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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 (mu-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.Fast X-ray fluorescence microscopy (mu-XRF) provides a high-throughput method to investigate the mechanisms controlling the accumulation and spatial variations of mineral elements in plant seeds. Refereed/Peer-reviewed

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.

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 distributionswere 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 inmetal(loid) spatial distribution provide valuable quantitative data on metal(loid) physiology, including uptake, transport, and toxicity in plant roots. Refereed/Peer-reviewed

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. Refereed/Peer-reviewed

Arsenic (As) is considered to be the environmental contaminant of greatest concern due to its potential accumulation in the food chain and in humans. Using novel synchrotron-based x-ray fluorescence techniques (including sequential computed tomography), short-term solution culture studies were used to examine the spatial distribution of As in hydrated and fresh roots of cowpea (Vigna unguiculata ‘Red Caloona’) seedlings exposed to 4 or 20 µm arsenate [As(V)] or 4 or 20 µm arsenite. For plants exposed to As(V), the highest concentrations were observed internally at the root apex (meristem), with As also accumulating in the root border cells and at the endodermis. When exposed to arsenite, the endodermis was again a site of accumulation, although no As was observed in border cells. For As(V), subsequent transfer of seedlings to an As-free solution resulted in a decrease in tissue As concentrations, but growth did not improve. These data suggest that, under our experimental conditions, the accumulation of As causes permanent damage to the meristem. In addition, we suggest that root border cells possibly contribute to the plant’s ability to tolerate excess As(V) by accumulating high levels of As and limiting its movement into the root.

The phytotoxicity of trace metals is of global concern due to contamination of the landscape by human activities. Using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy, the distribution and speciation of copper (Cu), nickel (Ni), and zinc (Zn) was examined in situ using hydrated roots of cowpea (Vigna unguiculata) exposed to 1.5 μm Cu, 5 μm Ni, or 40 μm Zn for 1 to 24 h. After 24 h of exposure, most Cu was bound to polygalacturonic acid of the rhizodermis and outer cortex, suggesting that binding of Cu to walls of cells in the rhizodermis possibly contributes to the toxic effects of Cu. When exposed to Zn, cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. While Ni concentrations were high in both the cortex and meristem, concentrations in the stele were comparatively low. To our knowledge, this is the first report of the in situ distribution and speciation of Cu, Ni, and Zn in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.

The phytotoxicity of Mn is important globally due to its increased solubility in acid or waterlogged soils. Short-term (≤24 h) solution culture studies with 150 µM Mn were conducted to investigate the in situ distribution and speciation of Mn in apical tissues of hydrated roots of cowpea [Vigna unguiculata (L.) Walp. cv. Red Caloona] using synchrotron-based techniques. Accumulation of Mn was rapid; exposure to 150 µM Mn for only 5 min resulting in substantial Mn accumulation in the root cap and associated mucigel. The highest tissue concentrations of Mn were in the root cap, with linear combination fitting of the data suggesting that ≥80% of this Mn(II) was associated with citrate. Interestingly, although the primary site of Mn toxicity is typically the shoots, concentrations of Mn in the stele of the root were not noticeably higher than in the surrounding cortical tissues in the short-term (≤24 h). The data provided here from the in situ analyses of hydrated roots exposed to excess Mn are, to our knowledge, the first of this type to be reported for Mn and provide important information regarding plant responses to high Mn in the rooting environment. Refereed/Peer-reviewed