Accumulation of a given metal in plants depends on a delicate and precise balance of various biological processes. Some plants have developed strategies that allow them to tolerate heavy metals in extreme conditions without suffering toxicity. This research focuses on the characterization of two metal transporters, a member of the P1B-type (ATPase) transporter family (HMA3) and a member the Natural Resistance Associated Macrophage Protein (NRAMP) Nramp1 family. These transporters have proposed roles in ion homeostasis and mineral nutrition. The work here sought to determine if these transporters might have characteristics that suggest a role in heavy metal transport and tolerance in metal hyperaccumulating plants. These proteins are very well conserved among different taxa. Nonetheless, as little as a single amino acid change has the potential to modify their capacity to take up non essential metals such as Cd, or Pb, and/or increase affinity for other mineral nutrients. These transporters were cloned from a non-accumulator (Arabidopsis thaliana L) and two ecotypes (Prayon and Ganges) of the hyperaccumulator Noccaea caerulescens (formerly= Thlaspi caerulescens). The full cDNA of an ortholog of either Nramp1 or HMA3 was expressed in yeast in order to provide a heterologous model to elucidate how polymorphisms between the orthologs might translate into functional differences between the protein sequences. A comparison of the HMA3 sequences to each other, or the Nramp1 sequences to each other, demonstrated that major motifs and domains in each protein were highly conserved but that there were numerous single amino acid polymorphisms. Few of these polymorphisms corresponded to positions in a protein that are known to be critical for transporter function. However, metal accumulation, tolerance and cell growth assays showed that the Nramp1 and HMA3 genes from Arabidopsis encoded proteins with the expected broad selectivity for divalent ion transport. In contrast, the genes from the Thlaspi ecotypes encoded proteins that showed more selectivity for ion transport. The Thlaspi ecotypes showed high selectivity for cadmium but the accumulation of other elements differed between the Thlaspi orthologs. These results suggest that the polymorphisms present in the Thlaspi sequences have produced differences in the transport characteristics of both the HMA3 and the Nramp1 transporters.

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L'utilisation des plantes pour décontaminer les sols pollués par les métaux est une solution respectueuse de l'environnement. Mais le développement de cette technique à grande échelle est encore limité en raison de l'indisponibilité de plantes avec les caractéristiques souhaitées (hyperaccumulation, biomasse élevée et croissance rapide). Les objectifs de ce travail étaient d'évaluer le potentiel de plusieurs cultivars de Pélargonium odorants pour l'extraction du Pb au champ, étudier la disponibilité du plomb en relation avec l'activité rhizosphérique et développer un protocole de transformation génétique. Parmi les six cultivars de Pélargonium odorants testés au champ, trois : Attar of Roses, Clorinda et Atomic Snowflake ont accumulé plus de 1000 mg kg-1 Pb, avec une forte biomasse. Pendant les expérimentations en conditions contrôlées, Attar of roses (le cultivar hyperaccumulateur) acidifie sa rhizosphère et augmente la concentration en COD significativement plus par rapport Concolor Lace (le cultivar non hyperaccumulateur), sans doute en réponse à la pollution métallique. Les concentrations en plomb dans les deux cultivars sont corrélées avec l'extraction au CaCl2. Les analyses par EXAFS et ESEM-EDS ont montré que le plomb présent dans les racines était principalement sous forme de complexes organiques alors que les sulfates de plomb prédominent dans le sol. Parallèlement à ces essais, un protocole de transformation génétique a été mis au point en vue de mieux comprendre les processus biochimiques impliqués dans l'hyperaccumulation et la fonction des gènes, Le système de régénération optimisé se base sur la pré-culture d'explants sur un milieu contenant 10 μM TDZ + 1 mg L-1 de chacun de BAP et NAA suivie par l'enlèvement de TDZ du milieu de culture. La kanamycine et l'hygromycine se sont avérés être de bons marqueurs sélectifs pour le Pélargonium. Deux souches d'Agrobacterium, C58 et EHA105 contenant des vecteurs binaires avec des gènes marqueurs hpt et nptII ont été choisis pour des expériences de transformation. Ils ont également le gène codant uidA séquence du gène rapporteur. Après l'infection avec C58, 4 et 107 plantes enracinées sur hygromycine ont été obtenues pour Attar of Roses et Atomic Snowflake, respectivement. Parmi ces plantes enracinées, les quatre plantes d'Attar et 82 d'Atomic Snowflake ont exprimé le Gus dans les feuilles, pétioles, les tiges et les racines comme prévu avec une séquence sous contrôle du promoteur constitutif CaMV 35S. De 20 plantes qui expriment le Gus, 7 plantes se sont avérées être positives après criblage par PCR. Après infection par EHA105, 23 et 133 plantes enracinées ont été obtenues après sélection sur kanamycine, mais aucune n'a démontré d'activité GUS. Seule des expériences d'empreintes par Southern blotting permettront de corréler le nombre d'insertions et niveau de l'expression dans ces différents événements de transformation. / Metal removal from contaminated soils using plants can provide an environment friendly solution. However, its