Elemental and metabolite profiling of nickel hyperaccumulators from New Caledonia

Phytochemistry 81 (2012) 80–89

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Elemental and metabolite profiling of nickel hyperaccumulators from New Caledonia Damien L. Callahan a,b,⇑, Ute Roessner a, Vincent Dumontet c, Alysha M. De Livera a, Augustine Doronila b, Alan J.M. Baker a, Spas D. Kolev b a b c

Metabolomics Australia, School of Botany, The University of Melbourne, Victoria 3010, Australia School of Chemistry, The University of Melbourne, Victoria 3010, Australia Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France

a r t i c l e

i n f o

Article history: Received 15 May 2012 Received in revised form 15 June 2012 Accepted 18 June 2012 Available online 12 July 2012 Keywords: Nickel hyperaccumulation Metabolomics GC–MS ICP New Caledonia Serpentine

a b s t r a c t 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. Ó 2012 Elsevier Ltd. All rights reserved.

1. 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 ⇑ Corresponding author at: School of Botany, The University of Melbourne, Victoria 3010, Australia. Tel.: +61 3 83444261; fax: +61 3 93475460. E-mail address: [email protected] (D.L. Callahan). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.06.010

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

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D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89 Table 1 Mean metal concentrations (g/kg) in soil samples from the 10 sites investigated (aqua regia digestion, n = 3) and pH of the corresponding aqueous soil extracts. Site

pH

Al

Cr

Mn

Fe

Co

Ni

Cu

Zn

1 2 3 4 5 6 7 8 9 10

6.4 6.4 7.0 5.9 6.1 5.0 n.a. 5.9 6.2 6.8

30 45 12 26 32 36 43 22 26 25

10 4 1 34 12 19 10 19 17 5

5.0 3.0 1.8 2.0 5.3 12 7.1 7.1 5.6 3.6

476 286 162 575 420 534 419 340 514 252

0.78 0.59 0.35 0.42 0.76 1.1 0.89 0.64 0.68 0.55

6.4 5.0 5.4 3.0 5.8 3.8 3.6 8.3 5.2 6.3

0.055 0.035 0.011 0.018 0.040 0.035 0.034 0.009 0.18 0.022

0.42 0.27 0.15 0.23 0.37 0.39 0.43 0.27 0.21 0.20

Table 2 Comparison of selected mean heavy metal soil concentrations (mg/kg) in the 10 New Caledonian sites (Table 1) with typical ultramafic and non-ultramafic soil concentrations.

Mean from this study Typical Serpentine Non-ultramafic soils (Hamon et al., 2004)

Cr

Mn

Fe (g/kg)

Ni

Co

Zn

Cu

12,960 135–23,950 <15–1000

5220 560–6870 850

397 5–460 0.5–500

5280 880–6190 <5–230

673 88–740 <10

294 10–800 <9–225

44 20 <4–120

Table 3 Mean metal concentrations (mg/kg) in dry leaf material, standard error in brackets n = 4–6. Elements with concentrations below the lowest calibration standard (0.1 mg/kg) are not included. Species

Ni

Co

Zn

Mn

Fe

Cu

Cr

H. austrocaledonicus G. pruinosa H. kanaliense G. hirsuta P. douarrei C. silivana G. bradfordii H. deplanchei S. acuminata

10,955 (1152) 3,406 (439) 3,987 (1362) 2,050 (580) 20,300 (1688) 273 (13) 6,736 (334) 585 (145) 15,038 (1092)

59 (13) 14 (23) 36 (16) 22 (7) 2 (0.8) 1.1 (0.4) 13 (1.5) 55 (38) 10 (2.2)

87 (18.6) 15 (2) 189 (45) 16 (1.3) 41 (8) 70 (10) 13 (0.4) 79 (22) 12 (2)

380 (30) 79 (18) 307 (115) 239 (97) 124 (21) 417 (21) 108 (12) 909 (286) 93 (23)

113 (44) 107 (25) 70 (10) 91 (18) 252 (55) 52 (12) 51 (2.4) 41 (3.5) 420 (157)

2 7 5 6 2 5 6 4 5

25 (4) 9 (3) 1 (0.2) 29 (12) 34 (9) 1 (0.3) 5 (0.3) 1 (0.1) 12 (4)

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

(0.2) (1) (0.8) (1) (1) (1) (0.4) (0.5) (1)

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. 2. Results and discussion 2.1. Elemental analysis by ICP-OES 2.1.1. 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 concentrations (L’Huillier and Edighoffer, 1996). A detailed characterization of the soils near the sampling site in the Parc Provincial de la Rivière Bleue (Sites 05,06,10) was carried out by Perrier et al. (2004a).

