Dissertations / Theses on the topic 'Hyperaccumulation de nickel'

Phylogenetic studies are providing powerful new insights into the evolution of complex traits. Metal hyperaccumulation is an unusual and complex physiological trait found in about 500 plant species and is associated with an exceptionally high degree of tolerance of metalliferous soils. Alyssum L. (Brassicaceae) is the largest known hyperaccumulator genus, comprising approximately 188 species distributed throughout the Mediterranean region and south-west Asia. Approximately one-quarter of these are largely restricted to areas of serpentine soils and have the ability to accumulate nickel to high concentrations in shoot tissue. This genus provides a good example in which to study the origins of a complex physiological trait, but its phylogeny is currently poorly understood. To produce a well-resolved phylogenetic tree to investigate the number and timing of origins of nickel hyperaccumulation within Alyssum, DNA sequences were generated for four chloroplast regions (matK, rps16–trnK, trnD–T and trnL–F) from 170 of 255 species in the tribe Alysseae. Additional sequencing was carried out for the chloroplast genes ndhF and rbcL and the nuclear gene PHYA. A Bayesian analysis employing a relaxed uncorrelated lognormal molecular clock and multiple fossil-age calibration points was carried out to reconstruct a time-calibrated phylogeny of this tribe using appropriate outgroups. Optimization of the nickel hyperaccumulation trait onto the resulting phylogenetic tree suggests that nickel hyperaccumulation arose twice in the Alysseae in the late Miocene/early Pliocene: 3.3–8.3 Mya in Alyssum and 6.3–8.8 Mya in Bornmuellera. The single origin in Alyssum is strongly associated with a significant acceleration in net species diversification rate, suggesting the ability to hyperaccumulate nickel could have provided a key evolutionary innovation facilitating rapid range expansion and subsequent species diversification. The scattered distribution of nickel hyperaccumulators across small island-like patches of serpentine soil suggests that allopatric speciation may have driven rapid diversification in this clade.

Au regard de l’amenuisement des ressources primaires et de l’augmentation de la production mondiale de déchets, le concept d’agromine propose de phytoextraire les métaux contenus dans les matériaux délaissés. La solution proposée dans ce concept s’inspire de la Nature (SBN) et des principes de l’agronomie et s’inscrit dans une démarche d’économie circulaire. Ainsi, les plantes hyperaccumulatrices (HA) sont capables de prélever les métaux à partir de leur système racinaire pour les stocker à de hautes concentrations dans leurs parties aériennes. Les enjeux de la thèse sont de valoriser des déchets ou matériaux secondaires par l’extraction des éléments d’intérêt qu’ils contiennent et d’identifier les plantes à même de pouvoir se développer sur ces milieux. L’objectif est de formuler à partir des matériaux choisis un substrat fonctionnel, c’est-à-dire capable de rendre un service de fourniture en nickel (Ni). Dans cette optique, le substrat doit permettre l’installation et le développement des HA pour parvenir au transfert des métaux vers les parties aériennes. Les travaux s’intéressent à une boue de phosphatation acide essentiellement composée de Fe, Zn, P et Mn et contenant 0,5% de Ni. Des tests de germination et de croissance ont été effectués avec différents substrats élaborés à partir de cette boue assemblée avec un mélange terreux. Le substrat retenu est constitué de 10% de boue et 90% de terre (m/m). Sur celui-ci, l’HA Alyssum murale produit une biomasse supérieure comparée à un sol témoin (sol ultramafique au même pH et contenant la même quantité de Ni biodisponible), malgré des signes de toxicité pour les plantes. Un des verrous majeurs est la forte toxicité due à la présence de 6% de Zn dans la boue. Deux voies d’amélioration du substrat sont expérimentées : i) l’utilisation d’amendements et ii) la disposition des matériaux dans le profil. L’amendement le plus efficace est un biochar de bois ; il améliore le développement des plantes et ainsi la quantité de Ni phytoextraite. De plus, en modifiant la disposition des matériaux au sein du profil par une répartition en couches, la production de biomasse et la phytoextraction sont améliorées. Ce dispositif permet de lever la toxicité liée au Zn. Il apparait essentiel de contrôler le pH du substrat lors d’une multi-contamination car l’immobilisation du métal varie selon l’élément. L’association du génie pédologique et du génie végétal a permis de formuler un substrat fonctionnel pour la récupération d’éléments d’intérêt tel que le Ni. Ces travaux démontrent la possibilité de valoriser des sous-produits appelés classiquement « déchets » pour obtenir une plus-value, diminuant aussi leur charge métallique et faisant émerger une nouvelle source de métaux « d’origine végétale » obtenue par l’agromine
In view of the depletion of primary resources and the increase in global waste production, the concept of agromining proposes phytoextracting the metals contained in abandoned materials. The solution proposed in this concept is inspired by Nature (NbS) and the principles of agronomy and is part of a circular economy. Thus, hyperaccumulator plants (HA) are able to collect metals from their root system and to store them at high concentrations in their aerial parts. The challenges of the thesis are to give value to waste or secondary materials by extracting the elements of interest that they contain and to identify the plants able to develop on these media. The objective is to formulate, from the chosen materials, a functional substrate, that is to say, capable of rendering a Ni supply service. From this point of view, the substrate must allow the installation and the development of the HAs in order to transfer the metals to the aerial parts. The work focuses on an acid phosphating sludge essentially composed of Fe, Zn, P and Mn and containing 0.5% Ni. Germination and growth tests were carried out with different substrates prepared from this sludge assembled with a soil sample mixture. The retained substrate consists of 10% sludge and 90% soil (w/w). On the latter, HA Alyssum murale produces a higher biomass compared to a control soil (ultramafic soil at the same pH and containing the same amount of bioavailable Ni), despite signs of toxicity to plants. One of the major locks is the high toxicity due to the presence of 6% Zn in the sludge. Two ways of improving the substrate are tested: i) the use of amendments and ii) the arrangement of materials in the profile. The most efficient amendment is a wood biochar; it improves the development of plants and thus the amount of phytoextracted Ni. In addition, by modifying the layout of the materials within the profile by a layered distribution, biomass production and phytoextraction are improved. This device makes it possible to remove Zn-related toxicity. It is essential to control the pH of the substrate during multi-contamination because the immobilization of the metal varies according to the element. The association of soil engineering and plant engineering has made it possible to formulate a functional substrate for the recovery of elements of interest such as Ni. This work demonstrates the possibility of upgrading by-products conventionally called "waste" in order to obtain a surplus value, also reducing their metallic charge and bringing about a new source of "plant-derived" metals obtained by agromining

L’agromine concerne la récupération de métaux stratégiques dans les sols métallifères par la culture de plantes hyperaccumulatrices de métaux (et metalloïdes). Le moteur de cette recherche était d'évaluer le potentiel des ressources végétales mexicaines pour le développement de l'agromine. Les principaux objectifs étaient d'identifier et d'étudier quelques espèces de plantes hyperaccumulatrices de métaux au Mexique, et d'évaluer l'agronomie d'une de ces espèces avec des caractéristiques prometteuses pour l’agromine. Nous avons d'abord effectué des explorations dans trois régions ultramafiques riches en nickel (Ni) du centre et du sud du Mexique. Malgré la disponibilité du nickel dans le sol et les conditions climatiques, aucune hyperaccumulation de Ni n'a été trouvée dans ces régions. Une deuxième stratégie basée sur la phylogénie végétale comme outil de prédiction de l'hyperaccumulation des métaux a été suivie. Au total, dix espèces hyperaccumulatrices de métaux ont été identifiées au cours de cette recherche (Rubiaceae et Violaceae) dans des sols riches en Ni influencés par l'activité volcanique, dans le sud-est du Mexique ; la majorité d’entre elles n’était pas identifiée comme hyperaccumulatrices. Nos études ont révélé deux des hypernickelophores les plus puissants détectés jusqu'à présent (>4% wt Ni) et deux nouveaux genres hyperaccumulateurs de nickel (Orthion et Mayanaea). Une attention particulière a été accordée à l'hypernickelophore Blepharidium guatemalense. Le phloème des feuilles, des racines, des tiges et des pétioles de cette plante est très riche en Ni, ce qui suggère un mécanisme de redistribution via le phloème. Différentes pratiques agronomiques ont été testées pour cette plante. La fertilisation inorganique a fortement augmenté l'absorption du Ni sans modifier la croissance ou la biomasse de la plante, tandis que la fertilisation organique a augmenté la biomasse de la plante avec un effet négligeable sur les concentrations de Ni dans les parties aériennes. Une parcelle avec une culture de 5 ans, qui a ensuite été récolté deux fois par an, produit le rendement maximal en Ni de 142 kg ha⁻¹ an⁻¹. Blepharidium guatemalense est un candidat de choix pour l'agromine du Ni en raison de ses caractéristiques appréciables : absorption extrêmement efficace du Ni, production élevée de biomasse, taux de croissance rapide, et facilité de reproduction
Agromining technology involves the recovery of strategic metals from metalliferous soils through the cultivation of metal(loid) hyperaccumulator plants. The impetus of this research was to evaluate the potential of Mexican plant resources for the future development of agromining. The main objectives were then to identify and to study some metal hyperaccumulator plant species in Mexico, and to assess the agronomy of one promising “metal crop” for agromining. We first undertook field explorations in three nickel-rich ultramafic regions of central and southern Mexico. Despite the availability of soil and climatic conditions, no nickel (Ni) hyperaccumulation was found in any of these regions. A second strategy based on plant phylogeny as a prediction tool for metal hyperaccumulation was followed. In total, ten plant metal hyperaccumulator species were identified during this research (Rubiaceae and Violaceae) in Ni-enriched soils influenced by volcanic activity in Southeastern Mexico; most of them were priorly unknown. Our studies revealed two of the strongest hypernickelophores reported so far (>4%wt Ni) and two new Ni hyperaccumulator genera (Orthion and Mayanaea). Special focus was given to the hypernickelophore tree Blepharidium guatemalense. The phloem on leaves, roots, stems and petioles of this plant are the richest in Ni suggesting an unusual re-distribution mechanism via the phloem. Different agronomic practices were tested for this plant. Synthetic fertilization strongly increased nickel uptake without any change in plant growth or biomass, whereas organic fertilization enhanced plant shoot biomass with a negligible effect on foliar Ni concentrations. A 5-year-old stand which was subsequently harvested twice per year produced the maximum Ni yield tree⁻¹ yr⁻¹, with an estimated total nickel yield of 142 kg ha⁻¹ yr⁻¹. Blepharidium guatemalense is a prime candidate for Ni agromining on the account of its valuable traits: extremely efficient Ni uptake, high biomass production, fast growth rate, and easy to reproduce

