Mapping Phosphorus Availability in Soil at a Large Scale and High Resolution Using Novel Diffusive Gradients in Thin Films Designed for X-ray Fluorescence Microscopy.

A novel binding layer (BL) as part of the diffusive gradients in thin films (DGT) technique was developed for the two-dimensional visualization and quantification of labile phosphorus (P) in soils. This BL was designed for P detection by synchrotron-based X-ray fluorescence microscopy (XFM). It differs from the conventional DGT BL as the hydrogel is eliminated to overcome the issue that the fluorescent X-rays of P are detected mainly from shallow sample depths. Instead, the novel design is based on a polyimide film (Kapton) onto which finely powdered titanium dioxide-based P binding agent (Metsorb) was applied, resulting in superficial P binding only. The BL was successfully used for quantitative visualization of P diffusion from three conventional P fertilizers applied to two soils. On a selection of samples, XFM analysis was confirmed by quantitative laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The XFM method detected significant differences in labile P concentrations and P diffusion zone radii with the P fertilizer incubation, which were explained by soil and fertilizer properties. This development paves the way for fast XFM analysis of P on large DGT BLs to investigate in situ diffusion of labile P from fertilizers and to visualize large-scale P cycling processes at high spatial resolution.

Foliar-applied manganese and phosphorus in deficient barley: Linking absorption pathways and leaf nutrient status.

Foliar fertilization delivers essential nutrients directly to plant tissues, reducing excessive soil fertilizer applications that can lead to eutrophication following nutrient leaching. Foliar nutrient absorption is a dynamic process affected by leaf surface structure and composition, plant nutrient status, and ion physicochemical properties. We applied multiple methods to study the foliar absorption behaviors of manganese (Mn) and phosphorus (P) in nutrient-deficient spring barley (Hordeum vulgare) at two growth stages. Nutrient-specific chlorophyll a fluorescence assays were used to visualize leaf nutrient status, while laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to visualize foliar absorption pathways for P and Mn ions. Rapid Mn absorption was facilitated by a relatively thin cuticle with a low abundance of waxes and a higher stomatal density in Mn-deficient plants. Following absorption, Mn accumulated in epidermal cells and in the photosynthetically active mesophyll, enabling a fast (6 h) restoration of Mn-dependent photosynthetic processes. Conversely, P-deficient plants developed thicker cuticles and epidermal cell walls, which reduced the penetration of P across the leaf surface. Foliar-applied P accumulated in trichomes and fiber cells above leaf veins without reaching the mesophyll and, as a consequence, no restoration of P-dependent photosynthetic processes was observed. This study reveals new links between leaf surface morphology, foliar-applied ion absorption pathways, and the restoration of affected physiological processes in nutrient-deficient leaves. Understanding that ions may have different absorption pathways across the leaf surface is critical for the future development of efficient fertilization strategies for crops in nutrient-limited soils.

Synchrotron-Based Imaging Reveals the Fate of Selenium in Striped Marsh Frog Tadpoles.

Synchrotron-based X-ray fluorescence microscopy (XFM) coupled with X-ray absorption near-edge structure (XANES) imaging was used to study selenium (Se) biodistribution and speciation in Limnodynastes peronii tadpoles. Tadpoles were exposed to dissolved Se (30 µg/L) as selenite (SeIV) or selenate (SeVI) for 7 days followed by 3 days of depuration. High-resolution elemental maps revealed that Se partitioned primarily in the eyes (specifically the eye lens, iris, and retinal pigmented epithelium), digestive and excretory organs of SeIV-exposed tadpoles. Speciation analysis confirmed that the majority of accumulated Se was converted to organo-Se. Multielement analyses provided new information on Se colocalization and its impact on trace element homeostasis. New insights into the fate of Se on a whole organism scale contribute to our understanding of the mechanisms and risks associated with Se pollution.

Zinc Accumulates in the Nodes of Wheat Following the Foliar Application of 65Zn Oxide Nano- and Microparticles.

