Phenomic profiling to reveal tolerance mechanisms and regulation of ascorbate-glutathione cycle in wheat varieties (Triticum aestivum L.) under arsenic stress.

The potential of arsenic (As) tolerant and sensitive varieties of wheat (Triticum aestivum L.) has yet to be explored despite of alarming situation of arsenic toxicity. To fill this gap, the study aimed to explore the role of antioxidants, phytochelatins, and ascorbate-glutathione for As tolerance in wheat. A total of eight varieties were exposed to different arsenate treatments (0, 1, 5, 10, 50, 100, 200, 500, 1000, 2000, and 10,000 µM) initially to screen effective treatment as well as contrasting varieties via Weibull distribution frequency for further analysis. The Weibull analysis found 200 µM as the most effective treatment in the present study. Selected varieties were analyzed for accumulation of total As and As speciation, oxidative stress (malondialdehyde, hydrogen peroxide), antioxidants (superoxide dismutase, catalase, peroxidase), phytochelatins, and ascorbate-glutathione cycle (glutathione-S-transferase, glutathione reductase, glutathione peroxidase, ascorbate peroxidase). Tolerant varieties showed less accumulation and translocation of total As, arsenate, and arsenite to the shoots compared with sensitive varieties under 200 µM treatment. Low concentration in tolerant varieties correlated with better growth and development response. Tolerant varieties showed higher induction of metabolites (glutathione, phytochelatins) compared to sensitive ones. Furthermore, tolerant varieties showed better performance of antioxidant and ascorbate-glutathione cycle enzymes in response to As exposure. The findings of the present study provided great insight into the wheat tolerance mechanism upon As exposure between contrasting varieties.

Silicate reduces cadmium uptake into cells of wheat.

Cadmium (Cd) is a health threat all over the world and high Cd content in wheat causes high Cd intake. Silicon (Si) decreases cadmium content in wheat grains and shoot. This work investigates whether and how silicate (Si) influences cadmium (Cd) uptake at the cellular level in wheat. Wheat seedlings were grown in the presence or absence of Si with or without Cd. Cadmium, Si, and iron (Fe) accumulation in roots and shoots was analysed. Leaf protoplasts from plants grown without Cd were investigated for Cd uptake in the presence or absence of Si using the fluorescent dye, Leadmium Green AM. Roots and shoots of plants subjected to all four treatments were investigated regarding the expression of genes involved in the Cd uptake across the plasma membrane (i.e. LCT1) and efflux of Cd into apoplasm or vacuole from the cytosol (i.e. HMA2). In addition, phytochelatin (PC) content and PC gene (PCS1) expression were analysed. Expression of iron and metal transporter genes (IRT1 and NRAMP1) were also analysed. Results indicated that Si reduced Cd accumulation in plants, especially in shoot. Si reduced Cd transport into the cytoplasm when Si was added both directly during the uptake measurements and to the growth medium. Silicate downregulated LCT1 and HMA2 and upregulated PCS1. In addition, Si enhanced PC formation when Cd was present. The IRT1 gene, which was downregulated by Cd was upregulated by Si in root and shoot facilitating Fe transport in wheat. NRAMP1 was similarly expressed, though the effect was limited to roots. This work is the first to show how Si influences Cd uptake on the cellular level.

Novel Field Data on Phytoextraction: Pre-Cultivation With Salix Reduces Cadmium in Wheat Grains.

Cadmium (Cd) is a health hazard, and up to 43% of human Cd intake comes from wheat products, since Cd accumulates in wheat grains. Salix spp. are high-accumulators of Cd and is suggested for Cd phytoextraction from agricultural soils. We demonstrate, in field, that Salix viminalis can remove Cd from agricultural soils and thereby reduce Cd accumulation in grains of wheat subsequently grown in a Salix-treated field. Four years of Salix cultivation reduce Cd concentration in the soil by up to 27% and in grains of the post-cultivated wheat by up to 33%. The higher the plant density of the Salix, the greater the Cd removal from the soil and the lower the Cd concentration in the grains of post-cultivated wheat, the Cd reduction remaining stable several years after Salix cultivation. The effect occurred in both sandy and clayey soil and in winter and spring bread wheat cultivars. Already one year of Salix cultivation significantly decrease Cd in post grown wheat grains. With this field experiment we have demonstrated that phytoextraction can reduce accumulation of a pollutant in post-cultivated wheat and that phytoextraction has no other observed effect on post-cultivated crops than reduced uptake of the removed pollutant.