successful application on a large scale is still limited due to unavailability of plants with desired set of characteristics i.e. hyperaccumulation, high biomass and rapid growth. The objective of this work was to assess the potential of scented Pelargonium cultivars for lead (Pb) extraction under field conditions, plant induced rhizosphere changes, soil factors influencing availability of Pb and to develop an efficient genetic transformation protocol for the selected cultivars. Of the six scented Pelargonium cultivars field-tested, three cultivars (Attar of Roses, Clorinda and Atomic Snowflake) accumulated more than 1000 mg Pb kg-1 DW, with high biomass reaching up to 45 tons ha-1 y-1 dry matter. During assays in controlled conditions, Attar of roses (Pb hyperaccumulator) significantly acidified its rhizosphere and increased Dissolved Organic Carbon (DOC) concentration as compared to Concolor Lace (non-accumulator), probably due to enhanced exudation in response to the metal stress. Lead concentrations in both cultivars were best correlated with CaCl2 extracted Pb. Extended X-ray Absorption Fine Structure (EXAFS) and Environmental Scanning Electron Microscopy-Energy Dispersive x-ray Spectroscopy (ESEM-EDS) demonstrated that Pb was mainly complexed to organic acids within plant tissues whereas the dominant form in soil was PbSO4. Parallel to the soil-plant Pb transfer assays, a genetic transformation protocol was optimized in view of better understanding biochemical processes involved in lead hyperaccumulation and gene function, in the future. The best regeneration scheme was based on the pre-culture of explants on 10 μM TDZ (Thidiazuron) in addition to 1 mg L-1 each of N6-benzylaminopurine (BAP) and α- naphthaleneacetic acid (NAA), followed by removal of TDZ from the culture medium. Kanamycin and hygromycin proved to be efficient selectable markers for genetic transformation. Two Agrobacterium strains, C58 and EHA105 harboring binary vectors carrying the selectable marker genes hpt and nptII were chosen for transformation experiments. They also contained the uidA gene coding sequence as reporter gene. After infecting with C58, 4 and 107 rooted plants on hygromycin-containing medium were obtained for Attar and Atomic cultivars, respectively. The four Attar plants and 82 Atomic plants expressed Gus in leaves, petioles, stems and roots as expected with a sequence driven by the 35S constitutive promoter. Polymerase Chain Reaction (PCR) screening was performed on Gus positive plants and 2 and 20 plants of Attar and Atomic were screened as PCR positive, respectively. After infection with EHA105, 23 and 133 rooted plants were obtained on kanamycin selection medium but none of these expressed Gus. Southern hybridization patterns will enable to correlate gene copy numbers to expression levels in these different events. The optimized protocols could be used for understanding molecular mechanisms of Pb accumulation and improvement in phytoextraction technique.

The continuity of the menace of mercury (Hg) is due to the continuous production and use of Hg and Hg containing products. Toxicity is just an outfall of use and exposure. Anthropogenic activities such as coal combustion and artisanal and small-scale gold mining have led to increasing Hg contamination and is the major source of Hg pollution into the environment that needs to be remediated. This study aimed to assess the phytoextraction capability of sweet sorghum (Sorghum bicolor) under different fertiliser treatments in Hg-contaminated soil. The potted experiment in a controlled environment included control S. bicolor and three phytoremediation treatments, i.e., Hg only; the addition of 4:1 green compost and; the addition of 0.2% NPK fertiliser. There were conspicuous signs of Hg phytotoxicity in plants with Hg only, namely wilting, senescent, inhibition of growth, and photosynthesis. There was stunted growth, but healthy plants observed in the treatment with the addition of green compost towards the end (day 60) of exposure. However, S. bicolor grew well until the last day of exposure in the treatment with the addition of 0.2% NPK fertiliser. Thus, this treatment showed the most effective phytoextraction potential of S. bicolor in Hg-contaminated soil. The effectiveness of S. bicolor in reducing the level of mercury was best assessed in the Hg bioavailable concentration in the spiked soil in which the Hg + NPK treatment has the lowest (0.77 mg kg−1). That resulted in the highest uptake (84.31%) percentage of Hg concentration recorded in the treatment with the addition of 0.2% NPK fertiliser compared to the other two treatments. The results suggest that the proportion of phosphate in the NPK fertiliser used, plays a huge role in the phytoextraction of Hg in the contaminated soil by S. bicolor. The Translocation Factor (TF) and Bioconcentration Factor (BCF), although higher within Days 20 and 40, was greater than 1 at the end of the exposure period suggesting a high probability that Hg was significantly transferred to the aerial parts of the plants. This is regarded as typical hyperaccumulator plant species. While S. bicolor was able to reduce the level of Hg in all three treatments, Hg + NPK treatment gave overall best results in physiological growth, the uptake, and reducing the level of Hg bioavailable in the spiked soil in terms of the effectiveness of phytoremediation method. / Environmental Sciences / M. Sc. (Environmental Science)