Shining a Light on How Soil Organic Carbon Behaves at Fine Scales under Long-Term Elevated CO2: An 8 Year Free-Air Carbon Dioxide Enrichment Study.

Building and protecting soil organic carbon (SOC) are critical to agricultural productivity, soil health, and climate change mitigation. We aim to understand how mechanisms at the organo-mineral interfaces influence SOC persistence in three contrasting soils (Luvisol, Vertisol, and Calcisol) under long-term free air CO2 enrichment conditions. A continuous wheat-field pea-canola rotation was maintained. For the first time, we provided evidence to a novel notion that persistent SOC is molecularly simple even under elevated CO2 conditions. We found that the elevated CO2 condition did not change the total SOC content or C forms compared with the soils under ambient CO2 as identified by synchrotron-based soft X-ray analyses. Furthermore, synchrotron-based infrared microspectroscopy confirmed a two-dimensional microscale distribution of similar and less diverse C forms in intact microaggregates under long-term elevated CO2 conditions. Strong correlations between the distribution of C forms and O-H groups of clays can explain the steady state of the total SOC content. However, the correlations between C forms and clay minerals were weakened in the coarse-textured Calcisol under long-term elevated CO2. Our findings suggested that we should emphasize identifying management practices that increase the physical protection of SOC instead of increasing complexity of C. Such information is valuable in developing more accurate C prediction models under elevated CO2 conditions and shift our thinking in developing management practices for maintaining and building SOC for better soil fertility and future environmental sustainability.

The role of plants in ironstone evolution: iron and aluminium cycling in the rhizosphere.

The Carajás plateaus in Brazil host endemic epilithic vegetation ("campo rupestre") on top of ironstone duricrusts, known as canga. This capping rock is primarily composed of iron(III) oxide minerals and forms a physically resistant horizon. Field observations reveal an intimate interaction between canga's surface and two native sedges (Rhynchospora barbata and Bulbostylis cangae). These observations suggest that certain plants contribute to the biogeochemical cycling of iron. Iron dissolution features at the root-rock interface were characterised using synchrotron-based techniques, Raman spectroscopy and scanning electron microscopy. These microscale characterisations indicate that iron is preferentially leached in the rhizosphere, enriching the comparatively insoluble aluminium around root channels. Oxalic acid and other exudates were detected in active root channels, signifying ligand-controlled iron oxide dissolution, likely driven by the plants' requirements for goethite-associated nutrients such as phosphorus. The excess iron not uptaken by the plant can reprecipitate in and around roots, line root channels and cement detrital fragments in the soil crust at the base of the plants. The reprecipitation of iron is significant as it provides a continuously forming cement, which makes canga horizons a 'self-healing' cover and contributes to them being the world's most stable continuously exposed land surfaces. Aluminium hydroxide precipitates ("gibbsite cutans") were also detected, coating some of the root cavities, often in alternating layers with goethite. This alternating pattern may correspond with oscillating oxygen concentrations in the rhizosphere. Microbial lineages known to contain iron-reducing bacteria were identified in the sedge rhizospheric microbiome and likely contribute to the reductive dissolution of iron(III) oxides within canga. Drying or percolation of oxygenated water to these anaerobic niches have led to iron mineralisation of biofilms, detected in many root channels. This study sheds light on plants' direct and indirect involvement in canga evolution, with possible implications for revegetation and surface restoration of iron mine sites.

Applications and opportunities of click chemistry in plant science.

The Nobel Prize in Chemistry for 2022 was awarded to the pioneers of Lego-like 'click chemistry': combinatorial chemistry with remarkable modularity and diversity. It has been applied to a wide variety of biological systems, from microorganisms to plants and animals, including humans. Although click chemistry is a powerful chemical biology tool, comparatively few studies have examined its potential in plant science. Here, we review click chemistry reactions and their applications in plant systems, highlighting the activity-based probes and metabolic labeling strategies combined with bioorthogonal click chemistry to visualize plant biological processes. These applications offer new opportunities to explore and understand the underlying molecular mechanisms regulating plant composition, growth, metabolism, defense, and immune responses.

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.