La Nouvelle-Calédonie possède une flore riche, diverse et unique au monde. Sa forte endémicité (74,7%) résulte en partie de l’origine gondwanienne de la flore et de la forte pression de sélection exercée par les sols ultramafiques, riches en éléments traces métalliques, dont le nickel. Si cet élément fait la richesse du pays par son exploitation minière, cette dernière, ainsi que l’anthropisation du territoire engendrent une détérioration des écosystèmes. Dans le cadre de la dynamique mondiale de conservation, de protection et de restauration de la biodiversité, il convient de caractériser au mieux la flore néo-calédonienne foisonnante de plantes uniques. Les graines, innovation des végétaux supérieurs assurant leur dissémination, sont un des points clés du succès de la domination mondiale des plantes supérieures et un outil indispensable à la restauration écologique. Notre étude s’est attachée à caractériser par une approche biochimique la biologie des graines de deux plantes endémiques exceptionnelles, Amborella trichopoda, soeur de toutes les plantes à fleurs et Psychotria gabriellae, une des plantes contenant le plus de nickel dans ses feuilles au monde.La caractérisation protéomique de la graine d’Amborella trichopoda a permis d’obtenir le premier protéome d’un embryon rudimentaire. L’étude de ce cortège protéique a apporté des éléments de réponse aux nombreuses questions que soulèvent les graines à dormance morphologique comme celles d’A. trichopoda. Notamment, nous avons montré que l’embryon rudimentaire a acquis un stade de maturité moléculaire (présence de protéines chaperons, de réserves lipidiques). L’étude phylogénique de ces protéines a permis de conforter la place basale d’A. trichopoda. La caractérisation de l’évolution du protéome au cours de la germination a quant à elle mise en évidence une utilisation massive des protéines de réserve avant la fin de la germination ce qui questionne la définition de la germination sensu stricto pour les espèces à embryon rudimentaire.Une précèdente étude du cortège protéique de P. gabriellae avait révélé la présence de protéines DING notamment impliquées dans l’homéostasie d’éléments minéraux via leur interaction avec des transporteurs de type ABC ou encore en séquestrant elles-mêmes le phosphore. Leur identification fut corrélée avec l’observation d’un gradient de nickel dans la graine visant à protéger l’embryon du caractère toxique de ce dernier. Les données recueillies au cours du présent travail ont permis de confirmer la présence de ces protéines dont l’origine eucaryotique fait cependant débat. Face à cette controverse, nous avons cherché à identifier la présence de bactéries chez la graine mature sèche. Quatre bactéries endophytes de graine ont été identifiées mais aucune ne semble produire de protéines DING. Le rôle de ces protéines dans la physiologie de la graine de P. gabriellae et dans l’adaptation au nickel restent à explorer.Par ailleurs, cette approche protéomique a été complétée par l’obtention des transcrits exprimés au cours de la formation de la graine de Psychotria gabriellae. Cette base de données représente une source de données génomique utile pour approfondir les mécanismes impliqués dans la mise en place de l’hyperaccumulation de nickel chez les plantes, mécanismes qui pourront un jour s’avérer utiles pour répondre à des questions de phytoremédiation
New Caledonia possesses one of the world most rich, diverse and unique flora. Its high endemism (74,7%) is partly due to the gondwanian origin of its flora and to the high speciation induced by the ultramafic soils rich in heavy metals, including nickel. If this element is the source of the country richness, its mining exploitation and human colonization of the land induce ecosystems degradation. The study and comprehension of the new Caledonian flora is essential to be able to preserve, protect and restore its rich biodiversity. Preservation and restoration both depend on seeds. They are the unit of dispersal of higher plants, and responsible of their world domination on flora. We focused our study on the biochemical characterisation of seed biology of two extraordinary species, Amborella trichopoda, the sister to all extant flowering plants and Psychotria gabriellae, one of the world most nickel hyperaccumulating plant.Proteomic characterisation of A. trichopoda seeds was the first study that documented a rudimentary embryo proteome. This approach provides a better understanding of the mechanisms involved in the control of dormancy and germination of seeds with morphological dormancy such as A. trichopoda. The results obtained allow us to highlight the molecular maturity of the rudimentary embryo, as well as confirming the basal position of Amborella trichopoda trough phylogenetic analyses of selected protein families. The characterisation of the protein evolution during germination highlights massive mobilisation of storage proteins before the end of germination sensu stricto, and suggests a new definition of germination for seeds with rudimentary embryo.Previous proteomics characterisation of P. gabriellae seeds revealed a high representation of DING proteins that are known to be involved with ABC type transporters or to bind phosphorus. This observation was associated with an observed gradient of nickel inside the seed presumably to protect the embryo from its toxicity. During this work, we confirmed the presence of this protein family in the seeds, from which the belonging to the eukaryotic kingdom remains a subject of debate. To answer about the origin of these proteins in seed, we tried to determine the presence or not of bacteria in the dry mature seed. Four endophytic bacteria were identified but none of them seems to produce such proteins. However, the physiological signification of these bacteria to account for physiological features of the Psychotria gabriellae seeds and their exceptional tolerance toward nickel toxicity remains to be established.Beside this proteomics approach, we sequenced a large number of transcripts expressed during Psychotria gabriellae seed formation. This database will enrich the very limited genomic data available for this specie. It will allow a better understanding of the mechanisms involved in nickel hyperaccumulation, and may highlight novel tools for phytoremediation

Des modèles prédictifs de prélèvement d’éléments traces métalliques (ETM) par des plantes hyperaccumulatrices sont à développer pour rendre la phytoextraction opérationnelle. Ce travail a pour objectif de développer, calibrer et valider un modèle biophysique combiné d’accumulation foliaire et de mise en solution du nickel lors de cultures de l’hyperaccumulateur Leptoplax emarginata sur un sol fertilisé et contaminé en Ni. Une partie de ce modèle intègre un facteur de bioconcentration lié à la transpiration (TSCF) qui caractérise le mode de transport principal du Ni à travers la racine et jusqu’aux feuilles, lors d’une cinétique couplée de production de biomasse foliaire et de transpiration. Sur des plantes intactes et transpirantes, nous avons déterminé un TSCFNi supérieur à 1 du fait : (i) d’une grande perméabilité des racines à la fois au nickel et à l’eau et (ii) d’un transport actif du Ni largement prédominant. A l’opposé, le TSCFNi du blé de Printemps, plante exclusive, était inférieur à 0,02, et le coefficient de réflection correspondant proche de 1, ce qui caractérise des racines perméables à l’eau mais quasiment pas au nickel. L’exceptionnelle capacité de L. emarginata à accumuler et à tolérer le nickel dans ses feuilles, et plus précisément dans ses épidermes, serait également attribuable à ses transpiration et production de protéines soufrées très élevées, tout particulièrement au niveau de ses feuilles les plus jeunes. Enfin, après avoir couplé notre modèle biophysique d’accumulation foliaire du nickel au modèle de mise en solution des ETM développé par Ingwersen et al. (2006), nous avons optimisé les paramètres du modèle, notamment les paramètres physico-chimiques, et avons validé notre modèle sur des données cinétiques conjointes de quantités de nickel accumulé dans les feuilles de l’hyperaccumulateur et de concentration en nickel dans la solution du sol. Les perspectives de ce travail sont (i) un approfondissement des relations entre l’accumulation foliaire du nickel (ou d’un autre ETM) par un hyperaccumulateur, la transpiration et la production de protéines soufrées permettant une complexation de l’ETM et (ii) une adaptation du modèle pour le terrain, ce qui nécessite notamment une meilleure utilisation du couplage production de biomasse foliaire/transpiration et une prise en compte des cinétiques d’humectation et de dessiccation du sol (équation de Richards de transport d’eau en conditions non saturées), ce qui conduira à la mise au point d’un modèle 1D (la profondeur du sol) d’accumulation foliaire et de mise en solution d’ETM
To make phytoextraction practically feasible, predictive models of metal uptake by hyperaccumulators need to be developed. The aim of this work was to design, calibrate and validate a biophysical combined model of nickel foliar accumulation and availability in soil solution during cultures of the hyperaccumulator Leptoplax emarginata on a fertilized and Ni-contaminated sandy topsoil. We succeed in this. Part of the model integrates a transpiration bioconcentration factor (TSCF) which characterized the main Ni transport through the root and to the leaves. We determined a TSCF value greater than 1 for L. emarginata, which was attributed to (i) a high root permeability to both Ni and water and (ii) a predominant Ni active transport. By contrast, Spring wheat was characterized by a TCSF value less than 0.02 and a reflection coefficient very near 1, indicating that its roots are permeable to water but quite unpermeable to nickel. The high capacity of L. emarginata to tolerate and accumulate Ni in their leaves should also be attributed to its large transpiration and sulfur accumulation, particularly in their youngest leaves. Perspectives of this work are (i) a detailed study on relations between Ni accumulation, transpiration and production of sulphur proteins and (ii) a field adaptation of the model taken into account water transport in unsaturated conditions, leading to design a combined 1D model of nickel foliar accumulation and availability in soil solution

Metal hyperaccumulating plants accumulate exceptionally high concentrations of metal ions in their above ground tissues and are defined as containing 1000 μg/g dry mass Co, Cu, Cr, Pb, Zn or Ni. This is remarkable because plants typically only require small amounts of these metals for survival, such as 0.004 μg/g Ni and 15-20 μg/g Zn. Scientific investigation has sought to understand the mechanisms underpinning hyperaccumulation in order to apply them in the phyto-technological processes of phytoremediation (removal of metal pollutants from the environment) and phytomining. However, little is known about the molecular mechanisms underlying Ni hyperaccumulation despite the fact that Ni hyperaccumulators account for almost three quarters of all known hyperaccumulating species. A comparative RNA-Seq experiment carried out on Ni accumulating and non-accumulating populations of the South African Ni hyperaccumulator Senecio coronatus (Asteraceae) identified a number of putative transport proteins that are constitutively upregulated in the hyperaccumulator plants. This MSc project focused on two of these, iron regulated 2 (ScIREG2) and iron regulated transporter 1 (ScIRT1), and aimed to validate the RNA-Seq derived nucleotide sequences, test for Ni transport activity and determine their sub-cellular localisation. Full-length ScIREG2 and ScIRT1 protein coding sequences were obtained using RT-PCR and conformed to the predicted sequences derived from the RNA-Seq data. Heterologous expression of ScIRT1 in yeast consistently conferred an increased Ni resistance phenotype to yeast across a variety of experimental conditions, suggesting that this protein is capable of transporting Ni, and may function as a Ni export protein in yeast. In contrast, the results obtained from heterologous expression of ScIREG2 were variable and thus inconclusive. An attempt was made to determine the subcellular localization of ScIRT1 using transient expression of an ScIRT1-YFP fusion protein in onion cells. While inconclusive, a YFP signal was detected in these cells, and appeared to localise to the plasma membrane. The work conducted serves as a pilot study to optimize the experimental systems necessary to identify Ni transporters from S. coronatus. These experimental systems can now be applied to characterise the remaining transport proteins identified in the RNA-Seq analysis.