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Ni in leaf samples

20,000

Ni (mg/Kg)

15,000

10,000

5,000

0

Species Fig. 1. Mean Ni concentrations ± SE (n = 4–7) in leaf samples from the 9 species collected. The horizontal line indicates the threshold for Ni hyperaccumulator classification at 1,000 mg/kg.

In comparison with typical concentrations found in other soils, the heavy metal concentrations in the soil samples collected from New Caledonia are very high (Table 2). Similar concentrations have been observed from previous an analyses (Yang et al., 1985; Brooks, 1987). The high concentrations of Cr, Mn, Co and Ni are typical for serpentine soils, and explain the highly specialized flora found in New Caledonia. 2.1.2. Leaf tissue The dry mass elemental concentrations were measured for the following metals Al, As, Ba, Be, Cd, Cr, Co Cu, Fe, Pb, Mn, Ni, and Zn. Of the metals quantified, Ni was the only one which showed significant accumulation. In fact, Ni is the major inorganic element in each species above the notional hyperaccumulation threshold (Table 3). The concentrations found are typical for plants found on ultramafic soils. The mean Co concentration was 21 mg/kg for all samples, with the highest concentration reaching 204 mg/kg in one sample of Homalium deplanchei. These concentrations are well above the normal levels of Co found in plants growing on non-serpentine soils, however, no species collected could be classified as hyperaccumulators of Co. Known hyperaccumulators of Ni were targeted for sampling, therefore it was expected that the mean Ni concentration would be much higher than normal (Reeves, 1992). The mean Ni concentrations from dried leaf material for each species are presented in Fig. 1. H. deplanchei was the only species collected which has not been previously reported with Ni concentrations above the 1,000 mg/kg Ni-hyperaccumulation classification. All other species collected were known Ni-hyperaccumulators (Jaffré et al., 1979a,b; Kersten et al., 1979; Reeves, 2003; Yang et al., 1985). The mean Ni concentrations in the samples collected ranged from 250–28,000 mg/kg (Fig. 1; see Supplementary 1 for the full data set). Two species, Casearia silvana and H. deplanchei, contained Ni in leaf tissue below the 1,000 mg/kg cut-off, although they were both

above the normal Ni concentration range of 0.1–10 mg/kg on a dry matter basis (Reeves, 1992). It is not surprising that some species are below the threshold, as wide variations can occur in field-collected samples. For example, Ni concentrations for H. deplanchei have been reported in the range from 10 to 1,850 mg/kg dry mass (Jaffré et al., 1979b). The other seven species contained Ni concentrations well above the 1,000 mg/kg threshold. As expected, Psychotria douarrei had the highest concentration of Ni (Davis et al., 2001; Jaffré et al., 1979b; Jaffré and Schmid, 1974; Kelly et al., 1975; Kersten et al., 1980; Lee et al., 1978). C. silvana contained the lowest Ni concentration, making it an appropriate species to use as a reference for the comparison of metabolite levels. Correlations between Ni and the other elements were also examined. Correlations were found between Fe and Ni (Fig. 2)

Fig. 2. Plot of the ln[Fe mg/kg] vs ln[Ni mg/kg] for all leaf samples.

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D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89 Table 4 Summary of the average Ni concentrations (mg/kg) in leaves, soil extracts (DTPA) and soil digests (aqua regia) from the 10 sampling sites in New Caledonia. Species

Site

Leaf Ni

DTPA Ni

Total Ni

Accumulation Factor

H. austrocaledonicus H. austrocaledonicus G. pruinosa G. pruinosa H. kanaliense G. hirsuta P. douarrei C. silvana G. bradfordii H. deplanchei P. acuminata P. acuminata

