Elsevier

Phytochemistry

Volume 81, September 2012, Pages 80-89
Phytochemistry

Elemental and metabolite profiling of nickel hyperaccumulators from New Caledonia

https://doi.org/10.1016/j.phytochem.2012.06.010Get rights and content

Abstract

Leaf material from nine Ni hyperaccumulating species was collected in New Caledonia: Homalium kanaliense (Vieill.) Briq., Casearia silvana Schltr, Geissois hirsuta Brongn. & Gris, Hybanthus austrocaledonicus Seem, Psychotria douarrei (G. Beauvis.) Däniker, Pycnandra acuminata (Pierre ex Baill.) Swenson & Munzinger (syn Sebertia acuminata Pierre ex Baill.), Geissois pruinosa Brongn. & Gris, Homalium deplanchei (Viell) Warb. and Geissois bradfordii (H.C. Hopkins). The elemental concentration was determined by inductively-coupled plasma optical emission spectrometry (ICP-OES) and from these results it was found that the species contained Ni concentrations from to 250–28,000 mg/kg dry mass. Gas chromatography mass spectrometry (GC–MS)-based metabolite profiling was then used to analyse leaves of each species. The aim of this study was to target Ni-binding ligands through correlation analysis of the metabolite levels and leaf Ni concentration. Approximately 258 compounds were detected in each sample. As has been observed before, a correlation was found between the citric acid and Ni concentrations in the leaves for all species collected. However, the strongest Ni accumulator, P. douarrei, has been found to contain particularly high concentrations of malonic acid, suggesting an additional storage mechanism for Ni. A size exclusion chromatography separation protocol for the separation of Ni-complexes in P. acuminata sap was also applied to aqueous leaf extracts of each species. A number of metabolites were identified in complexes with Ni including Ni-malonate from P. douarrei. Furthermore, the levels for some metabolites were found to correlate with the leaf Ni concentration. These data show that Ni ions can be bound by a range of small molecules in Ni hyperaccumulation in plants.

Graphical abstract

Elemental and metabolite profiling were used to discover small molecule metabolites involved in Ni-hyperaccumulation. LC–MS was then used to confirm the presence of these complexes.

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Highlights

► Nine species were studied from New Caledonia with leaf Ni concentrations between 250–28,000 mg/kg dw. ► Elemental profiling using ICP-OES showed these plants maintain their metal ion homeostasis. ► GC–MS profiling found a number of correlations between nickel and small molecule metabolites. ► LC–MS detected a range of Ni-complexes with these metabolites. Psychotria douarrei had the highest malonic acid level and Ni-malonate was detected by LC–MS.

Introduction

New Caledonia is located in the Melanesian region of the southwest Pacific. Unlike many South Pacific islands, it is not of volcanic origin, but is instead a fragment of the ancient continent of Gondwana. This separation occurred 80 million years ago giving rise to a long period of evolution resulting in an ecosystem with a high degree of endemism. Approximately 3,300 plant species have been identified in New Caledonia of which 75% are endemic (Myers et al., 2000). The main island, Grand Terre, is particularly rich in ultramafic soils, and when classifying species on these soils the degree of endemism increases to 90% (Jaffré, 1992). New Caledonia is the fourth largest producer of Ni ore world-wide, making it the island’s main industry. However, this has put intense pressure on the native ecosystems (L’Huillier and Edighoffer, 1996). As a result of deforestation, New Caledonia has now been identified as a ‘biodiversity hotspot’ due to the exceptional concentration of endemic species which are under threat from habitat loss with primary vegetation contracting to 28% of the original cover (Myers et al., 2000).

