The role of roots in the phytoextraction of bioavailable zinc

Phytoextraction is a non-invasive remediation method using plants. Phytotech Foundation and AGB in Berne are performing phytoextraction on an old landfill of a former hot dip galvanization plant, using sunflowers and tobaccos as extractors. Every year since 2007, total and soluble zinc concentrations in soil as well as zinc concentrations in shoot of the plants have been measured. To study the role of roots in phytoextraction, 14 root samples were harvested in autumn 2010 and their zinc concentration was measured. The results show that sunflowers were more efficient in fast transfer of zinc from root to shoot. They were also compared with the 4-year measurements and a mass balance was performed. This has shown that phytoextraction was so efficient that even the decrease in soluble zinc did not explain itself the concentrations in the plant. The roots may mobilize total zinc, which might re-load the soluble zinc pool in soil. In parallel, a pot experiment was performed at laboratory scale with young tobacco plants growing on polluted soils, monitoring root, stem, leaves, leachates and soil zinc concentrations. A mass balance was also performed, which confirmed the previous statement, even at young stage and for detailed values. Finally thanks to both experiments, it was suggested that there should be a potential concentration for each part of plants. Once this concentration reached, transfer of zinc could occur from the root to the stem, and then from the stem to the leaves where it is accumulated.

Non-GMO approach for the improvement of heavy metal accumulation and extraction of high yielding crop species for efficient phytoextraction of contaminated soil

Erika Nehnevajova

Some plants able to take up heavy metals from contaminated soils offer a possibility to clean up sites contaminated with heavy metals. Plants thus act as a solar driven pump, which can extract and concentrate heavy metals from the environment. Since most of the metal hyperaccumulating wild plants only produce very low biomass and most of plants producing high biomass accumulate only moderate amounts of metals, the current research is mainly focused on the overcoming of this deficiency to optimise metal phytoextraction. The main goal of this EC financed study was aimed at improvement of phytoextraction through improved metal extraction of high yielding oil crops, such as Helianthus annuus L. and Brassica juncea L. producing a high biomass. The use of their oil and biomass for technical purpose (lubricants, biogas and energy) allows to produce an additional value from this in situ decontamination technique and to improve the economical balance of phytoextraction. The enhancement of metal accumulation properties of sunflower and Indian mustard by non-GMO approach and of stimulated metal bioavailability in the soil were the main milestones of the present study. Classical fertilisers were used to lower soil pH, increasing metal availability from the soil and conventional biotechnological approaches were used as an alternative to genetic engineering to enhance both metal accumulation and extraction efficiency of oil crops. The first field experimentation (2002) was mainly focused on the screening of 15 commercial sunflower cultivars growing on a contaminated field in Rafz (Switzerland) to select the cultivar with the naturally highest potential to accumulate and extract metals from contaminated soil, and to assess the effect of classical fertilisers on metal accumulation/extraction of the sunflower cultivars. Highly significant differences of heavy metal accumulation and extraction were found between cultivars. Cadmium extraction varied by a factor of 4, Zn extraction by factor of 3 and Pb extraction by a factor of 14 between the cultivars with the highest and lowest metal extraction treated with the same fertiliser. Sulphate fertilisation significantly enhanced Zn and Pb extraction, whereas ammonium nitrate enhanced Cd extraction by most of the sunflower cultivars. Cultivar Salut showed the highest Cd, Zn and Pb extraction and was chosen for the next mutation breeding. Classical mutation breeding techniques were assumed to be efficient to improve the efficiency of metal accumulation of high yielding crops. Therefore, in vitro breeding was used to improve metal uptake of Indian mustard and chemical mutagenesis using ethyl methanesulphonate (EMS) was additionally applied for sunflowers, hybrid cultivar Salut and genetically homogenous inbred lines. Somaclonal variation of tissue culture was used as a source of genetic variability to increase the potential of metal accumulation in Indian mustard. After the selection of B. juncea callus cultures on a medium spiked with 10-200 μM of Cd or Pb, new somaclonal variants were regenerated from metal tolerant callus cells. A subsequent screening of 30 new B. juncea regenerants growing on a hydroponic medium spiked with toxic metals showed that 7 new somaclones (23 %) possessed a significantly higher shoot metal extraction than the control plants. The best regenerant showed a 6 times higher Cd, a 3 times higher Zn and a 4 times higher Pb extraction in the shoots than the control. Prior to the mutation breeding of sunflower, the effects of toxic metals on the growth, metal accumulation in shoot and root, metal translocation of control sunflower cultivars were investigated on a hydroponic medium spiked with toxic metal. Based on phytotoxic effects of roots and shoots the assessed sunflowers showed the following tolerance towards toxic metals: Pb >> Zn > Cd. The effect of the chemical mutagenesis on the efficiency of metal accumulation and extraction was first assessed on a hydroponic system, spiked with Cd, Zn and Pb. The results show that the EMS mutagenesis affected metal uptake, root and shoot metal accumulation and root to shoot metal translocation in mutant variants. We obtained M1 sunflower mutants with an enhanced metal accumulation, mutants with a lower metal accumulation and mutants without changed metal accumulation characteristics compared to the control plants. The next two field experiments in 2003 and 2004 were focused on the screening of 500 sunflower mutants of M1 generation and 300 sunflower mutants of M2 generation on the metal contaminated Rafz site to assess the effect of mutagenesis on yield, metal accumulation and extraction. The M1 sunflower mutants showed a 2-3 times higher Cd, Zn and Pb concentration in shoots, but a considerably reduced growth, as compared to control cultivars due to the phytotoxic effect of the mutagen. The sunflower mutants of M2 generation also showed a 2-3 times higher metal shoot accumulation than the control plants, and even more the metal extraction of the best M2 sunflower mutants "Giant Mutant 14/190/04" was increased 7.5 times for Cd, 8.2 times for Zn and 9.2 times for Pb extraction compared to the control plants. Theoretical calculations of phytoextraction potential of sunflower point out that the best sunflower mutant can produce up to 26 t dry matter yield per ha and remove 13.3 kg Zn per ha and year at the metal contaminated site in Rafz (Switzerland), that is a gain of factor 9 compared to Zn removal of control sunflowers. From a practical point of view this improvement looks very promising for boosting future phytoextraction research.

