Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots

The present study aimed to investigate the effects of root surface iron plaque on the uptake kinetics of arsenite and arsenate by excised roots of rice (Oryza sativa) seedlings. The results demonstrated that the presence of iron plaque enhanced arsenite and decreased arsenate uptake. Arsenite and arsenate uptake kinetics were adequately fitted by the Michaelis-Menten function in the absence of plaque, but produced poor fits to this function in the presence of plaque. Phosphate in the uptake solution did not have a significant effect on arsenite uptake irrespective of the presence of iron plaque; however phosphate had a significant effect on arsenate uptake. Without iron plaque, phosphate inhibited arsenate uptake. The presence of iron plaque diminished the effect of phosphate on arsenate uptake, possibly through a combined effect of arsenate desorption from iron plaque.     

The response of tomato (Lycopersicon esculentum) to different concentrations of inorganic and organic compounds of arsenic

Tomato plants were cultivated in greenhouse and water solutions of arsenite (As(III)), arsenate (As(V)), methylarsonic acid (MA) and dimethylarsinic acid (DMA) were applied individually into cultivation substrate at two As levels, 5 and 15 mg kg⁻¹ of the substrate. Comparing the availability of arsenic compounds increased in order arsenite = arsenate < MA < DMA where the arsenic contents in plants decreased during vegetation period. Within a single plant, the highest arsenic concentration was found in roots followed in decreasing order by leaves, stems, and fruits regardless of arsenic compound applied. Arsenic toxicity symptoms reflected in suppressed growth of plants and a lower number and size of fruits were most significant with DMA treatment. However, the highest accumulation of arsenic by plants growing in the soil containing DMA was caused by higher mobility of this compound in the soil due to its lower sorption affinity. Our results confirmed substantial role of transformation processes of arsenic compounds in soil in uptake and accumulation of arsenic by plants.     

Enhanced cadmium accumulation in maize roots—the impact of organic acids

Low molecular weight organic acids are important components of root exudates and therefore, knowledge regarding the mechanisms of cadmium (Cd) uptake and distribution within plants under the influence of organic acids, is necessary for a better understanding of Cd behavior in the plant–soil system. In this study, acetic and malic acids increased the uptake of Cd by maize (Zea mays L. cv. TY2) roots and enhanced Cd accumulation in shoots under hydroponic conditions. Concentration-dependent net Cd influx in the presence and absence of organic acids could be resolved into linear and saturable components. The saturable component followed Michaelis–Menten kinetics, which indicated that Cd uptake across the plasma membrane was transporter-mediated. While the K _m values were similar, the V _max values in the presence of acetic and malic acids were respectively 6.0 and 3.0 times that of the control. Zinc transporters were the most probable pathways for Cd accumulation. It was hypothesized that Cd(II)–organic acid complexes associated with the root zone, could decompose and liberate Cd²⁺ for subsequent absorption by maize roots; and that in the layer of the roots or within the root free space, depletion of Cd²⁺ was buffered by the presence of Cd(II)–organic acid complexes. Plant response to elevated Cd levels involved overproduction of organic acids in maize roots as a resistance mechanism to alleviate Cd toxicity.     

Uptake kinetics of different arsenic species by Brassica carinata

The time- and concentration-dependent uptake kinetics for arsenate and arsenite were determined in 15-day-old excised roots. In both cases, arsenite showed a mono-phasic influx with the isotherm data fitting a linear model better than a non-linear one. The time- and the concentration-dependent uptake of arsenate displayed a hyperbolic kinetic. Greater uptake of arsenate, compared with arsenite, was found especially at lower external substrate concentrations. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly inhibited in the presence of phosphate, whereas arsenite uptake was not affected.     

Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus.

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium "conditioned" by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.     

