Search

Previous studies have shown that the Nodulin 26-like intrinsic membrane protein (NIP) Lsi1 (OsNIP2;1) is involved in arsenite [As(III)] uptake in rice (Oryza sativa). However, the role of other rice NIPs in As(III) accumulation in planta remains unknown. In the present study, we investigated the role OsNIP3;2 in As(III) uptake in rice. When expressed in Xenopus laevis oocytes, OsNIP3;2 showed a high transport activity for As(III). Quantitative real-time RT-PCR showed that the expression of OsNIP3;2 was suppressed by 5 pM As(III), but enhanced by 20 and 100 pM As(III). Transgenic rice plants expressing OsNIP3;2pro-GUS showed that the gene was predominantly expressed in the lateral roots and the stele region of the primary roots. Transient expression of OsNIP3;2:GFP fusion protein in rice protoplasts showed that the protein was localized in the plasma membrane. Knockout of OsNIP3;2 significantly decreased As concentration in the roots, but had little effect on shoot As concentration. Synchrotron microfocus X-ray fluorescence showed decreased As accumulation in the stele of the lateral roots in the mutants compared with wild-type. Our results indicate that OsNIP3;2 is involved in As(III) uptake by lateral roots, but its contribution to As accumulation in the shoots is limited. [ABSTRACT FROM AUTHOR]

Knowledge of arsenic (As) accumulation in rice (Oryza sativa L.) is important for minimizing As transfer to the food chain. The aim of this study was to investigate the role of rice nodes in As storage and distribution. Synchrotron μX-ray fluorescence (μ-XRF) was used to map As distribution in the top node and internode of a lsi2 mutant defective in silicon/arsenite efflux carrier and its wild-type (WT) grown in soil. Lsi2 expression in different tissues during grain filling was investigated by quantitative RT-PCR. Arsenite or dimethylarsinic acid (DMA) was supplied to excised panicles to investigate the roles of Lsi2 and phytochelatins (PC) in As distribution. μ-XRF mapping revealed As storage in the phloem of different vascular bundles in the top node and internode. Soil-grown plants of lsi2 had markedly decreased As accumulation in the phloem compared with the WT. Lsi2 was strongly expressed, not only in the roots but also in the nodes. When excised panicles were exposed to As(III), the lsi2 mutant distributed more As to the node and flag leaf but less As to the grain compared with the WT, while there was no significant difference in DMA distribution. Inhibition of PC synthesis by l-buthionine-sulphoximine decreased As(III) deposition in the top node but increased As accumulation in the grain and flag leaf. The results suggest that rice nodes serve as a filter restricting As(III) distribution to the grain. Furthermore, Lsi2 plays a role in As(III) distribution in rice nodes and phytochelatins are important compounds for As(III) storage in the nodes. [ABSTRACT FROM AUTHOR]

Arsenic (As) accumulation in rice (Oryza sativa) may pose a significant health risk to consumers. Plants take up different As species using various pathways. Here, we investigated the contribution of the phosphate (Pi) transport pathway to As accumulation in rice grown hydroponically or under flooded soil conditions. In hydroponic experiments, a rice mutant defective in OsPHF1 (for phosphate transporter traffic facilitatorl) lost much of the ability to take up Pi and arsenate and to transport them from roots to shoots, whereas transgenic rice overexpressing either the Pi transporter OsPht1;8 (OsPT8) or the transcription factor OsPHR2 (for phosphate starvation response2) had enhanced abilities of Pi and arsenate uptake and translocation. OsPT8 was found to have a high affinity for both Pi and arsenate, and its overexpression increased the maximum influx by 3- to 5-fold. In arsenate-treated plants, both arsenate and arsenite were detected in the xylem sap, with the proportion of the latter increasing with the exposure time. Under the flooded soil conditions, the phf1 mutant took up less Pi whereas the overexpression lines took up more Pi. But there were no similar effects on As accumulation and distribution. Rice grain contained predominantly dimethylarsinic acid and arsenite, with arsenate being a minor species. These results suggest that the Pi transport pathway contributed little to As uptake and transport to grain in rice plants grown in flooded soil. Transgenic approaches to enhance Pi acquisition from paddy soil through the overexpression of Pi transporters may not increase As accumulation in rice grain. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters. [ABSTRACT FROM AUTHOR]

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%-83%) whereas As(III) predominated in phloem exudate (70%-94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem. [ABSTRACT FROM AUTHOR]

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyper-accumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 µM selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively upregulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine. [ABSTRACT FROM AUTHOR]

