Highlights-Zn uptake by N. caerulescens tranlocated to leaves.-Great ability of young N. caerulescens plants to accumulate Zn in shoots.-Decrease of Ca and P concentration with increasing amount of Zn treated.-Zn crystals found in leaf epidermal cells and root cortex of N. caerulescens.-In the plant tissues, P and S co-localized while Ca localized with Zn. AbstractUnderstanding the uptake mechanisms of heavy metals by hyperaccumulators is necessary for improving phytoextraction options to reduce metal toxicities in contaminated soils. In this study, the capacity of Zn uptake by the hyperaccumulator Noccaea caerulescens was investigated and compared to the non-hyperaccumulator Thlaspi arvense. The plants were grown under hydroponic conditions in a glasshouse. The distribution of Zn in the roots and leaves of these species was investigated by scanning electron microscopy with energy-dispersive X-ray analysis. Compared with the control with no Zn added, it was shown that prolonged Zn treatments decreased the biomass of both N. caerulescens and T. arvense. Since N. caerulescens requires Zn for growth, no Zn toxicity symptoms were observed, even when the Zn A C C E P T E D M A N U S C R I P T concentration in shoots reached 2.5% dry mass. T. arvense showed serious Zn toxicity only after two weeks of Zn treatment. Zn uptake by N. caerulescens was mainly translocated to the leaves while almost all of the Zn taken-up by T. arvense was retained in the roots. In N. caerulescens, increased concentrations of Zn in the shoots resulted in reductions in Ca and P concentrations by up to 50% and 35%, respectively. Zn-containing crystals were abundant in both the upper and lower epidermal cells of the leaves and in the cortex of the roots during the later growth phase.Co-localization of Ca and Zn, P and S were found in leaf and root tissues. The results suggest that Zn-rich crystals with an abundance of the Zn ligand in the roots and shoots, and colocalization and interaction between Zn and other ions, may have functional significance with respect to conferring particular attributes to N. caerulescens that are not present in the nonhyperaccumulator counterpart. An understanding of these species-specific differences has relevance from the perspective of offering some insight into how particular species could contribute to a strategy for the detoxification of Zn-contaminated sites.

Tolerance and accumulation of multiple trace elements simultaneously in hyperaccumulator species enable these plants to grow on sites contaminated with these elements. In this study, accumulation and tolerance to zinc (Zn) and lead (Pb) by the hyperaccumulator Noccaea caerulescens (Brassicaceae) was investigated using different Zn 2+ and Pb 2+ treatments in hydroponic culture. The results confirmed that N. caerulescens has a high capacity for Zn 2+ accumulation while having high levels of Pb 2+ tolerance. The younger plants were more tolerant to Zn 2+ and Pb 2+ than the older plants. The accumulation of Zn 2+ in shoots or roots was not significantly affected by treatment regime or plant age. Pb accumulated mainly in the roots (0.16-0.23 wt% dry mass), confirming substantial tolerance to Pb. The concentration of phosphorus (P) in older plant shoots decreased ∼25% in the plants treated with Zn 2+ , but enhanced ∼26% in the plants treated with Zn 2+ + Pb 2+. The high ratio of Zn to P in both fresh and dry leaves is suggestive of the formation of insoluble Zn-salts. The Zn 2+ distribution in living cells was examined using three selective fluorescent probes (Zinpyr-1, Newport Green DCF and Phen Green SK). The fluorescent probes showed that Zn 2+ was mainly located in the apoplastic space of the leaf epidermal cells. Selective fluorescent probes in combination with laser confocal microscopy proved a useful tool for elucidating cellular and tissue-level distribution of Zn 2+ in living plant cells at high resolution. However, the expected vacuolar sequestration of Zn 2+ was not observed, which may be explained by insufficient penetration of the fluorophores.

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 nonhyperaccumulator, 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. Znrich 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.ii 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. In this thesis, a number of novel techniques were succe...