Effects of phosphate on arsenate and arsenite sensitivity in two rice (Oryza sativa L.) cultivars of different sensitivity

Arsenate and arsenite sensitivity and arsenate influx tests were conducted for two rice cultivars of different arsenic sensitivity, Azucena and Bala. These were to establish if the mechanism of reduced arsenic sensitivity is achieved through an altered phosphate uptake system, as shown for Holcus lanatus. High phosphate treatments (≥50 μM) provided protection against both arsenate and arsenite. Unlike the H. lanatus tolerance mechanism, in the less sensitive cultivar Bala, arsenate influx did not decrease with phosphate treatment and phosphate transporters appeared to be constitutively upregulated; V_max for arsenate influx remain similar when Bala was grown in the presence or absence of phosphate (V_max - 0.90 and 0.63 nmol g⁻¹ f.wt min⁻¹ respectively). Although mean K_m appear different, Bala did not show lower affinity to arsenate than Azucena in the absence of phosphate (K_m - Azucena, 0.30 mM and Bala, 0.18), while in phosphate treatment, Bala arsenate affinity was half that observed for Azucena (K_m - Azucena, 0.14 and Bala, 0.36 mM). These were low compared to a 4 and 6 fold decrease seen for similar studies on H. lanatus in the absence and presence of phosphate. Phosphate-induced arsenic protection was observed but the mechanism does not resemble that of H. lanatus. Alternative mechanisms were discussed.     

Uptake kinetics of arsenic species in rice plants

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0-0.0532 mM) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid.     

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.     

Rapid reduction of arsenate in the medium mediated by plant roots

Microbes detoxify arsenate by reduction and efflux of arsenite. Plants have a high capacity to reduce arsenate, but arsenic efflux has not been reported. Tomato (Lycopersicon esculentum) and rice (Oryza sativa) were grown hydroponically and supplied with 10 microm arsenate or arsenite, with or without phosphate, for 1-3 d. The chemical species of As in nutrient solutions, roots and xylem sap were monitored, roles of microbes and root exudates in As transformation were investigated and efflux of As species from tomato roots was determined. Arsenite remained stable in the nutrient solution, whereas arsenate was rapidly reduced to arsenite. Microbes and root exudates contributed little to the reduction of external arsenate. Arsenite was the predominant species in roots and xylem sap. Phosphate inhibited arsenate uptake and the appearance of arsenite in the nutrient solution, but the reduction was near complete in 24 h in both -P- and +P-treated tomato. Phosphate had a greater effect in rice than tomato. Efflux of both arsenite and arsenate was observed; the former was inhibited and the latter enhanced by the metabolic inhibitor carbonylcyanide m-chlorophenylhydrazone. Tomato and rice roots rapidly reduce arsenate to arsenite, some of which is actively effluxed to the medium. The study reveals a new aspect of As metabolism in plants.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

Characterization of As efflux from the roots of As hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L.

In some plant species, various arsenic (As) species have been reported to efflux from the roots. However, the details of As efflux by the As hyperaccumulator Pteris vittata remain unknown. In this study, root As efflux was investigated for different phosphorus (P) supply conditions during or after a 24-h arsenate uptake experiment under hydroponic growth conditions. During an 8-h arsenate uptake experiment, P-supplied (P+) P. vittata exhibited much greater arsenite efflux relative to arsenate uptake when compared with P-deprived (P–) P. vittata, indicating that arsenite efflux was not proportional to arsenate uptake. In the As efflux experiment following 24 h of arsenate uptake, arsenate efflux was also observed with arsenite efflux in the external solution. All the results showed relatively low rates of arsenate efflux, ranging from 5.4 to 16.1% of the previously absorbed As, indicating that a low rate of arsenate efflux to the external solution is also a characteristic of P. vittata, as was reported with arsenite efflux. In conclusion, after 24 h of arsenate uptake, both P+ and P– P. vittata loaded/effluxed similar amounts of arsenite to the fronds and the external solution, indicating a similar process of xylem loading and efflux for arsenite, with the order of the arsenite concentrations being solution ≪ roots ≪ fronds.     

Phytochelatins are involved in differential arsenate tolerance in Holcus lanatus

Arsenate tolerance is conferred by suppression of the high-affinity phosphate/arsenate uptake system, which greatly reduces arsenate influx in a number of higher plant species. Despite this suppressed uptake, arsenate-tolerant plants can still accumulate high levels of As over their lifetime, suggesting that constitutive detoxification mechanisms may be required. Phytochelatins are thiol-rich peptides, whose production is induced by a range of metals and metalloids including arsenate. This study provides evidence for the role of phytochelatins in the detoxification of arsenate in arsenate-tolerant Holcus lanatus. Elevated levels of phytochelatin were measured in plants with a range of tolerance to arsenate at equivalent levels of arsenate stress, measured as inhibition of root growth. The results suggest that arsenate tolerance in H. lanatus requires both adaptive suppression of the high-affinity phosphate uptake system and constitutive phytochelatin production.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

The Effect of Arsenate and Other Inhibitors on Early Events during the Germination of Lettuce Seeds (Lactuca sativa L.)

