Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii
Résumé
Chlamydomonas reinhardtii is a common model organism for investigation of metal stress. This green alga produces phytochelatins in the presence of metal ions. The influence of cadmium is of main interest, because it is a strong activator of phytochelatin synthase. Cell wall bound and intracellular cadmium content was determined after exposition to 70μm CdCl 2, showing the main portion of the metal outside the cell. Nevertheless, imported cadmium was sufficient to cause significant changes in thiolpeptide metabolism and its transcriptional regulation. Modern analytical approaches enable new insights into phytochelatin (PC) distribution. A new rapid and precise UPLC-MS method allowed high-throughput PC quantification in algal samples after 1, 4, 24 and 48h cadmium stress. Initially, canonic PCs were synthesized in C.reinhardtii during cadmium exposition, but afterwards CysPCs became the major thiolpeptides. Thus, after 48h the concentration of the PC-isoforms CysPC 2-3 and CysGSH attained between 105 and 199nmolg -1 fresh weight (FW), whereas the PC 2-3 concentrations were only 15nmolg -1 FW. The relative quantification of γ-glutamyl transpeptidase (γ-GT) mRNA suggests the generation of CysPCs by glutamate cleavage from canonic PCs by γ-GT. Furthermore, a homology model of C.reinhardtii phytochelatin synthase was constructed to verify the use of crystal structures from Anabaena sp. phytochelatin synthase (PCS) for docking studies with canonical PCs and CysPCs. From the difference in energy scores, we hypothesize that CysPC may prevent the synthesis of canonical PCs by blocking the binding pocket. Finally, possible physiological reasons for the high abundance of CysPC compared with their canonic precursors are discussed. Chlamydomonas reinhardtii consecutively synthesized canonic and Cys-phytochelatins in presence of an ecologically relevant cadmium concentration of 70μM. Also the cadmium induced increase of phytochelatin synthase (PCS) transcription rate demonstrates the high impact of PCs in mediating cadmium detoxification. Furthermore, a homology model of C. reinhardtii phytochelatin synthase was constructed to prove if CysPCs can serve as competetive inhibitors of PCS. © 2011 Blackwell Publishing Ltd.