Grain Unloading of Arsenic Species in Rice

Rice (Oryza sativa) is the staple food for over half the world''s population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a ± stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.Paddy rice (Oryza sativa) is particularly effective, compared to other cereals, at accumulating arsenic (As) in shoot and grain (Williams et al., 2007b). Rice is the staple food for over half the world''s population (Fageria, 2007) and rice represents a significant dietary source of inorganic As, a class 1, nonthreshold carcinogen, particularly in Southeast Asia (Meharg et al., 2009). Inorganic As levels in rice grain are problematic even where soil As is at background levels, derived from geogenic sources (Lu et al., 2009; Meharg et al., 2009). However, widespread pollution of paddy soils with As, leading to further elevation of grain As, has occurred in some regions due to base and precious mining (Liao et al., 2005; Zhu et al., 2008), irrigation of paddies with As-elevated groundwaters (e.g. Meharg and Rahman, 2003; Williams et al., 2006), and the use of arsenical pesticides (Williams et al., 2007a). Unlike other cereal grains, paddy rice cultivation is dependent of soils being anaerobic, and it is this anoxia that gives rise to elevated As concentrations in the plant. Anaerobic soil conditions lead to the mobilization of As as arsenite, where under aerobic systems arsenate dominates (Xu et al., 2008). Arsenite is efficiently assimilated by rice roots through silicic acid transport pathway (Ma et al., 2008).Knowledge of As metabolism and partitioning within plants, particularly rice, is still developing rapidly (Zhao et al., 2009). Several studies have now shown that As in rice vegetative tissue and grain is predominantly speciated as inorganic As and the methylated species dimethylarsinic acid (DMA), with variable, though low, levels of monomethyl arsonic acid (MMA; Abedin et al., 2002a; Williams et al., 2005, 2006; Norton et al., 2009). Arsenate is an analog of phosphate and competes with phosphate for rice root uptake (Abedin et al., 2002a) while arsenite is taken up by rice roots via silicic acid transporters (Ma et al., 2008). Abedin et al. (2002b) demonstrated that the methylated species DMA and MMA are also taken up by rice plants although at a much slower rate than inorganic As, with the protonated neutral forms also transported through silicic acid pathway (Li et al., 2009). Arsenate is reduced to arsenite within the rice root (Xu et al., 2008; Zhao et al., 2009), which then enters the xylem via a silicic acid/arsenite effluxer (Ma et al., 2008; Zhao et al., 2009). Arsenite may be detoxified through complexation with thiol-rich peptides including phytochelatins (PCs) and glutathione followed by sequestration into vacuoles (Bleeker et al., 2006; Raab et al., 2007b; Zhao et al., 2009). Raab et al. (2007a) found that while methylated As species are taken up by rice roots much less efficiently than inorganic species, they appear to be translocated within the plant more efficiently. The comparative contributions of xylem and phloem transport, in translocation of As to the grain, are unknown.The main species within rice grain, along with DMA, are inorganic As, particularly arsenite, which may be complexed with thiols (Williams et al., 2005; Lombi et al., 2009). Nutrients are unloaded into the grain from the ovular vascular trace (OVT) into the nucellar tissue and from there are uploaded, via the apoplast into the filial tissue (the aleurone and the endosperm; Krishnan and Dayanandan, 2003). Lombi et al. (2009) recently suggested that this may represent a physiological barrier that As species cross with differential efficiency. However, the transport and unloading of As to/into the grain, which are key processes in terms of human exposure to this contaminant, are far from being fully understood.This study investigated the differential efficiency with which important As species are translocated and unloaded into the rice grain and the comparative contributions of phloem and xylem transport. Rice panicles were excised below the flag leaf node during grain development, 10 DPA, and treated to a hydroponically administered 48-h pulse of arsenite, arsenate, arsenite glutathione, or DMA. Total As concentrations in flag leaf, grain, and husk samples for each treatment were quantified by inductively coupled plasma mass spectroscopy (ICP-MS), and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy (XANES) analysis. To evaluate the contributions of phloem versus xylem transport, a stem-girdling treatment was applied, using steam to destroy phloem cells in a second set of panicles prior to a pulse of either DMA or arsenite. The spatial unloading of As species into the developing grain was examined by synchrotron x-ray fluorescence (XRF) mapping, and fluorescence microtomography for the DMA and arsenite treatments.     

