This study is the first to investigate the speciation and spatial distribution patterns of manganese (Mn) accumulated at elevated concentrations in Eucalyptus leaves by X-ray fluorescence (µ-XRF) and absorption near-edge spectroscopy (µ-XANES). Eucalyptus tereticornis is a tree species with great economic value and potential to accumulate and tolerate high Mn despite not being considered a hyperaccumulator. Seedlings grown under glasshouse conditions were irrigated with two Mn treatments: control Mn (9µM) and high Mn solution (1000µM). Biomass and total nutrient concentrations were assessed in roots, stems and leaves. Manganese, calcium (Ca) and potassium (K) spatial patterns were imaged by µ-SXRF in different foliar structures, and Mn speciation was conducted in these compartments by µ-XANES. Under high supply, Mn was distributed across the leaf mesophyll suggesting vacuolar sequestration in these cells. High Mn decreased cytosolic Ca by almost 50% in mesophyll cells, but K remained unaltered. Speciation suggests that a majority of the Mn fraction was complexed by organic ligands modeled as Mn-bound malate and citrate, instead of as free aqueous Mn2+ or oxidised forms. These two detoxification mechanisms: effective vacuolar sequestration and organic acid complexation, may be responsible for the impressively high Mn tolerance found in eucalypts.

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