Cellular proton dynamics in Elodea canadensis leaves induced by cadmium.

Our earlier investigations showed that Elodea canadensis shoots, grown in the presence of cadmium (Cd), caused basification of the surrounding medium. The present study was aimed to examine the proton dynamics of the apoplastic, cytosolic and vacuolar regions of E. canadensis leaves upon Cd exposure and to establish possible linkage between cellular pH changes and the medium basification. The changes in cytosolic calcium [Ca(2+)]cyt was also investigated as the [Ca(2+)]cyt and [pH]cyt homeostasis are closely linked. The cellular H(+) and Ca(2+) concentrations were monitored by fluorescence microscopy and ion-specific fluorescent dyes. Cadmium concentration of leaf-cell walls was measured after plant cultivation at different fixed levels of starting pH. The protoplasts from E. canadensis leaves were isolated by use of a newly developed enzymatic method. Upon Cd addition, both cytosolic and vacuolar pH of leaf protoplasts increased with a concomitant rise in the cytosolic Ca(2+) concentration. Time course studies revealed that changes in [Ca(2+)]cyt and [pH]cyt followed similar dynamics. Cadmium (0.5 µM) exposure decreased the apoplastic pH by 0.85 units. The maximum cell wall bound Cd-contents were obtained in plants grown at low starting pH. It is concluded that Cd treatment causes apoplastic acidosis in E. canadensis leaves associated with enhanced Cd binding to the cell walls and, consequently, reduced Cd influx into the cytosol.

Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants.

BACKGROUND AND AIMS: Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues. METHODS: Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of (109)Cd applied at 34 nm was investigated between root and shoot. KEY RESULTS: Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots. CONCLUSIONS: Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.

Cadmium triggers Elodea canadensis to change the surrounding water pH and thereby Cd uptake.

This study was aimed to investigate the influence of Elodea canadensis shoots on surrounding water pH in the presence of cadmium and the effect of plant-induced pH on cadmium uptake. The pH change in the surrounding nutrient solution and Cd uptake by Elodea shoots were investigated after cultivation of various plant densities (1, 3, 6 plants per 500 ml) in hydroponics at a starting pH of 4.0 and in the presence of different concentrations of cadmium (0, 0.1, 0.5 microM). Cadmium uptake was also investigated at different constant pH (4.0, 4.5, 5.5 and 6.5). To investigate if the pH change arose from photosynthetic activities, plants were grown under light, darkness or in the presence of a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and 0.5 microM cadmium in the solution. Elodea had an ability to increase the surrounding water pH, when the initial pH was low, which resulted in increased accumulation of Cd. The higher the plant density, the more pronounced was the pH change. The pH increase was not due to the photosynthetic activity since the pH rise was more pronounced under darkness and in the presence of DCMU. The pH increase by Elodea was triggered by cadmium.

Increased metal tolerance in Salix by nicotinamide and nicotinic acid.

We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of N-acetyl-cystein (NAC) and l-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se.

Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.

Fate of cadmium in Elodea canadensis.

Elodea canadensis is a submersed macrophytes, widely distributed in stormwater treatment ponds and able to remove heavy metals from water. This study examines the Cd uptake, translocation, and efflux patterns in Elodea. Several experiments were set up in a climate chamber. To study the root and shoot Cd uptake, living and dead roots and shoots were treated with (109)Cd in one- and two-compartment systems. Furthermore, to examine Cd translocation and distribution, either roots or shoots were treated with (109)Cd. Finally, the efflux of Cd from roots and shoots, respectively, to the external solution was studied after loading whole plants with (109)Cd. Results from the two compartment studies show that Cd is accumulated via direct uptake by both roots and shoots of Elodea. The Cd accumulation proved not to be metabolically dependent in Elodea, and the apoplastic uptake in particular was decreased by Cd pretreatment. In one week, up to 23% of the root uptake was translocated to the shoots, while about 2% of the Cd accumulated by shoots was translocated to the roots. Thus, slight dispersion of Cd is possible, while metal immobilization will not be directly mediated via the Elodea plant. The efflux experiment proved that both shoots of dead plants and roots of living plants had a faster efflux than did shoots of living plants. This information is relevant for an understanding of the fate of Cd in stormwater treatment ponds with Elodea.