The overlooked functions of trichomes: Water absorption and metal detoxication.

Trichomes are epidermal outgrowths on plant shoots. Their roles in protecting plants against herbivores and in the biosynthesis of specialized metabolites have long been recognized. Recently, studies are increasingly showing that trichomes also play important roles in water absorption and metal detoxication, with these roles having important implications for ecology, the environment, and agriculture. However, these two functions of trichomes have been largely overlooked and much remains unknown. In this review, we show that the trichomes of 37 plant species belonging to 14 plant families are involved in water absorption, while the trichomes of 33 species from 13 families are capable of sequestering metals within their trichomes. The ability of trichomes to absorb water results from their decreased hydrophobicity compared to the remainder of the leaf surface as well as the presence of special structures for collecting and absorbing water. In contrast, the metal detoxication function of trichomes results not only from the good connection of their basal cells to the underlying vascular tissues, but also from the presence of metal-chelating ligands and transporters within the trichomes themselves. Knowledge gaps and critical future research questions regarding these two trichome functions are highlighted. This review improves our understanding on trichomes.

Uptake, transformation, and environmental impact of zinc oxide nanoparticles in a soil-wheat system.

Zinc oxide nanoparticles (ZnO-NPs) are metal-based nanomaterials, but their long-term effects on plant growth and the soil environment in the field remain unclear with most previous studies using short-term laboratory and glasshouse studies. In this study, we used a field experiment to examine the long-term effects of ZnO-NPs in a soil-wheat (Triticum aestivum) system. It was found that although ZnO-NPs had no significant effect on either yield or the concentration of other nutrients within the grain, the application of ZnO-NPs significantly increased Zn concentrations. Indeed, for grain, the application of ZnO-NPs to both the soil and foliage (SFZnO) (average of 33.1 mg/kg) significantly increased grain Zn concentrations compared to the the control treatment (21.7 mg/kg). Using in situ analyses, nutrients were found to accumulate primarily in the crease tissue and the aleurone layer of the grain, regardless of treatment. Specifically, the concentration of Zn in the aleurone layer for the SFZnO treatment was 2-3 times higher than that in the control, being >300 mg/kg, whilst the Zn concentration in the crease tissue was ca. 600 mg/kg in the SFZnO treatment, being two times higher than for the control. Although the application of ZnO-NPs increased the total Zn within the grain, it did not accumulate within the grain as ZnO-NPs with this being important for food safety, but rather mainly as Zn-phytate, with the remainder of the Zn complexed with either cysteine or phosphate. Finally, we also observed that ZnO-NPs caused fewer changes to the soil bacterial community structure and that it had no nano-specific toxicity.

Fast X-ray fluorescence microscopy provides high-throughput phenotyping of element distribution in seeds.

The concentration, chemical speciation, and spatial distribution of essential and toxic mineral elements in cereal seeds have important implications for human health. To identify genes responsible for element uptake, translocation, and storage, high-throughput phenotyping methods are needed to visualize element distribution and concentration in seeds. Here, we used X-ray fluorescence microscopy (µ-XRF) as a method for rapid and high-throughput phenotyping of seed libraries and developed an ImageJ-based pipeline to analyze the spatial distribution of elements. Using this method, we nondestructively scanned 4,190 ethyl methanesulfonate (EMS)-mutagenized M1 rice (Oryza sativa) seeds and 533 diverse rice accessions in a genome-wide association study (GWAS) panel to simultaneously measure concentrations and spatial distribution of elements in the embryo, endosperm, and aleurone layer. A total of 692 putative mutants and 65 loci associated with the spatial distribution of elements in rice seed were identified. This powerful method provides a basis for investigating the genetics and molecular mechanisms controlling the accumulation and spatial variations of mineral elements in plant seeds.

What is missing to advance foliar fertilization using nanotechnology?

