Ecology of the hyperaccumulating plants

All three species are native to per-humid equatorial rainforest. Figure 1 shows P. balgooyi, P. securinegioides and R. bengalensis in the native habitat in Sabah (Malaysia) on the Island of Borneo. The soils on which they grow are derived from serpentinised ultramafic bedrock. Concentrations of Ni, Co and Mn are strongly enriched during soil formation. The mean soil pH is rather similar in the rhizosphere from all three species and circum-neutral (pH 6.4–7.1). Although total Ca and Mg concentrations vary, the exchangeable concentrations are similar between the rhizospheres. The soils are K-deficient with mean exchangeable K+ ranging from 57–120 μg g−1. The mean total Ni concentrations are high (2480–3110 μg g−1), but potentially plant available concentrations are moderate with 88–220 μg g−1 mean DTPA Ni2+ (Table 1).

Figure 1 Phyllanthus balgooyi: exuding phloem sap (A), branch with leaves (B), cut branch with phloem tissue (C); Phyllanthus securinegioides: branch with leaves and seed capsules (D), plant with inflorescences (E), seed capsules (F); Rinorea bengalensis: mature tree 22 m high (G), excised bark with phloem tissue (H), pith of twig (I). Full size image

Table 1 Elemental concentrations in the rhizosphere and in the bedrock in the natural habitat of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis values as ranges and means in μg g−1 and *wt% dry weight). Full size table

Elemental concentrations and distribution in hyperaccumulator plants

Bulk analysis by ICP-AES showed that in the three species foliar and tissue concentrations of most elements, except Ni, are mostly unremarkable. However, foliar K concentrations are high (mean 860–8740 μg g−1) considering the K-deficient nature of the soil (Table 2). In comparison with the soil Ca concentrations (mean 1330–1600 μg g−1 exchangeable Ca), foliar Ca-concentrations (mean 4290–8020 μg g−1) are strongly enriched, especially in R. bengalensis (Table 2). The high Ni concentrations (mean 5140–7630 μg g−1) in the branches, compared to the wood, can be attributed to the presence of phloem tissue, which is extremely high in Ni (mean 8940–65410 μg g−1). The phloem sap of P. balgooyi is one of the most unusual biological liquids, it contains 12.6–16.9% Ni, and is also enriched in Co (560–2340 μg g−1) and Zn (1900–3690 μg g−1), but low in major cations such as Ca (660–1960 μg g−1) and K (520–1530 μg g−1) (Table 3).

Table 2 Bulk elemental concentrations in plant tissues (leaves, twigs, bark, wood) and fluids (xylem and phloem) in Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis (values as ranges and means in μg g−1 dry weight). Full size table

Table 3 Bulk elemental concentrations in transport tissue and liquids (xylem and phloem) in Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis (values as ranges and means in μg g−1 dry weight). Full size table

Micro-PIXE analysis showed that the most important feature of Ni distribution in roots and stems of all three hyperaccumulators is the exceptional enrichment in the phloem (Tables 4 and 5, Figs 2 and 3). However, the species differ in the leaves – in P. balgooyi the highest Ni concentration is in phloem, but in P. securinegioides in the epidermis. Rinorea bengalensis has the highest Ni concentration in the spongy mesophyll but also high enrichment in the epidermis (Table 6, Fig. 4). There are also some other differences. In the roots Ni concentrations in the phloem vary from slightly less than 0.2 wt% in P. balgooyi to ca. 0.9 wt% in P. securinegioides and 5.6 wt% in R. bengalensis (Table 4, Fig. 2). In P. balgooyi there is also some enrichment in the epidermis, but the concentrations there are significantly lower than in the phloem (180 μg g−1). The lowest amounts of Ni were measured in cortex and xylem. In P. securinegioides and R. bengalensis the cortex contains more Ni than the epidermis and xylem (Table 4, Fig. 2). Phosphorus, S, K, Ca, and Zn show somewhat similar distribution pattern as Ni, with the highest amounts in the phloem in P. balgooyi and P. securinegioides. Chlorine is the only element with the highest enrichment in the cortex of P. balgooyi but in P. securinegioides and R. bengalensis the highest concentrations are in the phloem (Table 4, Supplementary Figures 3, 4 and 5). In R. bengalensis P and S show markedly different distribution compared to P. securinegioides and P. balgooyi, forming two enrichment rings in the cortex and xylem, separated by depletion zone in the phloem (Table 4, Supplementary Figures 3, 4 and 5). The highest concentrations of Cl, K, Ca, Cu, Zn, Rb and Sr were found in the phloem. In all three species Si, Ti, Cr, Mn, Fe and Co reach the highest concentrations in the root epidermis.