Silicon has opposite effects on the accumulation of inorganic and methylated arsenic species in rice

Background and aims

Rice (Oryza sativa) is a main source of human exposure to inorganic arsenic and mitigation measures are needed to decrease As accumulation in this staple crop. It has been shown that silicon decreases the accumulation of arsenite but, unexpectedly, increases the accumulation of dimethylarsinic acid (DMA) in rice grain. The aim of this study was to investigate why Si increases DMA accumulation.

Methods

Pot and incubation experiments were conducted to investigate how the addition of sparingly soluble silicate gel affected As speciation in the soil solution and the accumulation of different As species in rice tissues.

Results

Silicon addition significantly decreased the concentration of inorganic As (mainly arsenite) but increased the concentration of DMA in both the vegetative and reproductive tissues of rice. Silicon increased the concentration of DMA in the soil solution, whereas autoclaving soil decreased DMA concentration. Less DMA was adsorbed by the soil than arsenate and Si addition significantly inhibited DMA adsorption.

Conclusions

Silicon increased DMA accumulation and decreased arsenite accumulation in rice through different mechanisms. Silicic acid released from the silicate gel increased the availability of DMA for rice uptake by inhibiting DMA adsorption on the soil solid phase or by displacing adsorbed DMA. Although silicic acid also increased the concentration of inorganic As in the soil solution, this effect was much smaller than the inhibitory effect of Si on arsenite uptake by rice roots.     

Methylated arsenic species in plants originate from soil microorganisms

? Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. ? Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC-ICP-MS. ? Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil-grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. ? Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.     

Evidence for a role of phytochelatins in regulating arsenic accumulation in rice grain

Phytochelatins (PCs) play a crucial role in detoxifying cellular arsenic (As) through complexation of arsenite. Here, we investigated whether PCs influence As accumulation in rice grain by using six rice cultivars varying in grain As accumulation. The cultivars with low grain As had significantly higher PCs concentration in the shoots than the cultivars with high grain As, but lower glutathione concentration. Shoot PCs concentration correlated negatively with grain As accumulation. Foliar sprays with 0.5 mM l-buthionine-sulphoxime (BSO) on rice leaves at grain filling stage decreased GSH and PC accumulation in rice shoots by 40-63% and 20-55%, respectively, but did not significantly affect plant growth. Foliar sprays with BSO decreased shoot As concentration, while increased As concentrations in husk and brown rice significantly. These results suggest that PC complexation of arsenite in rice leaves reduces As translocation from leaves to grains, and implicate that manipulation of PC synthesis might mitigate As accumulation in rice grain.     

Differential toxicity and accumulation of inorganic and methylated arsenic in rice

Background and aims

Efficient accumulation of arsenic (As) in rice (Oryza sativa L.) poses a potential health risk to rice consumers. The aim of this study was to investigate the mechanisms of uptake, transport and distribution of inorganic arsenic (As_i) and dimethylarsinic acid (DMA) in rice plants.

Methods

Rice was exposed to As_i (As(V)) and DMA in hydroponics. High-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and synchrotron X-ray fluorescence (SXRF) microprobe were used to determine As concentration and the in situ As distribution.

Results

DMA induced abnormal florets before flowering and caused a sharp decline in the seed setting rate after flowering compared to As_i. Rice grains accumulated 2-fold higher DMA than As_i. The distribution of As_i concentration (root?>?leaf?>?husk?>?caryopsis) in As(V) treatments was different from that of the DMA concentration (caryopsis?>?husk?>?root?≥?leaf) in DMA treatments. SXRF showed that As_i mainly accumulated in the vascular trace of caryopsis with limited distribution to the endosperm, whereas DMA was observed in both tissues.