1 8 2 3 4 5 5 6 7 8 9 10

10810 13574 3033 4150 3987 2050 20300 273 6737 585 16802 14055

100.4 323 209.2 137.6 10.1 362 362 94.4 76.6 323 725 211.8

6400 8300 5000 5400 3000 5800 5800 3800 3600 8300 5200 6300

108 42 14 30 395 6 56 3 88 2 23 66

and Cr and Ni with Pearson correlations of 0.7 for loge transformed values. The Fe/Ni correlation has been documented previously in hyperaccumulators from New Caledonia (Boyd and Jaffre, 2009). Other correlations were observed such as Mn/Zn and Fe/Cr however these also match the soil concentrations whereas the Ni/Fe does not, providing more evidence that plants actively accumulate Ni over other elements. It is remarkable that these plants can maintain their metal-ion homeostasis in the presence of such extreme concentrations of Ni. For more details on serpentine plants refer to (Brooks, 1987). The accumulation factors of other elements are not in the same order as Ni. For Co the soil concentration is significantly (10 times) lower in comparison to Ni. For Cr the actual bioavailability is very low. From the DTPA results the bioavailable fraction of Cr is about 0.4 ppm in comparison to Ni, which range from 10–300 ppm so it is not expected to be accumulated in leaf tissue. Mn is a ubiquitous element in soils and it is also an essential micronutrient. Responses to Mn are not as dramatic as those of the much more toxic Ni and Co. Mn bioavailability is strongly influenced by soil pH and its effects (apart from very calcifuge plants) are not significant until soil pH is less than 4.2. Also, hyperaccumulators do have preference to particular metal ions and there are Mn-hyperaccumulators in New Caledonia (Fernando et al., 2007). These Ni hyperaccumlators may have a transporter or ligand which is selective to Ni however the mechanism for this selectivity is not known.

2.1.3. Ni accumulation factors from soils to leaves The 1,1,4,7,7-diethylenetriaminepentaacetic acid (DTPA) extractable Ni is considered to be a good indication of Ni bioavailability in ultramafic soils (L’Huillier and Edighoffer, 1996). Becquer et al. (2002) used ion exchange resin bags, and produced more reliable results in the estimation of the bioavailable Ni pool for serpentine soils in New Caledonia. There are many factors which affect the amount of soluble Ni in the soil, for example: microbial activity, soil structure, pH and redox potential (Becquer et al., 2002). It may be that hyperaccumulators have the ability to access higher pools of Ni than non-accumulators, therefore the ‘bioavailable’ Ni is different for each species and dependent on the uptake mechanism applied in each plant. For example, the Ni concentration in P. douarrei is an order of magnitude higher than that in Geissois hirsuta even though they were sampled in the same area. The total soil Ni concentration, DTPA extracted Ni and total leaf Ni concentration from the 10 sampling locations is summarized in Table 4. There was no correlation found between the Ni concentration in the leaf material on one hand and DTPA-extracted or the total Ni concentrations in the soil on the other. The DTPA-extracted concentrations were used to calculate the Ni accumulation factor for each species by dividing the total leaf Ni by the DTPA extracted soil

concentrations. The accumulation factors ranged from 2–395 (Table 4). This shows that the hyperaccumulators analyzed here actively concentrate Ni in their above-ground tissues. The differences in Ni concentration observed in the plants collected suggest that leaf Ni concentrations are most likely determined by the efficiency of metal ion sequestration from the soil and internal transport mechanisms rather than by the soil properties. The concentrations of the other essential micronutrients were not lower due to displacement by Ni. In fact, the three plants with the highest Ni concentrations (P. douarrei, P. acuminata and H. austrocaledonicum), also contained the highest Fe concentrations (Fig. 2).

Fig. 3. Hierarchical cluster analysis on the metabolite profiling data matrix, tight grouping of each species can be observed.

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D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89

Fig. 4. (Top) A plot of the loge RRR for aspartic acid vs loge Ni concentration for samples above the hyperaccumulator threshold; (bottom) loge RRR for citric acid vs. the loge Ni concentration (n = 4–7).

