Throughout the world, over 400 species of plants are known to accumulate large quantities of metals in their shoots (`hyperaccumulators'), but of these, relatively few accumulate manganese (Mn). We have identified for the first time an Australian native hyperaccumulator of Mn, Austromyrtus bidwillii (Benth.) Burrett (Myrtaceae). Concentrations of Mn up to 19 200 µg g-1 were measured in dried leaves of this rainforest tree, and young bark was found to contain up to 26 500 µg g-1 Mn. Approximately 40% of the Mn in the leaves is readily extracted with water, suggesting that some of the Mn is associated with water-soluble compounds such as organic acids. Organic acids present in appreciable amounts in leaf extracts of A. bidwillii were identified and quantified by HPLC and gas chromatography-mass spectrometry. The following organic acids (in order of concentration) were present: succinic > malic ≥ malonic > oxalic >> citric acid. The concentration of total organic acids was on average 123 000 µg g-1 dry tissue, which amounted to approximately three times the molar equivalent of Mn and two times the molar equivalent of total cations (Mn, Mg and Ca), demonstrating that organic anions were in excess. The Mn remaining after water extraction ((61 ± 3.9%) could be extracted with 0.2M HCl, suggesting that a significant portion of the Mn is associated with the cell wall (perhaps replacing Ca) or is present as other insoluble compounds.

Gossia bidwillii (Myrtaceae) is a manganese (Mn)-hyperaccumulating tree native to subtropical eastern Australia. It typically contains foliar Mn levels in excess of 1% dry weight. However, in G. bidwillii and other Mn-hyperaccumulating species, the cellular and subcellular localization of Mn has not been measured. Quantitative in vivo cryo-scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDAX) was used to localize Mn and other elements in tissue collected from mature trees growing in a natural population. Cryo-SEM showed that the leaf mesophyll is differentiated as a double-layer palisade mesophyll above spongy mesophyll. Transmission electron microscopy (TEM) revealed that the palisade and epidermal cells are highly vacuolated. EDAX data were used to estimate in situ vacuolar Mn concentrations of all cell types in fresh cryo-fixed leaf tissues. The highest average vacuolar Mn concentration of over 500 mM was found in the upper-layer palisade mesophyll, while the lowest concentration of around 100 mM was found in the spongy mesophyll. Qualitative in vivo cryo-SEM/EDAX was employed to further investigate the spatial distribution of Mn in fresh leaf tissues and young bark tissue, which was also found to have a high Mn concentration. It is concluded that Mn distribution in G. bidwillii is quantitatively different to metal distribution in other hyperaccumulating species where the highest localized concentrations of these elements occur in non-photosynthmetic tissues such as epidermal cells and associated dermal structures including trichomes and leaf hairs.

Summary• Little is known about the spatial distribution of excess manganese (Mn) in the leaves of tolerant plants. Recently, the first such study of a Mn hyperaccumulator showed that the highest localized Mn concentrations occur in the photosynthetic tissue. This is in contrast to reports based on localization of foliar accumulation of other heavy metals.• Here, four tree species, Gossia bidwillii , Virotia neurophylla , Macadamia integrifolia and Macadamia tetraphylla , which hyperaccumulate or strongly accumulate Mn, were studied. Cross-sectional foliar Mn localization was carried out in situ using proton-induced X-ray emission/energy dispersive X-ray analysis (PIXE/EDAX).• All four species contained photosynthetic tissues with multiple palisade layers. These were shown to be the primary sequestration sites for Mn. Mn was not detected in the epidermal tissues.• The findings of this study demonstrate a concurrence of three traits in four tree species, that is, accumulation of excess Mn in the leaves, its primary sequestration in the photosynthetic tissues, and multiple-layer palisade mesophyll.

Gossia bamagensis, G. fragrantissima, G. sankowsiorum, G. gonoclada and Maytenus cunninghamii were identified as 'new' Mn hyperaccumulators, while Gossia lucida and G. shepherdii are possible 'new' Mn hyperaccumulators. Of the three Myrtaceae genera examined, Mn hyperaccumulation appears restricted to Gossia, supporting its recent taxonomic revision. In the context of this present investigation and existing information, a reassesment of the general definition of Mn hyperaccumulation may be warranted. Morphological variation of Maytenus cunninghamii at two extremities was consistent with variation in Mn accumulation, indicating two possible 'new' subspecies. Although caution should be exercised in interpreting the data, surveying herbarium specimens by chemical analysis has provided an effective means of assessing foliar Mn accumulation. These findings should be followed up by field studies.

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