An urgent challenge within agriculture is to improve fertilizer efficiency in order to reduce the environmental footprint associated with an increased production of crops on existing farmland. Standard soil fertilization strategies are often not very efficient due to immobilization in the soil and losses of nutrients by leaching or volatilization. Foliar fertilization offers an attractive supplementary strategy as it bypasses the adverse soil processes, but implementation is often hampered by a poor penetration through leaf barriers, leaf damage, and a limited ability of nutrients to translocate. Recent advances within bionanotechnology offer a range of emerging possibilities to overcome these challenges. Here we review how nanoparticles can be tailored with smart properties to interact with plant tissue for a more efficient delivery of nutrients.

Nutrient accumulation and transcriptome patterns during grain development in rice.

Rice is an important source of calories and mineral nutrients for more than half of the world's population. The accumulation of essential and toxic mineral elements in rice grain affects its nutritional quality and safety. However, the patterns and processes by which different elements progressively accumulate during grain filling remain largely unknown. In the present study, we investigated temporal changes in dry matter, elemental concentrations, and the transcriptome in the grain of field-grown rice. We also investigated the effects of seed setting rate and the position of the grain within the rice panicle on element accumulation. Three different patterns of accumulation were observed: (i) elements including K, Mn, B, and Ca showed an early accumulation pattern; (ii) dry matter and elements including N, P, S, Mg, Cu, Zn, Mo, As, and Cd showed a mid accumulation pattern; and (iii) elements such as Fe showed a gradual increase pattern. These different accumulation patterns can be explained by the differences in the biogeochemical behavior of the various elements in the soil, as well as differences in plant nutrient redistribution, gene expression, and the sink-source relationship. These results improve our knowledge of the dynamics of elemental accumulation in rice grain and are helpful for identification of functional genes mediating the translocation of elements to grain.

Improved Zn bioavailability by its enhanced colocalization and speciation with S in wheat grain tissues after N addition.

Zinc bioavailability with the presence of other elements in wheat grains might be affected by fertilizers. A long-term field experiment was conducted to examine effects of N fertilizer on Zn bioavailability in wheat grain tissues, with changes in the concentrations, distribution, and speciation of Zn as well as P and sulfur S via synchrotron-based technology. Results showed that addition of N fertilizer was associated with changes in Zn concentrations and distributions in grain tissues, especially in the crease region and endosperm. Simultaneously, N addition enhanced Zn-S colocalization in the crease region and endosperm and lowered the P/Zn ratio and Zn-P colocalization. Addition of N fertilizer with P increased Zn-cysteine (9.2%) and decreased Zn-phytate (47.3%) in the crease region, leading to potentially higher grain Zn bioavailability. Thus, addition of N fertilizer improved concentrations and bioavailability of Zn, by coordinating the relationships among Zn, P and S within wheat grains.

Microspectroscopic visualization of how biochar lifts the soil organic carbon ceiling.

The soil carbon (C) saturation concept suggests an upper limit to the storage of soil organic carbon (SOC). It is set by the mechanisms that protect soil organic matter from mineralization. Biochar has the capacity to protect new C, including rhizodeposits and microbial necromass. However, the decadal-scale mechanisms by which biochar influences the molecular diversity, spatial heterogeneity, and temporal changes in SOC persistence, remain unresolved. Here we show that the soil C storage ceiling of a Ferralsol under subtropical pasture was raised by a second application of Eucalyptus saligna biochar 8.2 years after the first application-the first application raised the soil C storage ceiling by 9.3 Mg new C ha-1 and the second application raised this by another 2.3 Mg new C ha-1. Linking direct visual evidence from one-, two-, and three-dimensional analyses with SOC quantification, we found high spatial heterogeneity of C functional groups that resulted in the retention of rhizodeposits and microbial necromass in microaggregates (53-250 µm) and the mineral fraction (<53 µm). Microbial C-use efficiency was concomitantly increased by lowering specific enzyme activities, contributing to the decreased mineralization of native SOC by 18%. We suggest that the SOC ceiling can be lifted using biochar in (sub)tropical grasslands globally.

Non-target impacts of pesticides on soil N transformations, abundances of nitrifying and denitrifying genes, and nitrous oxide emissions.