Conclusions

DMA tended to accumulate in caryopsis and induced higher toxicity to the reproductive tissues resulting in markedly reduced grain yield, whereas As_i mainly remained in the vegetative tissues and had no significant effect on yield. DMA is more toxic than As_i to the reproductive tissues when both of them are at similar concentrations in nutrient solution.     

Relationship between leaf photosynthetic function at grain filling stage and yield in super high-yielding hybrid rice (Oryza sativa. L)

The characteristics of dry matter production before and after heading and the relationships between photosynthesis of flag leaves and dry matter accumulation in panicles were investigated on super high-yielding rice cv. Xieyou 9308 (the yield of up to 12 t/ha) with rice cv. Xieyou 63 as a control. The results showed that (i) the capacity of dry matter production before and after heading in Xieyou 9308, i.e. biomass and daily dry matter production, was remarkably higher than that in Xieyou 63, especially after heading; (ii) CO2 assimilation capacity in flag leaves in Xieyou 9308, namely Leaf Source Capacity (LSC), was also significantly higher than that in Xieyou 63, and the supply of photosynthate in leaves and the demand of grain filling were completely synchronous in Xieyou 9308, but photosynthetic function in flag leaves in Xieyou 63 declined sharply 20 days after heading and it was not enough to meet the demand of grain filling. These results confirmed that high efficient photosynthetic function in     

Relationship between leaf photosynthetic function at grain filling stage and yield in super high-yielding hybrid rice ( Oryza sativa . L)

The characteristics of dry matter production before and after heading and the relationships between photosynthesis of flag leaves and dry matter accumulation in panicles were investigated on super high-yielding rice cv. Xieyou 9308 (the yield of up to 12 t/ha) with rice cv. Xieyou 63 as a control. The results showed that (i) the capacity of dry matter production before and after heading in Xieyou 9308, i.e. biomass and daily dry matter production, was remarkably higher than that in Xieyou 63, especially after heading; (ii) CO₂ assimilation capacity in flag leaves in Xieyou 9308, namely Leaf Source Capacity (LSC), was also significantly higher than that in Xieyou 63, and the supply of photosynthate in leaves and the demand of grain filling were completely synchronous in Xieyou 9308, but photosynthetic function in flag leaves in Xieyou 63 declined sharply 20 days after heading and it was not enough to meet the demand of grain filling. These results confirmed that high efficient photosynthetic function in leaves after heading and its complete synchronization with grain filling are the key approaches to super high yield of rice.     

Relationship between leaf photosynthetic function at grain filling stage and yield in super high-yielding hybrid rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>. L)

The characteristics of dry matter production before and after heading and the relationships between photosynthesis of flag leaves and dry matter accumulation in panicles were investigated on super high-yielding rice cv. Xieyou 9308 (the yield of up to 12 t/ha) with rice cv. Xieyou 63 as a control. The results showed that (i) the capacity of dry matter production before and after heading in Xieyou 9308, i.e. biomass and daily dry matter production, was remarkably higher than that in Xieyou 63, especially after heading; (ii) CO₂ assimilation capacity in flag leaves in Xieyou 9308, namely Leaf Source Capacity (LSC), was also significantly higher than that in Xieyou 63, and the supply of photosynthate in leaves and the demand of grain filling were completely synchronous in Xieyou 9308, but photosynthetic function in flag leaves in Xieyou 63 declined sharply 20 days after heading and it was not enough to meet the demand of grain filling. These results confirmed that high efficient photosynthetic function in leaves after heading and its complete synchronization with grain filling are the key approaches to super high yield of rice.     

Arsenic biotransformation by Streptomyces sp. isolated from rice rhizosphere

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As‐contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S‐adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.     

