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
EPFL, 2005
Thèse EPFL

Résumé

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.

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Erika Nehnevajova
EPFL, 2005
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/56052?ln=fr

À propos de ce résultat

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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

Chargement

The contamination of agricultural land and groundwater by heavy metals is essentially linked to human activities. A major problem with heavy metals is that they cannot be biodegraded and therefore reside in the environment for long periods of time if they are not removed. Thus, depending of the kind and depth of contamination, different remediation techniques were developed. One of these methods, called "phytoextraction", uses the ability of so-called hyperaccumulating plants to extract high amounts of heavy metals. Accumulation of heavy metals in the environment is a serious concern for animal and human health. At the microscopic scale, heavy metals may have also deleterious effects on bacteria which are the key-players of the different nutrient turnovers in soils. Consequently, ecosystem functioning can be seriously perturbed and the long-term soil fertility may be threatened. The recent development of molecular biology greatly contributed to the discovery of the microbial diversity and its function in the soil. However, to date only a small number of studies used molecular methods to investigate the impacts of heavy metals on the bacterial community. In this thesis, a pot experiment was conducted under controlled conditions with one hyperaccumulating plant (Thlaspi caerulescens) grown in two different soils, a long-term and an artificially heavy metal-contaminated soil. The impact of heavy metals on the microbial community was then investigated with several molecular methods. Moreover, the decrease of the bioavailable heavy metals concentrations in soil due to plant uptake allowed to study the consequences on bacterial community structure and function. Based on the 16S ribosomal RNA and the corresponding gene (16S rDNA), four clone libraries were constructed to retrieve information on the structure of the microbial community and the potentially active part of the microbial community in the rhizosphere of Thlaspi caerulescens grown during three months in a long-term contaminated soil. The data obtained with the two clone libraries (rRNA and rDNA) from the rhizosphere of Thlaspi caerulescens were compared with the bulk soil data to identify any effect of the plant on the soil microbial community structure. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, α-Proteobacteria, β-Proteobacteria, Acidobacteria and the Planctomycetales. The taxa dominating the bacterial community structure in the bulk soil also dominated the rhizosphere community, indicating that the plant did not exert a major influence on the overall bacterial diversity. However, all dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries as compared to the rDNA libraries. On the contrary, sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA. Seventy per cent of these clone sequences were related to two subgroups of the Rubrobacteria, which was an indication that this group of bacteria was probably metabolically active in heavy metal-contaminated soils. Fluorescence in situ hybridization (FISH) was used for the in situ detection and quantification of selected bacterial groups previously detected in the clone libraries from the rhizosphere of Thlaspi caerulescens. By applying the most general probe EUB338, only 20% of the total rhizosphere microbial community could be detected. Based on this result, it was difficult to conclude with certitude which were the most dominant bacteria in the rhizosphere. However, despite this low detection rate, it was possible to detect the major groups present in the rhizosphere clone libraries using group-specific oligonucleotide probes. As part of our sequences were affiliated to two emerging bacterial groups, the Acidobacteria and the Rubrobacteria, two new probes were designed, Acido228 (specific for the subgroup 1 of the Acidobacterium division) and Rubro198 (specific for the all Rubrobacteria subclass), for the detection of these microorganisms. These two probes were first checked for their specificity with pure cultures and finally applied in the rhizosphere soil allowing for the first time the detection of these organisms in situ. Finally, the impact of heavy metals and a subsequent one-year phytoextraction with Thlaspi caerulescens on the soil microbial community was investigated in an artificially heavy metal-contaminated soil. All the different molecular and culture-dependent techniques used, denaturing gradient gel electrophoresis (DGGE), community level physiological profile (CLPP) and potential ammonium-oxidation measurement showed that the heavy metal addition induced drastic changes in the bacterial community. Moreover, the analysis of the different bacterial DGGE patterns (Bacteria, β-Proteobacteria and ammonia-oxidising bacteria) obtained during this experiment showed that one-year phytoremediation was not sufficient to recover the initial community present in the non-contaminated soil. However, with the CLPP analysis, it was possible to detect a stimulating effect of the plant on a part of the microbial community in both contaminated and non-contaminated soils. The most obvious result was obtained in the contaminated soil where the number of substrates metabolised increased significantly in the presence of the plant as compared to the unplanted contaminated samples. The measurement of the potential ammonium-oxidation was used as a criterion for soil quality. Although this test showed that the ammonia-oxidising bacteria were significantly stimulated in the planted non-contaminated soil samples, the positive effect of the plant on these bacteria was not sufficient to overcome the inhibition induced by the presence of the heavy metals in the contaminated soil, even after one year phytoremediation. To conclude, molecular methods in combination with culture-dependent techniques have proven in this study to be very useful for the detection of the changes induced by the heavy metals in the structure and the function of the microbial community. Moreover, the molecular techniques contributed to the identification of bacteria which could be potentially used for the bioremediation of contaminated soils thus offering new perspectives of investigation and technology development.