Interactions between sulfur (S) nutritional status and sulfate transporter expression in field-grown wheat (Triticum aestivum) were investigated using Broadbalk +S and -S treatments (S fertilizer withheld) at Rothamsted, United Kingdom. In 2008, 5, sulfate, selenium (Se), and molybdenum (Mo) concentrations and sulfate transporter gene expression were analyzed throughout development. Total S concentrations were lower in all tissues of -S plants, principally as a result of decreased sulfate pools. S, Se, and Mo concentrations increased in vegetative tissues until anthesis, and thereafter, with the exception of Mo, decreased until maturity. At maturity, most of the S and Se were localized in the grain, indicating efficient remobilization from vegetative tissues, whereas less Mo was remobilized. At maturity, Se and Mo were enhanced 7- and 3.7-fold, respectively, in -S compared with +S grain, while grain total S was not significantly reduced. Enhanced expression of sulfate transporters, for example Sultrl;1 and Sultr4;1, in -S plants explains the much increased accumulation of Se and Mo (7- and 3.7-fold compared with +S in grain, respectively). Sultr5;2 (mot1), thought to be involved in Mo accumulation in Arabidopsis (Arabidopsis thaliana), did not fully explain patterns of Mo distribution; it was expressed in all tissues, decreasing in leaf and increasing in roots under -S conditions, and was expressed in florets at anthesis but not in grain at any other time. hi conclusion, S fertilizer application has a marked impact on Mo and Se distribution and accumulation, which is at least partially a result of altered gene expression of the sulfate transporter family. [ABSTRACT FROM AUTHOR]

Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(HI) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As([II)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to L-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops. [ABSTRACT FROM AUTHOR]

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)I and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice Isil mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher Vmax than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsil in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsil mediates the uptake of undissociated methylated As in rice roots. [ABSTRACT FROM AUTHOR]

Zinc (Zn) hyperaccumulation is a constitutive property of Thlaspi caerulescens, whereas cadmium (Cd) hyperaccumulation varies greatly among different ecotypes. The molecular basis of this variation is unknown. Ecotypic differences in the sequences and expression of four representative ZIP family transporter genes were investigated. Genome analysis indicated the presence of at least two closely related copies of the TcIRT1 gene in both Ganges (high Cd accumulating) and Prayon (low Cd accumulating) ecotypes, with different copies being expressed in each, and, furthermore, the two genes potentially encode different length transcripts. The predominant transcript in Prayon was truncated, missing sequence coding for the putative metal-binding site and the five C-terminal transmembrane helices. The two ecotypes were grown hydroponically ±Fe and Cd, and mRNA abundance determined for four ZIP genes. The four ZIP genes studied (TcIRT1, TcIRT2, TcZNT1, and TcZNT5) were expressed in roots only. TcIRT1 expression (full-length in Ganges, TcIRT1-1G; truncated in Prayon, TcIRT1-2P) was enhanced by Fe deficiency or by exposure to Cd. TcIRT2 expression was induced by Fe deficiency, but was unaffected by Cd exposure. TcZNT5-G showed greater expression in Prayon compared with Ganges. The functions of TcIRT1 from Ganges and Prayon and the Arabidopsis homologue were analysed by heterologous expression in yeast. All three IRT1 genes were able to facilitate growth on low Fe concentrations. Cd sensitivity of yeast was conferred in the order AtIRT1>TcIRT1-1G>TcIRT1-2P (truncated). Cd uptake after 4 h was only detectable following complementation by AtIRT1. The results suggest that although TcIRT1-G may be involved in Cd hyperaccumulation in the Ganges ecotype of T. caerulescens, the transporter expressed in yeast does not have an enhanced ability to transport Cd compared with AtIRT1. Therefore, the unique Cd-accumulating ability of the T. caerulescens Ganges ecotype must be due to the levels of expression of the protein or to other factors such as interacting proteins. [ABSTRACT FROM PUBLISHER]

Astragalus bisulcatus and Stanleya pinnata hyperaccumulate selenium (Se) up to 1% of plant dry weight. In the field, Se was mostly present in the young leaves and reproductive tissues of both hyperaccumulators. Microfocused scanning x-ray fluorescence mapping revealed that Se was hyperaccumulated in trichomes in young leaves of A. bisulcatus. None of 10 other elements tested were accumulated in trichomes. Micro x-ray absorption spectroscopy and liquid chromatography-mass spectrometry showed that Se in trichomes was present in the organic forms methylselenocysteine (MeSeCys; 53%) and γ-glutamyl-MeSeCys (47%). In the young leaf itself, there was 30% inorganic Se (selenate and selenite) in addition to 70% MeSeCys. In young S. pinnata leaves, Se was highly concentrated near the leaf edge and surface in globular structures that were shown by energy-dispersive x-ray microanalysis to be mainly in epidermal cells. Liquid chromatography-mass spectrometry revealed both MeSeCys (88%) and selenocystathionine (12%) inside leaf edges. In contrast, both the Se accumulator Brassica juncea and the nonaccumulator Arabidopsis thaliana accumulated Se in their leaf vascular tissues and mesophyll cells. Se in hyperaccumulators appears to be mobile in both the xylem and phloem because Se-treated S. pinnata was found to be highly toxic to phloem-feeding aphids, and MeSeCys was present in the vascular tissues of a S. pinnata young leaf petiole as well as in guttation fluid. The compartmentation of organic selenocompounds in specific storage areas in the plant periphery appears to be a unique property of Se hyperaccumulators. The high concentration of Se in the plant periphery may contribute to Se tolerance and may also serve as an elemental plant defense mechanism. [ABSTRACT FROM AUTHOR]

