Pb-induced ultrastructural alterations and subcellular localization of Pb in two species of Lespedeza by TEM-coupled electron energy loss spectroscopy

Transmission electron microscopy (TEM) coupled electron energy loss spectroscopy (EELS) was conducted in the present study, in order to explore mechanisms involved in Pb tolerance and detoxification in two species of Lespedeza (Fabaceae). Both Lespedeza chinensis and Lespedeza davidii have been recently shown to be suitable for chelate-assisted Pb phytoremediation. Ultrastructural alterations, Pb localization and speciation in leaf and root cells of both species were investigated after Pb exposure. Additionally, the influence of EDTA which facilitates Pb translocation in plants was investigated. In general, deleterious effects were more pronounced in root cells of both species than in leaf cells. Similar ultrastructural alterations such as vacuolization and dilation of the nuclear envelopes were induced in leaf and root cells of both species by Pb treatment. The effects were intensified with the addition of EDTA. Autophagy occurred only in leaf cells of L. davidii after Pb and Pb plus EDTA treatment, and in root cells of both species after Pb treatment. Moreover, electron-dense deposits with variable shapes such as acicular, granulated, punctuated and amorphous, were present in cell walls, intercellular spaces, cytoplasm and vacuoles of root cells of both species after Pb and Pb plus EDTA treatment. These deposits contained Pb as identified via the Pb-M4,5 edge by EELS in a 120 kV TEM. As phosphorus was always measured at the sites of Pb deposition, it is likely that Pb in Lespedeza is stored as lead phosphate particularly in cell walls and vacuoles. The occasional presence of S and Ca in Pb depositions may point to additional other Pb compounds. Our studies suggest that Pb is transported both in apoplastic and symplastic pathways, independent of the presence or absence of EDTA. Different possible detoxification strategies such as sequestration in cell walls or compartmentalization in vacuoles exist both in L. chinensis and L. davidii. Only minor differences in ultrastructural alterations and Pb localization were observed between two species.

► Pb is localized in hyperaccumulator Lespedeza with TEM-coupled electron energy loss spectroscopy. ► Ultrastructural changes were shown to be more pronounced in root cells than leaf cells. ► Pb is transported in both apoplastic and symplastic pathways in Lespedeza. ► In roots Pb is sequestrated in various forms in two different species of Lespedeza. ► Lead phosphate is the main compound sequestrated in cell walls and vacuoles.