Table 4 Elemental concentrations (micro-PIXE, μg g−1 dry weight) in the morphological structures of the roots of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Full size table

Table 5 Elemental concentrations (micro-PIXE, μg g−1 dry weight) in the morphological structures of the stems of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Full size table

Figure 2: Elemental maps and morphological structure of root cross-sections of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Concentration scale in wt% dry weight or in μg g−1dry weight. X, xylem; P, phloem; C, cortex; E, epidermis. Scale bar – 100 μm. Full size image

Figure 3: Elemental maps and morphological structure of stem cross-sections of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Concentration scale in wt% dry weight or in μg g−1 dry weight. X, xylem; P, phloem; C, cortex; E, epidermis ; Pd, periderm; VC, vascular cambium; YX, younger xylem; OX, older xylem. Scale bar – 1000 μm. Full size image

Table 6 Elemental concentrations (micro-PIXE, μg g−1 dry weight) in the morphological structures of the leaves of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Full size table

Figure 4: Elemental maps and morphological structure of leaf cross-sections of Phyllanthus balgooyi, Phyllanthus securinegioides and Rinorea bengalensis. Concentration scale in wt% dry weight (for Ni, Ca, K, S, Cl) and μg g−1 dry weight (for Mn, Fe). UE, upper epidermis; LE, lower epidermis; X, xylem; P, phloem; PM, palisade mesophyll; SM, spongy mesophyll. Scale bar – 100 μm. Full size image

The elemental distribution in the stems, petioles and leaves of P. balgooyi were reported and discussed earlier18. The main feature in the stems is the extremely high Ni concentration in the phloem, followed by significant enrichment in the cortex (Table 5, Fig. 3). The distributions of Fe, Co, and Zn mirror that of Ni, but their concentrations are much lower. Although in the stems of P. securinegioides and R. bengalensis Ni also shows the highest concentrations in the phloem, some important differences were found. The xylem in P. securinegioides can be subdivided into younger and older parts, with the younger part significantly enriched in Ni in comparison with the older parts (500 μg g−1 and 140 μg g−1, respectively) and a very distinct, highly Ni-enriched (up to 2800 μg g−1) compressed pith (Table 5). In the stems of R. bengalensis the highest Ni concentrations are in the phloem, but equally high values were found in the meristematic cortical cells (Table 5, Fig. 3), of the order of 0.6 wt% in both cases. The remaining parts of cortex also show Ni enrichment (0.3 wt%) as well as the pith (0.28 wt%).

In P. securinegioides Ca shows a similar distribution pattern compared to P. balgooyi with the highest concentration in the phloem, In R. bengalensis it also shows enrichment in the phloem, but the highest concentrations are in the epidermis (Fig. 3, Table 5). Many small “dots” with high Ca amounts are visible in the pith of this species. In P. securinegioides K and S are also concentrated in the phloem area, but their distribution pattern is more “diluted” than that of Ni or Ca, and the highest concentration of K is rather in the vascular cambium region. In R. bengalensis the distribution of K shows similarities to that of Ni, but the highest concentration of this element is in the cortex.

