Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy

Abstract

The green micro-alga Chlamydomonas reinhardtii is commonly used as a model to investigate metallic stress in photosynthetic organisms. The aim of this study was to explore processes implemented by three C. reinhardtii strains to cope with cadmium (Cd), and particularly to evidence Cd sequestration in the cell. For that, we used a combination of subcellular fractionation and chemical imaging (micro X-ray fluorescence (μXRF) and transmission electron microscopy (TEM/X-EDS)) to identify subcellular compartments of Cd accumulation, and X-ray absorption spectroscopy (XAS) to determine chemical Cd speciation. C. reinhardtii wild type strain 11/32b (wt), a newly design strain (pcs1) expressing a modified phytochelatin synthase in the chloroplast and a cell wall less strain CC400 (cw15) were exposed to 70 μM Cd. At this Cd concentration, cell vitality was not affected, however, the strains showed various strategies to cope with Cd stress. In wt, most of Cd was diffused in the whole cell, and complexed by thiol ligands, while the other part was associated with phosphate in vacuolar Ca polyphosphate granules. Thiol ligands increased with exposure time, confirming their important role in Cd stress. In pcs1, Cd was also present as vacuolar Ca polyphosphate granules, and diffused in the cell as Cd-thiol complexes. In addition, while it should be regarded with caution, a minor proportion of Cd complexed by carboxyl groups, was potentially provided by starch produced around the pyrenoid and in the chloroplast. Results suggested that pcs1 uses thiol compounds such as PC to a lesser extent for Cd sequestration than wt. In cw15, an excretion of Cd, Ca polyphosphate granules has to be considered. Finally, Cd was detected in the pyrenoid of all strains.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga

“…This pyrenoidal sequestration of Cd could thus be responsible for Cd toxicity in C. reinhardtii . Furthermore, Cd was localized beside Ca and P in electron dense bodies in vacuoles suggesting a cadmium sequestration in calcium polyphosphate granules as confirmed in our previous study by μXANES analysis (Penen et al, ). It is important to note that the elemental composition of vacuoles and pyrenoid determined by NanoSIMS was confirmed by the X‐EDS measurements.…”

“…However, under control conditions, this modification induced already a decrease of cell vitality. Indeed, in our previous work (Penen et al, ), C. reinhardtii grown in classic TAP medium showed a higher growth rate (1.15 ± 0.03 day −1 ) and a higher production of chlorophyll (10.78 ± 0.38 μg ml −1 OD 730 nm −1 ) compared with the present study where microalgae have been cultivated in TAP EDDHA medium (Figure b,c). These effects of TAP EDDHA medium on cell vitality in control conditions could be explained by the changes in the availability of some trace elements compared with the classic TAP medium (Table ).…”

“…Cd L III ‐edge XANES was demonstrated powerful to distinguish sulphur from oxygen ligands due to absence of a peak at 3,539 eV in sulphur binding atoms (Isaure et al, ). Moreover, as indicated by arrows in Cd reference XANES spectra in Figure b,c, the second oscillation was shifted to higher energy values for Cd‐acetate and Cd‐malate where Cd is bound to carboxyl groups compared with Cd bound to phosphate groups in Cd‐phosphate and Cd‐phytate (Penen et al, ). This enabled a differentiation between the oxygen donor ligands in Cd‐carboxyl and Cd‐phosphate.…”

“…The main part of Cd was associated with phosphate ligands (44 ± 10%) suggesting that the calcium polyphosphate granules sequestered an important part of the metal in C. reinhardtii . Indeed, these granules were previously probed by μXANES and revealed the sequestration of Cd by phosphate ligands (Penen et al, ).…”

“…NanoSIMS mapping has been also used to determine metal distributions in plants and microalgae (Hong-Hermesdorf et al, 2014;Moore et al, 2010Moore et al, , 2011Moore et al, , 2012. As complementary techniques, synchrotron micro X-ray fluorescence (μXRF) and micro X-ray absorption spectroscopy (μXAS) can be combined and directly applied on frozen hydrated samples to locate metals and decipher their structural environment, respectively, allowing the determination of metal distribution and speciation in subcellular regions of microalgae (Adams et al, 2016;Leonardo et al, 2014;Leonardo et al, 2016;Penen et al, 2017;Wang, Lv, Ma, & Zhang, 2016).…”