Speciation of Nickel in a Hyperaccumulating Plant by High-Performance Liquid Chromatography−Inductively Coupled Plasma Mass Spectrometry and Electrospray MS/MS Assisted by Cloning Using Yeast Complementation

Abstract

A novel analytical approach based on a combination of multidimensional hyphenated techniques and cloning of the Ni-resistance gene using yeast complementation screens was developed for the identification of nickel species in a Thlaspi caerulescens hyperaccumulating plant. The presence of an unknown strong Ni complex was demonstrated by size exclusion HPLC-capillary electrophoresis with ICPMS detection. The Ni-containing peak was characterized by electrospray MS (m/z 360) and shown by collision-induced dissociation MS to be a chelate with a tricarboxylic amino acid ligand. To identify the species and demonstrate its functional character, a cDNA library was constructed from T. caerulescens, expressed in the yeast, and screened on a toxic Ni2+ medium. The extract from the surviving transformant culture gave identical HPLC-ICPMS, CZE-ICPMS, and ES MS/MS data and contained a cDNA insert homologous to the nicotianamine synthase gene. This observation allowed the identification of nicotianamine as the nickel-binding ligand. The presence of the Ni-nicotianamine complex was ultimately demonstrated by comparing tandem mass spectra of the plant and yeast extracts with those of a synthetic standard.

Metal and metalloid speciation in plants: Overview, instrumentation, approaches and commonly assessed elements

“…This study recommended that future studies involve a Quadrupole Time of Flight Mass Spectrometer (QTOF-MS) or ESI-MS for more sensitive and accurate measurements of less concentrated Ni complexes. Similar experiments were conducted by Vacchina et al [4], regarding a different plant species, T. caerulescens [4] using SE-HPLC-ICP-MS and SE-HPLC-ESI-MS. It was found that metal complexes could be identified through the complementary use of HPLC-ICP-MS, CZE-ICPMS and ESI-MS-MS.…”

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens

“…Other metal chelators such as organic acids or amino acids, particularly histidine and nicotianamine have also been connected to metal tolerance or hyperaccumulation in plants (Krämer et al 1996;Sarret et al 2002;Vacchina et al 2003;Weber et al 2004).…”

Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in archaea

“…The NA level in plants can be manipulated through genetic engineering to produce crops with higher iron contents or with increased tolerance to low iron availability (9,10). The action of NA is not restricted to iron homeostasis, but extends to that of other metal ions such as Cu 2ϩ , Zn 2ϩ , Mn 2ϩ , and Ni 2ϩ (10)(11)(12)(13)(14)(15). For example, one of the most pronounced defects in the chloronerva mutant is a lack of root-to-shoot transport of Cu that induces a severe shortage in leaves with the consequence that Cu-containing enzymes are less active.…”

“…For example, one of the most pronounced defects in the chloronerva mutant is a lack of root-to-shoot transport of Cu that induces a severe shortage in leaves with the consequence that Cu-containing enzymes are less active. NA has also been shown to be the main metabolite in Cd 2ϩ and Zn 2ϩ hyperaccumulation in Arabidopsis haleri (16) and largely contributes to Ni 2ϩ chelation and long distance transport in Thlaspi caerulescens, a natural hyperaccumulator plant (12,17). Aside from its roles in metal homeostasis, NA inhibits the angiotensin I-converting enzyme in spontaneously hypertensive rats and may therefore reduce high blood pressure in humans (18,19).…”