Due to the high nickel concentrations, serpentine soils provide a very restrictive and selective environment for plant life. Some plants, termed “Ni-hyperaccumulators”, are adapted to live on these heavy-metal-enriched soils without toxicity symptoms. Ni-hyperaccumulators are increasingly important for research on metal tolerance, homeostasis and biotechnological applications. This project aims to investigate nickel accumulation in taxa and populations of Odontarrhena, a genus of tribe Alysseae (Brassicaceae) that includes over 85 species many of which are Ni-hyperaccumulators. Based on a previous systematic study conducted on poorly-known populations of Odontarrhena native to Albania we performed a molecular study to characterize taxa and populations of this genus. To this purpose we used DNA sequencing and the AFLP-fingerprint technique to reconstruct the species phylogenetic relationships and the population differentiation patterns in relation to their distribution, ploidy level, intensity of anthropic site disturbance, altitude, soil type and metal concentration population (Ni, Cr, Co, Ca, Mg). We found significant population differentiation, dominance of within-population variation, no isolation by geographic distance and existence of six genetic groups variously represented across the six taxa possibly due to hybridization especially in disturbed sites. Next, we compared metal concentrations in native Odontarrhena populations from Albania in relation to their soil of origin. We determined the concentration of the most important trace metals (Ni, Co, Cr, Mg, Ca, K, Fe and Mn) in soil, plant roots and shoots of five taxa from 20 different outcrops. We found large differences in mineral element concentrations in soils and also between the plants; shoot Ni concentrations in Albanian Odontarrhena taxa depend on soil Ni concentrations but not on species identity. For O. chalcidica, the most widely distributed species, this “environmental fingerprint” was found not only for Ni, but also for Ca and Mg. After these investigations on native populations from the natural environment, we designed an experimental study in controlled conditions. Plant seedlings of seven taxa and 11 populations of Odontarrhena from serpentine and non-serpentine sites of the Balkan peninsula and Italy were cultivated in hydroponics with increasing NiSO4 concentrations to determine plant growth and Ni accumulation. These plantlets were analyzed to test inter- and intra-specific differences in nickel tolerance and accumulation, in relation to Ni levels in the soils and in wild plants. We found a metal stimulatory effect on growth that was present in the low-dose zone and significantly fitted the Brain-Cousens hormetic model. Taxa showed broad variation in tolerance, with the most tolerant plants requiring the highest Ni concentration for optimal growth. Our data suggested that tolerance is associated with hyperaccumulation ability. Among the obligate and facultative serpentinophytic species of Odontarrhena that have been investigated we found a notable exception, O. sibirica, a facultative serpentinophyte in which accumulation ability was enigmatic from previous studies. We addressed this issue using observational and experimental methods as in our previous researches. We found that Ni-concentrations in the native populations sampled on serpentine soils in Greece were always much lower than the hyperaccumulation threshold. When cultivated together with other Ni-accumulating Odontarrhena species on the same natural ultramafic soil, O. sibirica was the only one unable to accumulate the metal. When grown in hydroponics at different NiSO4 levels Ni-accumulation occurred only at higher concentrations which, however, had a toxic effect. This peculiar combination of Ni-response traits could be the result of a partial evolutionary loss of ability with respect to all other Ni-accumulating congeneric species. For its unique characteristics, O. sibirica could therefore represent a unique model system for further studies on the evolutionary dynamics, physiological mechanisms and genetic control of metal accumulation and homeostasis. In a parallel study, we investigated photosynthesis responses of the same plants using an experimental approach. In non-hyperaccumulator plants, toxicity symptoms to above 10 μg g-1 DW nickel concentrations in soils can include inhibition of photosynthesis, impaired nitrogen assimilation and disturbed enzyme activity. However, there is a complete lack of information about how Ni-hyperaccumulators reconcile that extraordinary amount of metal in their shoots with an efficient photosynthetic activity, or at least on which photosynthetic parameters the excess of Ni impacts less in these plants. We measured Ni effects on growth, root and shoot metal accumulation and several photosynthetic parameters, such as gas exchange, chlorophyll fluorescence analyses and pigments content in three Odontarrhena taxa (two hyperaccumulators, one not) grown in hydroponics and exposed to three NiSO4 treatments. We found that Ni-hyperaccumulators species are photosynthetically more efficient under Ni excess in respect to the non-accumulating species. In fact, Ni treatment in O. chalcidica increased not only the photochemical efficiency of PSII and the CO2 assimilation rate, but also the stomatal conductance. Finally, this project focused on the determination of the activity of the enzyme urease, the only Nimetalloenzyme known so far in plants, in selected Odontarrhena taxa. The hypothesis to test was whether the high basal requirement for this micronutrient in these plants could be linked to a depletion of the Ni cytosolic pool at low external metal concentration, due to hyperaccumulation mechanism and impairing urease activity. To this purpose, enzyme activity and Ni shoot concentration were determined in plants of accumulating and non-accumulating taxa of Odontarrhena cultivated on Ni-rich serpentine soil and on garden soil, as well as in samples of O. bertolonii cultivated in hydroponics at increasing Ni concentrations. Odontarrhena hyperaccumulators showed similar urease activity when grown on both kinds of soils, with no relation between the enzyme activity and the leaf Ni accumulation. Contrarily, clear indications came from the experiment in controlled conditions, where the presence of Ni determined a progressive stimulation, in respect to control samples, of the activity of the enzyme, associated with an increase in shoot metal concentration. A significant relationship was found between the levels of urease activity and the amount of Ni accumulated in the leaves. Therefore, the already known Ni-stimulated growth of O. bertolonii at increasing metal concentrations in the low-dose zone could be explained by a Ni-induced activity of urease, associable to an enhanced nitrogen metabolism, unless other still unknown physiological functions of Ni in hyperaccumulating plants.

L'hyperaccumulation est un phénomène qui a été découvert il y a 45 ans seulement, il a fait l'objet de nombreuses recherches, en raison du comportement inhabituel des métaux dans l'écosystème et en raison du potentiel de solutions fondées sur la nature qui en découle. La description du cycle biogéochimique du Ni des plantes hyperaccumulatrices est nécessaire pour élucider leur rôle écologique dans leur environnement naturel, mais aussi pour comprendre leur comportement potentiel dans des cultures tropicales d’agromine. L'exploitation agricole et l'exportation de la biomasse riche en Ni interrompront le cycle du Ni. Il est donc important de comprendre les mécanismes qui régissent le cycle biogéochimique du Ni dans les systèmes naturels et de cultures : Quels sont les cycles du Ni et leur impact sur le fonctionnement écologique des forêts tropicales d’hyperaccumulateurs ? Quelle est la vitesse des flux de Ni à travers les compartiments sol-plante et quelle est la dynamique et le renouvellement du Ni dans un système tropical d'hyperaccumulateurs ? À quelle vitesse une culture tropicale Agromine peut-elle épuiser le Ni dans le sol ? Comment pouvons-nous gérer la fertilisation des sols pour une culture d’agromine tropicale de Ni durable ? Les objectifs de ce doctorat étaient donc les suivants (i) d'étudier le cycle biogéochimique d'une forêt naturelle de Phyllanthus rufuschaneyi afin d'évaluer les flux de Ni dans l'écosystème ; (ii) de manipuler un tel écosystème afin d'effectuer un test de sensibilité de l'écosystème pour le flux suivant : retour de la litière au sol ; de l'absence de retour (exportation) à un doublement du retour ; (iii) d'optimiser le système de culture de P. rufuschaneyi pour l'agromine du Ni. Deux peuplements parallèles de P. rufuschaneyi ont été instrumentés, surveillés et comparés pendant deux ans (2018 et 2019), (i) une forêt secondaire naturelle de 100 m2 et (ii) un champ densément planté dans lequel les retours de litière au sol ont été calibrés ; de l'absence de retour (exportation) à un doublement du retour. Cette étude n'a pas révélé l'allopathie des plantes hyperaccumulatrices tropicales. Les plantes hyperaccumulatrices de Ni influent sur la constitution des stocks de Ni disponibles dans les couches arables. Le cycle du Ni est principalement régi par des flux internes, notamment la dégradation et le recyclage de la litière. Ce pourcentage de renouvellement du Ni ne semble pas être influencé par le taillis à court terme mais par la présence initiale de litière sur le sol. La fertilisation NPK n'a aucun effet sur le rendement en Ni à court terme (75 kg Ni ha-1 an-1), même si la fertilisation en azote a tendance à réduire le rendement en Ni de P. rufuschaneyi. Il convient de tenir compte du renouvellement du nickel lors de la conception des cultures d’agromine tropicales. Une étude plus approfondie de l’altération permettrait de préciser son rôle dans la reconstitution des stocks de Ni disponible et de nutriments
Hyperaccumulation is a phenomenon that was only discovered 45 years ago, it has been the focused of very intensive research because of the unusual behaviour of metals in the ecosystem and also because it offers a vast potential for nature-based solutions. Describing the Ni biogeochemical cycle within the soil-hyperaccumulator plants ecosystem is necessary to elucidate the ecological role of hyperaccumulator plants in their natural environment, but also to understand their potential behaviour under tropical agromining systems. Agromining and exporting Ni rich-biomass will interrupt the cycle. It is therefore important to understand the mechanisms which govern the Ni biogeochemical cycle in both natural and agromining systems: What are the Ni cycles (internal and external) and their impact on the ecological functioning of tropical hyperaccumulator forest? How rapid are the Ni fluxes across the soil-plant compartments, and what is the turnover of Ni in a hyperaccumulator tropical system? How fast can a tropical Agromining crop deplete Ni in soil? How can we manage soil fertilisation for a sustainable tropical Ni agromining crop? Therefore, the objectives were: (i) to study the biogeochemical cycling of a natural forest of Phyllanthus rufuschaneyi in order to assess and evaluate the natural fluxes of Ni in the ecosystem; (ii) to manipulate such an ecosystem in order to perform a sensitivity test of the ecosystem for the following flux: litter return to the soil; (iii) to optimize the cropping system of P. rufuschaneyi for Ni agromining. Two parallel stands of P. rufuschaneyi were instrumented, monitored and compared over two years (2018 and 2019), (i) a natural secondary 100-m2 forest and (ii) a densely planted field in which litter returns to the soil were calibrated; from no return (export) to a doubling of the return.This study did not prove allelopathy of tropical hyperaccumulator plants, despite the extreme influence of Ni hyperaccumulators in building up available Ni stocks in topsoils. Nickel cycle was mainly driven by internal fluxes, i.e. degradation and recycling of the hyperaccumulator biomass. The percentage of Ni recycled by litterfall tended to decrease with increasing litter addition to the soil and was not influenced by coppicing, at least in the short term. Major nutrient (NPK) fertilisation did not affect Ni yield (i.e. 75kg Ni ha-1 yr-1) in the short term either, even if N fertilisation reduced Ni concentrations in leaves and plant biomass production. Nickel turnover should be taken into account when designing tropical agromining crops and natural secondary forests are a good surrogate to evaluate the long term impacts of agromining. Further study of the weathering processes would help to precise the contribution of bedrock and soil mineral horizons in the Ni and nutrient budgets of the system