Using zinc (Zn) foliar fertilizers to enhance the grain quality of wheat (Triticum aestivum) can be an effective alternative or supplement to Zn soil fertilizers. However, knowledge about the mechanisms of Zn absorption and translocation following foliar application is scarce. Here, autoradiography and γ-spectrometry were used to investigate the behavior of 65Zn applied to wheat leaves as soluble 65Zn chloride (65ZnCl2), chelated 65Zn (65ZnEDTA), 65Zn oxide nanoparticle (65ZnO-NP) suspensions, and 65ZnO microparticle (65ZnO-MP) suspensions. The largest amount of 65Zn absorption occurred in 65ZnCl2 treated leaves. However, this treatment (65ZnCl2) also had the lowest proportion of absorbed 65Zn translocated away from the treated leaf after 15 d due to leaf scorching (p = 0.0007). Foliar-applied 65ZnO-NPs and 65ZnO-MPs had the lowest absorption, but 65ZnO-NPs had the highest relative translocation. 65Zinc EDTA was intermediate, with higher 65Zn absorption than 65ZnO treatments but similar translocation. Regardless, the majority of the foliar-applied 65Zn remained in the treated leaf for all treatments. Furthermore, 65ZnO-NPs and 65ZnO-MPs accumulated in plant nodes, suggesting that Zn was absorbed as dissolved 65Zn and particulate 65ZnO. Overall, the form and amount of absorbed 65Zn affected translocation.

Development and evaluation of a new colorimetric DGT technique for the 2D visualisation of labile phosphate in soils.

A new colorimetric technique for the measurement of labile phosphate in soils using the diffusive gradients in thin films (DGT) technique was developed in this study. This technique can determine the mass of phosphate accumulated on the precipitated Zr-oxide based binding gel by forming the blue colour following the standard molybdate-ascorbic acid method. The optimal reaction temperature and coloration time were 20 °C (room temperature) and 26 min. After determining a well-fitted calibration equation, the technique was able to measure phosphate concentration up to 2.5 mg/L for 24 h deployment with a detection limit of 10.1 µg/L. Two-dimensional quantitative visualisation of phosphate diffusion in three phosphorus (P) fertilised soils were obtained using the colorimetric technique. The results from the colorimetric DGT technique were compared to the elution DGT technique and Colwell P extraction. The DGT techniques (colorimetric and elution) and Colwell P measurements demonstrated similar patterns of phosphate diffusion in soil. Both DGT techniques showed similar phosphate concentration along the concentric rings around the fertiliser application. A new, convenient, and fast DGT colorimetric technique was developed, and successfully used to measure the distribution of potentially available phosphate in soils. The new technique is less laborious than current techniques as it does not require any pre-treatment of the binding gel layers or heating during scanning, thus providing faster results. Therefore, the technique may be more suitable for in-field applications and can be used to investigate the in situ diffusion of potentially available phosphate from fertilisers, and relate this to the plant uptake of P.

Plant-Available Phosphorus in Highly Concentrated Fertilizer Bands: Effects of Soil Type, Phosphorus Form, and Coapplied Potassium.

Phosphorus (P) is increasingly being applied in concentrated bands to satisfy plant nutrient requirements. To quantify changes in plant-available P in the fertosphere of highly concentrated fertilizer bands, we conducted a soil-fertilizer incubation experiment using seven soil types, three highly water-soluble P sources [monocalcium phosphate (MCP), monoammonium phosphate (MAP), and diammonium phosphate (DAP)], and coapplication of potassium chloride (KCl). First, we found that soil properties were important in influencing P availability. For a calcareous soil, availability was generally low irrespective of treatment, presumably due to precipitation of the fertilizer as Ca-P minerals. For all six noncalcareous soils, fertosphere pH was critical in determining potential P availability, with decreasing pH values decreasing availability, presumably due to precipitation of Al- and Fe-P minerals. Second, given the importance of pH, we also found that the form of P supplied (MCP, MAP, or DAP) had a pronounced effect on P availability due to associated changes in fertosphere pH. Finally, we also found that the coapplication of K also decreased P availability in some soils. We conclude that the selection of the P source is of utmost importance when fertilizers are placed as highly concentrated bands and that soil properties also need to be considered.

Bioimaging Techniques Reveal Foliar Phosphate Uptake Pathways and Leaf Phosphorus Status.