Uptake of Cd and Zn by intact seedlings of two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens was characterized using radioactive tracers. Uptake of Cd and Zn at 2 °C was assumed to represent mainly apoplastic binding in the roots, whereas the difference in uptake between 22 °C and 2 °C represented metabolically dependent influx. There was no significant difference between the two ecotypes in the apoplastic binding of Cd or Zn. Metabolically dependent uptake of Cd was 4.5‐fold higher in the high Cd‐accumulating ecotype, Ganges, than in the low Cd‐accumulating ecotype, Prayon. By contrast, there was only a 1.5‐fold difference in the Zn uptake between the two ecotypes. For the Ganges ecotype, Cd uptake could be described by Michaelis–Menten kinetics with a Vmax of 143 nmol g−1 root FW h−1 and a Km of 0.45 μM. Uptake of Cd by the Ganges ecotype was not inhibited by La, Zn, Cu, Co, Mn, Ni or Fe(II), and neither by increasing the Ca concentration. By contrast, addition of La, Zn or Mn, or increasing the Ca concentration in the uptake solution decreased Cd uptake by Prayon. Uptake of Ca was larger in Prayon than in Ganges. The results suggest that Cd uptake by the low Cd‐accumulating ecotype (Prayon) may be mediated partly via Ca channels or transporters for Zn and Mn. By contrast, there may exist a highly selective Cd transport system in the root cell membranes of the high Cd‐accumulating ecotype (Ganges) of T. caerulescens. [ABSTRACT FROM PUBLISHER]

Nickel uptake and cellular compartmentation were investigated in three Ni hyperaccumulators: Alyssum bertolonii (Desv), Alyssum lesbiacum (Candargy) and Thlaspi goesingense (Hálácsy). The three species showed similar hyperaccumulation of Ni, but T. goesingense was less tolerant to Ni than the two Alyssum species. An addition of 500 mg Ni kg−1 to a nutrient‐rich growth medium significantly increased shoot biomass of all three species, suggesting that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Energy‐dispersive X‐ray microanalysis (EDXA) was performed on frozen‐hydrated tissues of leaves (all species) and stems (Alyssum only). In all species analysed, Ni was distributed preferentially in the epidermal cells, most likely in the vacuoles, of the leaves and stems. In stems, there was a second peak of Ni in the boundary cells between the cortical parenchyma and the vascular cylinder. The non‐glandular trichomes on the leaf surfaces of the two Alyssum species were highly enriched with Ca, but contained little Ni except in the base. In the leaves of T. goesingense, the large elongated epidermal cells contained more Ni than the cells of the stomatal complexes. The role of cellular compartmentation in Ni hyperaccumulation is discussed. [ABSTRACT FROM PUBLISHER]

Astragalus bisulcatus and Stanleya pinnata hyperaccumulate selenium (Se) up to 1% of plant dry weight. In the field, Se was mostly present in the young leaves and reproductive tissues of both hyperaccumulators. Microfocused scanning x-ray fluorescence mapping revealed that Se was hyperaccumulated in trichomes in young leaves of A. bisulcatus. None of 10 other elements tested were accumulated in trichomes. Micro x-ray absorption spectroscopy and liquid chromatography-mass spectrometry showed that Se in trichomes was present in the organic forms methylselenocysteine (MeSeCys; 53%) and γ-glutamyl-MeSeCys (47%). In the young leaf itself, there was 30% inorganic Se (selenate and selenite) in addition to 70% MeSeCys. In young S. pinnata leaves, Se was highly concentrated near the leaf edge and surface in globular structures that were shown by energy-dispersive x-ray microanalysis to be mainly in epidermal cells. Liquid chromatography-mass spectrometry revealed both MeSeCys (88%) and selenocystathionine (12%) inside leaf edges. In contrast, both the Se accumulator Brassica juncea and the nonaccumulator Arabidopsis thaliana accumulated Se in their leaf vascular tissues and mesophyll cells. Se in hyperaccumulators appears to be mobile in both the xylem and phloem because Se-treated S. pinnata was found to be highly toxic to phloem-feeding aphids, and MeSeCys was present in the vascular tissues of a S. pinnata young leaf petiole as well as in guttation fluid. The compartmentation of organic selenocompounds in specific storage areas in the plant periphery appears to be a unique property of Se hyperaccumulators. The high concentration of Se in the plant periphery may contribute to Se tolerance and may also serve as an elemental plant defense mechanism. [ABSTRACT FROM AUTHOR]