The distribution pattern of S in R. bengalensis is somewhat similar to that of Ni, with the highest concentrations in the phloem and cortex. In P. securinegioides P is another element forming an enrichment “ring” in the cambium region (Table 5, Supplementary Figure 6). In R. bengalensis this element has a very different distribution pattern, with the highest concentrations in the pith; xylem is the second region of its enrichment, although less pronounced (Table 5, Supplementary Figure 6). Silicon, Ti, Cr, Mn, Fe and Co are only enriched in the epidermal area of stems, both in P. securinegioides and R. bengalensis. Cl is concentrated in the phloem, but it is not evenly distributed in this region (Supplementary Figure 6). In P. securinegioides it forms an enrichment ring in the outer phloem and inner cortex, but in R. bengalensis such a ring is visible in the innermost part of phloem.

In comparison with the leaves of P. balgooyi, Ni concentrations in the epidermal in P. securinegioides and R. bengalensis are much higher than in the phloem (Table 6, Fig. 4), although there is some enrichment in the phloem, relative to the adjoining xylem in P. securinegioides. In P. securinegioides the upper epidermis is notably richer in Ni (up to 4 wt%, on average) than the lower epidermis with the adjoining part of spongy mesophyll (on average 1.9 wt%). In R. bengalensis the highest concentrations are in the spongy mesophyll and the adjoining lower epidermis. The upper epidermis has slightly lower concentrations, as well as palisade mesophyll. Among elements showing enrichment in epidermal regions, Mn in P. securinegioides is only concentrated in the upper epidermis, while in R. bengalensis such enrichment extends to the adjoining palisade mesophyll. Iron enrichment in the epidermal regions is much more “patchy” and less pronounced. Calcium in P. securinegioides shows the highest enrichment in the phloem, but in both species it is also concentrated in the mesophyll showing a “grainy” inhomogeneous distribution. The distributions of P and Zn are shown in the Supplementary Figure 7.

Chemical speciation of Ni2+ in hyperaccumulator plants

X-ray Absorption spectroscopy (XAS) was used to investigate the in situ Ni speciation in various tissues across the three plant species. The Ni K-edge XANES spectra of the tissues are presented in Fig. 5 (traces C-P) and visual inspection reveals that the spectra vary little beyond variations in noise level that arise as a result of the varying Ni concentration in the respective tissues. A principal component analysis (PCA) of these fourteen plant spectra suggested that between two and four significant components were present, while visualisation of the PCA eigenvectors (not shown) indicated that the variation was limited to <8350 eV.

Figure 5 Ni K-edge X-ray absorption near edge spectra for A 1:10 Ni:citrate in aqueous solution; B 1:1 Ni:citrate in aqueous solution; C R. bengalensis mid-vein tissue; D R. bengalensis leaf tissue; E R. bengalensis xylem; F R. bengalensis phloem tissue; G P. balgooyi xylem; H P. balgooyi wood tissue; I P. balgooyi root sheath tissue; J P. balgooyi phloem tissue; K P. balgooyi leaf tissue; L P. securinegioides xylem; M P. securinegioides wood tissue; N P. securinegioides root tissue; O P. securinegioides phloem tissue; P P. securinegioides leaf tissue. Vertical lines are drawn at 8361 and 8400 eV to aid visual comparison of key features in the spectra. The spectra are shifted vertically for clarity. Full size image

The plant XANES spectra were compared against a number of aqueous solution model complexes of Ni and physiological relevant ligands as well as two insoluble Ni compounds (Fig. 6). Several of the model spectra were easily distinguished from the plant spectra by visual inspection, including complexes with N- or S-donor ligands (Fig. 6 - trace H), the solids (trace G) as well as the complexes with nicotianamine, oxalate or phytate (trace F). The remaining spectra of complexes dominated by carboxylate binding matched the plant-based spectra far more closely, but could be differentiated by features centred at either ~8361 and 8400 eV. The lower energy feature varied in intensity between the different models, being more intense and well resolved in the citrate (1:10 Ni:citrate–trace B), tartrate and malonate spectra (trace D), but less so in the remaining spectra including the plant-based example in A.