Global demand for phosphorus (P) requires new agronomic practices to address sustainability challenges while increasing food production. Foliar P fertilization could increase P use efficiency; however, leaf entry pathways for inorganic phosphate ion (Pi) uptake remain unknown, and it is unclear whether foliar P applications can meet plant nutrient demands. We developed two techniques to trace foliar P uptake in P-deficient spring barley (Hordeum vulgare) and to monitor the effectiveness of the treatment on restoring P functionality. First, a whole-leaf P status assay was developed using an IMAGING PAM system; nonphotochemical quenching was a proxy for P status, as P-deficient barley developed nonphotochemical quenching at a faster rate than P-sufficient barley. The assay showed restoration of P functionality in P-deficient plants 24 h after foliar P application. Treated leaves reverted to P deficiency after 7 d, while newly emerging leaves exhibited partial restoration compared with untreated P-deficient plants, indicating Pi remobilization. Second, vanadate was tested as a possible foliar Pi tracer using high-resolution laser ablation-inductively coupled plasma-mass spectrometry elemental mapping. The strong colocalization of vanadium and P signal intensities demonstrated that vanadate was a sensitive and useful Pi tracer. Vanadate and Pi uptake predominantly occurred via fiber cells located above leaf veins, with pathways to the vascular tissue possibly facilitated by the bundle sheath extension. Minor indications of stomatal and cuticular Pi uptake were also observed. These techniques provided an approach to understand how Pi crosses the leaf surface and assimilates to meet plant nutrient demands.

Transformation of Calcium Phosphates in Alkaline Vertisols by Acidified Incubation.

Acid-soluble soil phosphorus (P) is a potential resource in P-limited agricultural systems that may become critical as global P sources decrease in the future. The fate of P in three alkaline Vertisols, a major agricultural soil type, after acidic incubation was investigated using synchrotron-based K-edge X-ray absorption near-edge structure (XANES) spectroscopy, geochemical modeling, wet chemistry soil extraction, and a P sorption index. Increases in labile P generally coincided with decreased stability and dissolution of calcium phosphate (CaP) minerals. However, only a minor proportion of the CaP dissolved in each soil was labile. In two moderate-P soils (800 mg P kg-1), XANES indicated that approximately 160 mg kg-1 was repartitioned to sorbed phases at pH 5.1 of one soil and at pH 4.4 of the second; however, only 40 and 28% were labile, respectively. In a high-P soil (8900 mg P kg-1), XANES indicated a decrease in P of 1170 mg kg-1 from CaP minerals at pH 3.8, of which approximately only 33% was labile. Phosphorus mobilized by agricultural practices without concurrent uptake by plants may be repartitioned to sorbed forms that are not as plant-available as prior to acidification.

The effect of different pyrolysis temperatures on the speciation and availability in soil of P in biochar produced from the solid fraction of manure.

Biochar application to agricultural land has been proposed as a means for improving phosphorus (P) availability in soil. The purpose of the current study was to understand how pyrolysis temperature affects P speciation in biochar and how this affects availability of P in the amended soil. Biochar was produced at different temperatures from digestate solids. The primary species of P in digestate solids were simple calcium phosphates. However, a high co-occurrence of magnesium (Mg) and P, indicated that struvite or other magnesium phosphates may also be important species. At low temperatures, pyrolysis had little effect on P speciation; however, as the temperature increased above 600 °C, the P gradually became more thermodynamically stable in species such as apatite. At very high temperatures above 1000 °C, there were indications of reduced forms of P. Biochar production decreased the immediate availability of P in comparison with the original digestate solids. However, for biochar produced at low temperatures, availability quickly increased to the same levels as in the digestate solids. For biochar produced at higher temperatures, availability remained depressed for much longer. The low availability of P in the biochar produced at high temperatures can probably be explained by the formation of less soluble P species in the biochar. In contrast, the transient decrease of availability of the P in the biochar produced at low temperatures can be explained by mechanisms, such as sorption on biochar, which gradually decreases because of oxidation of the biochar surfaces or changes in pH around the biochar particles.

XANES Demonstrates the Release of Calcium Phosphates from Alkaline Vertisols to Moderately Acidified Solution.

