accumulating ferns have distinct mechanisms for As uptake modulating As cycling in As-rich environments.

pH-Dependent Bioavailability, Speciation, and Phytotoxicity of Tungsten (W) in Soil Affect Growth and Molybdoenzyme Activity of Nodulated Soybeans.

Increasing use of tungsten (W)-based products opened new pathways for W into environmental systems. Due to its chemical alikeness with molybdenum (Mo), W is expected to behave similarly to its "twin element", Mo; however, our knowledge of the behavior of W in the plant-soil environment remains inadequate. The aim of this study was to investigate plant growth as well as W and nutrient uptake depending on soil chemical properties such as soil pH and texture. Soybean ( Glycine max cv. Primus) was grown on two acidic soils differing in soil texture that were either kept at their natural soil pH (pH of 4.5-5) or limed (pH of ≥7) and amended with increasing concentrations of metallic W (control and 500 and 5000 mg kg-1). In addition, the activity of molybdoenzymes involved in N assimilation (nitrate reductase) and symbiotic N2 fixation (nitrogenase) was also investigated. Our results showed that the risk of W entering the food web was significantly greater in high-pH soils due to increased solubility of mainly monomeric W. The effect of soil texture on W solubility and phytoavailability was less pronounced compared to soil pH. Particularly at intermediate W additions (W 500 mg kg-1), symbiotic nitrogen fixation was able to compensate for reduced leaf nitrate reductase activity. When W soil solution concentrations became too toxic (W 5000 mg kg-1), nodulation was more strongly inhibited than nitrogenase activity in the few nodules formed, suggesting a more-efficient detoxification and compartmentalization mechanism in nodules than in soybean leaves. The increasing presence of polymeric W species observed in low-pH soils spiked with high W concentrations resulted in decreased W uptake. Simultaneously, polymeric W species had an overall negative effect on nutrient assimilation and plant growth, suggesting a greater phytotoxicity of W polymers. Our study demonstrates the importance of accounting for soil pH in risk assessment studies of W in the plant-soil environment, something that has been completely neglected in the past.

Root exudation of phytosiderophores from soil-grown wheat.

For the first time, phytosiderophore (PS) release of wheat (Triticum aestivum cv Tamaro) grown on a calcareous soil was repeatedly and nondestructively sampled using rhizoboxes combined with a recently developed root exudate collecting tool. As in nutrient solution culture, we observed a distinct diurnal release rhythm; however, the measured PS efflux was c. 50 times lower than PS exudation from the same cultivar grown in zero iron (Fe)-hydroponic culture. Phytosiderophore rhizosphere soil solution concentrations and PS release of the Tamaro cultivar were soil-dependent, suggesting complex interactions of soil characteristics (salinity, trace metal availability) and the physiological status of the plant and the related regulation (amount and timing) of PS release. Our results demonstrate that carbon and energy investment into Fe acquisition under natural growth conditions is significantly smaller than previously derived from zero Fe-hydroponic studies. Based on experimental data, we calculated that during the investigated period (21-47 d after germination), PS release initially exceeded Fe plant uptake 10-fold, but significantly declined after c. 5 wk after germination. Phytosiderophore exudation observed under natural growth conditions is a prerequisite for a more accurate and realistic assessment of Fe mobilization processes in the rhizosphere using both experimental and modeling approaches.

Accurate LC-ESI-MS/MS quantification of 2'-deoxymugineic acid in soil and root related samples employing porous graphitic carbon as stationary phase and a ¹³C4-labeled internal standard.

For the first time the phytosiderophore 2'-deoxymugineic acid (DMA) could be accurately quantified by LC-MS/MS in plant and soil related samples. For this purpose a novel chromatographic method employing porous graphitic carbon as stationary phase combined with ESI-MS/MS detection in selected reaction monitoring was developed. Isotope dilution was implemented by using in-house synthesized DMA as external calibrant and ¹³C4-labeled DMA as internal standard (concentration levels of standards 0.1-80 µM, determination coefficient of linear regression R² > 0.9995). Sample preparation involved acidification of the samples in order to obtain complete dissociation of metal-DMA complexes. Excellent matrix related LOD and LOQ depending on different experimental setups were obtained in the range of 3-34 nM and 11-113 nM, respectively. Standard addition experiments and the implementation of the internal ¹³C4-DMA standard proved the accuracy of the quantification strategy even in complex matrices such as soil solution. The repeatability of the method, including sample preparation, expressed as short- and long term precision was below 4 and 5% RSD, respectively. Finally, application in the context of plant and soil research to samples from rhizosphere sampling via micro suction cups, from soil solutions and soil adsorption/extraction studies revealed a DMA concentration range from 0.1 to 235 µM.

Localized flux maxima of arsenic, lead, and iron around root apices in flooded lowland rice.

