Tommy Landberg

USE OF WILLOW IN PHYTOEXTRACTION

ABSTRACT Willow is shown to be able to accumulate high levels of heavy metal ions such as cadmium and zinc. The properties of willow to be used in phytoextraction therefore have been studied. In this article, results from different studies on heavy metals and willow have been used to demonstrate the capacity and possibility of using Salix in phytoextraction.

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.

Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis

Summary Many stress mechanisms, such as antioxidative mechanisms, are known to be activated by heavy metals. The aim of this work was to investigate whether oxidative stress and related mechanisms were expressed differently in plants with different resistances to heavy metals. Different clones of Salix viminalis with different resistances to Cd, Cu, and Zn were cultivated hydroponically in the presence of 7 μmol/L Cd, 3 μmol/L Cu or 70 μmol/L Zn for 20 days. The clones were then compared with regard to the concentrations of free radicals, estimated by measuring thiobarbituric acid-reactive material (TBA-rm) and glutathione (GSH). The activity of the enzymes, aspartate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) was also analysed. Salicylic acid was also measured since it is known to be involved in antioxidative activities. The results showed that some differences could be observed between resistant and sensitive clones. The SOD activity was higher in untreated resistant clones compared with the sensitive ones. Under metal treatment, however, the SOD activity was similar. Furthermore, TBA-rm was higher in shoots of resistant clones compared to sensitive ones, while the opposite was found in roots.

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.

Acacia nilotica L. Bark Removes Toxic Elements from Solution: Corroboration from Toxicity Bioassay Using Salix viminalis L. in Hydroponic System

ABSTRACT The present investigation deals with the advantages and potential of the Acacia nilotica bark as an adsorbent of toxic metals. Bark (1 g) when added to 100 ml of aqueous solution containing 10 μg ml-1 metal solution exhibited different metal adsorption values for different metals. The order of metal adsorption being Cr ≥ Ni > Cu > Cd > As > Pb. A similar trend of metal adsorption was observed when the bark is reused (1st recycle) Cr> Ni > Cu > Cd > Pb and also in the column sorption. In order to verify the metal removal property of A. nilotica bark, toxicity bioassay with Salix viminalis stem cuttings in hydroponic system augmented with Cd, Cr, and Pb together with A. nilotica bark powder was carried out. The results of toxicity bioassay confirmed the metal adsorption property of the bark powder. The functions of toxicity studies include leaf area, root length and number of new root primordia produced per stump. The leaf area, root length, and the number of new root primordia increased considerably in the presence of A. nilotica bark. The order of metal toxicity for leaf area and new root primordial is Cd > Cr > Pb. However, for root length the order of metal toxicity is Cr > Cd > Pb. The metal budgets of the leaf and root confirmed that the bark powder had adsorbed substantial amount of toxic metals and thus alleviates the toxicity imposed by the various tested elements. Hence, the utility of A. nilotica bark in developing and designing innovative technology for the clean up of toxic elements in aqueous solutions and possible scope for its use in phytoremediation are discussed.

Role of rhizosphere mechanisms in Cd uptake by various wheat cultivars

In each wheat type, cultivars have different propensities to accumulate Cd in their grains, likely depending on Cd uptake by roots and/or Cd distribution in the plant. This study investigates the processes in the root–soil interface and their role in high or low grain Cd accumulation. Twenty-four cultivars of spring bread, winter bread, durum, and spelt wheat with different grain Cd accumulation levels were investigated regarding removal of Cd from soil, pH, Cd and organic acids in root exudates, and cation-exchange capacity of roots (rootCEC). In addition, we investigated 109Cd uptake from a nutrient solution resembling soil solution. The removal of Cd from the rhizosphere soil increased, likely due to increased rootCEC with increased grain Cd accumulation propensity, except in spring bread wheat. The 109Cd uptake from solution did not differ between high and low grain Cd accumulators. If the soil Cd concentration was elevated, rootCEC increased, as did pH, and succinic acid levels in the exudates, while lactic and citric acid levels in root exudates decreased. This work indicates that high grain Cd accumulators take up more Cd from soil than do low accumulators. But not by a different capacity to take up Cd from soil solution. The higher rootCEC in high accumulating cultivars may influence the release of Cd from the soil particles.

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.

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.

Influence of silicon on arsenic uptake and toxicity in lettuce

Lettuce grown in soil is found to contain high concentrations of arsenic (As). This paper investigates the uptake and speciation of As in lettuce as well as the influenceof silicon (Si) on As uptake, since Si may decrease it. Lettuce plants were cultivated in nutrient solution containing arsenite or arsenate with or without silicate. The uptake and distribution of As between roots and shoots, As accumulation in cell walls, As speciation, and toxic effects on growth were analysed. Results indicate that arsenite was more toxic to lettuce than was arsenate. Silicate decreased arsenate toxicity but had little effect on arsenite toxicity. In contrast, Si decreased arsenite uptake more than arsenate uptake. The concentration of arsenate was higher than that of arsenite in the plants independent of the As species added. When arsenate was added, the As concentration in shoots was half of that in the roots and this distribution did not change with Si addition. When arsenite was added, approximately 10% of As was found in the shoots and 90% in the roots; this pattern changed in the presence of Si, and As became evenly distributed in the plant. In both roots and shoots, approximately 40% of the As was found in the cell wall fraction; when arsenite was added, the presence of Si increased this fraction to 47%, but only in the shoots. The extraction efficiency when analysing the As species was lower in shoots than in roots, especially in the presence of arsenite and Si. The opposite was found for As concentration in pellets after extraction. This indicated variation in the binding strength of arsenite and arsenate between roots and shoots and between Si- and non-Si-treated plants.

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.