Reliable, high-throughput and low-cost next-generation sequencing technologies have invigorated genetic research into non-model organisms over the last decade. In this work, RNA-Seq was employed to obtain the first-ever transcriptomes of two groups of closely related plant taxa possessing distinctive complex physiological traits, namely metal hyperaccumulation and C4 photosynthesis. Metal hyperaccumulator plants possess an extraordinary ability to take up trace elements from the soil and accumulate them to high concentrations in their shoots, probably to serve as a type of elemental defence against natural enemies. Taxonomically, the most common form of metal hyperaccumulation, nickel hyperaccumulation, is encountered on nickel-rich ultramafic (serpentine) soils, and is found with the highest frequency (ca. 51 species) in the genus Alyssum (family Brassicaceae). Here, the genetic basis and evolutionary history of nickel tolerance and hyperaccumulation was investigated in the Alyssum serpyllifolium Desf. species complex, which contains both serpentine and non-serpentine populations of unresolved phylogenetic relationships on the Iberian Peninsula. Genome scans for outlier loci and differential expression analyses identified a number of candidate hyperaccumulator genes common to two serpentine populations found in Portugal and Spain, but the majority of adaptive variation was of local origin. Phylogenetic and population-genetic inferences based on neutral and putatively adaptive loci suggested that the key genes for the nickel hyperaccumulation trait evolved once and spread across serpentine populations early in the history of this species, with no genetic isolation but continued recent gene flow between serpentine and non-serpentine populations. To test the power of next-generation sequencing for analysing the genetic basis of a separate complex trait, a cross-species comparison was performed using RNA-Seq of two congeneric tropical species, the C4 plant Alternanthera pungens Kunth and the C3 plant Alternanthera philoxeroides (Mart.) Griseb. f. angustifolia Suess. (family Amaranthaceae). These species were cultivated at two different temperatures and showed significant differences in levels of overall gene expression plasticity and isoform switching in certain photosynthesis genes, which it is proposed may explain the observed difference in the ability of these two species to acclimate to low and high growth temperatures.

Les plantes hyperaccumulatrices peuvent accumuler des concentrations extraordinaires de métaux dans leurs parties aériennes (e.g. Ni, Zn et Cd). Cette thèse a été entreprise afin d’élucider : 1) comment le Ni est absorbé par les racines des hyperaccumulateurs, et 2) comment il circule dans les différents organes via le xylème et le phloème. Les travaux ont utilisé des cultures hydroponiques avec l’hyperaccumulateur de Ni et Zn Noccaea caerulescens en présence de concentrations faibles et élevées de Ni et Zn et en interaction avec Fe et Co ; des analyses isotopiques et d’expression de gènes ont été conduites. Les résultats ont montré que l’hyperaccumulateur N. caerulescens possède un système de transport du Ni à faible affinité et à haute efficacité. L’absorption du Ni semble impliquer principalement les transporteurs de Zn et Fe. Le transport par le xylème est la principale voie d'accumulation du Ni dans les jeunes feuilles et les feuilles âgées. Mais la translocation par le phloème est aussi une source importante de Ni pour les jeunes feuilles. Dans le phloème, le Ni est principalement chélaté par des acides organiques, de type malate. La thèse ouvre des perspectives pour l’optimisation des procédés de phytoextraction et d’agromine des sols contaminés
Hyperaccumulating plants are capable of accumulating extraordinary concentrations of heavy metals, e.g. Ni, Zn and Cd, in their shoots. This thesis was conducted to assess: 1) how roots of hyperaccumulators absorb Ni, and 2) how Ni circulates in different organs via xylem and phloem. Methods used were hydroponic cultures with the Ni/Zn hyperaccumulator Noccaea caerulescens in the presence of low and high Ni and Zn solutions, and in competition with Fe, Co, and Rb and Sr. Isotope fractionation in the plant, and gene expression of the Zn transporter ZIP10 and the Fe transporter IRT1 were studied. Results showed that the hyperaccumulator N. caerulescens takes up Ni mainly via low-affinity transport system, which seemed to be Zn and Fe transporters. Xylem transport is the main source for Ni accumulation in both young and old leaves, while phloem translocation also acts as an important source for young leaves. Ni is enriched in phloem sap and mainly chelated by organic acids especially malate during phloem translocation

The contamination of metals like Nickel (Ni) in the soil represents a serious threat worldwide. To counteract this phenomenon, hyperaccumulator plant species, able to remove metal from soil and store it at high concentration in shoots, are employed for metal phytoremediation purposes. Native microbial communities occurring in the rhizosphere of hyperaccumulators often promote plant growth and metal uptake. So far, each abiotic and biotic rhizospheric components (soil, root system and microbiota) have been used without considering the reciprocal interactions and the responses to Ni stress as a whole. The present study aims to develop for the first time an innovative and multidisciplinary approach to examine the rhizosphere of Ni-hyperaccumulators as a holistic model, promoting the plant development and the Ni uptake. This integrated system is feasible owing to the collaboration with the Laboratory of Micology and the Laboratory of Microbiology of Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa. Among metalliferous soils, specific attention was given to serpentinitic soil which display extremely hostile conditions (nutrient shortage and concentration of metals - e.g., Ni - highly toxic) for most plants except for some hyperaccumulator species. Early response to Ni in plant development was assessed with micro- and mesocosm germination tests under Ni stress in the Ni-hyperaccumulator species Alyssoides utriculata (L.) Medik, Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. and Odontarrhena bertolonii (Desv.) L. Cecchi & Selvi and on the related non-accumulator species Alyssum montanum L. and Thlaspi arvense L., used for comparison. Afterwards, the response to increasing Ni concentrations in terms of root surface area, root and shoot biomass and photosynthetic efficiency was evaluated. Subsequently, A. utriculata was selected as a good candidate to study rhizospheric components because of its Ni-facultative hyperaccumulation traits and its ability to thrive in harsh metalliferous soils. Related rhizosphere and bare soil samples were collected from serpentine and non-serpentine sites. Plant and soil samples were processed and analysed with specific attention to isolation and identification of culturable microbiota, then selected for their Ni-tolerance and Plant Growth Promoting (PGP) traits. Later, most performing Ni tolerant bacterial and fungal strains were tested by means of co-growth methods to estimate their reciprocal behaviour in a mixed culture to be used as inoculum in the rhizosphere of A. utriculata. Results demonstrate that increasing Ni concentrations can induce marked inhibition of germination in hyperaccumulator species, despite their accumulation ability. However, hyperaccumulator species exhibit a positive response in terms of root surface area, biomass and photosynthetic efficiency, compared to non-hyperaccumulator species in which there is a dose-response effect by Ni, except for T. arvense in pot test. In particular, A utriculata reveals an increased aboveground biomass and sample vitality in pot test, suggesting an adaptation to harsh environmental conditions. Microbiota isolates are more abundant in non-serpentinitic and rhizospheric soil, without selectivity between microorganisms and Ni. Some bacterial and fungal strains (Pseudomonas sp. SERP1, Streptomyces sp. SERP4 and Penicillium ochrochloron Biourge Serp03S, Trichoderma harzianum Rifai Serp05S respectively) reveal high Ni tolerance (up to 20 nM) and PGP traits. In particular SERP1 and Serp03S display a mutual synergism in co-growth methods and they could be promising candidates as natural chelators in the rhizosphere of A. utriculata, to enhance plant development and Ni uptake. This research represents the first step of integrated plant-microbiota tool, in the perspective to improve Ni uptake from polluted soil, using native Ni-hyperaccumulator species and associated rhizobiota, although further investigations are required to ascertain the efficiency of the field application.

