Heavy metal stress and sulfate uptake in maize roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.     

AtKuP1: a dual-affinity K+ transporter from Arabidopsis.

Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil.     

Does salicylic acid regulate antioxidant defense system, cell death, cadmium uptake and partitioning to acquire cadmium tolerance in rice?

Salicylic acid (SA) may accelerate the cell death of cadmium-stressed roots to avoid cadmium (Cd) uptake by plants or may play positive roles in protecting the stressed roots from Cd-induced damage. To test these hypotheses, we performed a series of split-root hydroponic experiments with one-half of rice (Oryza sativa L. cv. Jiahua 1) roots exposed to 50 microM Cd and the other half not exposed. The objectives were to elucidate the effects of SA pretreatment on the time-dependent changes of H(2)O(2) levels in roots, antioxidant defense system in different organs, root cell death and the dynamic distribution of Cd in the plants. In the split-root system, a higher Cd uptake rate was observed in the Cd-stressed portions of roots compared with the treatment with the whole roots exposed to Cd. Furthermore, an appreciable amount of Cd was translocated from the Cd-exposed roots to the unexposed roots and trace amounts of Cd were released into the external solution. The split-root method also caused the two root portions to respond differently to Cd stress. The activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; and catalase, CAT) were significantly suppressed in the Cd-treated roots, hence leading to H(2)O(2) burst, lipid peroxidation, cell death and growth inhibition. By contrast, in the non-Cd-treated roots, the activities of enzymes (SOD, CAT, and POD) and root growth were persistently stimulated during the experimental period. The H(2)O(2) accumulation and lipid peroxidation were also induced in the non-Cd-treated roots, but they were significantly lower than those of the Cd-treated roots. The concentrations of glutathione (GSH) and non-protein thiols (NPT) in the Cd-treated roots were significantly higher than those of the untreated roots. SA pretreatment elevated enzymatic and non-enzymatic antioxidants, and the concentrations of GSH and NPT in roots and shoots, hence leading to alleviation of the oxidative damage as indicated by the lowered H(2)O(2) and MDA levels. Furthermore, SA pretreatment mitigated the Cd-induced growth inhibition in both roots and shoots and increased transpiration compared with non-SA-pretreatment under Cd exposure. It is concluded that Cd can be partly transferred from the Cd-exposed roots to Cd-unexposed roots and that cell death can be accelerated in the Cd-stressed roots in response to Cd stress. The SA-enhanced Cd tolerance in rice can be attributed to SA-elevated enzymatic and non-enzymatic antioxidants and NPT, and to SA-regulated Cd uptake, transport and distribution in plant organs.     

Iron acquisition by phytosiderophores contributes to cadmium tolerance

Based on the ability of phytosiderophores to chelate other heavy metals besides iron (Fe), phytosiderophores were suggested to prevent graminaceous plants from cadmium (Cd) toxicity. To assess interactions between Cd and phytosiderophore-mediated Fe acquisition, maize (Zea mays) plants were grown hydroponically under limiting Fe supply. Exposure to Cd decreased uptake rates of 59Fe(III)-phytosiderophores and enhanced the expression of the Fe-phytosiderophore transporter gene ZmYS1 in roots as well as the release of the phytosiderophore 2'-deoxymugineic acid (DMA) from roots under Fe deficiency. However, DMA hardly mobilized Cd from soil or from a Cd-loaded resin in comparison to the synthetic chelators diaminetriaminepentaacetic acid and HEDTA. While nano-electrospray-high resolution mass spectrometry revealed the formation of an intact Cd(II)-DMA complex in aqueous solutions, competition studies with Fe(III) and zinc(II) showed that the formed Cd(II)-DMA complex was weak. Unlike HEDTA, DMA did not protect yeast (Saccharomyces cerevisiae) cells from Cd toxicity but improved yeast growth in the presence of Cd when yeast cells expressed ZmYS1. When supplied with Fe-DMA as a Fe source, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing a cauliflower mosaic virus 35S-ZmYS1 gene construct showed less growth depression than wild-type plants in response to Cd. These results indicate that inhibition of ZmYS1-mediated Fe-DMA transport by Cd is not related to Cd-DMA complex formation and that Cd-induced phytosiderophore release cannot protect maize plants from Cd toxicity. Instead, phytosiderophore-mediated Fe acquisition can improve Fe uptake in the presence of Cd and thereby provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe.     

