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Cell imaging of the metallic stress induced by cadmium in the green micro-alga Chlamydomonas reinhardtii by synchrotron-based techniques (µXRF/XAS) and nanoSIMS





The green micro-alga Chlamydomonas reinhardtii is commonly used as a model for the study of the metallic stress in photosynthetic organisms. Tolerance mechanisms against stress induced by cadmium are not well understood. In order to determine these mechanisms, subcellular location and in situ speciation have been determined in three C. reinhardtii strains exposed to cadmium in mixotrophic conditions (CO2 + Acetate) : (i) a wild type strain (wt), (ii) a cell-wall less strain (cw15) which is deficient in cell-wall, (iii) the pcs1 strain which overexpresses the cytosolic enzyme phytochetlatin synthase (PCS) directly in the chloroplast. Cadmium toxicity has been determined by the monitoring of growth and chlorophyll, starch content in micro-algae. Then, cadmium location at subcellular level has been performed using three complementary techniques (subcellular fractionation, µXRF and TEM/X-EDS). In situ cadmium speciation has been studied by µXAS and XAS. Finally, elemental and isotopic imaging by nanoSIMS has allowed to complete elemental distribution in the cells and to determine the impact of cadmium on the assimilation of carbon. (i) The results of this work show that the wt strain is the most sensitive strain to cadmium stress among the three studied strains with a growth and chlorophyll content decrease. When wt cells do not show signs of toxicity, cadmium is mainly sequestered in the whole cell by thiolated ligands and in polyphosphate granules. After an exposure to high concentration of cadmium, intracellular cadmium is mainly bound to carboxylated ligands, probably induced by oxidative stress. Moreover, cadmium located in the pyrenoid blocks inorganic carbon (CO2) assimilation and increases organic carbon (acetate) assimilation which is stored as starch. (ii) The overexpresssion of PCS in the pcs1 strain induces a strong production of starch around the pyrenoid and proctects the chlorophyll against cadmium stress. Although the synthesis of phytocheltins was potentially strong, half of the intracellular cadmium is sequestered in polyphosphate granules and in starch. (iii) Unlike cell-walled cells, the cw15 strain is the most tolerant strain and does not accumulate the totality of available cadmium. Similarly to wt strain, intracellular cadmium is mainly sequestered by thiolated ligands and in polyphosphate granules. The observation of polyphosphate granules excreted by the micro-algae allows the hypothesis of the excretion of vacuolar cadmium, inducing a constant flux of cadmium through the cells. In conclusion, cadmium sequestration by sulfur ligands, potentially by thiolated polypeptides, is the main tolerance mechanism implemented by C. renhardtii. However, cadmium sequestration in polyphosphate granules seems to allow a better tolerance against cadmium stress.

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