In wetland-adapted plants, such as rice, it is typically root apexes, sites of rapid entry for water/nutrients, where radial oxygen losses (ROLs) are highest. Nutrient/toxic metal uptake therefore largely occurs through oxidized zones and pH microgradients. However, the processes controlling the acquisition of trace elements in rice have been difficult to explore experimentally because of a lack of techniques for simultaneously measuring labile trace elements and O2/pH. Here, we use new diffusive gradients in thin films (DGT)/planar optode sandwich sensors deployed in situ on rice roots to demonstrate a new geochemical niche of greatly enhanced As, Pb, and Fe(II) mobilization into solution immediately adjacent to the root tips characterized by O2 enrichment and low pH. Fe(II) mobilization was congruent to that of the peripheral edge of the aerobic root zone, demonstrating that the Fe(II) mobilization maximum only developed in a narrow O2 range as the oxidation front penetrates the reducing soil. The Fe flux to the DGT resin at the root apexes was 3-fold higher than the anaerobic bulk soil and 27 times greater than the aerobic rooting zone. These results provide new evidence for the importance of coupled diffusion and oxidation of Fe in modulating trace metal solubilization, dispersion, and plant uptake.

Rhizobium symbiosis improves amino acid and secondary metabolite biosynthesis of tungsten-stressed soybean (Glycine max).

The industrially important transition metal tungsten (W) shares certain chemical properties with the essential plant micronutrient molybdenum and inhibits the activity of molybdoenzymes such as nitrate reductase, impacting plant growth. Furthermore, tungsten appears to interfere with metabolic processes on a much wider scale and to trigger common heavy metal stress response mechanisms. We have previously found evidence that the tungsten stress response of soybeans (Glycine max) grown with symbiotically associated N2-fixing rhizobia (Bradyrhizobium japonicum) differs from that observed in nitrogen-fertilized soy plants. This study aimed to investigate how association with symbiotic rhizobia affects the primary and secondary metabolite profiles of tungsten-stressed soybean and whether changes in metabolite composition enhance the plant's resilience to tungsten. This comprehensive metabolomic and proteomic study presents further evidence that the tungsten-stress response of soybean plants is shaped by associated rhizobia. Symbiotically grown plants (N fix) were able to significantly increase the synthesis of an array of protective compounds such as phenols, polyamines, gluconic acid, and amino acids such as proline. This resulted in a higher antioxidant capacity, reduced root-to-shoot translocation of tungsten, and, potentially, also enhanced resilience of N fix plants compared to non-symbiotic counterparts (N fed). Taken together, our study revealed a symbiosis-specific metabolic readjustment in tungsten-stressed soybean plants and contributed to a deeper understanding of the mechanisms involved in the rhizobium-induced systemic resistance in response to heavy metals.

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.

Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation.

Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients.

Visualizing and quantifying 33P uptake and translocation by maize plants grown in soil.

Phosphorus (P) availability severely limits plant growth due to its immobility and inaccessibility in soils. Yet, visualization and measurements of P uptake from different root types or regions in soil are methodologically challenging. Here, we explored the potential of phosphor imaging combined with local injection of radioactive 33P to quantitatively visualize P uptake and translocation along roots of maize grown in soils. Rhizoboxes (20 × 40 × 1 cm) were filled with sandy field soil or quartz sand, with one maize plant per box. Soil compartments were created using a gravel layer to restrict P transfer. After 2 weeks, a compartment with the tip region of a seminal root was labeled with a NaH2 33PO4 solution containing 12 MBq of 33P. Phosphor imaging captured root P distribution at 45 min, 90 min, 135 min, 180 min, and 24 h post-labeling. After harvest, 33P levels in roots and shoots were quantified. 33P uptake exhibited a 50% increase in quartz sand compared to sandy soil, likely attributed to higher P adsorption to the sandy soil matrix than to quartz sand. Notably, only 60% of the absorbed 33P was translocated to the shoot, with the remaining 40% directed to growing root tips of lateral or seminal roots. Phosphor imaging unveiled a continuous rise in 33P signal in the labeled seminal root from immediate post-labeling until 24 h after labeling. The highest 33P activities were concentrated just above the labeled compartment, diminishing in locations farther away. Emerging laterals from the labeled root served as strong sinks for 33P, while a portion was also transported to other seminal roots. Our study quantitatively visualized 33P uptake and translocation dynamics, facilitating future investigations into diverse root regions/types and varying plant growth conditions. This improves our understanding of the significance of different P sources for plant nutrition and potentially enhances models of plant P uptake.

Chemical imaging reveals environmental risk of minor tungsten and lead shotgun pellet constituents during weathering in soil.