Calcium phosphate (CaP) minerals may comprise the main phosphorus (P) reserve in alkaline soils, with solubility dependent on pH and the concentration of Ca and/or P in solution. Combining several techniques in a novel way, we studied these phenomena by progressively depleting P from suspensions of two soils (low P) using an anion-exchange membrane (AEM) and from a third soil (high P) with AEM together with a cation-exchange membrane. Depletions commenced on untreated soil, then continued as pH was manipulated and maintained at three constant pH levels: the initial pH (pHi) and pH 6.5 and 5.5. Bulk P K-edge X-ray absorption near-edge structure (XANES) spectroscopy revealed that the main forms of inorganic P in each soil were apatite, a second more soluble CaP mineral, and smectite-sorbed P. With moderate depletion of P at pHi or pH 6.5, CaP minerals became more prominent in the spectra compared to sorbed species. The more soluble CaP minerals were depleted at pH 6.5, and all CaP minerals were exhausted at pH 5.5, showing that the CaP species present in these alkaline soils are soluble with decreases of pH in the range achievable by rhizosphere acidification.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 µg g(-1) iron (Fe) and 16 µg g(-1) zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 µg g(-1) Fe and 28 µg g(-1) Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 µg g(-1) in glasshouse settings; in contrast, under field conditions, 4 µg g(-1) was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 µg g(-1) Fe and 45.7 µg g(-1) Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Synchrotron-based X-ray absorption near-edge spectroscopy imaging for laterally resolved speciation of selenium in fresh roots and leaves of wheat and rice.

Knowledge of the distribution of selenium (Se) species within plant tissues will assist in understanding the mechanisms of Se uptake and translocation, but in situ analysis of fresh and highly hydrated plant tissues is challenging. Using synchrotron-based fluorescence X-ray absorption near-edge spectroscopy (XANES) imaging to provide laterally resolved data, the speciation of Se in fresh roots and leaves of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) supplied with 1 µM of either selenate or selenite was investigated. For plant roots exposed to selenate, the majority of the Se was efficiently converted to C-Se-C compounds (i.e. methylselenocysteine or selenomethionine) as selenate was transported radially through the root cylinder. Indeed, even in the rhizodermis which is exposed directly to the bulk solution, only 12-31% of the Se was present as uncomplexed selenate. The C-Se-C compounds were probably sequestered within the roots, whilst much of the remaining uncomplexed Se was translocated to the leaves-selenate accounting for 52-56% of the total Se in the leaves. In a similar manner, for plants exposed to selenite, the Se was efficiently converted to C-Se-C compounds within the roots, with only a small proportion of uncomplexed selenite observed within the outer root tissues. This resulted in a substantial decrease in translocation of Se from the roots to leaves of selenite-exposed plants. This study provides important information for understanding the mechanisms responsible for the uptake and subsequent transformation of Se in plants.

Selenopeptides and elemental selenium in Thunbergia alata after exposure to selenite: quantification method for elemental selenium.

Three month old Thunbergia alata were exposed for 13 days to 10 µM selenite to determine the biotransformation of selenite in their roots. Selenium in formic acid extracts (80 ± 3%) was present as selenopeptides with Se-S bonds and selenium-PC complexes (selenocysteinyl-2-3-dihydroxypropionyl-glutathione, seleno-phytochelatin2, seleno-di-glutathione). An analytical method using HPLC-ICPMS to detect and quantify elemental selenium in roots of T. alata plants using sodium sulfite to quantitatively transform elemental selenium to selenosulfate was also developed. Elemental selenium was determined as 18 ± 4% of the total selenium in the roots which was equivalent to the selenium not extracted using formic acid extraction. The results are in an agreement with the XAS measurements of the exposed roots which showed no occurrence of selenite or selenate but a mixture of selenocysteine and elemental selenium.

Speciation of metal(loid)s in environmental samples by X-ray absorption spectroscopy: a critical review.