2.2. GC–MS metabolite profiling The data produced from the GC–MS analysis of the leaf material was used for a comparative analysis of a metabolite across the different samples. Relative response ratios (RRR’s) were calculated using the metabolite peak area divided by the internal standard area (ribitol for TMS, norleucine for TBS) and sample mass (mg). Two different derivatization chemistries were used for GC–MS analysis to increase comprehensiveness of the number of metabolites detectable (see Material and Methods, Jacobs et al., 2007). The combination of data from the two derivatization techniques resulted in a list of 258 compounds, of which 103 were positively identified by mass spectral libraries. The average RRR all metabolites in each sample was calculated (see Supplementary 2 for identified metabolite RRRs multiplied by 1e6). The metabolite list produced a large data matrix, 258 metabolites  55 samples. Missing values which were found to be below the instrumental detection limits were replaced by half of the minimum value of the entire data matrix while the remaining missing values were treated as missing for statistical analyses (Xia and Wishart, 2011). The data were then log transformed to remove heteroscedasticity. Hierarchical cluster analysis of the full metabolite matrix produced discrete clusters for replicate samples of each species (Fig. 3). This showed that clear distinctions can be made between species based on their metabolite profiles. It was expected that large differences exist between metabolic profiles between species (Roessner et al., 2006), and the aim of this study was to determine if any of these

differences could be related to the Ni concentrations and the associated metabolites within the leaves. Pearson correlations of the log transformed metabolite responses and log Ni concentration levels were then calculated for each of the Hyperaccumulator species. A number of associations were identified in which the metabolite RRRs were plotted versus the Ni concentrations. A Pearson correlation coefficient r = 0.62 (pvalue<0.05) was found between the average Ni concentration and citric acid for all species except P. douarrei which had a within species correlation coefficient of 0.75 with p-value<0.05.(Fig. 4) but much lower concentrations. This agreed with the data reported by (Lee et al., 1978). This agreement was still surprising as citric acid is neither a strong or selective Ni -binding ligand (lg K = 5.4) compared with the ubiquitous plant chelator nicotianamine (NA; lg K = 16.1) (Martell and Smith, 1974; Von Wiren et al., 1999). However, of the 258 metabolites analyzed, citric acid has again been shown to be associated with Ni in hyperaccumulators. Unfortunately NA was not identified in these extracts. Other studies on the outlier P. douarrei have shown that an organic acid other than citric acid is responsible for Ni co-ordination (Kersten et al., 1980). (Kersten et al., 1980) showed that 63% of Ni is complexed by malic acid. The fold change calculation is another method used to find differences in metabolic profiles, and in this case the moderate Ni accumulator C. silvana was used as a reference. The fold changes values were calculated as the exponential of the difference between the metabolite log RRR for each species and log RRR for C.

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D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89 II

100

[Ni -EDTA] 33.76

(a)

+

Relative Abundance

Base peak chromatogram

II

[Ni -HBED] 46.89 50

II

[Ni -citrate] 35.76

+

+

0 0

10

30

20

40

Time (min)

Relative Abundance

100

349.1

(b) II

[Ni -EDTA]

100

+

II

[Ni -HBED]

50

50

351.1

445.1

(c) +

447.1

366.0 449.1 0 325

335

345

355

365

375

0 425

435

m/z 100

II

[Ni -citrate]

465

100

(e)

331.0 II + [Ni -citrate.2CH3CN]

+

50

50

442.9

333.0

443.8 0 425

455

m/z

440.9

(d)

445

335.0 0

435

445

455

m/z

320 325 330 335 340 345

m/z

Fig. 5. (a) Base peak SEC chromatogram resulting from the separation of Ni-EDTA, citrate, HBED solution; (b) spectrum of Ni-EDTA [NiII(C10H15N2O8)]+ RT 33.8 min; (c) spectrum of NiII-HBED [NiII(C20H23N2O6)]+ RT 46.9 min; (d,e) Ni-citrate [NiII(C6H8O7)(C6H7O7)]+, [NiII(C6H7O7)(CH3CN)2.

silvana (Callow et al., 2000). When examining the fold change values it was found that P. douarrei had more than ten times malonic acid than the average for the other species. (Kersten et al., 1979), our data also found malic acid to be present. However, from the fold change calculation, malonic acid in P. douarrei has a much higher relative difference when compared to all other species. It is possible that both malic acid and malonic acid rather than citric acid are responsible for Ni-binding in P. douarrei. A number of other associations with Ni were found using correlation and fold change analyses. For example, an overall correlation (r = 0.68, p-value<0.05) was found when the average leaf Ni concentration was plotted versus the average aspartic acid RRR for each species above the hyperaccumulation threshold (Fig. 4). Aspartic acid has a high association constant (lg K = 7.16) relative