The effect of arsenate, arsenite, 2,4-dinitrophenol, and anaerobiosis on early events in seed germination was investigated using both intact and punched seeds of lettuce (Lactuca sativa L.). It was found that punching the seed removes penetration barriers to the entrance of inhibitors without an undue loss of germination or light responses. The kinetics of the action of germination inhibitors were established by 2-hour pulse experiments. Arsenate and 2, 4-dinitrophenol have very different kinetics. The inhibition of germination in punched seeds by arsenate given in conjunction with phosphate compared with the lack of inhibition of arsenate plus phosphate on the growing seedling, suggest a distinct metabolic change in the germinating embryo at some time between the onset of germination and subsequent seedling growth.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Active Silicon Uptake by Wheat

The absorption of Si by wheat, Triticum aestivum L. ‘Yecora Rojo,’ followed Michaelis–Menten kinetics over a concentration range of 0.004–1.0 mM. K_m and V_max were determined using linear transformations and the calculated curve fitted the data closely. The absorption resulted in accumulation ratios of 200/1 or more. In keeping with that finding, this study also demonstrated that Si uptake by wheat is under metabolic control, being severely restricted by dinitrophenol (DNP) and potassium cyanide (KCN). Silicon uptake by wheat was not significantly affected by phosphate ions, but the chemical analog Ge exerted a direct competitive effect on Si uptake, and vice versa.     

Organic acids enhance the uptake of lead by wheat roots

The uptake and bioavailability of lead (Pb) in soil–plant systems remain poorly understood. This study indicates that acetic and malic acids enhance the uptake of Pb by wheat (Triticum aestivum L.) roots under hydroponic conditions. The net concentration-dependent uptake influx of Pb in the presence and absence of organic acids was characterized by Michaelis–Menten type nonsaturating kinetic curves that could be resolved into linear and saturable components. Fitted maximum uptake rates (V _max) of the Michaelis–Menton saturable component in the presence of acetic and malic acids were, respectively, 2.45 and 1.63 times those of the control, while the Michaelis–Menten K _m values of 5.5, 3.7 and 2.2 μM, respectively, remained unchanged. Enhanced Pb uptake by organic acids was partially mediated by Ca²⁺ and K⁺ channels, and also depended upon the physiological function of the plasma membrane P-type ATPase. Uptake may have been further enhanced by an effectively thinner unstirred layer of Pb adjacent to the roots, leading to more rapid diffusion towards roots. X-ray absorption spectroscopic studies provided evidence that the coordination environment of Pb in wheat roots was similar to that of Pb(CH₃COO)₂·3H₂O in that one Pb atom was coordinated to four oxygen atoms via the carboxylate group.     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis.

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.     

Mechanisms of arsenic hyperaccumulation in<Emphasis Type="Italic"> Pteris</Emphasis> species: root As influx and translocation

Several species of fern from the Pteris genus are able to accumulate extremely high concentrations of arsenic (As) in the fronds. We have conducted short-term unidirectional As influx and translocation experiments with ⁷³As-radiolabeled arsenate, and found that the concentration-dependent influx of arsenate into roots was significantly larger in two of these As-hyperaccumulating species, Pteris vittata (L.) and Pteris cretica cv. Mayii (L.), than in Nephrolepis exaltata (L.), a non-accumulating fern. The arsenate influx could be described by Michaelis-Menten kinetics and the kinetic parameter K _m was found to be lower in the Pteris species, indicating higher affinity of the transport protein for arsenate. Quantitative analysis of kinetic parameters showed that phosphate inhibited arsenate influx in a directly competitive manner, consistent with the hypothesis that arsenate enters plant roots on a phosphate-transport protein. The significantly augmented translocation of arsenic to the shoots that was seen in these As hyperaccumulator species is proposed to be due to a combination of the increased root influx and also decreased sequestration of As in the roots, as a larger fraction of As could be extracted from roots of the Pteris species than from roots of N. exaltata. This leaves a larger pool of mobile As available for translocation to the shoot, probably predominantly as arsenite.Abbreviations As ^V Arsenate - As ^III Arsenite - K _m Michaelis-Menten constant - P _i Phosphate - V _max Maximum rate of an enzyme-catalyzed reaction