Agriculture is the leading contributor to global nitrous oxide (N2O) emissions, mostly from soils. We examined the non-target impacts of four pesticides on N transformations, N cycling genes and N2O emissions from sugarcane-cropped soil. The pesticides, including a herbicide glyphosate (GLY), an insecticide imidacloprid (IMI), a fungicide methoxy ethyl mercuric chloride (MEMC) and a fumigant methyl isothiocyanate (MITC), were added to the soil and incubated in laboratory at 25 °C. The soil microcosms were maintained at two water contents, 55 % and 90 % water holding capacity (WHC), to simulate aerobic and partly anaerobic conditions, respectively. Half of the soil samples received an initial application of KNO3 and were then maintained at 90 % WHC for 38 d, whilst the other half received (NH4)2SO4 and were maintained at 55 % WHC for 28 d followed by 10 d at 90 % WHC to favour denitrification. Responses of individual functional genes involved in nitrification and denitrification to the pesticides and their relationships to N2O emissions varied with time and soil water. Overall, MITC had pronounced repressive effects on AOA and AOB amoA gene abundances and gross nitrification. Under 55 % WHC during the initial 28 d, N2O emissions were low for all treatments (≤62 µg N kg-1 soil). However, under 90 % WHC (either during the first 28 d or the increase in water content from 55 to 90 % WHC after 28 d) the cumulative N2O emissions increased markedly. Overall, under 90 % WHC the cumulative N2O emissions were 19 (control) to 79-fold (MITC) higher than under 55% WHC; with the highest emissions observed in the MITC treatment (3140 µg N kg-1 soil). This was associated with increases in gross nitrate consumption rates and abundances of denitrifying genes (nirK, nirS and qnorB). Therefore, to minimise N2O emissions, MITC should not be applied to field under wet conditions favouring denitrification.

Tandem Probe Analysis Mode for Synchrotron XFM: Doubling Throughput Capacity.

Synchrotron-based X-ray fluorescence microscopy (XFM) analysis is a powerful technique that can be used to visualize elemental distributions across a broad range of sample types. Compared to conventional mapping techniques such as laser ablation inductively coupled plasma mass spectrometry or benchtop XFM, synchrotron-based XFM provides faster and more sensitive analyses. However, access to synchrotron XFM beamlines is highly competitive, and as a result, these beamlines are often oversubscribed. Therefore, XFM experiments that require many large samples to be scanned can penalize beamline throughput. Our study was largely driven by the need to scan large gels (170 cm2) using XFM without decreasing beamline throughput. We describe a novel approach for acquiring two sets of XFM data using two fluorescence detectors in tandem; essentially performing two separate experiments simultaneously. We measured the effects of tandem scanning on beam quality by analyzing a range of contrasting samples downstream while simultaneously scanning different gel materials upstream. The upstream gels were thin (<200 µm) diffusive gradients in thin-film (DGT) binding gels. DGTs are passive samplers that are deployed in water, soil, and sediment to measure the concentration and distribution of potentially bioavailable nutrients and contaminants. When deployed on soil, DGTs are typically small (2.5 cm2), so we developed large DGTs (170 cm2), which can be used to provide extensive maps to visualize the diffusion of fertilizers in soil. Of the DGT gel materials tested (bis-acrylamide, polyacrylamide, and polyurethane), polyurethane gels were most suitable for XFM analysis, having favorable handling, drying, and analytical properties. This gel type enabled quantitative (>99%) transmittance with minimal (<3%) flux variation during raster scanning, whereas the other gels had a substantial effect on the beam focus. For the first time, we have (1) used XFM for mapping analytes in large DGTs and (2) developed a tandem probe analysis mode for synchrotron-based XFM, effectively doubling throughput. The novel tandem probe analysis mode described here is of broad applicability across many XFM beamlines as it could be used for future experiments where any uniform, highly transmissive sample could be analyzed upstream in the "background" of downstream samples.

Translocation of Foliar Absorbed Zn in Sunflower (Helianthus annuus) Leaves.