Water management,rice varieties and mycorrhizal inoculation influence arsenic concentration and speciation in rice grains

A pot experiment was carried out to investigate the effects of water management and mycorrhizal inoculation on arsenic (As) uptake by two rice varieties, the As-resistant BRRI dhan 47 (B47) and As-sensitive BRRI dhan 29 (B29). Grain As concentration of B47 plants was significantly lower than that of B29, and grain As concentration of B47 was higher under flooding conditions than that under aerobic conditions. In general, mycorrhizal inoculation (Rhizophagus irregularis) had no significant effect on grain As concentrations, but decreased the proportion of inorganic arsenic (iAs) in grains of B47. The proportion of dimethylarsinic acid (DMA) in the total grain As was dramatically higher under flooding conditions. Results demonstrate that rice variety selection and appropriate water management along with mycorrhizal inoculation could be practical countermeasures to As accumulation and toxicity in rice grains, thus reducing health risks of As exposure in rice diets.     

Arsenic species excretion after controlled seafood consumption

Influence of controlled consumption of marine fish on the urinary excretion of arsenite, arsenate, dimethylarsinic and monomethylarsonic acid (DMA, MMA) was investigated in two experiments. Arsenic species were separated by anion-exchange chromatography and detected with hydride-technique atomic absorption spectrometry (detection limit 1, 10, 2, 2 microg/l). Firstly, 13 probands ate different types of seafood after having refrained from any seafood for 1 week. DMA levels rose from 3.4+/-1.3 microg/g creatinine (n=12; a day before seafood) to a mean peak level of 28.2+/-20.6 microg/g (n=13; 10-23 h after; P<0.001; max. 77.7 microg/g). No other species were excreted before the meal, but small amounts of arsenite (8.5% positive; max. 1.7 microg/g) and MMA (1.2%; 1.6 microg/g) within 2 days after it (n=82). Consumption of white herring caused the highest DMA levels. Secondly, eight probands ingested white herring (dose 3.5 g/kg; DMA content 32.1+/-15.3 ng/g wet weight; n=36). No arsenite, arsenate and MMA was found in the urine or in the herring tissues. The mean DMA mass excreted after the meal (65.3+/-22.0 microg/24 h) was about 6-fold higher than the sum of base DMA excretion (3.0+/-1.7 microg/24 h) and the ingested DMA mass (7.9+/-2.7 microg). This indicates that the elevated DMA excretion after herring consumption is not caused by the metabolism of inorganic arsenic but of other arsenic species present in the fish tissue, e.g. arsenobetaine or fat-soluble arsenic species.     

Dietary Sources of Methylated Arsenic Species in Urine of the United States Population,NHANES 2003–2010

Background

Arsenic is an ubiquitous element linked to carcinogenicity, neurotoxicity, as well as adverse respiratory, gastrointestinal, hepatic, and dermal health effects.

Objective

Identify dietary sources of speciated arsenic: monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA).

Methods

Age-stratified, sample-weighted regression of NHANES (National Health and Nutrition Examination Survey) 2003–2010 data (∼8,300 participants ≥6 years old) characterized the association between urinary arsenic species and the additional mass consumed of USDA-standardized food groups (24-hour dietary recall data), controlling for potential confounders.

Results

For all arsenic species, the rank-order of age strata for median urinary molar concentration was children 6–11 years > adults 20–84 years > adolescents 12–19 years, and for all age strata, the rank-order was DMA > MMA. Median urinary molar concentrations of methylated arsenic species ranged from 0.56 to 3.52 µmol/mol creatinine. Statistically significant increases in urinary arsenic species were associated with increased consumption of: fish (DMA); fruits (DMA, MMA); grain products (DMA, MMA); legumes, nuts, seeds (DMA); meat, poultry (DMA); rice (DMA, MMA); rice cakes/crackers (DMA, MMA); and sugars, sweets, beverages (MMA). And, for adults, rice beverage/milk (DMA, MMA). In addition, based on US (United States) median and 90th percentile consumption rates of each food group, exposure from the following food groups was highlighted: fish; fruits; grain products; legumes, nuts, seeds; meat, poultry; and sugars, sweets, beverages.