Tungsten (W)-based shots are considered more environmentally safe than lead (Pb)-based shots, but knowledge about the W-shot fate in the soil environment is still limited, especially in terms of minor constituents such as iron, copper, and nickel (Ni). Contaminant behaviour in soil strongly depends on pH; in turn, the corrosion of metal composites may affect the pH locally. The aim of this study was to compare Pb- and W-shot weathering dynamics in soil (silt loam, pH 6.3) and reveal the interplay of shot weathering-induced pH-changes on the mobility of elements using in situ chemical imaging (Diffusive gradients in thin films for labile elements, planar optodes for soil pH) and batch incubation experiments over time (16 months). Despite our expectation to find acidification due to W oxidation, we observed a pH increase by 0.2 units in extracted soil solutions and by 0.6 units in the soil around W-shots as Ni dissolved from the binder phase of the shot. After 10 weeks, release of labile Ni was 3-times higher compared to W despite the low Ni content in the shot (7 %, m/m). Pb-shot oxidation increased soil solution pH by 0.5 units which likely supported mobility of Pb-shot-derived antimony (Sb). Steep gradients of labile W and Pb and soil solution concentrations <0.8 µmol L-1 indicated that transfer from shot to soil was low. Contrastingly, labile Ni and Sb were found up to ~4 mm from the shot surface and in higher soil solution concentrations as suggested by the shot constitution, indicating higher mobility of minor as compared to major shot constituents. After 16 months, 36 % of total Ni were dissolved in the soil solution highlighting the environmental relevance of minor shot constituents in Pb-shot alternatives after short term weathering in soil.

Surveying the mugineic acid family: Ion mobility - quadrupole time-of-flight mass spectrometry (IM-QTOFMS) characterization and tandem mass spectrometry (LC-ESI-MS/MS) quantification of all eight naturally occurring phytosiderophores.

Phytosiderophores (PS) are root exudates released by grass species (Poaceae) that play a pivotal role in iron (Fe) plant nutrition. A direct determination of PS in biological samples is of paramount importance in understanding micronutrient acquisition mediated by PS. To date, eight plant-born PS have been identified; however, no analytical procedure is currently available to quantify all eight PS simultaneously with high analytical confidence. With access to the full set of PS standards for the first time, we report comprehensive methods to both fully characterize (IM-QTOFMS) and quantify (LC-ESI-MS/MS) all eight naturally occurring PS belonging to the mugineic acid family. The quantitative method was fully validated, yielding linear results for all eight analytes, and no unwanted interferences with soil and plant matrices were observed. LOD and LOQ values determined for each PS were below 11 and 35 nmol L-1, respectively. The method's precision under reproducibility conditions (intra- and inter-day) of measurement was less than 2.5% RSD for all analytes. Additionally, all PS were annotated with high-resolution mass spectrometric fragment spectra and further characterized via drift tube ion mobility-mass spectrometry. The collision cross-sections obtained for primary ion species yielded a valuable database for future research focused on in-depth PS studies. The new quantitative method was applied to analyse root exudates from Fe-controlled and deficient barley, oat, rye, and sorghum plants. All eight PS, including mugineic acid (MA), 3"-hydroxymugineic acid (HMA), 3"-epi-hydroxymugineic acid (epi-HMA), hydroxyavenic acid (HAVA), deoxymugineic acid (DMA), 3"-hydroxydeoxymugineic acid (HDMA), 3"-epi-hydroxydeoxymugineic acid (epi-HDMA) and avenic acid (AVA) were for the first time successfully identified and quantified in root exudates of various graminaceous plants using a single analytical procedure. These newly developed methods can be applied to studies aimed at improving crop yield and micronutrient grain content for food consumption via plant-based biofortification.

Analysis of iron-phytosiderophore complexes in soil related samples: LC-ESI-MS/MS versus CE-MS.

Phytosiderophores (PS) form stable complexes with various transition metals. These ligands are exuded by the roots of graminacous plants as a mechanism for mobilizing and acquiring soil iron. To investigate iron mobilization and transport, a novel LC method in combination with ESI-MS/MS for the determination of three Fe(III)-complexes with mugineic acid (MA), 2'-epi-MA and 2'-deoxymugineic acid (DMA) has been developed. Liquid chromatographic separation was realized using a silica-based mixed-mode reversed phase/weak-anion exchange type stationary phase and a 50 mM ammonium acetate buffer, pH 6.5. Baseline separation of the two complex diastereomers Fe(III)-MA and Fe(III)-epi-MA could be achieved. ESI-MS/MS detection allowed for simultaneous quantification of the complexes and the free ligands. Limits of detection were determined to be 0.001 and 0.05 µM for DMA and Fe(III)-DMA, respectively. The analytical figures of merit of the novel method were evaluated and compared with a CE-ESI-MS method that we had published earlier. The LC-ESI-MS/MS method has been successfully applied to real samples derived from preliminary extraction experiments.

Harnessing belowground processes for sustainable intensification of agricultural systems.

