Study Of Mechanisms For Cu Tolerance And Accumulation In Elsholtzia Splendens

One of the most important issues in the field of environment engineering is theremediation of heavy metal-polluted soils. Some plants are known as hyperaccumulatoror tolerant plants which are able to accumulate considerable amount of heavy metalssuch as Ni, Zn, Cd or Cu in their roots or shoots. Therefore, the mechanisms fortolerance and accumulation of heavy metals in these plants are of interest for thescientists in the field of environmental, plant and biological sciences all over the world.From the ecological, plant physiological and molecular biological points of view,respectively, scientists are devoting themselves in revealing the mysteries of the specialabilities in plants.Elsholtzia splendens is one of these plants, which is reported as a Cu-tolerant plant,usually found in copper mining areas of southern China. The Cu concentration in theleaves can reach 1000 mg kg^-1 in solution cultivation. The plant is considered to be anappropriate choice for phytoextracting the highly concentrated Cu inmetal-contaminated soils. Corresponding research is being undertaken with respect tothe mechanisms for Cu tolerance and accumulation. In the present study, furtherinvestigation on the Cu tolerance and accumulation was conducted with respect to cellwall precipitation of Cu, ligands involved in Cu chelation and elemental distributionwithin the plant leaves. On the other hand, new quantification strategies on imagingmass spectrometry using laser ablation inductively coupled plasma mass spectrometry(LA-ICP-MS) of essential and toxic elements in biological tissues were developed tostudy the uptake of heavy but also nutrient elements.It was illustrated that the precipitation of Cu on cell walls was one of the importantmechanisms for Cu tolerance and accumulation. E. splendens had high absorptioncapacity for Cu on the cell walls. More Cu was absorbed on the root cell walls than onthe shoot cell walls. Esterification of carboxyl groups and methylation of amino groupsof the cell walls resulted in reduction in the amount of Cu absorbed by about 64% and25%, respectively. Cu was bound with the carboxyl, amino and hydroxyl groups in thecell walls revealed by Fourier transform infrared spectroscopy. Synchrotron radiationX-ray absorption spectroscopy was used to investigate the Cu species on the cell walls.The percentages of potential Cu ligands were estimated by fitting the XANES spectra with linear combinations of the references'. Cu in cell walls was bound withoxalate-like ligands, histidine-like ligands, as well as aqueous Cu.Free histidine and cysteine were found to be potential ligands chelating with Cu inE. splendens. Sixteen free amino acids were. analyzed to investigate the correlationsbetween free amino acids and Cu stress. The synthesis of free histidine and cysteine wasinduced by high Cu content in the nutrient solution. The concentrations of both aminoacids were significantly correlated with Cu concentrations in the nutrient solution and intissues of the plant. The two amino acids were involved in the chelation of Cu in theplant. Particularly, the histidine concentration in stems after 500μM Cu treatment waseleven times more than it in the stems without treatment, indicating that free histidineprobably participated in the transport of Cu from the roots to the shoots.Cu- and Zn-containing proteins in the roots of E. splendens were investigated bymetaliomic studies using 2D gel electrophoresis (2D-PAGE) for protein separation andLA-ICP-MS for screening of metal-containing proteins. Twelve proteins were inducedor up-regulated, while another seventeen proteins were disappeared or down-regulatedby Cu treatment. LA-ICP-MS was an effective technique to imaging metals andnon-metals in protein spots on gels after electrophoresis with silver staining, ¹⁰⁷Ag⁺ wasused to indicate the location and the relative content of the proteins. SeveralCu-containing proteins were found, as well as Zn-, Mn- or Mg-containing proteins werepresent in the gels. Further studies with respect to protein identification and theirfunctions should be carried out.New quantification strategies on imaging mass spectrometry using LA-ICP-MS ofessential and toxic elements in biological tissues were developed to study the uptake ofheavy but also nutrient elements. In the present work, it was demonstrated thatLA-ICP-MS is a powerful analytical technique for directly imaging both macro- andmicronutrients in the plant leaves without sample preparation, and the resolution of thistechnique is sufficient for all the elements measured (Cu, K, Mg, Mn, P, S and B). Themethod was able to quantify the analytical data with synthetic laboratory standardcalibration. The established quantitative imaging LA-ICP-MS method will provide aneasy and effective tool in the field of the phytosphere with respect to distribution ofelements, and will open up a new window onto in vivo ionomic imaging of plant tissues.LA-ICP-MS was successfully applied together with tracer experiments using enriched⁶⁵Cu isotope tracer to investigate the accumulation of Cu and other essential elements of interest (K, Mg, Mn, P, S and B) in the leaves of E. splendens after ⁶⁵Cu treatment. Cuaccumulation and the effect of Cu stress on essential element uptake in the newlyformed leaves can be clearly observed by quantitative imaging, while no significantchanges were found in the fully grown leaves due to the short-term treatment. The⁶⁵Cu/⁶³Cu isotope ratios measured by LA-ICP-MS increased after ⁶⁵Cu treatment. Theresults indicated the path of Cu uptake via the petiole and main veins of the leaves.Together with tracer experiments, quantitative imaging LA-ICP-MS was successfullyapplied for studying the uptake and accumulation of toxicological and essentialelements in plant leaves.