Element specificity is one of the key factors underlying the widespread use and acceptance of X-ray absorption spectroscopy (XAS) as a research tool in the environmental and geo-sciences. Independent of physical state (solid, liquid, gas), XAS analyses of metal(loid)s in complex environmental matrices over the past two decades have provided important information about speciation at environmentally relevant interfaces (e.g. solid-liquid) as well as in different media: plant tissues, rhizosphere, soils, sediments, ores, mineral process tailings, etc. Limited sample preparation requirements, the concomitant ability to preserve original physical and chemical states, and independence from crystallinity add to the advantages of using XAS in environmental investigations. Interpretations of XAS data are founded on sound physical and statistical models that can be applied to spectra of reference materials and mixed phases, respectively. For spectra collected directly from environmental matrices, abstract factor analysis and linear combination fitting provide the means to ascertain chemical, bonding, and crystalline states, and to extract quantitative information about their distribution within the data set. Through advances in optics, detectors, and data processing, X-ray fluorescence microprobes capable of focusing X-rays to micro- and nano-meter size have become competitive research venues for resolving the complexity of environmental samples at their inherent scale. The application of µ-XANES imaging, a new combinatorial approach of X-ray fluorescence spectrometry and XANES spectroscopy at the micron scale, is one of the latest technological advances allowing for lateral resolution of chemical states over wide areas due to vastly improved data processing and detector technology.

In situ speciation and distribution of toxic selenium in hydrated roots of cowpea.

The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based x-ray absorption spectroscopy and x-ray fluorescence microscopy (two-dimensional scanning [and associated mathematical model] and computed tomography), the speciation and distribution of toxic Se were examined within hydrated roots of cowpea (Vigna unguiculata) exposed to either 20 µM selenite or selenate. Based upon bulk solution concentrations, selenate was 9-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations approximately 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100%) than for selenate (26%), the absolute concentration of organoselenium was actually approximately 5-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly: the highest tissue concentrations were in the endodermis and cortex approximately 4 mm or more behind the apex when exposed to selenate but in the meristem (approximately 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provides valuable data in understanding Se uptake, transport, and toxicity.

Quantitative determination of metal and metalloid spatial distribution in hydrated and fresh roots of cowpea using synchrotron-based X-ray fluorescence microscopy.

Many metals and metalloids, jointly termed metal(loid)s, are toxic to plants even at low levels. This has limited the study of their uptake, distribution, and modes of action in plant roots grown at physiologically relevant concentrations. Synchrotron-based X-ray fluorescence microscopy was used to examine metal(loid)s in hydrated cowpea (Vigna unguiculata L.) roots exposed to Zn(II), Ni(II), Mn(II), Cu(II), Hg(II), Se(IV), Se(VI), As(III), or As(V). Development of a mathematical model enabled in situ quantitative determination of their distribution in root tissues. The binding strength of metals influenced the extent of their movement through the root cylinder, which influenced the toxic effects exerted-metals (e.g. Cu, Hg) that bind more strongly to hard ligands had high concentrations in the rhizodermis and caused this tissue to rupture, while other metals (e.g. Ni, Zn) moved further into the root cylinder and did not cause ruptures. When longitudinal distributions were examined, the highest Se concentration in roots exposed to Se(VI) was in the more proximal root tissues, suggesting that Se(VI) is readily loaded into the stele. This contrasted with other metal(loid)s (e.g. Mn, As), which accumulated in the apex. These differences in metal(loid) spatial distribution provide valuable quantitative data on metal(loid) physiology, including uptake, transport, and toxicity in plant roots.

Characterization of leached phosphorus from soil, manure, and manure-amended soil by physical and chemical fractionation and Diffusive Gradients in Thin films (DGT).

We are challenged to date to fully understand mechanisms controlling phosphorus (P) mobilization in soil. In this study we evaluated physical properties, chemical reactivity, and potential bioavailability of P mobilized in soil during a leaching event and examined how the amounts and properties of leached P were influenced by surface application of cattle manure. Leaching experiments on manure itself, and on intact soil columns (14.1 cm inner dia., 25 cm height) before and after manure application, were carried out at an irrigation rate of 1 mm h(-1) for 48 h. High concentrations of dissolved reactive P (DRP) were found in manure leachates (up to 32 mg L(-1)), whereas concentrations of P in soil leachates were low both before and after manure application (around 0.04 mg L(-1) before application and up to 0.4 mg L(-1) afterward). This result indicates that the soil retained most of the P added with manure. Manure particles themselves were also largely retained by the soil. Combined physical (centrifugation) and chemical (molybdate reactiveness) fractionation of leached P showed that leachates in the manure treated soils were dominated by dissolved unreactive P (DUP), mainly originating from manure. However, centrifugation only removed a small fraction of total particles from the leachates, indicating that the so-called dissolved fraction may be associated with low density particulate matter. Deployment of Diffusive Gradients in Thin films (DGT) devices in the leachates proved to be a good approach for measuring reactive P in soil leachates. The results indicated that total reactive P (TRP) gave a better estimate of potentially bioavailable P than both total P (TP) and DRP in these experiments.