Nickel hyperaccumulation is a unique plant adaption that has led to roughly 390 plant taxa being able to not only withstand the toxicity associated with Ni but actively translocate it to aerial tissues. However, the underlining molecular mechanisms that drive Ni hyperaccumulation remain unclear. Senecio coronatus, a Ni hyperaccumulator, is a novel species as both hyperaccumulating and non-accumulating populations can be found on the serpentine soils of the Barberton Greenstone Belt, South Africa. A comparative RNA-seq analysis on these populations of S. coronatus revealed that ScIRT1 and ScIREG2 , putative homologues of the Arabidopsis transporters, AtIRT1 and AtIREG2 which are capable of transporting Ni, showed much higher expression in the hyperaccumulating populations compared to the non-hyperaccumulating populations, suggesting a potential role in Ni hyperaccumulation. It was thus necessary to investigate whether ScIRT1 and ScIREG2 encode functional homologues of these Arabidopsis transporters. To accomplish this, irt1 and ireg2 mutants were obtained from a T-DNA insertion seed collection and their homozygosity was then determined by PCR genotyping. Since a lack of iron induces IRT1 and IREG2 expression, loss of gene expression of homozygous irt1 and ireg2 mutants by means of reverse transcriptase PCR on plant roots grown hydroponically in the absence of Fe was then done to establish full knock-out status. From this, homozygous mutants were identified, however, absence of gene expression for irt1 and ireg2 mutants was not clear. In addition to validating homozygosity, phenotypic characterisation, with the aim of developing reliable assays to be used in complementation analysis, was done by growing homozygous mutants and Col-0 in hydroponic media deficient in Fe and supplemented with Ni. The assays revealed that under Fe-deficient and Ni-supplemented conditions, a reduction in root biomass was a more reliable phenotypic characteristic for ireg2 mutants than root length or shoot biomass. In contrast, for irt1, no observable phenotype was established under Fe-deficiency conditions. In parallel, Gateway cloning was employed to create expression clones where ScIRT1 and ScIREG2 protein coding expression was to be driven by native Arabidopsis promoters pAtIRT1 and pAtIREG2 (i.e. pAtIRT1:ScIRT1 and pAtIREG2:ScIREG2) respectively for complementation of the Arabidopsis irt1 and ireg2 mutants. The open reading frames of the S. coronatus genes and the Arabidopsis promoters were PCR amplified, cloned into appropriate pDONR221 vectors, and sequence verified. The ScIREG2 clone however, revealed point mutations and could not be used. pAtIRT1 was successfully recombined with ScIRT1 to generate a two-fragment expression clone which was verified by DNA sequencing. Thus herein, the foundations for ScIRT1 and ScIREG2 complementation experiments have been established.

The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane.

Soil contamination with heavy metal(loid)s is a major environmental problem that requires effective and affordable remediation technologies. The utilisation of plants to remediate heavy metal(loid)s contaminated soils has attracted considerable interest as a low cost green remediation technology. The process is referred to as phytoremediation, and this versatile technology utilises plants to phytostabilise and/or phytoextract heavy metal(loid)s from contaminated soils, thereby effectively minimising their threat to ecosystem, human and animal health. Plants that can accumulate exceptionally high concentrations of heavy metal(loid)s into above-ground biomass are referred to as hyperaccumulators, and may be exploited in phytoremediation, geobotanical prospecting and/or phytomining of low-grade ore bodies. Despite the apparent tangible benefits of utilising phytoremediation techniques, a greater understanding is required to comprehend the ecophysiological aspects of species suitable for phytoremediation purposes. A screening study was instigated to assess phytoremediation potential of several fern species for soils contaminated with cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn). Hyperaccumulation was not observed in any of the studied species, and in general, species excluded heavy metal uptake by restricting their translocation into aboveground biomass. Nephrolepis cordifolia and Hypolepis muelleri were identified as possible candidates in phytostabilisation of Cu-, Pb-, Ni- or Zn-contaminated soils and Dennstaedtia davallioides appeared favourable for use in phytostabilisation of Cu- and Zn-contaminated soils. Conversely, Blechnum nudum, B. cartilagineum, Doodia aspera and Calochlaena dubia were least tolerant to most heavy metals and were classified as being least suitable for phytoremediation purposes Ensuing studies addressed the physiology of arsenic (As) hyperaccumulation in a lesser known hyperaccumulator, Pityrogramma calomelanos var. austroamericana. The phytoremediation potential of this species was compared with that of the well known As hyperaccumulator Pteris vittata. Arsenic concentration of 3,008 mg kg–1 dry weight (DW) occurred in P. calomelanos var. austroamericana fronds when exposed to 50 mg kg–1 As without visual symptoms of phytotoxicities. Conversely, P. vittata was able to hyperaccumulate 10,753 mg As kg–1 DW when exposed to 100 mg kg–1 As without the onset of phytotoxicities. In P. calomelanos var. austroamericana, As was readily translocated to fronds with concentrations 75 times greater in fronds than in roots. This species has the potential for use in phytoremediation of soils with As levels up to 50 mg kg–1. Localisation and spatial distribution of As in P. calomelanos var. austroamericana pinnule and stipe tissues was investigated using micro-proton induced X-ray emission spectrometry (µ-PIXE). Freeze-drying and freeze-substitution protocols (using tetrahydrofuran [THF] as a freeze-substitution medium) were compared to ascertain their usefulness in tissue preservation. Micro-PIXE results indicated that pinnule sections prepared by freeze-drying adequately preserved the spatial elemental distribution and tissue structure of pinnule samples. In pinnules, µ-PIXE results indicated higher As concentration than in stipe tissues, with concentrations of 3,700 and 1,600 mg As kg–1 DW, respectively. In pinnules, a clear pattern of cellular localisation was not resolved whereas vascular bundles in stipe tissues contained the highest As concentration (2,000 mg As kg–1 DW). Building on these µ-PIXE results, the chemical speciation of As in P. calomelanos var. austroamericana was determined using micro-focused X-ray fluorescence (µ-XRF) spectroscopy in conjunction with micro-focused X-ray absorption near edge structure (µ-XANES) spectroscopy. The results suggested that arsenate (AsV) absorbed by roots was reduced to arsenite (AsIII) in roots prior to transport through vascular tissues as AsV and AsIII. In pinnules, AsIII was the predominant species, presumably as aqueous-oxygen coordinated compounds. Linear least-squares combination fits of µ-XANES spectra showed AsIII as the predominant component in all tissues sampled. The results also revealed that sulphur containing thiolates may, in part sequester accumulated As. The final aspect of this thesis examined several ecophysiological strategies of Ni hyperaccumulation in Hybanthus floribundus subsp. floribundus, a native Australian perennial shrub species and promising candidate in phytoremediation of Ni-contaminated soils. Micro-PIXE analysis revealed that cellular structure in leaf tissues prepared by freeze-drying was adequately preserved as compared to THF freeze-substituted tissues. Elemental distribution maps of leaves showed that Ni was preferentially localised in the adaxial epidermal tissues and leaf margin, with concentration of 10,000 kg–1 DW in both regions. Nickel concentrations in stem tissues obtained by µ-PIXE analysis were lower than in the leaf tissues (1,800 mg kg–1 vs. 7,800 mg kg–1 DW, respectively), and there was no clear pattern of compartmentalisation across different anatomical regions. It is possible that storage of accumulated Ni in epidermal tissues may provide Ni tolerance to this species, and may further act as a deterrent against herbivory and pathogenic attack. In H. floribundus subsp. floribundus seeds, µ-PIXE analysis did not resolve a clear pattern of Ni compartmentalisation and suggests that Ni was able to move apoplastically within the seed tissues. The role of organic acids and free amino acids (low molecular weight ligands [LMW]) in Ni detoxification in H. floribundus subsp. floribundus were quantified using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC). Nickel accumulation stimulated a significant increase in citric acid concentration in leaf extracts, and based on the molar ratios of Ni to citric acid (1.3:1–1.7:1), citric acid was sufficient to account for approximately 50% of the accumulated Ni. Glutamine, alanine and aspartic acid concentrations were also stimulated in response to Ni hyperaccumulation and accounted for up to 75% of the total free amino acid concentration in leaf extracts. Together, these LMW ligands may complex with accumulated Ni and contribute to its detoxification and storage in this hyperaccumulator species. Lastly, the hypothesis that hyperaccumulation of Ni in certain plants may act as an osmoticum under water stress (drought) was tested in context of H. floribundus subsp. floribundus. A 38% decline in water potential and a 68% decline in osmotic potential occurred between water stressed and unstressed plants, however, this was not matched by an increase in accumulated Ni. The results suggested that Ni was unlikely to play a role in osmotic adjustment in this species. Drought stressed plants exhibited a low water use efficiency which might be a conservative ecophysiological strategy enabling survival of this species in competitive water-limited environments.