to other amino acid (lg K = 5.1–5.95) excluding histidine and cysteine (Martell and Smith, 1974). Correlations within replicates of each species were also examined. See Supplementary 3 for a summary of the associations identified with Ni for those metabolites which were found to have Pearson correlation coefficients greater than 0.9 in magnitude and p-values less than 0.05. It is important to note that these are only correlations, and may not imply any causality. These data do, however, suggest possible Ni binding ligands in addition to citric acid, which may be the focus of future studies on Ni hyperaccumulation. Controlled glasshouse studies are the next step required for this profiling approach. There are also a number of unidentified compounds which show correlations with Ni. Identification of these compounds may reveal new selective for Ni ligands involved

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D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89

Table 5 Ni-complexes found in SEC-MS; ligand (L). For the m/z calculations it was assumed that there was a loss of one proton to form a singly charged cation. Species

Ligand

Stoichiometry

m/z

RT (min)

P. douarrei

Maleic acid

Ni.2L.CH3CN Ni.2L.2CH3CN Ni.L.2CH3CN Ni.2L.2CH3CN Ni.2L.CH3CN Ni.2L Ni.3L Ni.L Ni.L.2CH3CN, Ni.2L Ni.L Ni.2L.2CH3CN Ni.3L Ni.L Ni.L.2CH3CN Ni.L.2CH3CN, Ni.2L complexes Ni.L Mixture of complexes Ni.L.2CH3CN Ni.2L Ni.L.2CH3CN, Ni.2L Ni.2L Ni.L.2CH3CN, Ni.2L complexes Ni.L Ni.L.2CH3CN, Ni.2L of complexes Ni.2L Ni.L

330 371 321 289 330 445 369 360 331,441,537 360 349 372 360 333 331,441,537 360 331,441,537 333 441 331,441 445 331,441,537 360 331,441,537 505 360

42 42 48 48 42 38 36 37 36 37 33 33 37 29 36 37 36 28 36 36 47 36 37 36 34 37

Salicylic acid Fumaric acid

G.pruinosa G. bradfordii

G. hirsuta

H. deplanchei H. austrocaledonicus strocaledonicum C. silvana H. kanaliense

P. acuminata

Ferulic acid Malonic acid Nicotianamine Citric acid Nicotianamine Serine Nicotianamine Galacturonic Citric acid Nicotianamine Citric acid Ferulic acid Citric acid Citric acid Ketogluconic acid Citric acid Nicotianamine Citric acid New organic acid Nicotianamine

in Ni storage within the leaves of hyperaccumulators. However, the identification of these compounds, which usually involves isolation, purification and structural determination by MS and 1D and 2D NMR spectroscopy, was outside the scope of the present study. An organic acid (2,4,5-trihydroxy-3-methoxy-hexan-1,6-dioic acid) which was found to co-ordinate Ni in the latex of P. acuminata was also detected in the leaf material by GC–MS. This ligand was only found in P. acuminata samples (Callahan et al., 2008). 2.3. SEC-MS of leaf extracts SEC was found to be the most appropriate method for the separation of the metal complexes. These complexes do not retain using reversed phase as they are charged and very polar. Hydrophilic interaction chromatography also produced poor results with broad peaks and disassociation of the weak complexes. A large number of ions containing the isotope pattern of Ni were present in each chromatogram of the leaf extracts. Many of the m/z observed were species specific. The most prominent Ni-containing ion detected in all samples was Ni-citrate (Fig. 5). Ni-NA was detected in P. acuminata, P. douarrei, Geissois bradfordii, G. hirsuta and Homalium kanaliense. The identity of the Ni-NA complex was confirmed using CID (Supplementary 4). The Ni-NA complex was observed in the leaf material of P. acuminata but not the latex (See (Callahan et al., 2008). This is contradictory to the results of earlier studies (Schaumlöffel et al., 2003) which reported the identification of the Ni-NA complex in the latex of P. acuminata. Also, the Ni-aldaric acid complex with m/z 505 was detected in the leaves of P. acuminata by LC–MS (Callahan et al., 2008). When the leaves of P. acuminata were harvested, the green latex exuded from the point of incision, therefore the detection of the complexed form of the organic acid is not surprising. These results show that Ni is transported to the laticifers in complexes with organic acids. The chromatograms of each plant species were searched using the masses of compounds with high fold change values in the metabolite profiling data. A number of compounds with the expected masses were found (Table 5). P. douarrei, which did not have a high RRR for citric acid, did contain a number of Ni-complexes with other organic acids. The Ni-complexes listed in Table 5

