The isotopic discrimination of copper in soil-plant systems: examining sources, uptake and translocation pathways

Abstract

Copper (Cu) is an essential micronutrient for plants and many microorganisms, playing a key role in electron transport during photosynthesis, lignin formation and cell wall metabolism. However, when Cu is present at elevated concentrations it can cause toxicity with impacts on the growth, reproduction and survival of aquatic and terrestrial organisms. The biogeochemical cycle of Cu in aquatic and terrestrial environments can be influenced by numerous biological (e.g. root rhizosphere) and physicochemical (e.g. redox, pH) properties. A better understanding of Cu biogeochemical cycling is required to ensure optimal Cu supply to organisms. As such, there is an increasing need for the development of new analytical tools that can be used in complex environmental systems to examine this. This thesis investigates the use of Cu stable isotopes to yield new information on the behaviour of Cu in soil-plant systems. Copper has two stable isotopes, ⁶³Cu and ⁶⁵Cu, and the different partitioning of these two isotopes between Cu pools (known as fractionation) can provide information on the reactions and mechanisms involved in Cu transport from one pool to another. Stable isotope data from plant growth studies were coupled with solid phase speciation and dialysis solution speciation to yield a better understanding of the isotopic signature of bioavailable Cu and the mechanisms by which Cu is absorbed into, and translocated throughout, plants. The effect of Cu complexation by soluble organic matter was quantified to assess whether the isotopic signature of bioavailable ‘free’ Cu differed to that of the total soil solution Cu. This is important as fractionation between soil solution and plants cannot be accurately measured if the isotopic signature of the available pool of Cu is not accurately known. Copper isotope fractionation was examined in solutions of both synthetic organic ligands and Suwannee River fulvic acid (SRFA) using Donnan dialysis to separate the free and complexed Cu pools. The results showed that Cu contained within the organic complex was enriched in the heavy isotope, with the magnitude of fractionation proportional to the strength of the Cu-ligand bond. These results highlight the importance of determining the isotopic signature of the bioavailable ‘free’ Cu when looking at plant uptake mechanisms, as the isotopic signature of the total solution Cu is different to that of free Cu if Cu is partly complexed with organic ligands, as is usually the case in environmental samples. When using Cu isotope fractionation to assess root absorption mechanisms, it is important to consider the contribution of Cu adsorbed to the cell wall. In order to assess the isotope fractionation involved with Cu adsorption onto plant cell walls, four-week old plants and seedlings of Fe-acquisition Strategy I and Strategy II species were exposed to various concentrations of Cu for short periods of time. Adsorbed Cu was then desorbed from four-week old tomato and oat plants, using 3 different desorption techniques to determine which root washing technique quantitatively released adsorbed Cu while not extracting symplastic Cu. The results showed that the root wash procedure based on cation exchange using La and Ca was the best extractant to exclusively target the apoplastic Cu, while EDTA and HCl extractants showed signs of symplastic Cu removal. No significant isotope fractionation was found during adsorption onto the surface of monocotyledonous (monocot, Strategy II) plant roots, but adsorption onto the surface of dicotyledonous (dicot, Strategy II) plant roots yielded Cu isotope fractionations on the order of that seen during Cu complexation with fulvic acid (Δ⁶⁵Cu root-solution = ca. 0.2‰). The results suggested that a difference in the type and/or strength of Cu binding sites on the cell walls exists for monocot and dicot species, and highlight the importance of root washing when assessing isotope fractionation due to root absorption. The fractionation of Cu stable isotopes during uptake into plant roots and translocation to shoots was used to gain new information on Cu acquisition mechanisms by plants. Copper isotope fractionation values were coupled with intact tissue speciation techniques (X-ray absorption spectroscopy, XAS) to examine the uptake, translocation and speciation of Cu in a dicot (tomato) and monocot (oat) plant species. Plants were grown in solution culture where Cu was maintained as free Cu by regular replacement of the nutrient solution, so that complexation-induced isotope fractionation in the solution did not complicate the determination of fractionation due to plant uptake. The iron (Fe) conditions were varied to test whether the stimulation of Fe acquiring mechanisms can affect Cu uptake in plants. The results showed that isotopically light Cu was preferentially incorporated into tomatoes (Δ⁶⁵Cu whole plant-solution= ca. -1‰), whereas oats showed minimal isotopic fractionation, with no effect on isotope fractionation with changing Fe conditions in either species. The presence of isotopically light Cu in tomatoes was attributed to a reductive uptake mechanism. The heavier isotope was preferentially translocated to shoots in tomato, while oat plants showed no significant fractionation during translocation. The translocation fractionation observed for tomatoes was suggested to be linked to an oxidation and organic complexation with nicotianamine, as both of oxidation and complexation processes lead to heavy isotope enrichment. The majority of Cu in roots and leaves of both speciesexisted as sulphur-coordinated Cu(I) species indicating glutathione/cysteine-rich proteins. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation was not redox-selective and different translocation pathways exist between monocot and dicot plant species. The results presented in this thesis provide significant new information on the behaviour of Cu isotopes in soil-plant systems. For the first time it has been shown that Cu complexation with soluble organic matter and adsorption onto plant roots can cause notable isotopic fractionation, with the organic complex or root surface enriched in the heavy isotope. The most significant findings of this research relate to differences observed between Cu uptake and translocation mechanisms between monocot and dicot species, elucidated from Cu isotope fractionations and XAS analysis. These data open the door to future research into Cu source tracing using isotopic signatures, further investigations into Cu behaviour in soil solutions in-situ, as well as field studies looking at Cu uptake and translocation mechanisms in plants grown in soil environments.