EPFL2005

Increased tolerance of sunflower mutant seedlings to Cd and Zn in hydroponic culture

Erika Nehnevajova, Jean-Paul Schwitzguebel

Cadmium and zinc show similarities in their chemistry, geochemistry and environmental properties. Whereas Cd is a phytotoxic non-essential element, Zn is an essential trace element for plant growth and development. Zinc plays a fundamental role in several key cellular functions, such as protein metabolism, gene expression, chromatin structure, photosynthetic carbon metabolism and indole acetic acid metabolism (Vallee and Falchuk, 1993; Marschner, 1995; Bonnet et al., 2000; Cakmak and Braun, 2001). Zinc is also an important component of many enzymes and proteins (Broadley et al., 2007). Cadmium can readily inhibit most of the Zn-dependent processes by binding to the membrane and to enzyme active sites, thus inactivating their functions (Aravind and Prasad, 2005). However, increasing Zn concentrations can replace such wrongly bound Cd (Van Assche and Clijsters, 1990; Shaw et al., 2004). Cadmium has been shown to cause many morphological, physiological, biochemical and structural changes in plants, such as growth inhibition, reduction in photosynthesis, transpiration or water imbalance (Sanità di Toppi and Gabbrielli, 1999; Schützendübel et al., 2001; Benavides et al., 2005; Mishra et al., 2006). Cadmium and high zinc concentration affect plant growth and metabolism, but the intensity of their toxic effects depends on plant species and the way and duration of metal exposure. Plants called hyperaccumulators are able to tolerate and accumulate extraordinary levels of trace elements in their above-ground tissue without developing any toxicity symptoms (Baker and Brooks, 1989; Reeves and Baker, 2000). Their high metal accumulation capability makes them interesting for the decontamination technique called phytoextraction, which uses plants for environmental clean-up (Salt et al., 1998). However, their low biomass production strongly limits the real application of this soil decontamination strategy. The ideal plant for phytoextraction should provide both a high biomass and a high tolerance and metal accumulation (Schwitzguébel et al., 2002). High biomass producing plants with improved tolerance to trace elements and with an enhanced metal accumulation capacity should be good candidates for metal removal from contaminated area. Previous studies have shown enhanced tolerance and metal accumulation properties in transgenic tobacco plants (Pomponi et al., 2006; Gorinova et al., 2007; Wojas et al., 2008). However, the main disadvantage of genetic engineering is still public acceptance and free-land application. In vitro breeding techniques have also been successfully used to obtain tobacco lines with a considerably enhanced Cd, Zn and Cu tolerance and metal accumulation (Herzig et al., 1997; Guadagnini et al., 1999; Herzig et al., 2003). In addition, the chemical mutagen ethyl methane sulphonate has been used to develop new sunflower lines with a significantly enhanced biomass and metal uptake on a metal contaminated field (Nehnevajova et al., 2007, 2009). Although these field experiments have revealed the potential of sunflower mutants for metal phytoextraction, their tolerance to trace elements and the effect of Cd and Zn on growth and photosynthetic pigments were not studied within previous free-land experimentation. The aim of the present work was thus to assess the tolerance to Cd and excess Zn of selected sunflower mutant lines with improved biomass and metal uptake capacity, by measuring different physiological parameters. For such a purpose, sunflower seedlings of eight selected mutant lines were cultivated under hydroponic conditions in the presence and in the absence of Cd and Zn to study the effect of these trace elements on (1) growth parameters, such as root elongation, root and shoot dry mass; (2) chlorophyll content; (3) carotenoid content; (4) Cd and Zn accumulation capacity of sunflower mutants; and (5) a possible correlation between Cd and Zn accumulation in sunflower shoots and roots.

2009

Feasibility Of Labile Zn Phytoextraction Using Enhanced Tobacco And Sunflower: Results Of Five- And One-Year Field-Scale Experiments In Switzerland

Erika Nehnevajova, Charlotte Pfistner, Jean-Paul Schwitzguebel

Phytoextraction with somaclonal variants of tobacco and sunflower mutant lines (non-GMs) with enhanced metal uptake and tolerance can be a sustainable alternative to conventional destructive decontamination methods, especially for stripping bioavailable zinc excess in topsoil. The overall results of a 5-year time series experiment at field scale in north-eastern Switzerland confirm that the labile Zn pool in soil can be lowered by 45-70%, whereas subplots without phytoextraction treatment maintained labile Zn concentrations. In 2011, the phytoextraction experiment site was enlarged by a factor of 3, and the labile 0.1M NaNO3 extractable Zn concentration in the soil was reduced up to 58% one period after harvest. A Mass Balance Analysis confirmed soil Zn decontamination in line with plant Zn uptake. The plants partially take Zn from the non-labile pool of the total. The sustainability of Zn phytoextraction in subplots that no longer exceed the Swiss trigger value is now assessed over time. In contrary to the phytoextraction of total soil Zn which needs a long cleaning up time, the bioavailable Zn stripping is feasible within a few years period.