do not cover all Ni-containing ions observed. There were many intense ions which could not be identified. Many of these could prove interesting with further analysis. It is difficult to determine the significance of many of the unknown Ni-complexes detected by SEC-MS as complexes could form during the extraction process. 3. 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, 1978). The association constant of citrate with Ni is 5.4 (Martell and Smith, 1974), much lower than for the multidentate ligand nicotianamine (lg K = 16.1) (Benes et al., 1983), so it is surprising that citric acid is involved in sequestering Ni. However, of the organic acids identified by GC–MS, citric acid has the highest association constant with Ni and is always present in high abundance; the metabolic cost of using citric acid as a chelator is thus low. This analysis has also identified other small molecules which show a positive relationship with Ni, in particular a strong association was found with aspartic acid. Aspartic acid has a high association constant (lg K = 7.16) relative to other amino acids (lg K = 5.1–5.95), only histidine and cysteine are higher (Martell and Smith, 1974). Also, it appears that P. douarrei, which contains the highest leaf Ni concentration, uses malonic acid instead of citric acid for sequestration. The results presented here have provided new potential targets for future studies on Ni hyperaccumulation. 4. Experimental 4.1. Plant and soil sampling The following 9 species were collected from 10 sites in southern New Caledonia: H. kanaliense, C. silvana, G. hirsuta, Hybanthus

D.L. Callahan et al. / Phytochemistry 81 (2012) 80–89

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 were washed with deionized water then frozen in liquid nitrogen and stored at 80 °C. Leaves were frozen as soon as possible in order to halt metabolic processes which would change metabolite levels. Samples were then freeze-dried and stored in desiccant for transport to Australia. The soils samples were generally dark brown and of alluvial origin. The thin leaf litter layer was removed and the top 30 cm was sampled. 4.2. ICP-OES leaf and soil analysis For leaf and total soil metal concentration the following elements were quantified: Al, As, Ba, Be, Cd, Cr, Co Cu, Fe, Pb, Mn, Ni and Zn using a Varian Vista ICP-OES (Varian Inc., Melbourne, Victoria, Australia). The ICP conditions used were: power 1 kW, plasma flow 15 L/min, auxiliary flow 1.5 L/min, nebuliser flow 0.9 L/min. Instrument data were evaluated using Vista Pro ICP Expert 4.1.0. Quantification was achieved using an external calibration curve method with standards in the range of 0.1–60.0 mg/L. Leaf material was re-dried in an oven at 60 °C overnight before being ground to a fine powder. Approximately 0.2 g of each dried and ground leaf sample was accurately weighed into digestion tubes (75 mL). A number of duplicate samples were weighed to check reproducibility. Concentrated HNO3 (5 mL) was added to each tube and then refluxed (3 h, 130 °C) to give a clear solution. After cooling, each sample was diluted to 75 mL with deionized water. A subsequent dilution (10–100 mL) was made to the P. douarrei digests. This was required to reduce the Ni concentration in the digests to fit within the linear range of the standard calibration curve. The total elemental profile in the soils sample was determined using aqua regia digestion (3:1 conc HCl:HNO3). Soil samples were dried in an oven (5 days; 60 °C). Each sample was ground to a fine powder using a rock breaker. Approximately 0.2 g of each dried and ground soil sample was weighed accurately into digestion tubes (75 mL). Each soil sample was analyzed in triplicate. Aqua regia (5 mL) was added to each tube and the suspensions heated (3 h, 130 °C). Tubes were cooled and diluted up to 75 mL with deionized water and mixed well. Tubes were allowed to settle overnight, and then a 1 in 10 mL dilution was made to each digest. 1,1,4,7,7-diethylenetriaminepentaacetic acid (DTPA) extractions were carried out in the Soil Analysis Laboratories at IRD Nouméa following the method outlined by (Perrier et al., 2004b). 4.3. GC–MS profiling Two derivatization techniques were used for the metabolite analysis, trimethylsilylation (TMS) and tert-butyldimethylsilylation (TBS). Both derivatize polar functional groups. Review of chemical GC based derivatization can be found in (Birkemeyer et al., 2003; Halket et al., 2005). Separate GC–MS protocols are used for the analysis of the different derivatives. The freeze-dried leaf samples were ground in liquid N2 using a mortar and pestle then accurately weighed (20 mg) into Eppendorf tubes (2 mL). Methanol (500 lL) and the internal standards ribitol (10 lL; 0.2 mg/mL for TMS) and norleucine (20 lL 0.2 mg/mL for TBS) were added and tubes vortexed. Samples were then extracted by gentle shaking for 15 min at 70 °C, then centrifuged (15 min, 14,000 rpm). The supernatant was transferred to a new Eppendorf tube and water (500 lL) added. For TBS derivatization, aliquots (50 lL) were transferred to Eppendorf tubes and dried in vacuo at

