Florent Penen

Marie-Pierre Isaure

Dirk Dobritzsch

Hiram Castillo-Michel

Etienne Gontier

Philippe Le Coustumer

Julien Malherbe

Dirk Schaumlöffel
email: dirk.schaumloeffel@univ-pau.fr

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga #

Keywords: Biogeochemical carbon cycle, CO2 fixation, toxic metals, cadmium stress, subcellular imaging, NanoSIMS, synchrotron µXRF, µXANES, TEM/X-EDS, isotopic labeling, isotope ratio

Mixotrophic micro-organisms are able to use organic as well as inorganic carbon sources and, thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of CO2 fixation by toxic metals such as cadmium -classified as a priority pollutantcould contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic micro-organisms is of high interest.

We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labeled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulfur ligands, impaired CO2 fixation to the benefit of acetate assimilation. TEM/X-EDS and µXRF/µXANES at Cd LIII-edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, NanoSIMS analysis of the 13 C/ 12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73% to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

INTRODUCTION

The increase of carbon dioxide concentration in the atmosphere over the past decades has been associated with global climate change. Therefore, investigation and understanding of the biogeochemical carbon cycle is of primordial importance. The terrestrial and oceanic biosphere plays a major role in the global carbon cycle by the production and conversion of organic matter [START_REF] Field | Primary Production of the Biosphere: Integrating Terrestrial and Oceanic Components[END_REF]. Plants have developed a process for CO2 fixation into organic carbon using the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) which is regarded to be the most abundant protein in the biosphere [START_REF] Ellis | The most abundant protein in the world[END_REF]. Microalgae are small eukaryotic organisms where RuBisCO is concentrated in the specific organelle called the pyrenoid [START_REF] Badger | The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO2-concentrating mechanisms in algae[END_REF]. Pyrenoid containing algae have been identified as being responsible for 28 -44% of the global carbon fixation which underlines the key role of microalgae in the biogeochemical carbon cycle [START_REF] Mackinder | A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle[END_REF].

Eukaryotic algae are classically considered as photoautotrophic using light and inorganic compounds for energy and carbon fixation, or heterotrophic using only organic compounds.

Recently, mixotrophy has been integrated into aquatic biogeochemical cycle modeling [START_REF] Caron | Mixotrophy stirs up our understanding of marine food webs[END_REF][START_REF] Ward | Marine mixotrophy increases trophic transfer efficiency, mean organism size, and vertical carbon flux[END_REF]. Mixotrophic organisms are able to combine photoautotrophic growth conditions as well as heterotrophic nutrition. They are dominant among micro-algal communities in oligotrophic and eutrophic environments [START_REF] Burkholder | Mixotrophy, a major mode of nutrition for harmful algal species in eutrophic waters[END_REF][START_REF] Hartmann | Mixotrophic basis of Atlantic oligotrophic ecosystems[END_REF]. To date, the consideration of mixotrophy in biogeochemical carbon cycle models is still at the beginning [START_REF] Caron | Mixotrophy stirs up our understanding of marine food webs[END_REF]. Recently it has been shown that mixotrophy globally lead to an increase of the size of organisms in the aquatic food chain and thus enhances carbon sinking into the deep ocean by 35% [START_REF] Ward | Marine mixotrophy increases trophic transfer efficiency, mean organism size, and vertical carbon flux[END_REF].

Cadmium is a highly toxic metal and is considered as a priority pollutant in ecosystems [START_REF] Campbell | Cadmium-A priority pollutant[END_REF]. In high polluted natural waters micromolar cadmium concentrations were found such as in the Tinto river in Spain (40 -620 µM) [START_REF] Aguilera | Tolerance to cadmium in Chlamydomonas sp. (Chlorophyta) strains isolated from an extreme acidic environment, the Tinto River (SW, Spain)[END_REF] while for other natural waters lower Cd concentration are described, e.g. 2.4 -17.8 nM in polluted

