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Publications - Stress and Develop Biology

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Publications

Sarret, G.; Harada, E.; Choi, Y.-E.; Isaure, M.-P.; Geoffroy, N.; Fakra, S.; Marcus, M. A.; Birschwilks, M.; Clemens, S.; Manceau, A.; Trichomes of Tobacco Excrete Zinc as Zinc-Substituted Calcium Carbonate and Other Zinc-Containing Compounds Plant Physiol. 141, 1021-1034, (2006) DOI: 10.1104/pp.106.082743

Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signs in plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn using Zinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn + Ca increased the trichome density and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates of submicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinate amounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that the large majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite and vaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separate grains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. The presence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy. Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. The proportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containing biogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. This study illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metal homeostasis processes in plants.
Publications

Harada, E.; von Roepenack-Lahaye, E.; Clemens, S.; A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to γ-glutamylcysteine and lacks phytochelatin synthase activity Phytochemistry 65, 3179-3185, (2004) DOI: 10.1016/j.phytochem.2004.09.017

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to γ-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC–MSMS analysis was unequivocally identified as γ-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to γ-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.
Publications

Weber, M.; Harada, E.; Vess, C.; Roepenack-Lahaye, E. v.; Clemens, S.; Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors Plant J. 37, 269-281, (2004) DOI: 10.1046/j.1365-313x.2003.01960.x

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri . We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT‐PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time‐of‐flight mass spectrometry (CapLC‐ESI‐QTOF‐MS)‐based NA analysis procedure and consistently found higher NA levels in roots of A. halleri . Expression of a NA synthase in Zn2+‐hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri . Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.
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