NanoSIMS and EPMA analysis of nickel localisation in leaves of the hyperaccumulator plant Alyssum lesbiacum

Abstract

Certain plants known as ‘metal hyperaccumulators’ can accumulate exceptional concentrations of elements such as zinc, manganese, nickel, cobalt, copper, selenium, cadmium or arsenic in their above ground tissue. In members of the genus Alyssum, nickel concentrations can reach values as high as 3% of leaf dry biomass. These plants must possess very effective mechanisms for the transport, chelation and sequestration of such elements within their tissues to avoid the toxic effects of free metal ions. Evidence from a number of different techniques suggests that nickel is concentrated primarily in the outermost, epidermal tissue of leaves of Alyssum hyperaccumulators, but there is currently no consensus on the principal sites of nickel sequestration. In this study, high resolution secondary ion mass spectrometry (NanoSIMS) analysis has been performed on longitudinal sections of Alyssum lesbiacum leaves. Elemental maps were obtained which revealed the high concentrations of nickel in the peripheral regions of the large unicellular stellate leaf hairs (trichomes) and in the epidermal cell layer. Electron probe microanalysis (EPMA) was used to provide independent confirmation of elemental distribution in the specimens, but the superior spatial resolution and high chemical sensitivity of the NanoSIMS technique provided a more detailed image of elemental distribution in these biological specimens at the cellular level.

Introduction

Certain terrestrial plants can accumulate very high concentrations of potentially toxic metallic elements in their leaves and stems without suffering from any impairment of growth. Over 400 species of these so-called “metal hyperaccumulator” plants are now known [1], [2]. These species tend to be restricted in their natural distribution to metalliferous soils, and they typically contain metal concentrations in their above ground tissues about two orders of magnitude higher than the great majority of plant species [3], [4]. The threshold values for metal hyperaccumulattion have been defined as 1% of dry shoot biomass for zinc and manganese, 0.1% for nickel, cobalt, copper and selenium, and 0.01% for cadmium and arsenic although values as high as 5 and 3% of dry biomass have been recorded for zinc and nickel, respectively [1], [2]. Due to the high degree of metal tolerance shown by hyperaccumulator plants, it is possible that they might find application in various forms of bioremediation treatment for the decontamination of metal-polluted soils [5], [6], [7], [8].

To avoid the toxic effects of free metal ions, hyperaccumulator plants must possess effective mechanisms for the transport, chelation and sequestration of such elements within their tissues. Many of the membrane proteins responsible for metal transport throughout the plant have now been identified [9], [10], [11], as have some of the principal ligands involved in metal binding in different phases of the cell [3], [9], [12], [13]. For example, uptake of nickel by hyperaccumulator plants is associated with a specific and proportional synthesis of the aminocarboxylic acid histidine, which strongly coordinates the incoming nickel and facilitates its translocation through the plant [14], [15]. However, there is less certainty about the ultimate sites of metal sequestration within the shoots of hyperaccumulator plants. Consequently, there is a need for the application of novel techniques to help determine the sites of metal deposition in biological tissues at high resolution.

Within the shoot tissues of hyperaccumulator plants, metals may be stored in several distinct cellular compartments. Quantitatively the largest compartment is the central vacuole, which may occupy as much as 90% of cell volume in mature cells. In terrestrial plants, the cell vacuole typically contains high concentrations of hydroxycarboxylic acids (e.g., malic, citric, isocitric, malonic, tartaric acids); these can bind metal ions with moderately high affinity, but are thought unlikely to be responsible for the chemical specificity of metal hyperaccumulation [3], [12], [14]. Most enzymes of central metabolism are localised in the cell cytoplasm, and functional groups on proteins must be protected from inactivation by metal ions. This is achieved by appropriate high-affinity ligands (including low-molecular-weight metabolites, peptides and proteins), which buffer the cytoplasmic pool of transition metals and maintain the concentration of free metal ions at very low values. Finally, significant metal binding may also occur in the extracellular matrix of the cellulosic cell wall, in which considerable quantities of metal ions can be bound by the polygalacturonic acid residues of non-esterified pectins, and by wall-localised proteins.

Considerable work has been performed on nickel-hyperaccumulator plants to try to determine the principal sites of metal localisation within the shoots, but a consistent picture of nickel distribution within these tissues has not yet emerged. Using subcellular fractionation techniques, Krämer et al. [16] reported that 67–73% of the nickel in the leaves of Thlaspi goesingense is associated with the cell wall. In contrast, Küpper et al. [17] investigated metal localisation by a direct method involving energy dispersive X-ray (EDX) analysis of rapidly frozen hydrated material performed on a scanning electron microscope (SEM) with a cryostage attachment. These authors reported nickel to be localised predominantly in the vacuoles rather than the cell wall of leaves of Alyssum bertolonii, Alyssum lesbiacum and T. goesingense. Furthermore, Küpper et al. [17] found that for the three hyperaccumulator plants they studied, all showed preferential accumulation of nickel in the vacuoles of the outermost, epidermal cell layer of leaves; in contrast the mesophyll cells forming the ground tissue and cells in the central vascular tissue contained much less nickel, a pattern of metal accumulation that may be interpreted as protecting photosynthetic activity in the chloroplasts-containing mesophyll cells. Apart from differences in methodology, these contrasting results might also be a function of the precise conditions under which the plants were cultivated and the developmental age of the tissues examined.