Conclusions

In a nationally representative sample of the US civilian, noninstitutionalized population, fish (adults), rice (children), and rice cakes/crackers (adolescents) had the largest associations with urinary DMA. For MMA, rice beverage/milk (adults) and rice cakes/crackers (children, adolescents) had the largest associations.     

Phosphorus remobilization from rice flag leaves during grain filling: an RNA‐seq study

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA‐seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up‐regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX‐MFS1 (a putative P transporter) and SPX‐MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up‐regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.     

The Rice Aquaporin Lsi1 Mediates Uptake of Methylated Arsenic Species

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] 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 lsi1 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 V_max 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 Lsi1 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 Lsi1 mediates the uptake of undissociated methylated As in rice roots.Arsenic (As) contamination affects millions of people worldwide, particularly in South Asia where As-contaminated groundwater has been extracted for drinking (Chakraborti et al., 2002; Nordstrom, 2002). Recent studies have shown that foods, especially rice (Oryza sativa), are an important source of inorganic As to populations dependent on a rice diet (Kile et al., 2007; Ohno et al., 2007; Mondal and Polya, 2008). Paddy rice is more efficient than other cereal crops in accumulating As (Williams et al., 2007). This is because anaerobic conditions in submerged paddy soils lead to mobilization of arsenite [As(III); Takahashi et al., 2004; Xu et al., 2008], which is then taken up by rice roots mainly through the highly efficient transport pathway for silicon (Si; Ma et al., 2008). The relatively high accumulation of As in rice is of concern, as it may pose a significant health risk (Zhu et al., 2008; Meharg et al., 2009).A number of As species may be present in soil depending on soil conditions and the history of As contamination. These include arsenate [As(V)], As(III), and methylated As species such as monomethylarsonic acid [MMA(V): CH₃AsO(OH)₂] and dimethylarsinic acid [DMA(V): (CH₃)₂AsO(OH)]. As(V) is the main species in aerobic soils, while As(III) dominates in anaerobic environments such as flooded paddy soils. Both MMA(V) and DMA(V) have been found in paddy soils (Takamatsu et al., 1982), which may have been derived from microbial and algal biomethylation and/or past uses of methylated As compounds. MMA(V), as sodium or calcium salt, and DMA(V), as sodium salt or free acid (also called cacodylic acid), are herbicides widely used for weed control on cotton (Gossypium hirsutum), orchards, and lawns, or as a defoliant of cotton (U.S. Environmental Protection Agency, 2006). Conversion of cotton fields for the production of paddy rice in the United States may be a reason for the high levels of methylated As reported for the U.S. rice (Meharg et al., 2009).The mechanism of As(V) uptake by plants through the phosphate transport system has been well established (for review, see Zhao et al., 2009). In contrast, As(III) is taken up into the cells by aquaglyceroporins in Escherichia coli, yeast (Saccharomyces cerevisiae), and mammalian tissues (for review, see Bhattacharjee and Rosen, 2007). Recent studies have shown that several plant aquaporin channels belonging to the Nodulin 26-like Intrinsic Protein (NIP) subfamily are permeable to As(III) when expressed heterologously in yeast (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008). The rice Si transporter Lsi1 (OsNIP2;1; Ma et al., 2006) is also permeable to As(III) when expressed in yeast or Xenopus laevis oocytes (Ma et al., 2008). Furthermore, the lsi1 rice mutant lost 57% of the influx capacity for As(III) compared to the wild type in short-term assays, suggesting that Lsi1 is an important entry route for As(III) (Ma et al., 2008). In rice roots, a second Si transporter, Lsi2, functions as an efflux carrier to transport Si efflux from the exodermis and endodermis cells toward the stele for xylem loading (Ma et al., 2007). This transporter also mediates As(III) efflux; two independent lsi2 mutants had 73% to 91% lower concentrations of As(III) in the xylem sap than their wild types (Ma et al., 2008). The shared uptake pathway between Si (silicic acid) and As(III) (arsenous acid) is consistent with their physiochemical properties; both are present predominantly as undissociated neutral molecules at the normal environmental and physiological pH range (pK_a = 9.2, >99% undissociated at pH ≤ 7.0), and the two molecules have similar sizes.The uptake mechanisms of methylated As species by plant roots are not known. Previous studies showed that both MMA(V) and DMA(V) can be taken up by roots and translocated to shoots in a number of plant species (Marin et al., 1992; Carbonell-Barrachina et al., 1998, 1999; Burló et al., 1999). Marin et al. (1992) found that uptake by rice followed the order of As(III) > MMA(V) > As(V) > DMA (V), although DMA(V) was more efficiently translocated from roots to shoots than other As species. Raab et al. (2007) reported large variations in the absorption and translocation efficiencies for As(V), MMA(V), and DMA(V) among the 46 plant species tested. On average, root absorption of As(V) was 2.5- and 5-times higher than MMA(V) and DMA(V), respectively. The translocation efficiency, defined as the shoot-to-root concentration ratio after 24-h exposure, was highest for DMA(V) (0.8), followed by MMA(V) (0.3) and As(V) (0.09). The concentration-dependent uptake kinetics of MMA(V) in rice roots could be described by the Michaelis-Menten equation, whereas the limited uptake of DMA(V) appeared to be linear in relation to the increasing concentration in the uptake medium (Abedin et al., 2002). Abbas and Meharg (2008) showed that DMA(V) uptake by maize (Zea mays) seedlings was enhanced by more than 10-fold by a pretreatment of phosphorus starvation; this compared with only 2-fold increase in As(V) uptake. They thought that DMA(V) might be taken up by the phosphate transporters, or that phosphorus starvation altered expression of a range of membrane transporters or even membrane permeability itself.In addition to the root uptake of methylated As species, some plants appear to be able to biomethylate As, but the pathway and enzymology remains unclear (Wu et al., 2002; Zhao et al., 2009). In microbes, As methylation follows the Challenger pathway involving repeated steps of As reduction and oxidative methylation (Bentley and Chasteen, 2002). As(V) is first reduced to As(III), which is methylated by S-adenosylmethyltransferase using S-adenosyl-l-Met as the methyl donor. The product of this reaction is pentavalent MMA(V), which is reduced by a reductase to trivalent MMA(III) with thiols (e.g. glutathione). Methylation and reduction steps continue to produce di- and trimethyl As compounds. MMA(III) and DMA(III) are intermediates in the As methylation pathway, which is not very stable (Gong et al., 2001). In rice grain, DMA(V) is the main form of methylated As, and can account for up to 80% of the total As (Zavala et al., 2008; Meharg et al., 2009). In light of the significant presence of methylated As in rice, it is important to elucidate the transport and assimilation pathways of these As species in plants.In this study, we present evidence that MMA(V) and DMA(V) are taken up by rice roots, at least partly, through the NIP aquaporin channel Lsi1, and that this process is strongly pH dependent. We also show that MMA(V) can be reduced to MMA(III) in planta.     

