Zinc and lead accumulation characteristics and in vivo distribution of Zn²⁺ in the hyperaccumulator Noccaea caerulescens elucidated with fluorescent probes and laser confocal microscopy

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 Zn²⁺ and Pb²⁺ treatments in hydroponic culture. The results confirmed that N. caerulescens has a high capacity for Zn²⁺ accumulation while having high levels of Pb²⁺ tolerance. The younger plants were more tolerant to Zn²⁺ and Pb²⁺ than the older plants. The accumulation of Zn²⁺ 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 Zn²⁺, but enhanced ∼26% in the plants treated with Zn²⁺ + Pb²⁺. The high ratio of Zn to P in both fresh and dry leaves is suggestive of the formation of insoluble Zn-salts. The Zn²⁺ 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 Zn²⁺ 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 Zn²⁺ in living plant cells at high resolution. However, the expected vacuolar sequestration of Zn²⁺ was not observed, which may be explained by insufficient penetration of the fluorophores.