Grain accumulation of selenium species in rice (Oryza sativa L.).

Efficient Se biofortification programs require a thorough understanding of the accumulation and distribution of Se species within the rice grain. Therefore, the translocation of Se species to the filling grain and their spatial unloading were investigated. Se species were supplied via cut flag leaves of intact plants and excised panicle stems subjected to a ± stem-girdling treatment during grain fill. Total Se concentrations in the flag leaves and grain were quantified by inductively coupled plasma mass spectrometry. Spatial accumulation was investigated using synchrotron X-ray fluorescence microtomography. Selenomethionine (SeMet) and selenomethylcysteine (SeMeSeCys) were transported to the grain more efficiently than selenite and selenate. SeMet and SeMeSeCys were translocated exclusively via the phloem, while inorganic Se was transported via both the phloem and xylem. For SeMet- and SeMeSeCys-fed grain, Se dispersed throughout the external grain layers and into the endosperm and, for SeMeSeCys, into the embryo. Selenite was retained at the point of grain entry. These results demonstrate that the organic Se species SeMet and SeMeSeCys are rapidly loaded into the phloem and transported to the grain far more efficiently than inorganic species. Organic Se species are distributed more readily, and extensively, throughout the grain than selenite.

A review of recent developments in the speciation and location of arsenic and selenium in rice grain.

Rice is a staple food yet is a significant dietary source of inorganic arsenic, a class 1, nonthreshold carcinogen. Establishing the location and speciation of arsenic within the edible rice grain is essential for understanding the risk and for developing effective strategies to reduce grain arsenic concentrations. Conversely, selenium is an essential micronutrient and up to 1 billion people worldwide are selenium-deficient. Several studies have suggested that selenium supplementation can reduce the risk of some cancers, generating substantial interest in biofortifying rice. Knowledge of selenium location and speciation is important, because the anti-cancer effects of selenium depend on its speciation. Germanic acid is an arsenite/silicic acid analogue, and location of germanium may help elucidate the mechanisms of arsenite transport into grain. This review summarises recent discoveries in the location and speciation of arsenic, germanium, and selenium in rice grain using state-of-the-art mass spectrometry and synchrotron techniques, and illustrates both the importance of high-sensitivity and high-resolution techniques and the advantages of combining techniques in an integrated quantitative and spatial approach.

Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants.

The ability to locate and quantify elemental distributions in plants is crucial to understanding plant metabolisms, the mechanisms of uptake and transport of minerals and how plants cope with toxic elements or elemental deficiencies. High-resolution secondary ion mass spectrometry (SIMS) is emerging as an important technique for the analysis of biological material at the subcellular scale. This article reviews recent work using the CAMECA NanoSIMS to determine elemental distributions in plants. The NanoSIMS is able to map elemental distributions at high resolution, down to 50 nm, and can detect very low concentrations (milligrams per kilogram) for some elements. It is also capable of mapping almost all elements in the periodic table (from hydrogen to uranium) and can distinguish between stable isotopes, which allows the design of tracer experiments. In this review, particular focus is placed upon studying the same or similar specimens with both the NanoSIMS and a wide range of complementary techniques, showing how the advantages of each technique can be combined to provide a fuller data set to address complex scientific questions. Techniques covered include optical microscopy, synchrotron techniques, including X-ray fluorescence and X-ray absorption spectroscopy, transmission electron microscopy, electron probe microanalysis, particle-induced X-ray emission and inductively coupled plasma mass spectrometry. Some of the challenges associated with sample preparation of plant material for SIMS analysis, the artefacts and limitations of the technique and future trends are also discussed.