A comprehensive model of purine uptake by the malaria parasite Plasmodium falciparum: identification of four purine transport activities in intraerythrocytic parasites

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.     

Cadmium uptake in isolated adrenocortical cells of rainbow trout and yellow perch

Cadmium uptake was studied in isolated adrenocortical cells of rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens) to test the hypothesis that the greater sensitivity of trout cells to Cd-induced disruption of cortisol secretion observed in previous studies is correlated to higher level of metal accumulation. There was no evidence for interspecies differences in accumulation level, and a specific transport mechanism of similar affinity has been characterized in both fish species. However, inhibition of Cd uptake by calcium was observed in rainbow trout exclusively. The free metal ion Cd(2+) and chlorocomplexes CdCl(n)(2-n) both contribute to Cd accumulation with different level of contribution between fish species. We conclude that interspecies differences in sensitivity to Cd endocrine disrupting effect are not necessarily related to different levels of metal accumulation but would rather be linked to transport pathways and metal speciation. Cadmium/calcium competition for uptake could be a determinant of the early Cd-induced impaired cortisol secretion in trout but not perch cells.     

Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad).

Pea (Pisum sativum L. cv. Azad) plants exposed to 4 and 40 microM of Cd for 7 d in hydroponic culture were analysed with reference to the distribution of metal, the accumulation of biomass and the metal's effects on antioxidants and antioxidative enzymes in roots and leaves. Cd-induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. An enhanced level of lipid peroxidation and an increased tissue concentration of hydrogen peroxide (H2O2) in both roots and leaves indicated that Cd caused oxidative stress in pea plants. Roots and leaves of pea plants responded differently to Cd with reference to the induction of enhanced activities of most of the enzymes monitored in the present study. These differential responses to Cd were further found to be associated with levels of Cd to which the plants were exposed. Cd-induced enhancement in superoxide dismutase (SOD) activity was more at 40 microM than at 4 microM in leaves. While catalase (CAT) prominently increased in leaves both at 4 and 40 microM Cd, ascorbate peroxidase (APX) showed maximum stimulation at 40 microM Cd in roots. Enhancement in glutathione reductase (GR) activity was also more at 40 microM than at 4 microM Cd in roots. While glutathione peroxidase (GPOX) activity decreased in roots and remained almost unmodified in leaves, glutathione S-transferase (GST) showed pronounced stimulation in both roots and leaves of pea plants exposed to 40 microM Cd. Increased activities of antioxidative enzymes in Cd-treated plants suggest that they have some additive function in the mechanism of metal tolerance in pea plants.     

Phytochelatin and cadmium accumulation in wheat

Cadmium (Cd) is a nonessential heavy metal that can be harmful at low concentrations in organisms. Therefore, it is necessary to decrease Cd accumulation in the grains of wheats aimed for human consumption. In response to Cd, higher plants synthesize sulphur-rich peptides, phytochelatins (PCs). PC–heavy metal complexes have been reported to accumulate in the vacuole. Retention of Cd in the root cell vacuoles might influence the symplastic radial Cd transport to the xylem and further transport to the shoot, resulting in genotypic differences in grain Cd accumulation. We have studied PC accumulation in 12-day-old seedlings of two cultivars of spring bread wheat (Triticum aestivum), and two spring durum wheat cultivars (Triticum turgidum var. durum) with different degrees of Cd accumulation in the grains. Shoots and roots were analysed for dry weight, Cd and PC accumulation. There were no significant differences between the species or the varieties in the growth response to Cd, nor in the distributions of PC chain lengths or PC isoforms. At 1 μM external Cd, durum wheat had a higher total Cd uptake than bread wheat, however, the shoot-to-root Cd concentration ratio was higher in bread wheat. When comparing varieties within a species, the high grain Cd accumulators exhibited lower rates of root Cd accumulation, shoot Cd accumulation, and root PC accumulation, but higher shoot-to-root Cd concentration ratios. Intraspecific variation in grain Cd accumulation is apparently not only explained by differential Cd accumulation as such, but rather by a differential plant-internal Cd allocation pattern. However, the higher average grain Cd accumulation in the durum wheats, as compared to the bread wheats, is associated with a higher total Cd accumulation in the plant, rather than with differential plant-internal Cd allocation. The root-internal PC chain length distributions and PC–thiol-to-Cd molar ratios did not significantly differ between species or varieties, suggesting that differential grain Cd accumulation is not due to differential PC-based Cd sequestration in the roots.     