Arsenic speciation in turnip as affected by application of chicken manure bearing roxarsone and its metabolites

Roxarsone (ROX) is widely used as a feed additive in intensive animal production. While animals are fed with ROX, the most commonly detectable As forms in fresh manures include ROX and small quantities of its metabolites such as arsenate (As(V)), arsenite (As(III)), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). A pot experiment was conducted to investigate the uptake, translocation and distribution of ROX, As(V), As(III), MMA and DMA in turnips, with the soil amended with 2% and 3% (w/w) chicken manure (CM) bearing ROX and its metabolites. Soil without any fertilizer was the control. The results show that only As(V) and As(III) were detected in turnip control samples. As(V), As(III) and DMA were found in all CM applied samples, but not ROX or MMA. This implies that turnip cannot take up ROX directly and accumulate MMA at detectable levels. The contents of DMA in tubers and the three As species in shoots increased with the CM rate in contrast to reduced levels of As(V) and As(III) in tubers. Increased CM rate enhanced the translocation of the three As species, especially for DMA, from tubers to shoots. DMA was the major form (42.9–61.4% in tubers and 38.1–76.3% in shoots), followed by As(III), in turnip plants fertilized with CM. The results indicate that ROX and its metabolites in animal manures can be introduced into human food chain by the way ROX → animal → manure → soil → crop.     