Doctor of Philosophy(PhD)
Soil contamination with heavy metal(loid)s is a major environmental problem that requires effective and affordable remediation technologies. The utilisation of plants to remediate heavy metal(loid)s contaminated soils has attracted considerable interest as a low cost green remediation technology. The process is referred to as phytoremediation, and this versatile technology utilises plants to phytostabilise and/or phytoextract heavy metal(loid)s from contaminated soils, thereby effectively minimising their threat to ecosystem, human and animal health. Plants that can accumulate exceptionally high concentrations of heavy metal(loid)s into above-ground biomass are referred to as hyperaccumulators, and may be exploited in phytoremediation, geobotanical prospecting and/or phytomining of low-grade ore bodies. Despite the apparent tangible benefits of utilising phytoremediation techniques, a greater understanding is required to comprehend the ecophysiological aspects of species suitable for phytoremediation purposes. A screening study was instigated to assess phytoremediation potential of several fern species for soils contaminated with cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn). Hyperaccumulation was not observed in any of the studied species, and in general, species excluded heavy metal uptake by restricting their translocation into aboveground biomass. Nephrolepis cordifolia and Hypolepis muelleri were identified as possible candidates in phytostabilisation of Cu-, Pb-, Ni- or Zn-contaminated soils and Dennstaedtia davallioides appeared favourable for use in phytostabilisation of Cu- and Zn-contaminated soils. Conversely, Blechnum nudum, B. cartilagineum, Doodia aspera and Calochlaena dubia were least tolerant to most heavy metals and were classified as being least suitable for phytoremediation purposes Ensuing studies addressed the physiology of arsenic (As) hyperaccumulation in a lesser known hyperaccumulator, Pityrogramma calomelanos var. austroamericana. The phytoremediation potential of this species was compared with that of the well known As hyperaccumulator Pteris vittata. Arsenic concentration of 3,008 mg kg–1 dry weight (DW) occurred in P. calomelanos var. austroamericana fronds when exposed to 50 mg kg–1 As without visual symptoms of phytotoxicities. Conversely, P. vittata was able to hyperaccumulate 10,753 mg As kg–1 DW when exposed to 100 mg kg–1 As without the onset of phytotoxicities. In P. calomelanos var. austroamericana, As was readily translocated to fronds with concentrations 75 times greater in fronds than in roots. This species has the potential for use in phytoremediation of soils with As levels up to 50 mg kg–1. Localisation and spatial distribution of As in P. calomelanos var. austroamericana pinnule and stipe tissues was investigated using micro-proton induced X-ray emission spectrometry (µ-PIXE). Freeze-drying and freeze-substitution protocols (using tetrahydrofuran [THF] as a freeze-substitution medium) were compared to ascertain their usefulness in tissue preservation. Micro-PIXE results indicated that pinnule sections prepared by freeze-drying adequately preserved the spatial elemental distribution and tissue structure of pinnule samples. In pinnules, µ-PIXE results indicated higher As concentration than in stipe tissues, with concentrations of 3,700 and 1,600 mg As kg–1 DW, respectively. In pinnules, a clear pattern of cellular localisation was not resolved whereas vascular bundles in stipe tissues contained the highest As concentration (2,000 mg As kg–1 DW). Building on these µ-PIXE results, the chemical speciation of As in P. calomelanos var. austroamericana was determined using micro-focused X-ray fluorescence (µ-XRF) spectroscopy in conjunction with micro-focused X-ray absorption near edge structure (µ-XANES) spectroscopy. The results suggested that arsenate (AsV) absorbed by roots was reduced to arsenite (AsIII) in roots prior to transport through vascular tissues as AsV and AsIII. In pinnules, AsIII was the predominant species, presumably as aqueous-oxygen coordinated compounds. Linear least-squares combination fits of µ-XANES spectra showed AsIII as the predominant component in all tissues sampled. The results also revealed that sulphur containing thiolates may, in part sequester accumulated As. The final aspect of this thesis examined several ecophysiological strategies of Ni hyperaccumulation in Hybanthus floribundus subsp. floribundus, a native Australian perennial shrub species and promising candidate in phytoremediation of Ni-contaminated soils. Micro-PIXE analysis revealed that cellular structure in leaf tissues prepared by freeze-drying was adequately preserved as compared to THF freeze-substituted tissues. Elemental distribution maps of leaves showed that Ni was preferentially localised in the adaxial epidermal tissues and leaf margin, with concentration of 10,000 kg–1 DW in both regions. Nickel concentrations in stem tissues obtained by µ-PIXE analysis were lower than in the leaf tissues (1,800 mg kg–1 vs. 7,800 mg kg–1 DW, respectively), and there was no clear pattern of compartmentalisation across different anatomical regions. It is possible that storage of accumulated Ni in epidermal tissues may provide Ni tolerance to this species, and may further act as a deterrent against herbivory and pathogenic attack. In H. floribundus subsp. floribundus seeds, µ-PIXE analysis did not resolve a clear pattern of Ni compartmentalisation and suggests that Ni was able to move apoplastically within the seed tissues. The role of organic acids and free amino acids (low molecular weight ligands [LMW]) in Ni detoxification in H. floribundus subsp. floribundus were quantified using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC). Nickel accumulation stimulated a significant increase in citric acid concentration in leaf extracts, and based on the molar ratios of Ni to citric acid (1.3:1–1.7:1), citric acid was sufficient to account for approximately 50% of the accumulated Ni. Glutamine, alanine and aspartic acid concentrations were also stimulated in response to Ni hyperaccumulation and accounted for up to 75% of the total free amino acid concentration in leaf extracts. Together, these LMW ligands may complex with accumulated Ni and contribute to its detoxification and storage in this hyperaccumulator species. Lastly, the hypothesis that hyperaccumulation of Ni in certain plants may act as an osmoticum under water stress (drought) was tested in context of H. floribundus subsp. floribundus. A 38% decline in water potential and a 68% decline in osmotic potential occurred between water stressed and unstressed plants, however, this was not matched by an increase in accumulated Ni. The results suggested that Ni was unlikely to play a role in osmotic adjustment in this species. Drought stressed plants exhibited a low water use efficiency which might be a conservative ecophysiological strategy enabling survival of this species in competitive water-limited environments.

Phytoextraction du nickel dans les sols ultramafiques d’Albanie La phytoextraction minière est un procédé de récupération des métaux des sols minéralisés naturels ou pollués à l’aide de plantes hyperaccumulatrices. Elle est une alternative à l’agriculture vivrière des zones ultramafiques. L’objectif de la thèse est le développement d’une technologie de phytoextraction extensive du Ni avec Alyssum murale sur les Vertisols ultramafiques. Pour cela, il s’agissait : i) d’identifier les plantes hyperaccumulatrices les plus efficaces dans le prélèvement du Ni et comprendre les relations entre le prélèvement du métal et sa biodisponibilité, ii) de déterminer les types de sols adaptés à la phytoextraction du Ni et iii) de définir et optimiser un itinéraire agronomique adapté pour l’espèce retenue et pour les conditions édaphiques. Dans ce but, des prospections géobotaniques ont été conduites en Albanie et en Grèce. Puis une étude in situ des facteurs qui influencent la biodisponibilité du Ni et le comportement des plantes sur une toposéquence ultramafique a été mise en place. Enfin un essai agronomique de quatre années sur un site ultramafique d’Albanie (Pojske) a permis de tester la fertilisation, le contrôle des adventices par herbicide et la date de récolte pour optimiser le rendement d’extraction du Ni. Les résultats montrent que parmi l’ensemble des espèces présentes naturellement sur les serpentines des Balkans, A. markgrafii et A. murale ont le plus fort taux d’accumulation du Ni. Les Vertisols ultramafiques présentent une disponibilité élevée du Ni favorable à la phytoextraction minière. La biomasse d’A murale est augmentée de 0,2 t ha-1 à 6,0 t ha-1 à partir des traitements agronomiques et le rendement de phytoextraction de Ni par A. murale est de 23 à 69 kg ha-1. Alyssum murale peut être envisagée comme une culture pérenne et la fertilisation permet d’augmenter la compétitivité de la plante sans affecter les concentrations de Ni dans les parties récoltées
Phytomining is a process for recovering metals with hyperaccumulating plants from natural or polluted soils. It is an alternative to conventional farming in ultramafic areas. The aim of the thesis is the development of an extensive phytoextraction technology with Alyssum murale on ultramafic Vertisols. Therefore, work was conducted to i) identify the most effective Ni hyperaccumulators, and understand the relationship between metal uptake and bioavailability, ii) identify soil types suitable for phytoextraction, and iii) define and optimize agronomic practices adapted to the plant species and the edaphic conditions. Hence, geobotanical surveys were conducted in Albania and Greece. Then an in situ study was run on an ultramafic toposequence to assess the factors that influence Ni bioavailability and behavior of plants. Finally a four-year field trial was carried out on an ultramafic site in Albania (Pojske) where fertilization, weed control by herbicide, and harvest date were tested to optimize the efficiency of Ni extraction. The results showed that A. markgrafii and A. murale exhibit the highest rate of Ni accumulation among all species of Balkan serpentines. The ultramafic Vertisols have a high Ni availability phytoextraction and are favourable for phytomining. A. murale biomass increased from 0.2 t ha-1 to 6.0 t ha-1 due to optimization of agronomic treatments, and performance of phytoextraction from 23 to 69 kg ha-1. Alyssum murale can be seen as a perennial crop, and fertilization increases the competitiveness of the plant without affecting the Ni concentrations in the harvested parts

Certaines plantes, dites hyperaccumulatrices, ont la capacité de se développer sur des sols riches en métaux et d’accumuler ces métaux à des concentrations élevées. L’incinération de la biomasse produit des cendres qui contiennent de 10 à 25% en masse de Ni. Ce travail s’inscrit dans la continuité d’une recherche menée par l’équipe depuis plusieurs années, qui a donné lieu notamment à un brevet sur la production du sel double sulfate de nickel et d’ammonium hexahydraté (ANSH) à partir de la biomasse d’Alyssum murale. Le manuscrit comprend d’abord une synthèse bibliographique sur la phytomine, allant des hyperaccumulateurs aux procédés de valorisation, essentiellement centrée sur le nickel. Ensuite, ont été comparées quinze plantes hyperaccumulatrices (des genres Alyssum, Leptoplax et Bornmuellera) provenant d’Albanie ou de Grèce, en vue de leur application pour la phytomine. Les teneurs en nickel ont été mesurées dans les différents organes des plantes et dans les cendres obtenues par combustion. Les trois genres ont de l’intérêt pour l’application, les plantes contiennent 1 à 3% en masse de nickel et les cendres 15 à 20 %. Le procédé hydrométallurgique de production d’ANSH a été étudié étape par étape en vue d’optimiser chaque étape pour produire un sel très pur tout en économisant matière et énergie et minimisant la production d’effluents et de déchets. Ce travail a conduit à l’amélioration du procédé de départ. Enfin, de nouvelles pistes ont été proposées pour conduire à de nouveaux procédés et produits du nickel. Les résultats obtenus et la dynamique actuelle autour de la phytomine montrent l’intérêt de cette approche et annoncent son développement imminent
Some plants, known as hyperaccumulators, are able to develop on metal containing soils and to accumulate these metals at high concentrations in shoots. Biomass incineration leads to ash containing 10 to 25 wt % nickels, greater than in some mineral ores. This work follows a research that has been carried out by the team for several years, which has resulted in a patent on the hydrometallurgical production of the double salt ammonium and nickel hexahydrate (ANSH) from the biomass of Alyssum murale. It aims at improving the synthesis method of this salt in order to upscale it at the pilot scale and explore new methods leading to new products. The manuscript begins with a bibliographic review on phytomining from hyperaccumulators to metal recycling processes, essentially focused on nickel. Then ca 15 hyperaccumulator plants (genus Alyssum, Leptoplax and Bornmuellera) collected in Greece or Albania have been compared, in the objective of phytomining. Nickel concentrations were measured in the plant organs and in the ashes after combustion. The three types of plants are of great interest for the technology, they contain 1 to 3 wt % of nickel and the ashes 15 to 20%. The hydrometallurgical process of ANSH production was investigated step by step to optimize each step to produce a salt of high purity, to decrease materials and energy consumption and to minimize effluent and waste production. The process was thus improved. Eventually, new ideas have been tested for new processes and nickel products. The obtained results and the current dynamics prove the interest of phytomining and announce its imminent development