Figure 6 Ni K-edge X-ray absorption near edge spectra for A P. balgooyi phloem tissue (black trace) and 1:1 Ni:citrate in aqueous solution (green trace); B Ni:nitrate in aqueous solution (i.e. [Ni(H 2 O) 6 ]2+-black trace) and 1:10 Ni:citrate in aqueous solution (green trace); C, 1:6 Ni:glutathione in aqueous solution at pH ~5–6 (black trace), 1:6 Ni:acetate in aqueous solution at pH ~5–6 (green trace), 1:6 Ni:aconitate in aqueous solution at pH ~5–6 (red trace), 1:6 Ni:succinate in aqueous solution at pH ~5–6 (blue trace)–all traces essentially identical; D 1:6 Ni:tartrate in aqueous solution at pH ~5–6 (black trace), 1:6 Ni:malate in aqueous solution at pH ~5–6 (green trace); E 1:6 Ni:acetylacetone in aqueous solution at pH ~5–6 (black trace), 1:6 Ni:malonate in aqueous solution at pH ~5–6 (green trace); F 1:6 Ni:nicotianamine in aqueous solution at pH ~5–6 (black trace), 1:6 Ni:oxalate in aqueous solution at pH ~5–6 (green trace), 1:6 Ni:phytate in aqueous solution at pH ~5–6 (red trace); G NiO (s) (black trace) and Ni(OH) 2(s) (green trace) diluted to 1000 ppm in boron nitride; H 1:6 Ni:cysteine in aqueous solution at pH ~8 (black trace), 1:6 Ni:glutathione in aqueous solution at pH ~8 (blue trace), 1:6 Ni:histidine in aqueous solution at pH ~8 (red trace). Vertical lines are drawn at 8361 and 8400 eV to aid visual comparison of key features in the spectra. The spectra are shifted vertically for clarity. Full size image

Variation in the higher energy feature was more subtle, being either: broad and rounded (1:10 Ni:citrate–trace B); narrower and rounded (malate and tartrate in D); split and resolved symmetrically (the four traces in C); split and resolved asymmetrically (acetylacetonate and malonate in trace E); or broad and unresolved (the plant and 1:1 Ni:citrate model in trace A).

Meanwhile, the spectrum of hexaquo Ni2+ showed intermediate intensity of the peak at 8361 eV and a broad unresolved feature centred on 8400 eV. While this region was very similar to the plant-based spectra, the white line peak (~8349 eV) intensity for the hexaquo complex was significantly greater than in either the plant or carboxylate model spectra, as well as appearing at slightly higher energy as previously reported by others44.

In summary, the plant XANES spectra were well matched by the spectrum of the 1:1 solution of Ni(II) and citrate at pH 5.5, and could be distinguished from the spectra of all other model complex spectra by a combination of features at the white line, 8361 and ~8400 eV. The XANES spectra for pH adjusted and unadjusted Ni:citrate were consistent.

We did attempt a least-squares fitting of the plant spectra with a selection of the model complex XANES spectra but abandoned this approach for several reasons. As noted above, a principal component analysis was not conclusive regarding the number of model compounds that should be included in the linear regression and fit results generated with different numbers of components varied markedly. In addition, and perhaps most importantly, the spectrum of the 1:1 Ni:citrate solution was recorded at a different beamline (the AS XAS beamline; as the ANBF was permanently decommissioned shortly after the spectra of the plants and other models were recorded) than the spectra of the plant tissues. Because the spectral resolution of the two beamlines was not necessarily identical (also potentially affecting energy calibration) the spectrum of the 1:1 citrate model could not be rigorously included in the statistical analyses, despite being apparently the best match by eye.