EPFL2003

Chargement

Phytoextraction of heavy metals by hyperaccumulating and non hyperaccumulating plants

Claudia Cosio

Although phytoextraction using hyperaccumulating plants is seen as a promising technique, a lack of understanding of the basic physiological, biochemical, and molecular mechanisms involved in heavy metal hyperaccumulation prevents the optimization of the phytoextraction technique and its further commercial application. The best-long term strategy for improving phytoextraction is therefore to understand and exploit the biological processes involved in metal acquisition, transport and shoot accumulation in plants. In order to compare Cd allocation in leaves and the role of the leaf cells in hyperaccumulating and non hyperaccumulating plants, we used a wide range of techniques and approaches on contrasting ecotypes of Thlaspi caerulescens, Arabidopsis halleri, Arabidopsis thaliana and one high biomass crop Salix viminalis. As a first step, the extent of hyperaccumulation and tolerance was studied in different plants. Five Swiss T. caerulescens populations were compared for their tolerance and Cd hyperaccumulation to two well studied populations: T. caerulescens Ganges (France) and T. caerulescens Prayon (Belgium). We showed that the behaviour of the populations in hydroponics was linked to the characteristics of their soil of origin. However, growing plants in hydroponics with 1 μM Cd seemed to be adequate to discriminate the various populations tested for hyperaccumulation. Cadmium effect on morphological parameters and Cd accumulation in S. viminalis leaves was also monitored. Salix viminalis was surprisingly tolerant to Cd and high Cd concentration in shoots. Because it may give an indication of the tolerance mechanisms employed by the different plants, we studied the general Cd allocation in leaves of hyperaccumulating (Ganges) and non-hyperaccumulating plants (Prayon, S. viminalis). Surprinsingly, when grown in hydroponics the only differences found between Prayon and Ganges at the leaf level was concentration of Cd found in the storage sinks. Results also showed similarities between T. caerulescens and S. viminalis in Cd storage, although the Cd concentrations found in leaves differed greatly. Both point-like accumulation and accumulation at the edges of the leaves were observed. A link could be established with visible symptoms (necrosis). At last differences in Cd localization between young and mature leaves were observed. These differences were attributed to physiological differences between leaves. Salix viminalis and T. caerulescens were both able to reduce Cd toxicity by allocating Cd in less sensitive tissues. Cadmium was found inside the cells and in the cell walls of the leaves of T. caerulescens, but mainly in cell walls and to a lesser extent in the symplasm in S. viminalis. Metal allocation in both plants indicated that the plant general growth strategy governed metal accumulation. We further investigated the role of the leaf cells in allocating Cd in leaves of Ganges, A. halleri and Prayon by characterizing Cd uptake in mesophyll protoplasts. Results indicated that differences in metal uptake could not be explained by different constitutive transport capacities at the leaf protoplast level. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of Ganges, whereas it decreased Cd accumulation in A. halleri protoplasts. The experiment with competitors eventually showed that probably more than one single transport system are carrying Cd in parallel into the cell. Metal allocation indicates that the principle of metal storage in metabolically less sensitive plant parts governs metal accumulation. Vacuolar compartmentalization and cell wall binding in leaves could therefore both play a role in accumulation of heavy metals. Based on these various results, we suggested that metal storage in plant demands the involvement of more than one compartment. Further work is however needed to assess many steps of the trafficking of metals that remain enigmatic.

EPFL2004

Chargement

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

Erika Nehnevajova, Jean-Paul Schwitzguebel

2009

EPFL2003

Phytoextraction of heavy metals by hyperaccumulating and non hyperaccumulating plants

Claudia Cosio

EPFL2004