Another distinctive structural feature of nickel-hyperaccumulator plants in the genus Alyssum is the presence of large, unicellular stellate hairs (trichomes) in the epidermis, which can form a sufficiently dense covering to give the shoot surface a silvery-whitish appearance, see Fig. 1. The EDX analyses of Küpper et al. [17] apparently indicated exclusion of nickel from the bulk of the trichomes of A. bertolonii and A. lesbiacum. However, evidence was obtained by SEM/EDX for a locally high concentration of nickel in the basal part of the trichome, which was confirmed by staining with the nickel reagent dimethylglyoxime [17]. These results differ somewhat from those of Krämer et al. [18], who used proton-induced X-ray emission (PIXE) with a spatial resolution of approximately 1 μm to examine dehydrated and fixed leaf specimens of A. lesbiacum prepared by molecular distillation drying. The PIXE results indicated that nickel is sequestered to a considerable degree in the epidermal trichomes, although this technique did not allow for the finer subcellular analysis needed to explore the mechanisms of sequestration. Again, the discrepancies in these reports concerning the sites of highest nickel concentration in leaves of Alyssum may be methodological. Dehydration and fixation prior to elemental mapping, as used by Krämer et al. [18], may lead to redistribution of mobile ions, even when the rate of sample dehydration is minimised. In localising nickel histochemically, Küpper et al. [17] immersed leaves in dimethyglyoxime for 3 h, then dissected and observed them optically. However, the trichomes of Alyssum are covered by a thick, waxy, hydrophobic cuticle, which may have hindered penetration of the dye; on detaching the trichomes from the leaf surface, the basal region was exposed to the dye but the thick waxy cuticle of the external surface remained intact, so the colorimetric reaction with nickel may have been favoured in the basal region of the trichomes.

There has also been a report by Psaras et al. [19], based on a study with SEM/EDX, that the epidermal trichomes of Leptoplax emarginata (a species closely related to Alyssum) do not accumulate nickel. These authors studied nickel accumulation in the leaf epidermis of dried herbarium material of eight hyperaccumulator species. In all species, nickel was apparently excluded from the trichomes and specialised stomatal guard cells. In some species, nickel was present at low levels in the subsidiary cells adjacent to the stomatal guard cells, while the sites of higher nickel accumulation were observed in epidermal cells away from the stomatal complex. Psaras et al. [19] suggested that these results indicate that nickel is not compatible with the specialised functions of cells in the stomatal complex, and concluded that nickel is sequestered mainly in the highly vacuolate and metabolically less active cells of the epidermis. However, the apparent absence of nickel from the trichomes reported by Psaras et al. [19] is in direct contradiction to both the PIXE results of Krämer et al. [18] and the colorimetric results of Küpper et al. [17]. In contrast, in the nickel hyperaccumulator Thlaspi montanum var. siskiyouense – a species that lacks epidermal trichomes – it has also been observed by SEM/EDX that nickel is present in subsidiary cells of the stomatal complex, but is apparently absent from both the stomatal guard cells and the unspecialised epidermal cells [20].

In 2004, Broadhurst et al. [21] reported the co-localisation of high levels of nickel, manganese and calcium in certain regions of the trichomes of several species of Alyssum using SEM/EDX analysis. It was reported that nickel is stored either in Alyssum leaf epidermal cell vacuoles or in the basal portion only of the stellate trichomes, with no appreciable nickel detectable in the arms (rays) or surface features (nodules). However, calcium was strongly concentrated in the trichome rays and nodules [21], [22]. More recently, McNear et al. [23] have used synchrotron-based fluorescence and absorption-edge computed microtomography to investigate speciation of nickel in leaves of Alyssum, although this technique lacks the spatial resolution required to map intracellular details of metal localisation at the subcellular level. The authors suggest that nickel is concentrated mainly in the vascular tissue, epidermal trichomes and basal compartments of trichomes, and is absent from the photosynthetically active mesophyll cells. Colocalisation of nickel with cobalt, zinc and manganese was observed in some specimens, suggesting that sites of metal deposition may be partly determined by pathways of delivery of metals to the shoot from the root in the transpiration stream [21], [23], [24].

To date, therefore, there is no complete consensus on the principal sites of nickel storage in the shoots of hyperaccumulator plants. New high resolution analytical methods would be valuable for obtaining independent evidence on sites of metal sequestration in plant tissues, especially at the sub-cellular level. Dynamic secondary ion mass spectrometry (SIMS) microscopy may provide such a method, although it has not yet been applied extensively to biological materials. It has been known for some 20 years that this technique may offer significant advantages for particular kinds of analysis, including exceptionally high sensitivities and high chemical specificity. Research in the life sciences using SIMS analysis began in 1982 with the work of Burns et al. [25], and 10 years later the whole of vol. 74 in the journal Biology of the Cell was dedicated to SIMS analysis of biological materials. When combined with the submicrometer lateral resolution of the latest generation of SIMS instrumentation, this technique can provide insight into many areas of biological research in which intracellular chemical variations in three dimensions are likely to be of critical importance.

The application of SIMS analysis to biological materials has been tested in a broad spectrum of studies; e.g., cancer research [26], [27] and the study of nucleotidic base analogues used in antiviral or anticancer therapy [28], [29]. In a SIMS study of soybean leaf Glycine max, Grignon et al. [30] showed that the easily diffusible, mobile ions potassium, calcium and magnesium in leaf tissue may be redistributed during chemical fixation and adsorbed onto certain cellular organelles. Recently, Hallegot et al. [31] reported results on the analysis of hair samples using high resolution SIMS, illustrating how the distribution of trace elements can be determined in organic samples.

In the present study, we have investigated the application of high resolution SIMS to the localisation of nickel in tissues of the hyperaccumulator plant A. lesbiacum, to determine whether the combination of analytical sensitivity and high spatial resolution (sub-150 nm) offered by this technique can provide new insight into nickel localisation at the cellular and subcellular levels in these tissues.