We also compared the extended X-ray absorption structure (EXAFS) data of the plant tissues (Supplementary Figure 1 - all recorded at ANBF) with the EXAFS data from a selection of the most closely matched model spectra (Supplementary Figure 2 - recorded at the AS) according to the XANES results. We note that the EXAFS data will be less sensitive to differences in the spectral resolution of the two beamlines. Visual inspection of the k-space data to 10 Å−1 in Supplementary Figure 1 reveals that the EXAFS for these samples are essentially identical with minor variations noted between 3–4 and 5–6 Å−1, particularly for the leaf samples (black traces F and I). More significant variations were noted in these ranges for the model complexes EXAFS data shown in Supplementary Figure 2 with those EXAFS and variations similar to those previously reported by others22,44. Only the EXAFS of the malonate complex was clearly distinguishable from that of the plant samples (due to a strong oscillation in the EXAFS at ~3.5 Å−1) but the EXAFS of the plants was closest to that of the 1:1 citrate model (asymmetrical oscillation between 3–4 Å−1 and a weak “shoulder” on the low-k side of the oscillation between 5–6 Å−1). Again, attempts to fit the plant sample EXAFS with a linear combination of the model complex EXAFS was abandoned because variation in the number of fitted models dramatically altered the fit results.

Taken together, the XANES and EXAFS data of the plants indicates that the predominant species present in the tissues is a 1:1 Ni complex with citrate and the remainder of the coordination sphere consisting of water ligands. The minor variations in the spectra in the leaf tissues can be explained by the presence of a 1:2 complex of Ni with citrate, producing a slightly more intense peak in the XANES at 8361 eV (Fig. 5–traces P and K) and, somewhat less clearly, a variation in the EXAFS between 5–6 Å−1. It is certainly feasible that the relative proportion of these two species may vary between plant species and between particular tissues in those species. We believe that the more complicated mixtures of ligands identified by other groups in other Ni hyperaccumulators, including malate complexes and others, may be as a result of the fact that they did not include the 1:1 Ni:citrate complex spectrum in their fitting, and the evidence for the presence of these other complexes based purely on XAS data is weak.

Gas-chromatography (GC-MS) was used for untargeted metabolite profiling in the freeze-dried plant tissues and phloem sap samples. Relative response ratios (RRR’s) were calculated using the metabolite peak area divided by the internal standard area and sample mass. This resulted in a list of 95 compounds, of which mass spectral libraries positively identified 60 compounds (Table 7), however the method cannot detect any Ni-complexes. GC-MS was also used to quantify the six most abundant metabolites determined in the metabolomic profiles; these were malate, citrate, fructose, glucose, sucrose and catechin in the P. balgooyi phloem sap (Table 8). The sugars are of interest, as ref. 48 reported the formation of Ni:citrate:sucrose complexes. Ion chromatography (IC) was then used to quantify anions in the P. balgooyi phloem sap and as such compile a mass balance of ligands and anions available to complex Ni2+ (Table 9). The pH of the thawed phloem sap was pH 6.54, which aligned well with spot tests taken in the field on fresh sap (~pH 6.0–6.5). The use of LC-MS enabled the identification Ni2+ containing complexes. A large number of ions containing the isotope pattern of Ni2+ were present in the chromatograms of the extracts of the plant species. Figure 7 shows the mass spectrum of Ni-citrate [M.L 2 -H]+ from P. balgooyi phloem sap (black trace) and theoretical isotope pattern for Ni-citrate (red boxes). The most prominent Ni2+ containing ion detected in all samples was Ni2+ citrate [M.L 2 -H]+.

Table 7 Major metabolites and identified Ni-complexes in leaf and phloem extracts of P. balgooyi, P. securinegioides and R. bengalensis. Full size table

Table 8 Concentrations of citric acid, malic acid, fructose, sucrose, glucose and catechin in leaf and phloem extracts of P. balgooyi, P. securinegioides and R. bengalensis. Full size table

Table 9 Mass-balance including major cations and anions in the phloem sap of P. balgooyi. Full size table

Figure 7 Mass spectrum of Ni-citrate [M.L 2 -H]+ from P. balgooyi phloem sap (black trace) and theoretical isotope pattern for Ni-citrate (red boxes). Full size image

The mass balance presented in Table 9 shows that the mean anion concentration is 0.46 wt% versus a mean of 76.4 wt% citrate to balance a mean of 15.6 wt% Ni. Taking into account relevant ionic charges, the quantities of citrate together with the relatively minor concentrations of cations are sufficient to complex and ionically balance virtually all of Ni2+ present in the phloem sap of P. balgooyi.