Targeting Hormone-Related Pathways to Improve Grain Yield in Rice: A Chemical Approach

Sink/source relationships, regulating the mobilization of stored carbohydrates from the vegetative tissues to the grains, are of key importance for grain filling and grain yield. We used different inhibitors of plant hormone action to assess their effects on grain yield and on the expression of hormone-associated genes. Among the tested chemicals, 2-indol-3-yl-4-oxo-4-phenylbutanoic acid (PEO-IAA; antagonist of auxin receptor), nordihydroguaiaretic acid (NDGA; abscisic acid (ABA) biosynthesis inhibitor), and 2-aminoisobutyric acid (AIB; ethylene biosynthesis inhibitor) improved grain yield in a concentration dependent manner. These effects were also dependent on the plant developmental stage. NDGA and AIB treatments induced an increase in photosynthesis in flag leaves concomitant to the increments of starch content in flag leaves and grains. NDGA inhibited the expression of ABA-responsive gene, but did not significantly decrease ABA content. Instead, NDGA significantly decreased jasmonic acid and jasmonic acid-isoleucine. Our results support the notion that the specific inhibition of jasmonic acid and ethylene biosynthesis resulted in grain yield increase in rice.     

Effect of sucrose level on rice grain deposition in detached panicle system

In detached IR36 rice panicles incubated in liquid medium for 10 days in 1.0–2.0% sucrose and 0.6% glutamine, final panicle weight increased but percentage and weight of grain protein decreased with 1.0–1.75% sucrose. Soluble sugars increased in stem and hull but not in developing grain. With five other rice panicles and IR36 panicles differing in grain size incubated in liquid culture containing 0.075% glutamine for 7 days, percentage grain protein was again lower in 1.5% sucrose than in 1.0% sucrose, with correspondingly heavier grain weight in four cases. Free-sugar levels of developing grains were lower in detached panicles than in the field grain samples in both experiments. Thus, sucrose level has a depressing effect on protein accumulation in the developing rice grain. Lysine content of grain protein decreased with increase in protein content.     

Rice is more efficient in arsenite uptake and translocation than wheat and barley

Rice is efficient at arsenic (As) accumulation, thus posing a potential health risk to humans and animals. Arsenic bioavailability in submerged paddy soil is enhanced due to mobilisation of arsenite, but rice may also have an inherently greater ability to take up and translocate arsenite than other cereal crops. To test this hypothesis, rice, wheat and barley were exposed to 5 µM arsenate or arsenite for 24 h. Arsenic uptake and distribution, and As speciation in the xylem sap and nutrient solution were determined. Regardless of the As form supplied to plants, rice accumulated more As in the shoots than wheat or barley. Arsenite uptake by rice was double of that by wheat or barley, whereas arsenate uptake was similar between rice and wheat and approximately a third smaller in barley. The efficiency of As translocation from roots to shoots was greater when plants were supplied with arsenite than with arsenate, and in both treatments rice showed the highest translocation efficiency. Arsenite was the main species of As (86–97%) in the xylem sap from arsenite-treated plants of all three species. In the arsenate-treated plants, 84%, 45% and 63% of As in the xylem sap of rice, wheat and barley, respectively, was arsenite. Arsenite efflux to the external medium was also observed in all three plant species exposed to arsenate. The results show that rice is more efficient than wheat or barley in arsenite uptake and translocation, probably through the highly efficient pathway for silicon.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.