Foliar zinc (Zn) fertilization is an important approach for overcoming crop Zn deficiency, yet little is known regarding the subsequent translocation of this foliar-applied Zn. Using synchrotron-based X-ray fluorescence microscopy (XFM) and transcriptome analysis, the present study examined the translocation of foliar absorbed Zn in sunflower (Helianthus annuus) leaves. Although bulk analyses showed that there had been minimal translocation of the absorbed Zn out of the leaf within 7 days, in situ analyses showed that the distribution of Zn in the leaf had changed with time. Specifically, when Zn was applied to the leaf for 0.5 h and then removed, Zn primarily accumulated within the upper and lower epidermal layers (when examined after 3 h), but when examined after 24 h, the Zn had moved to the vascular tissues. Transcriptome analyses identified a range of genes involved in stress response, cell wall reinforcement, and binding that were initially upregulated following foliar Zn application, whereas they were downregulated after 24 h. These observations suggest that foliar Zn application caused rapid stress to the leaf, with the initial Zn accumulation in the epidermis as a detoxification strategy, but once this stress decreased, Zn was then moved to the vascular tissues. Overall, this study has shown that despite foliar Zn application causing rapid stress to the leaf and that most of the Zn stayed within the leaf over 7 days, the distribution of Zn in the leaf had changed, with Zn mostly located in the vascular tissues 24 h after the Zn had been applied. Not only do the data presented herein provide new insight for improving the efficiency of foliar Zn fertilizers, but our approach of combining XFM with a transcriptome methodological system provides a novel approach for the study of element translocation in plants.

Widespread Occurrence of the Highly Toxic Dimethylated Monothioarsenate (DMMTA) in Rice Globally.

Arsenic (As) accumulation in rice is of global concern for human health and international trade. Rice is typically reported to contain inorganic As (iAs) and dimethylated arsenate (DMA), with current food guidelines limiting toxic iAs but not less-toxic DMA. Here, we show that the highly toxic dimethylated monothioarsenate (DMMTA) is also found in rice worldwide and has been unknowingly determined as less-toxic DMA by previous routine analytical methods. Using enzymatic extraction followed by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) analysis with a C18 column, DMMTA was detected in rice grains (n = 103) from a field survey from China and in polished rice grains (n = 140) from a global market-basket survey. Concentration ranged from <0.20 to 34.8 µg/kg (median 10.3 µg/kg), accounting for 0 to 21% of total As. A strong linear correlation was observed in all rice samples between DMA and DMMTA (being 30 ± 8% of DMA) concentrations. This robust relationship allows an estimation of DMMTA in rice grains from the DMA data reported in previous market-basket surveys, showing a general global geographical pattern with DMMTA concentration increasing from the equator toward high-latitude regions. Based on the global occurrence and potential high toxicity, DMMTA in rice should be considered in health risk assessments and for setting food regulations.

Genetic biofortification of wheat with zinc: Opportunities to fine-tune zinc uptake, transport and grain loading.

Zinc (Zn) is an important micronutrient in the human body, and health complications associated with insufficient dietary intake of Zn can be overcome by increasing the bioavailable concentrations in edible parts of crops (biofortification). Wheat (Triticum aestivum L) is the most consumed cereal crop in the world; therefore, it is an excellent target for Zn biofortification programs. Knowledge of the physiological and molecular processes that regulate Zn concentration in the wheat grain is restricted, inhibiting the success of genetic Zn biofortification programs. This review helps break this nexus by advancing understanding of those processes, including speciation regulated uptake, root to shoot transport, remobilisation, grain loading and distribution of Zn in wheat grain. Furthermore, new insights to genetic Zn biofortification of wheat are discussed, and where data are limited, we draw upon information for other cereals and Fe distribution. We identify the loading and distribution of Zn in grain as major bottlenecks for biofortification, recognising anatomical barriers in the vascular region at the base of the grain, and physiological and molecular restrictions localised in the crease region as major limitations. Movement of Zn from the endosperm cavity into the modified aleurone, aleurone and then to the endosperm is mainly regulated by ZIP and YSL transporters. Zn complexation with phytic acid in the aleurone limits Zn mobility into the endosperm. These insights, together with synchrotron-X-ray-fluorescence microscopy, support the hypothesis that a focus on the mechanisms of Zn loading into the grain will provide new opportunities for Zn biofortification of wheat.

Interaction of different-sized ZnO nanoparticles with maize (Zea mays): Accumulation, biotransformation and phytotoxicity.