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room temperature. Two chloroform extractions (400 lL then 300 lL) of the remaining supernatant were then carried out to remove lipophilic components. For TMS derivatization, aliquots (5 lL) of the aqueous phase were then transferred to Eppendorf tubes and dried in vacuo at room temperature. 4.4. TMS derivatization The dried residue was redissolved with the addition of methoxyamine hydrochloride (20 lL, 30 mg/mL in pyridine) and shaken (37 °C; 2 h). This was followed by trimethylsilylation with Nmethyl-N-[trimethylsilyl]trifluoroacetamide (40 lL; 37 °C; shaken 30 min). An RT standard mixture (5 lL of 0.029% (v/v) n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotriacontane, n-hexatriacontane dissolved in pyridine) was added prior to trimethylsilylation. Samples (1 lL) were then injected via the splitless mode onto a GC column using a hot needle technique. 4.5. TBS derivatization The dried residue from initial extraction was re-dissolved and shaken using the same method as TMS (above). This was followed by the addition of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (40 lL). The RT standard mixture used for TMS (5 lL) was added prior to tert-butyldimethylsilation. The samples were incubated at 65 °C for 45 min, then injected (1 lL) onto the GC column in the splitless mode. 4.6. GC–MS conditions The GC–MS system used comprised an AS 3000 autosampler, a Trace gas chromatograph Ultra and a DSQ quadrupole MS with an EI source (ThermoElectron Corporation, Austin, USA). The MS was tuned according to the manufacturer0 s recommended procedure using tris-(perfluorobutyl)-amine (FC43). The GC–MS method used for the analysis of both the TMS- and TBS-derivatized samples were as folows. Derivatives were separated on a 30 m VF-5MS column (0.25 mm i.d.; 10 m Integra guard column; 0.25 lm film thickness; Varian Inc., Victoria, Australia) with a helium carrier gas (1 mL/min). For TMS derivatives the following oven temperature program was used: start temperature at 70 °C followed by a 1 °C/min oven temperature ramp to 76 °C, then a 7 °C/min ramp to 325 °C and finally held at 325 °C for 10 min. For TBS derivatives the following oven program was used: start temperature at 100 °C and hold for 1 min then 1 °C/min ramp to 106 °C followed by a 7 °C/min ramp to 325 °C and finally held for 10 min at 325 °C. The following MS conditions were used: injection temperature 230 °C, MS transfer line 280 °C and ion source 250 °C. Mass spectra were recorded at 2 scan/s, with a scanning range of 70–600 amu. The GC–MS system was equilibrated for 1 min at initial temperature prior to injection of the next sample. 4.7. Data analysis AMDIS (Automated Mass spectral Deconvolution and Identification System; NIST, Gaithersburg, USA) deconvolution was carried out on a chromatogram for each species to identify as many known and unknown chromatographic peaks as possible. The eluting derivatives were identified using the National Institute of Standards and Technology (NIST, 2008) commercial mass spectra library, the public domain mass spectra library of the Max-PlanckInstitute for Plant Physiology, Golm, (Kopka et al., 2005) and a library constructed at the Metabolomics Australia, School of Botany, The University of Melbourne. Processing methods were created using the Xcalibur program (ThermoFinnigan, Manchester, UK) which determined the areas under each peak found following

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deconvolution in AMDIS. The processing method used the mass spectral pattern and RT relative to n-alkanes to enable reliable identification in all samples. In the processing method setup, metabolites which were not identified through library searching were labeled with their corresponding RT values.