Plants which store metals in their leaves at concentrations toxic to other organisms are known as hyperaccumulators. The threshold for Ni hyperaccumulation in plants is defined as a concentration greater than 1,000 mg Ni/kg in the leaf dry mass of plants growing in the field (Baker et al., 1999). Using this definition, approximately 50 taxa are known to hyperaccumulate Ni in New Caledonia (Jaffré et al., 1979b, Reeves et al., 1996). This number is second only to Cuba, where approximately 130 hyperaccumulators have been identified (Reeves et al., 1996). In Cuba, the older serpentine soils which are 10–30 million years old contain 81% of the serpentine endemic species compared with the younger soils that are approximately 1 million years old (Reeves et al., 1996). This trend appears to occur also in the New Caledonian serpentine flora (Jaffré, 1980). Areas not covered during the maximum advance of the Pleistocene ice caps (1,808,000 to 12,000 years before present) have afforded longer time spans for nickel tolerant plant populations to adapt to the metal rich soils. For this reason it is thought that Ni hyperaccumulation could be an evolutionary characteristic occurring in long undisturbed floras, not subjected to previous glaciations (Reeves et al., 1983), and hence explaining why all hyperaccumulators of Ni are located in areas where glaciation did not occur.

The work presented here is the first GC- and LC–MS based metabolomics study on Ni-hyperaccumulators from New Caledonia. The aim of metabolomics is to determine the absolute or relative amounts of all metabolites (the metabolome) within a sample to create a molecular profile or fingerprint of an organism (Halket et al., 2005). The number of compounds detected depends on the instrumentation used, most importantly chromatographic resolution and the mass accuracy and scanning speed of the detector. Lee et al. (1978) used GC–MS to determine absolute concentrations of citric acid in a range of hyperaccumulators from New Caledonia and Europe, and found a correlation between Ni and citric acid (Lee et al., 1978). Citric acid has also been shown to be the most important Ni-binding compound, out of 120 metabolites in the Ni-rich latex of Pycnandra acuminata Ni-citrate had by far the highest concentration (Callahan et al., 2008). Nine species from New Caledonia, representing a range of Ni-accumulating ability, were collected and their elemental and GC–MS metabolic profiles were compared to determine if common (or known) mechanisms of Ni sequestration operated within the leaves of these species. Since the initial study by Lee et al. (1978), GC–MS technology has advanced dramatically - now hundreds of metabolites can be detected in one GC–MS chromatogram when using the appropriate mass spectral libraries. In the context of the major improvements in this technology, the aim of the present study was to determine if other metabolites apart from citric acid were involved in the hyperaccumulation of Ni. Metabolites which correlated with the Ni concentration were then searched for using size exclusion chromatography–mass spectrometry (SEC-MS) of aqueous plant extracts. Nickel complexes were identified using the characteristic isotope pattern of nickel.

Section snippets

Soils

New Caledonian soils are derived from ultramafic (serpentine) substrates and therefore they contain relatively high concentrations of Mg, Fe, Co, Ni, and Cr. Table 1 lists the total soil metal concentrations obtained using aqua regia digestion at the 10 sampling sites where plant samples were taken. The areas sampled were mostly covered by tropical rainforest which have evolved on the metal-rich serpentine soils. These soils have been found toxic to crops not adapted to these high metal

Conclusions

This is the first study where comprehensive metabolite profiling and soil analysis have been used to study the phytochemistry of New Caledonian Ni hyperaccumulator plants. Of the 9 different species compared and 258 compounds analyzed the common metabolite, apart from in P. douarrei, associated with Ni is citric acid. This has now been reported a number of times (Bhatia et al., 2005, Boominathan and Doran Pauline, 2003, Homer et al., 1991, Lee et al., 1977, Lee et al., 1978). The association

Plant and soil sampling

The following 9 species were collected from 10 sites in southern New Caledonia: H. kanaliense, C. silvana, G. hirsuta, Hybanthus austrocaledonicus, P. douarrei, P. acuminata, Geissois pruinosa, H. deplanchei and G. bradfordii. The species collected represent a wide range of known Ni accumulation levels. When available, six randomly selected leaf samples were collected of each species, along with samples of the associated soils. Great care was taken to avoid leaf contamination from soil. Leaves

Acknowledgments

Damien Callahan acknowledges the Australian Research Council (ARC) (Linkage Project LP0347205) and The University of Melbourne for the provision of the PhD and Melbourne abroad travel scholarship. The authors also acknowledge financial support from the A. D. Rowden White Foundation, the ARC and the Victorian Institute for Chemical Sciences for the purchase of the ICP and MS instruments used in this study. We would also like to thank IRD Nouméa (New Caledonia), The Laboratoire des Plantes

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