L’agromine est une filière destinée à valoriser des métaux dispersés dans des sols ou autres matrices, à l’aide de plantes hyperaccumulatrices (HA). La première étape consiste à cultiver ces plantes pour obtenir des rendements élevés en métaux et la seconde, à produire des composés métalliques d’intérêt à partir de la biomasse. L’agromine a surtout été développée pour valoriser le nickel (Ni). Jusqu’à présent, la biomasse était brûlée pour concentrer le métal et éliminer les matières organiques. L’enjeu de cette recherche est de concevoir des procédés de récupération du Ni par extraction directe depuis la biomasse, sans brûler la plante. Il s’agit de comprendre les processus impliqués lors de l’extraction du Ni de la biomasse sèche à l’aide d’un solvant et déterminer les formes chimiques des espèces en solution. A partir de là seront mises en œuvre des opérations de séparation adaptées, pour isoler le Ni sous une forme intéressante pour des applications ultérieures. Les expériences de lixiviation à l’eau à 20 °C, menées avec deux HA contrastées, ont démontré qu’il était possible de transférer en solution jusqu’à 80% du Ni présent dans les tissus des plantes. Celui-ci est accompagné des ions majeurs et de composés organiques. L’analyse des composés et la modélisation des équilibres chimiques en solution ont montré que le Ni était complexé à plus de 95% par des ligands organiques, acides carboxyliques, porteurs du Ni dans la plante, ainsi que des complexants plus forts. A partir de ces résultats, des procédés de séparation ont été sélectionnés : la précipitation sélective et l’adsorption sur résine complexante. Ils ont permis de récupérer respectivement 75 et plus de 95% du nickel sous forme sulfure ou composé carboxylique. En revanche, la purification à l’aide de décanoate n’a pas permis d’isoler le Ni. Ainsi, ce travail a permis de mieux comprendre l’extraction du Ni directement à partir de plantes, la spéciation du Ni en solution multiconstituant en présence de ligands organiques, et de valoriser le nickel par des voies jusqu’alors inexplorées avec ce type de matière première
Agromining is a chain allowing the recovery of metals dispersed in soils or other matrices, using hyperaccumulator plants (HA). The first step is to grow these plants to achieve high yields of metals and the second to produce metal compounds of interest from the plant biomass. Agromining has mainly been developed to value nickel (Ni). Until now, biomass was burnt to concentrate the metal and remove organic matter. The challenge of this research is to design processes for Ni recovery by direct extraction from biomass, without burning the plant. It will allow a better understanding of the processes involved in the extraction of Ni from dry biomass using a solvent and the determination of the the speciation in the solution. Then, appropriate separation operations will be implemented to isolate the Ni in an interesting form for subsequent applications.Water leaching experiments, run at 20 ° C with two contrasted HAs, demonstrated that up to 80% of Ni could be transferred from the plant tissues to the solution. Ni is accompanied by major ions and organic compounds. The analysis of these compounds and the modeling of the chemical equilibria in solution showed that more than 95% of Ni was complexed by organic ligands, carboxylic acids (Ni carriers in the plant) as well as stronger complexing agents. From these results, separation processes were selected: selective precipitation and adsorption on complexing resin. They made it possible to recover respectively 75 and more than 95% of the nickel in sulphide or carboxylic compound forms. In contrast, purification with decanoate did not isolate the Ni.Thus, this work has made it possible to better understand the extraction of Ni directly from plants, the speciation of Ni in a multicomponent solution in the presence of organic ligands, and to valorize nickel by ways previously unexplored with this type of material

Includes bibliographical references.
Heavy metal (HM) accumulator plants possess the ability to actively hyperaccumulate and detoxify exceptionally high concentrations of metals in their aboveground tissues, without exhibiting any apparent signs of toxicity. Despite nickel (Ni) hyperaccumulator plants representing the largest percentage of known metal accumulator taxa (over 75%), the underlying genetic and molecular basis of Ni accumulation remains unclear. A prominent difficulty in understanding Ni hyperaccumulation has been the severe lack of intraspecific variation in the trait. Hence, the study of a single species exhibiting a significant degree of variation is highly desirable. as it avoids the use of inter-species comparative studies mostly utilized to date. The Ni hyperaccumulator Senecio coronatus (Asteraceae) has been reported to contain a significant degree of phenotypic plasticity with respect to the amount accumulated and subsequent cellular distribution of Ni. This apparent intraspecific variation means that S. coronatus may represent a useful system in which to study Ni hyperaccumulation. No population genetics study has been carried out to date on this species, and the evolutionary relationships between hyper and non- accumulator populations were unknown. Here, results are presented from a genetic analysis of 15 naturally occurring S. coronatus populations. Analysis of molecular variance (AMOVA) and phylogenetic analysis (based on non-coding nuclear and plastid markers) suggest that Ni accumulation may have evolved twice within S. coronatus, as hyperaccumulator plants from site Kaapsehoop, cluster with non-accumulating serpentine populations and demonstrate distinct genetic differentiation from other accumulator populations. Four populations were selected for a preliminary comparative shoot proteome analysis by means of two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE) to identify proteins potentially involved in Ni hyperaccumulation. This analysis identified nine chloroplastic proteins involved in plant energy production and metabolism as overexpressed in hyperaccumulator plants from Agnus Mine and Kaapsehoop, compared to hypertolerant non-accumulator and non-serpentine plants from Galaxy Mine and Pullen Farm, respectively. However, no difference in photosynthetic efficiency, as determined by chlorophyll fluorescence measurements, was detected between these populations.

L’agromine est une filière destinée à valoriser des métaux dispersés dans des sols ou autres matrices, à l’aide de plantes hyperaccumulatrices (HA). La première étape consiste à cultiver ces plantes pour obtenir des rendements élevés en métaux et la seconde, à produire des composés métalliques d’intérêt à partir de la biomasse. L’agromine a surtout été développée pour valoriser le nickel (Ni). Jusqu’à présent, la biomasse était brûlée pour concentrer le métal et éliminer les matières organiques. L’enjeu de cette recherche est de concevoir des procédés de récupération du Ni par extraction directe depuis la biomasse, sans brûler la plante. Il s’agit de comprendre les processus impliqués lors de l’extraction du Ni de la biomasse sèche à l’aide d’un solvant et déterminer les formes chimiques des espèces en solution. A partir de là seront mises en œuvre des opérations de séparation adaptées, pour isoler le Ni sous une forme intéressante pour des applications ultérieures. Les expériences de lixiviation à l’eau à 20 °C, menées avec deux HA contrastées, ont démontré qu’il était possible de transférer en solution jusqu’à 80% du Ni présent dans les tissus des plantes. Celui-ci est accompagné des ions majeurs et de composés organiques. L’analyse des composés et la modélisation des équilibres chimiques en solution ont montré que le Ni était complexé à plus de 95% par des ligands organiques, acides carboxyliques, porteurs du Ni dans la plante, ainsi que des complexants plus forts. A partir de ces résultats, des procédés de séparation ont été sélectionnés : la précipitation sélective et l’adsorption sur résine complexante. Ils ont permis de récupérer respectivement 75 et plus de 95% du nickel sous forme sulfure ou composé carboxylique. En revanche, la purification à l’aide de décanoate n’a pas permis d’isoler le Ni. Ainsi, ce travail a permis de mieux comprendre l’extraction du Ni directement à partir de plantes, la spéciation du Ni en solution multiconstituant en présence de ligands organiques, et de valoriser le nickel par des voies jusqu’alors inexplorées avec ce type de matière première
Agromining is a chain allowing the recovery of metals dispersed in soils or other matrices, using hyperaccumulator plants (HA). The first step is to grow these plants to achieve high yields of metals and the second to produce metal compounds of interest from the plant biomass. Agromining has mainly been developed to value nickel (Ni). Until now, biomass was burnt to concentrate the metal and remove organic matter. The challenge of this research is to design processes for Ni recovery by direct extraction from biomass, without burning the plant. It will allow a better understanding of the processes involved in the extraction of Ni from dry biomass using a solvent and the determination of the the speciation in the solution. Then, appropriate separation operations will be implemented to isolate the Ni in an interesting form for subsequent applications.Water leaching experiments, run at 20 ° C with two contrasted HAs, demonstrated that up to 80% of Ni could be transferred from the plant tissues to the solution. Ni is accompanied by major ions and organic compounds. The analysis of these compounds and the modeling of the chemical equilibria in solution showed that more than 95% of Ni was complexed by organic ligands, carboxylic acids (Ni carriers in the plant) as well as stronger complexing agents. From these results, separation processes were selected: selective precipitation and adsorption on complexing resin. They made it possible to recover respectively 75 and more than 95% of the nickel in sulphide or carboxylic compound forms. In contrast, purification with decanoate did not isolate the Ni.Thus, this work has made it possible to better understand the extraction of Ni directly from plants, the speciation of Ni in a multicomponent solution in the presence of organic ligands, and to valorize nickel by ways previously unexplored with this type of material

Selective accumulation of certain metals (elements) to exceptionally high concentrations in plants is intriguing. Approximately 425 species of so-called metal hyperaccumulators are currently known, of which about 75% hyperaccumulate nickel. Stackhousia tryonii Bailey (Stackhousiaceae) - a rare, herbaceous, serpentine-endelnic species - is one of the three nickel hyperaccumulators reported from Australia. This thesis reports research aimed at two broad aspects: propagation and ecophysiology of Ni hyperaccumulation in S. tryonii. Protocols were developed for seed germination, vegetative propagation and micropropagation and with the view to producing sufficient plants for use in the current study. Four-year-old S. tryonii seeds had poor germination (< 25%). However, this species was relatively easy to propagate via stem cuttings and micropropagation methods, as it possessed very high regenerative capacity (one explant produced up to 18 shoots within 4 weeks). Micropropagated shoots also responded well to ex vitro rooting, and were successfully hardened under controlled conditions. These propagation protocols could be useful to underpin conservation programs and mine site revegetation. The examination of natural populations of S. tryonii for arbuscular mycorrhizal colonisation suggested that S. tryonii is a favourable host. A moderately high colonisation (29-39%) of roots by arbuscular mycorrhizal fungi suggested a possible role of these fungi in improved nutrition of S. tryonii in typically nutrient-poor serpentine soils. A positive relationship between root colonisation and leaf Ni concentration suggested that mycorrhizal fungi might be involved in increased influx of Ni into the roots, which is readily transported and localised in the tissues. Spore density was very low (3-4 spores 100 g-¹dry soil, for two depths) in the associated serpentine soils and the dominant mycorrhizal species were: Glomus albidum, aggregatum, G. intraradices and G. tenebrosum. Based on five key soil characteristics (viz. pH, Ca, Mg, Ni and P), the study sites were segregated into four groups using hierarchical cluster analysis. Considerable variation existed in tissue Ni (and other elements) concentrations, both within and between populations and followed the order: leaf> root> stem. Localisation and spatial distribution of nickel, within both vegetative (leaf and stem) and reproductive (fruit) tissues were investigated using two microanalytical techniques [viz., micro-proton-induced x-ray emission spectrometry (micro-PIXE; nuclear microprobe) and scanning electron microscope with energy-dispersive x-ray spectroscopy (SEM-EDXS)]. In leaf and stem tissues, Ni was localised within epidermal and sub-epidermal tissues, palisade/mesophyll tissues, vascular bundles and/or pith. In contrast, in fruits, this metal was partitioned to the fruit wall (pericarp), while endospermic and cotyledonary tissues contained very little Ni. Accumulation of higher levels of Ni within the pericarp does not appear to inhibit seed germination in S. tryonii. To elucidate physiological mechanisms o fNi detoxification in S. tryonii, organic acids (leaf tissue) and free amino acids (xylem sap) were quantified using HPLC. Nickel concentration in the leaf tissues increased from 3695 g g-¹to 13,717 g g-¹with soil nickel supplementation, of which > 60% was extracted with dilute acid (0.025 M HCI). Oxalic, citric and malic acids were detected and quantified in the leaf tissue. Malic acid was the dominant organic acid, and based on a Ni to malic acid ratio (between 0.2:1 and 1:1), malic acid appears to play a major role in detoxification/transport and storage of Ni in S. tryonii. The total amino acid concentrations in the xylem sap decreased with nickel treatment. Glutamine was the major amino acid in both the low- and high- nickel treated plants. A role of amino acids in nickel complexation and transport in S. tryonii could not be established. The possibility of hyperaccumulated Ni acting as an osmoticum under waterstress (drought) in serpentine soils was also investigated. Drought severely affected the growth and overall biomass of the plants. However, survival of plants at the lowest levels of soil moisture (i. e. 20% of field capacity) suggested that it possesses an efficient water regulation mechanism. The results indicated possible involvement of Ni in osmotic adjustment under drought stress.