Appendix A. Supplementary data

4.8. SEC MS

References

The chromatographic conditions were adapted from Schaumlöffel et al. (2003). A size exclusion column (SEC; Superdex peptide 300  10 mm; Pharmacia Biotech) was used for the separation of Ni-complexes. Instead of collecting fractions for MS analysis the eluant from the SEC was directly transferred to the ESI source. The LC-MS system used comprised a Finnigan Surveyor LC pump, Surveyor autosampler and Finnigan LCQ Deca XP Max quadrupole ion trap MS, with an Ion max ESI source (ThermoFinnigan, San Jose, USA). Chromatograms and mass spectra were evaluated using the Xcalibur software (ThermoElectron, Manchester, UK). MS/MS and high resolution‘‘zoom’’ scans were triggered by isotope ratios or ion abundance. This improved the ability to detect Ni containing compounds that may have been missed through manual processing of data. Mobile phase used was 5 mM ammonium acetate and acetonitrile to improve desolvation. The mobile phase composition was optimized for separation efficiency using a synthetic mixture of Ni-complexes: Ni-EDTA, Ni-citrate and Ni-HBED. The isocratic mobile phase used was a 90:10 mixture of ammonium acetate (10 mM; pH 7.0) and acetonitrile at a flow rate of 0.4 mL/min. The resulting chromatogram and mass spectra obtained using this protocol is shown in Fig. 5a. For each discrete chromatographic peak a number of gas phase ions with a different Ni:ligand stoichiometry were present. For example, in the MS at the retention time for Ni-citrate, singly charged cations of [Ni(H3cit)(CH3CN)]+, [Ni2(H2cit)(H3cit)]+, [Ni2(H3cit)3]+ could be detected (e.g. Fig. 5d,e). MS/ MS of these ions helped to determine which complex was a weakly bound cluster ion formed in the ESI source or a tightly bound to Ni ligand. This aided the identification of the unknown ligand. To maximize the signal intensity for the eluting Ni-complexes the MS was tuned using synthetic Ni-citrate solution infused in-line with the eluant off the SEC column using a Tpiece connector. The MS source conditions (source position, sheath gas flow, auxiliary gas flow, capillary temperature) and ion optics (automatic tune) were then optimised with respect to the signal intensity of the [Ni(H3cit)(H4cit)]+ cation m/z 441. This resulted in the following optimal source conditions: sheath gas, 45 arbitrary units; auxiliary gas, 10 arbitrary units; spray voltage, 4.0 kV; capillary temperature, 300 °C. CID was carried out at 30% normalized collision energy. Mass spectra were recorded at 5 lscans/scan, with a scanning range of 100–1000 amu.

Baker, A.J.M., McGrath, S.P., Reeves, R.D., Smith, J.A.C., 1999. A review of the biological resource for possible exploitation in the phytoremediation of metalpolluted soils. In: Bañuelos, G.S., Terry, N. (Eds.), Phytoremediation of Contaminated Soil and Water. Lewis Publishers, Boca Raton, FL, pp. 85–107. Becquer, T., Rigault, F., Jaffré, T., 2002. Nickel bioavailability assessed by ion exchange resin in the field. Commun. Soil Sci. Plant Anal. 33, 439–450. Benes, I., Schreiber, K., Ripperger, H., Kircheiss, A., 1983. On the normalizing factor for the tomato mutant chloronerva. 13. Metal complex formation by nicotianamine, a possible phytosiderophore. Experientia 39, 261–262. Bhatia, N.P., Walsh, K.B., Baker, A.J.M., 2005. Detection and quantification of ligands involved in nickel detoxification in a herbaceous Ni hyperaccumulator Stackhousia tryonii Bailey. J. Exp. Bot. 56, 1343–1349. Birkemeyer, C., Kolasa, A., Kopka, J., 2003. 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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 Médicinales ICSN-CNRS (New Caledonia) for field support and DTPA soil analysis and The Province Sud from New Caledonia for the access to biodiversity. Thanks also to Terry Speed for helpful discussions related to the statistical analyses, and Suganthi Suren, Metabolomcis Australia for GC-MS analysis.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem. 2012.06.010.

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