Ralf Kneer

Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata

The nickel content in different parts of the hyperaccumulating tree Sebertia acuminata was analysed by atomic absorption spectroscopy. Nickel was found to be mainly located in laticifers. The total nickel content of a single mature tree was estimated to be 37 kg. By gel filtration and NMR spectroscopy, citric acid was unequivocally identified as counter ion for about 40% of this metal present. Nitrate was assumed to be a further partner for a complete ionic balance. Phytochelatins were not found to be involved in nickel detoxification in Sebertia. The localization of nickel complexes inside the laticifers was demonstrated by light microscopy as well as by scanning electron microscopy in combination with an EDX system for the analysis of elements. A repellent effect of the plant sap was observed on the fruit fly Drosophila melanogaster indicating that in hyperaccumulating plants nickel functions as an agent to prevent predation.

Phytochelatins protect plant enzymes from heavy metal poisoning

Challenging Rauvolfia serpentina suspension cells with sub-phytotoxic levels (100 μM) CdCl2 containing 109Cd2+ and subsequent gel filtration of the carefully prepared cell-free extracts shows that the bulk (97%) of the radioactivity is present as a phytochelatin complex. Less than 3% is associated with the high molecular weight fraction, no Cd2+ is found in free-form and there was no indication of the occurrence of a metallothionein complex. It is concluded that heavy metal ions entering cells at sub-lethal concentrations are totally complexed by phytochelatins (and to a much lesser extent to some high molecular weight proteins). It was shown that a series of metal-sensitive plant enzymes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, nitrate reductase, ribulose-1,5-diphosphate carboxylase and urease tolerate Cd2+ in the form of a phytochelatin complex from 10- to 1000-fold the amount as compared with the free metal ion. Free phytochelatin peptides reactivate metal-poisoned nitrate reductase in vitro up to 1000-fold better than chelators such as glutathione and citrate, showing the extraordinary sequestering potential of these peptides.

Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin

In fungi, cellular resistance to heavy metal cytotoxicity is mediated either by binding of metal ions to proteins of the metallothionein type or by chelation to phytochelatin-peptides of the general formula (γ-Glu-Cys)n-Gly. Hitherto, only one fungus, Candida glabrata has been shown to contain both metal inactivating systems. Here we show by unambiguous FAB-MS analysis that both a metallothionein-free mutant of Saccharomyces cerevisiae as well as a wildtype strain synthesize phytochelatin (PC2) upon exposure to 250 μM Cd2+ ions. The presence of Zn and/or Cu ions in the nutrient broth also induces PC2 synthesis in this organism. By 109Cd exchange and subsequent monobromobimane fluorescence HPLC, it could be shown that the presence of Cd2+ in the growth medium also induces phytochelatin synthesis in Neurospora crassa, which contains metallothioneins.

Evidence for discrete Cd(SCys)4 units in cadmium phytochelatin complexes from EXAFS spectroscopy

The principal structural features of multinuclear complexes of cadmium(II) with the plant-peptide phytochelatin have been deduced from Cd-EXAFS (EXAFS = extended X-ray absorption fine structure) data: (i) Cd(SCys)4 coordination, (ii) in contrast to Cd-metallothioneins, no formation of cadmium ‘clusters’ and (iii) non-coordinating carboxylate groups.

Discrimination of Three Pegaga (Centella) Varieties and Determination of Growth-Lighting Effects on Metabolites Content Based on the Chemometry of 1H Nuclear Magnetic Resonance Spectroscopy

The metabolites of three species of Apiaceae, also known as Pegaga, were analyzed utilizing (1)H NMR spectroscopy and multivariate data analysis. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) resolved the species, Centella asiatica, Hydrocotyle bonariensis, and Hydrocotyle sibthorpioides, into three clusters. The saponins, asiaticoside and madecassoside, along with chlorogenic acids were the metabolites that contributed most to the separation. Furthermore, the effects of growth-lighting condition to metabolite contents were also investigated. The extracts of C. asiatica grown in full-day light exposure exhibited a stronger radical scavenging activity and contained more triterpenes (asiaticoside and madecassoside), flavonoids, and chlorogenic acids as compared to plants grown in 50% shade. This study established the potential of using a combination of (1)H NMR spectroscopy and multivariate data analyses in differentiating three closely related species and the effects of growth lighting, based on their metabolite contents and identification of the markers contributing to their differences.