Phytoextraction of zinc by the hyperaccumulator Noccaea caerulescens: accumulation, distribution and multi-element interactions

Abstract

Phytoextraction involves the removal of heavy metals from contaminated sites using hyperaccumulators such as Noccaea caerulescens F.K.Meyer (formerly Thlaspi caerulescens) which is a well-known Zn hyperaccumulator and a model species for hyperaccumulator studies. This PhD thesis investigates the zinc response of the hyperaccumulator in terms of accumulation, distribution and multi-element interactions. The impact of Zn, Pb and Cu on the growth, Zn accumulation and distribution, and interaction between the metals and between the metals and other plant nutrients were examined in soution culture to provide insights into the physiological and biochemical mechanisms of metal hyperaccumulation and detoxification. Noccaea caerulescens were treated with a range of Zn concentrations to investigate uptake, tolerance and distribution of Zn, and these were compared with the responses in a related non-hyperaccumulator, Thlaspi arvense. Noccaea caerulescens showed a high Zn requirement for growth and a higher Zn accumulation and tolerance, with in excess of 2.5 % Zn in the shoot dry mass occurring without visible toxicity symptoms. Thlaspi arvense displayed Zn toxicity symptoms within two weeks of comparable Zn treatment. Increasing shoot Zn concentrations in N. caerulescens reduced Ca and P concentrations by up to 50% and 35%, respectively. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDS) showed that Zn-containing crystals were abundant in leaf epidermal cells and the cortex of the roots during the later growth phase of N. caerulescens. The impact of Pb and Cu on Zn phytoextraction in N. caerulescens was investigated and these metals in solution were found to inhibit both growth and Zn accumulation when present in combination with Zn. Plants were more sensitive to Cu than Pb with Cu being more readily transported to the shoots while almost all Pb was retained in the roots. Concentrations of other essential nutrients such as Ca, Mg, P, S, Fe, and K were decreased in shoots but increased in roots when there was a prolonged period of metal exposure. Higher levels of Zn accumulation decreased the concentrations of Ca, Mg, S and Fe in shoots. Sub-cellular elemental localization analysis using SEM-EDS revealed that Zn accumulated primarily inside the vacuoles of leaf epidermal cells. Zn-rich crystals were observed in the SEM freeze-dried leaf samples of the control and the Zn plus Pb treatment after extended exposure, but no crystals were found in the cryo-SEM leaf samples. Copper and Pb were not detected in the Zn-rich crystals of the freeze-dried samples or the cryo-SEM samples. A fluorescent probe technique was developed and applied to investigate the distribution of Zn ion in fresh plant tissues. Noccaea caerulescens was found to have not only a high capacity for Zn accumulation, but also a high tolerance to Pb. Both these characteristics were more prominent in younger plants. Zinc in combination with Pb had less impact on plant growth than Zn alone. Translocation of Zn from roots to shoots was reduced in older plants and/or long-term heavy metal exposure. A high ratio of Zn to P in leaves led to the formation of insoluble salts of Zn (Zn-phosphate and Zn-phytate). Fluorescence probe analysis of Zn2+ ion distributions in tissues and cells revealed that Zn2+ ions were located primarily in the leaf epidermal cells and commonly bound to the cell wall in both epidermal and mesophyll cells. Zinc ion distribution was not specific to any particular cell type in roots. Quantitatively, Zn ions were highest in the leaf epidermis at a concentration of 0.282 M (2.5 times higher than total Zn in fresh leaves) and were from 0.0561 to 0.282 M in the root epidermis. Confocal laser scanning microscopy in combination with selective fluorescent probes proved a useful tool for elucidating cellular and tissue-level distribution of Zn2+ in living plant cells at high resolution. In this thesis, a number of novel techniques were successfully employed to achieve the research objectives. It has been established that N. caerulescens requires high Zn for growth and can accumulate very high concentrations of Zn when exposed to high levels of Zn in solution at particular growth stages. Plant growth and Zn accumulation are adversely affected by elevated Cu concentrations, but the species has a degree of tolerance to Pb exposure. For exploiting the potential value of N. caerulescens as a phytoextraction and remediation option for Zn-enriched or contaminated substrates, it would seem that the species suitability for purpose will be maintained where there is a concomitant presence of elevated Pb, but less so if Cu contamination levels are also high.