Among hyperaccumulators, i.e. plants able to accumulate extremely high concentrations of heavy metals (HMs) in shoots, the Brassicaceae Arabidopsis halleri and Noccaea caerulescens, zinc (Zn)/cadmium (Cd) and Zn/Cd/nickel (Ni) hyperaccumulators respectively, represent two of the most interesting species, due to the huge variability between different ecotypes and populations in metal tolerance and accumulation. In this PhD work, two independent approaches were used aiming for a deeper understanding of metal hypertolerance and hyperaccumulation. The first approach exploits N. caerulescens ecotype Monte Prinzera (MP, Italy), native of a serpentine soil, that is able to hypertolerate and hyperaccumulate Ni in addition to Zn. Molecular mechanisms responsible for Ni tolerance and accumulation are still mostly unknown, although several studies suggested that metal transporters, also essential for metal homeostasis, have a fundamental role in HM tolerance and accumulation. Therefore, the vacuolar transporters MTP1 and NRAMP4 and of plasma membrane transporter ZNT1 were taken in consideration, thanks to their proposed role in Ni tolerance and accumulation in N. caerulescens MP (Visioli et al., 2014). At first, the expression of all three genes in N. caerulescens MP exposed to different Ni concentrations was analysed and compared with those of T. arvense (non-accumulator) and N. caerulescens ecotype Ganges (GA, Zn/Cd hyperaccumulator). Higher expression levels of all genes were found in N. caerulescens MP under normal and Ni excess conditions, confirming the different regulation of metal transporter proteins in hyperaccumulator plants and suggesting their role in Ni tolerance. The Ni transport properties of both NcNRAMP4 and NcZNT1 was tested by yeast complementation assays, using a WT strain of S. cerevisiae; according to the results previously found for the other Ni hyperaccumulator plant N. japonica (Mizuno et al., 2005), NcNRAMP4 reduced recombinant yeast survival under Ni treatment and the opposite result was found for NcZNT1, indicating a possible direct involvement of both proteins in Ni transport. To better understand the role of NcNRAMP4 and NcZNT1 in Ni tolerance and accumulation, plants of Arabidopsis thaliana were transformed with constructs carrying CaMV35S::NRAMP4 and CaMV35S::ZNT1 and the transgenic lines were crossed to obtain plants overexpressing both genes. Single 35S::NRAMP4 and double 35S::NRAMP4/35S::ZNT1 lines displayed bigger shoot compared to WT plants and the same results were also found for two of the three lines overexpressing ZNT1, although with a minor impact compared to the other gene. The impact of both genes on Ni tolerance and accumulation in planta was also elucidated by in vivo and in vitro analysis. Compared to WT plants, transgenic lines of A. thaliana expressing NcNRAMP4, NcZNT1 or NcNRAMP4/NcZNT1 in combination showed higher tolerance to Ni excess in vivo, thanks to a reduction of Ni accumulation in shoots. In addition, single 35S::NcNRAMP4 and double 35S::NcNRAMP4/35S::ZNT1 transgenic lines displayed longer roots in vitro, under standard and Ni excess condition compared to the WT, although no significative differences were found regarding 35S::ZNT1 overexpressing lines. These data may suggest that NRAMP4 and ZNT1 could participate in the transport and accumulation of Ni in N. caerulescens MP, although probably these proteins are more associated with the transport of other micronutrients than Ni itself. The possible involvement of the vacuolar transporter MTP1 in Ni hyperaccumulation and hypertolerance in N. caerulescens MP was also investigated. Gene expression analysis have initially confirmed the high constitutive levels of MTP1 expression previously found in different hyperaccumulator-hypertolerant plants belonging to Noccaea genus (Assunção et al., 2001; van de Mortel et al., 2006) under normal and stressful conditions, although in N. caerulescens MP this gene seem to be downregulate upon Ni treatment. In both N. caerulescens GA and MP two different CDSs of MTP1 gene, characterized by different length, were found in cDNA and genomic DNA; these proteins, called MTP1-long and MTP1-short, based on the presence or absence of the conserved His-loop region, are localized in the vacuolar compartment, as confirmed by subcellular localization experiments, suggesting their role in metal detoxification. The metal binding properties of both NcMTP1-long and NcMTP1-short were tested by yeast complementation assays using zrc1cot1 double mutant yeast of S. cerevisiae, which lacks these two vacuolar proteins, and WT strain of S. cerevisiae considering AtMTP1 as control for Zn transport ability (Desbrosses-Fonrouge et al., 2005). The two MTP1 forms displayed different metal specificity regarding Zn, Ni and Co, suggesting their possible different involvement in detoxification of metal excess in N. caerulescens MP. Therefore, A. thaliana mtp1+/+ homozigous mutants were transformed with constructs carrying CaMV35S::MTP1-long and CaMV35S::MTP1-short and also CaMV35S::AtMTP1 as control to elucidate their different metal binding specificity in planta. The transgenic lines were selected and the biological effect of MTP1 in planta will be investigated. With the second approach the attention is focused on the identification of miRNAs involved in the response to Zn excess. In eukaryotes, these small non-coding RNAs are essential for the regulation of gene expression during development and stress response, as well as nutrient homeostasis. miRNAs are small regulatory RNAs (20-24 nt length), with important role in plant development and response to many abiotic and biotic stress in Arabidopsis and other plant species. Moreover, several works have led the attention on metal -responsive miRNAs, which are responsible for the morphological and metabolical adaptation of environmental cues in plants, representing an interesting starting point for further analysis. To identify miRNAs putatively involved in response to Zn excess in Arabidopsis, miRNA-seq analysis was performed on miRNAs extracted from shoots of A. thaliana, treated and not treated with an excess of Zn for one week, and from untreated A. halleri. This hyperaccumulator plants was used in order to evaluate the existence of a possible different regulation mechanisms which regulates miRNA-target interaction compared to the non-hyperaccumulator A. thaliana. 100 miRNAs resulted to be differentially modulated in A. halleri compared to A. thaliana, including metal responsive miRNAs. Some of them were experimentally validated by Northern Blot Analysis and Real Time RT-PCR, in particular miRNAs with a role in plant development (miR157, miR159, miR390) and in nutrient homeostasis regulation (miR395, miR398, miR408), confirming the data obtained by miRNA-seq analysis and suggesting that some miRNAs could have an important role in metal hypertolerance and hyperaccumulation. Then, the attention was focuses on miR398b and miR408, two conserved miRNAs principally involved in the regulation of copper homeostasis in A. thaliana and other plant species; several works have elucidated their role in response to many abiotic stresses, including oxidative stress and heavy metals (Yamasaki et al., 2009; Ma et al. 2015; Pilon et al., 2017; Jalmi et al., 2018); moreover, the coordinate action of both miRNAs in planta have been suggested to be required for Cd basal tolerance in Arabidopsis (Gayomba et al., 2013; Gielen et al., 2016), representing an interesting starting point for further analysis. In order to investigate the possible involvement of both miR398b and miR408 in response to Zn excess, the promoter sequence of both miRNAs was amplified from genomic DNA of A. thaliana and A. halleri and fused to GUS reporter gene. A bioinformatic analysis was initially done comparing the entire sequences obtained by A. thaliana and A. halleri, and several DNA motifs involved in response to abiotic stresses were identified. GUS assay on A. thaliana transgenic lines expressing pAtMIR398::GUS, pAhMIR398b::GUS, pAtMIR408::GUS, pAhMIR408::GUS have shown that both miRNAs are expressed in roots and shoots of Arabidopsis, particularly in the vasculature tissues, suggesting their mobility into the whole plant. In addition, GUS expression was also evaluated on transgenic plants grown under Zn and Cu treatment, to better elucidate a possible competition between Cu and Zn, as previously proposed (Remans et al., 2012), and study the response of these two miRNAs under metal exposure. Both miRNAs resulted to be modulated by high concentration of these two micronutrients, suggesting a competition between Zn and Cu as previously proposed (Remans et a., 2012). Finally, the effect of the overexpression of pre-miR408 of A. halleri upon Zn treatment was investigated, since it is known its fundamental role for the adaptation to environmental cues in plants as demonstrated for the homologous of A. thaliana (Ma et al., 2015; Zhang and Li, 2013; Song et al., 2018). Compared to WT plants, transgenic lines overexpressing the precursor sequence of miR408 of A. halleri displayed higher shoot biomass and a significative reduction of root length, suggesting that high expression of miR408 could modify Zn tolerance in planta. Further investigations will be also required to better elucidate the possible role of miRNAs in response to Zn excess in Arabidopsis.