Increasing food demand coupled with climate change pose a great challenge to agricultural systems. In this review we summarize recent advances in our knowledge of how plants, together with their associated microbiota, shape rhizosphere processes. We address (molecular) mechanisms operating at the plant-microbe-soil interface and aim to link this knowledge with actual and potential avenues for intensifying agricultural systems, while at the same time reducing irrigation water, fertilizer inputs and pesticide use. Combining in-depth knowledge about above and belowground plant traits will not only significantly advance our mechanistic understanding of involved processes but also allow for more informed decisions regarding agricultural practices and plant breeding. Including belowground plant-soil-microbe interactions in our breeding efforts will help to select crops resilient to abiotic and biotic environmental stresses and ultimately enable us to produce sufficient food in a more sustainable agriculture in the upcoming decades.

A quick and simple spectrophotometric method to determine total carbon concentrations in root exudate samples of grass species.

Purpose: Root exudates are key components driving belowground interaction between plant, microbes and soil. High-end analytical approaches provide advanced insights into exudate metabolite diversity, however, the amount of total carbon (C) released by roots should always be determined as the most basic parameter when characterizing root exudation as it (i) provides quantitative information of C exuded into the surrounding soil and (ii) allows to relate the abundance of individual exudate compounds to total C released. Here we propose a simple and quick, spectrophotometry-based method to quantify total dissolved organic carbon (DOC) concentration in exudation samples that is based on measuring the absorption of a pre-filtered but otherwise untreated exudate sample at 260 nm (DOC260). Method: Exudate samples collected from different grass genotypes (Zea mays, Oryza sativa, Hordeum vulgare) grown in various experimental settings (soil, hydroponic) were analysed with the DOC260 assay and results were compared with C concentrations obtained by liquid TOC-analyser. Conclusion: We demonstrated that the DOC260 method allowed for quick and inexpensive measurements of total dissolved organic carbon concentrations in exudate samples from grass species grown under nutrient sufficient as well as under P deficient conditions. Interestingly, DOC260 failed to predict DOC concentrations in exudate samples from plants grown under Zn and Fe deficiency suggesting a strong shift in metabolite composition under micronutrient deficiency. Even though the applicability of the DOC260 method remains to be tested on exudate samples originating from dicots and plants exposed to other environmental stresses (e.g. pathogen attack, heavy metal stress, etc), it will help to increase our understanding of root exudation and related rhizosphere processes in the future.

LC-MS analysis of low molecular weight organic acids derived from root exudation.

A sensitive method for quantification of citric, fumaric, malic, malonic, oxalic, trans aconitic, and succinic acid in soil- and root-related samples is presented. The method is based on a novel, fast, and simple esterification procedure and subsequent analysis via liquid chromatography-mass spectrometry. Derivatization comprises in situ generation of HCl, which catalyzes the Fischer esterification with benzyl alcohol. As a key advance, the esterification with the aromate allows reversed-phase separation and improves electrospray ionization efficiency. The method provided procedural detection limits of 1 nM for citric, 47 nM for fumaric, 10 nM for malic, 10 nM for malonic, 16 nM for oxalic, 15 nM for succinic, and 2 nM for aconitic acid utilizing 500 µL of liquid sample. The working range was 3 nM to 10 µM for citric acid, 158 nM to 10 µM for fumaric acid, 34 nM to 10 µM for malic acid, 33 nM to 10 µM for malonic acid, 53 nM to 10 µM for oxalic acid, 48 nM to 10 µM for succinic acid, and 6 nM to 10 µM for aconitic acid. Quantification of the analytes in soil-related samples was performed via external calibration of the entire procedure utilizing (13)C-labeled oxalic and citric acid as internal standards. The robustness of the method was tested with soil extracts and samples from hydroponic experiments. The latter concerned the regulation of phosphorus solubilization via plant root exudation of citric, malic, and oxalic acid.

Complexation of metals by phytosiderophores revealed by CE-ESI-MS and CE-ICP-MS.

CE-ESI-MS and CE-ICP-MS were implemented for studying three phytosiderophores (mugineic acid, epi-mugineic acid and deoxymugineic acid) and their metal complexes. Free ligands and ferric complexes were analyzed using the first methodology, while six free metals (Co(II), Cu(II), Fe(III), Mn(II), Ni(II) and Zn(II)) together with the corresponding complexes were investigated by the latter technique. CE separation was realized at a voltage of +25 kV employing a BGE containing 20 mM ammonium bicarbonate at pH 7.2. Both techniques revealed limits of detection in the high nM to low microM range. Standard additions to hydroponic samples of H. distichon, cv. Bodega (spring barley) showed regression coefficients for the metal-ligand complexes ranging from 0.984 to 0.999. Additionally, results of a competitivity study allowed the determination of relative metal-phytosiderophore complex stability constants of deoxymugineic/mugineic acid.