High-affinity K+ uptake in pepper plants

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.     

The Sinorhizobium meliloti ABC transporter Cho is highly specific for choline and expressed in bacteroids from Medicago sativa nodules

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (KD, 2.7 microM) and acetylcholine with a low affinity (KD, 145 microM) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.     

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.     

Effect of spermine on the phytochelatin concentration and composition in cadmium-treated roots ofCanavalia lineata seedlings

Effect of spermine on the phytochelatins (PCs) in cadmium-treated roots ofCanavalia lineata seedlings was studied. With the treatment of spermine, total nonprotein thiol (SH) contents decreased by 55% in roots of Cd-treated plants. Glutathione (GSH) synthetase activity was inhibited by 36.8% in roots and cysteine synthase was also inhibited by 9.5% while y-GluCys synthetase activity was not affected. From the PC-Cd complex analyses by gel column chromatography, it was found that Cd+spermine-treated roots contain an additional PC that has low affinity for Cd, in addition to Cd-induced PC whose SH:Cd ratio is 1:1. Spermine affected the PC concentration and composition in the Cd-treated roots ofC. lineata seedlings.     

Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature

Xylem transport of sulfate regulates distribution of sulfur in vascular plants. Here, we describe SULTR3;5 as an essential component of the sulfate transport system that facilitates the root-to-shoot transport of sulfate in the vasculature. In Arabidopsis (Arabidopsis thaliana), SULTR3;5 was colocalized with the SULTR2;1 low-affinity sulfate transporter in xylem parenchyma and pericycle cells in roots. In a yeast (Saccharomyces cerevisiae) expression system, sulfate uptake was hardly detectable with SULTR3;5 expression alone; however, cells coexpressing both SULTR3;5 and SULTR2;1 showed substantial uptake activity that was considerably higher than with SULTR2;1 expression alone. The V(max) value of sulfate uptake activity with SULTR3;5-SULTR2;1 coexpression was approximately 3 times higher than with SULTR2;1 alone. In Arabidopsis, the root-to-shoot transport of sulfate was restricted in the sultr3;5 mutants, under conditions of high SULTR2;1 expression in the roots after sulfur limitation. These results suggested that SULTR3;5 is constitutively expressed in the root vasculature, but its function to reinforce the capacity of the SULTR2;1 low-affinity transporter is only essential when SULTR2;1 mRNA is induced by sulfur limitation. Consequently, coexpression of SULTR3;5 and SULTR2;1 provides maximum capacity of sulfate transport activity, which facilitates retrieval of apoplastic sulfate to the xylem parenchyma cells in the vasculature of Arabidopsis roots and may contribute to the root-to-shoot transport of sulfate.     

Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4-dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na+-dependent, cocaine-sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC50 = 15 microM). Pretreatment with 10 microM xylamine did not inhibit norepinephrine transport if 10 microM cocaine or 100 microM norepinephrine was also present during the pretreatment period or if Na+ was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [3H]xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (Km = 13 microM), and it was inhibited by cocaine (IC50 = 0.6 microM), desipramine (IC50 less than 1 nM), and norepinephrine (IC50 = 1 microM). Several proteins became prominently labeled when intact PC12 cells were incubated with [3H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [3H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na+-free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [3H]Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [3H]xylamine-labeled proteins is associated with the norepinephrine transporter.     

Expression and transport characterisation of the wheat low-affinity cation transporter (LCT1) in the methylotrophic yeast Pichia pastoris

The low-affinity cation transporter (LCT1) from wheat (Triticum aestivum) was expressed in the methylotrophic yeast Pichia pastoris and its transport characteristics studied employing Ca(45) and Cd(109). A clone (LCT1#3) with the highest uptake of 14pmol of Ca/10(6)cells/10min when exposed to 100microM Ca(45) was chosen for further Ca(45) and Cd(109) transport characteristics. We report for the first time a K(m) for Ca by LCT1 of 0.43+/-0.15mM Ca activity which confirms LCT1 to be a low affinity transporter. Interestingly, the expression of LCT1 in Pichia resulted in reduced Cd(109) uptake compared to wild type cells, when cells were exposed to >or=60microM Cd. This is the first report of the ability of a heterologously expressed transporter to reduce the activity of endogenous transporter proteins to transport Cd. To our knowledge, this is the first demonstration of functional expression of a plant ion transporter using P. pastoris.