This study aimed to investigate the biotransformation of ZnO nanoparticles (NPs) in maize grown in hydroponics for ecotoxicity assessment. Maize seedlings grown for 14 days were exposed to a solution of 9 nm ZnO NPs, 40 nm ZnO NPs, and ZnSO4 at a Zn concentration of 300 mg L-1 for 1, 3, and 7 days, respectively. The results of in-situ Zn distribution in maize (Zea mays) showed that 9 nm ZnO NPs could quickly enter the roots of maize and reach the center column transport system of the stem. The results of transmission electron microscopy combined with energy dispersive X-ray spectroscopy revealed that ZnO NPs were accumulated in the vacuoles of the roots, and then transformed and transported through vesicles. Simulated studies showed that low pH (5.6) played a critical role in the transformation of ZnO NPs, and organic acids (Kf = 1011.4) could promote particle dissolution. Visual MINTEQ software simulated the species of Zn after the entry of ZnO NPs or Zn2+ into plants and found that the species of Zn was mainly Zn2+ when the Zn content of plants reached 200-300 ppm. Considering that the lowest Zn content of the roots in treatments was 1920 mg kg-1, combination of the result analysis of root effects showed that the toxicity of roots in most treatments had a direct relationship with Zn2+. However, treatment with 9 nm ZnO NPs exhibited significantly higher toxicity than ZnSO4 treatment on day 1 when the Zn2+ concentration difference was not significant, which was mainly due to the large amount of ZnO NPs deposited in the roots. To the authors' knowledge, this study was the first to confirm the process of biotransformation and explore the factors affecting the toxicity of ZnO NPs in depth.

Dynamics of Dimethylated Monothioarsenate (DMMTA) in Paddy Soils and Its Accumulation in Rice Grains.

Arsenic species transformation in paddy soils has important implications for arsenic accumulation in rice grains and its safety to the consumers. Methylated thioarsenates including highly toxic dimethylated monothioarsenate (DMMTA) have been detected in paddy soils, but their production and dynamics remain poorly understood. In the present study, we first optimized a HPLC-ICP-MS method to quantify methylated thioarsenate species. Using this method together with 10 mM diethylenetriamine pentaacetate (DTPA) to preserve As speciation, we investigated methylated thioarsenate species in porewaters of seven As-contaminated soils incubated under flooded conditions and of two paddy fields. DMMTA was the main methylated thioarsenate species in the porewaters in both incubated soils and paddy fields, with concentrations ranging from 0.2 to 36.2 µg/L and representing ca. 58% of its precursor dimethylarsenate (DMA). The temporal production and dynamics of DMMTA were linked with the DMA concentrations. When soils were drained, DMMTA was converted to DMA. In the two paddy fields, DMMTA concentrations in rice grains were 0.4-10.1 µg/kg. Addition of sulfur fertilizer and rice straw incorporation increased grain DMMTA by 9-28%. These results suggest that DMMTA is an important As species in paddy soils and can accumulate in rice grains, presenting a risk to food safety and human health.

High phosphorus fertilization changes the speciation and distribution of manganese in wheat grains grown in a calcareous soil.

The physiological disorders in humans resulting from the excess dietary intake of manganese (Mn) via whole-grain food has attracted considerable attention. However, the speciation and bioavailability of Mn in wheat grains and their response to different phosphorus (P) fertilization rates are still unclear. In the current study, using a long-term field trial with P application rates of 0, 21.8, 43.6, 65.5 and 87.3 kg/ha, we examined changes in the concentration, distribution, and speciation of Mn of wheat grains using synchrotron-based X-ray fluorescence microscopy and X-ray absorption spectroscopy. The total Mn concentration in grains was found to be increased by phosphorus fertilization, especially in embryo in the form of Mn(II), but this phosphorus fertilization also decreased Mn concentrations in the nucellar projection. In this study, the speciation of Mn in different wheat grain tissues was examined, and results indicate that in calcareous soils, high rates of P fertilizers can increase Mn concentrations in wheat grain, including Mn which is likely to be of high bioavailability, and thus may increase the risk for human to expose to high Mn intake via whole-grain food.