Influence of nutrient levels on uptake and effects of mercury, cadmium, and lead in water spinach.

In Southeast Asia the aquatic macrophyte water spinach (Ipomoea aquatica Forsk.) is a popular vegetable that is cultivated in freshwater courses. These often serve as recipients for domestic and other sorts of wastewater that often contain a variety of pollutants, such as heavy metals. In addition, fertilizers are frequently used where water spinach is cultivated commercially for the food market. To estimate the importance of ambient nutrient concentrations for accumulation of mercury (Hg), cadmium (Cd), and lead (Pb) in water spinach, plants were exposed to nutrient solutions of different strength and with varying metal concentrations. Metal-induced toxic effects, which might possibly affect the yield of the plants, were also studied. The lower the nutrient strength in the medium was, the higher the metal concentrations that accumulated in the different plant parts and the lower the metal concentration in the medium at which metal-induced toxic effects occurred. Accordingly, internal metal concentrations in the plants were correlated to toxic effects. Plants exposed to metals retained a major proportion of the metals in the roots, which had a higher tolerance than shoots for high internal metal concentrations.

A new method to detect cadmium uptake in protoplasts.

The mechanism for cadmium (Cd2+) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl2 to the external medium, reflecting an uptake of Cd2+. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using 109Cd2+, and showed approximately the same maximal concentration of Cd2+ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.

Differences in structure of adventitious roots in Salix clones with contrasting characteristics of cadmium accumulation and sensitivity.

Various clones of Salix spp. have contrasting characteristics of accumulation, translocation to shoots and of sensitivity to cadmium (Cd). The aim was to investigate the structure of adventitious roots and find out if differences between groups of clones in root anatomy accounted for differences in relation to Cd. Stem cuttings of eight clones of Salix spp. with different combinations of high or low Cd accumulation, translocation of Cd to shoots and sensitivity to Cd, were cultivated for 3 weeks in hydroponics containing 100 micro M Ca(NO(3))(2). No Cd was added in this experiment. Equal-sized roots were selected for hand-sectioning and fluorescence staining to detect the beginning of Casparian band formation and suberin lamellae deposition in endodermis. In addition, root apices were fixed, embedded, sectioned longitudinally and transversally, and stained. The image analysis system LUCIA was used for quantitative evaluation of tissue differences. The structure of adventitious root apices showed an intermediate-open type of root apical meristem and the clones differed in organization of root apices. Clones with low accumulation of Cd and high Cd tolerance had smaller meristematic zones and more extensive vacuolation of cells in the root apices than clones characterized by high accumulation of, and high sensitivity to, Cd. The apoplastic barriers, exo- and endodermis, were developed relatively close to the apex. In both layers the first ontogenetic stage, the Casparian band development, was followed by the second stage, the suberin lamellae deposition. This process started in the endodermis, preferentially against phloem poles, which is a common phenomenon also in other plant species. However, preferential development of exodermis in the sectors against phloem poles was observed in this study for the first time in plants. Development of endodermal Casparian bands in clones characterized by high accumulation of Cd occurred more distant from the root tip than in clones with low accumulation. Furthermore, the suberin lamellae were more distant from the root tip in clones with high translocation of Cd compared with those with low translocation. This indicates that apoplastic movement of Cd into the stele and the upward translocation may vary due to the endodermal anatomy. The proportion of root apoplastic barriers, exodermis and endodermis as well as epidermis to other root tissues was significantly increased in clones with higher tolerance to Cd ions, indicating the importance of these tissues in protection of the root against toxic effects of Cd.