Macedonian rivers [START_REF] Ramani | Surface Water Characterization of Three Rivers in the Lead/Zinc Mining Region of Northeastern Macedonia[END_REF] and only 0.2 -2 nM in a Swiss river [START_REF] Xue | Cadmium speciation and complexation by natural organic ligands in fresh water[END_REF]. Also, in polluted soils and soil solutions high cadmium concentrations were measured, for example, 5.4 -45.5 µM Cd in soil solutions of soils close to a chemical engineering plant in Northern Taiwan [START_REF] Lee | Effect of chemical amendments on the concentration of cadmium and lead in long-term contaminated soils[END_REF]) and 284 mg Cd per kg in paddy soils from Thailand [START_REF] Simmons | Elevated levels of cadmium and zinc in paddy soils and elevated levels of cadmium in rice grain downstream of a zinc mineralized area in Thailand: implications for public health[END_REF]. Moreover, Cd has been

shown to be biomagnified in some aquatic food webs [START_REF] Croteau | Trophic transfer of metals along freshwater food webs: Evidence of cadmium biomagnification in nature[END_REF][START_REF] Ikemoto | Biomagnification of Trace Elements in the Aquatic Food Web in the Mekong Delta, South Vietnam Using Stable Carbon and Nitrogen Isotope Analysis[END_REF][START_REF] Rouleau | Cadmium accumulation in coastal demersal fish[END_REF][START_REF] Signa | Diet and habitat use influence Hg and Cd transfer to fish and consequent biomagnification in a highly contaminated area: Augusta Bay (Mediterranean Sea)[END_REF]. At the microbial level, this accumulation occurs in communities between bacteria and eukaryote organisms [START_REF] Worden | Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes[END_REF]. In view of the importance of mixotrophy in the aquatic carbon cycle, the impact of toxic metals such as Cd on carbon assimilation in microalgae is of growing interest.

Chlamydomonas reinhardtii is a unicellular green alga which is found worldwide in soils and fresh water as its natural habitat. This alga is used as a model for many cellular processes [START_REF] Dent | Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii[END_REF][START_REF] Goto | Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii[END_REF][START_REF] Harris | The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use[END_REF][START_REF] Merchant | Between a rock and a hard place: Trace element nutrition in Chlamydomonas[END_REF][START_REF] Merchant | TAG, You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Merchant | The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions[END_REF][START_REF] Rochaix | The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas[END_REF][START_REF] Silflow | Assembly and Motility of Eukaryotic Cilia and Flagella. Lessons from Chlamydomonas reinhardtii[END_REF] including metallic stress [START_REF] Hanikenne | Chlamydomonas reinhardtii as a eukaryotic photosynthetic model for studies of heavy metal homeostasis and tolerance[END_REF]. Cadmium is known to inhibit its sexual reproduction [START_REF] Goodenough | The role of calcium in the Chlamydomonas reinhardtii mating reaction[END_REF], to limit photosynthesis activity [START_REF] Faller | Mechanism of Cd2+ toxicity: Cd2+ inhibits photoactivation of Photosystem II by competitive binding to the essential Ca2+ site[END_REF][START_REF] Nagel | In Vitro Evolution and Preliminary Characterization of a Cadmium-Resistant Population of Chlamydomonas reinhardtii[END_REF], 1995;[START_REF] Voigt | The donor side of photosystem II is impaired in a Cd2+-tolerant mutant strain of the unicellular green algaChlamydomonas reinhardtii[END_REF][START_REF] Voigt | A Cadmium-tolerant Chlamydomonas Mutant Strain Impaired in Photosystem II Activity[END_REF] and to induce oxidative stress [START_REF] Aksmann | Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene[END_REF][START_REF] Vega | Effect of abiotic stress on photosynthesis and respiration in Chlamydomonas reinhardtii: Induction of oxidative stress[END_REF]. In order to reduce free intracellular Cd 2+ , the microalga synthesizes thiol peptides such as phytochelatins [START_REF] Bräutigam | Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii[END_REF][START_REF] Gekeler | Algae sequester heavy metals via synthesis of phytochelatin complexes[END_REF] and sequesters Cd bound to polyphosphate granules into vacuoles which were compared to acidocalcisomes [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF][START_REF] Ruiz | The Polyphosphate Bodies of Chlamydomonas reinhardtii Possess a Proton-pumping Pyrophosphatase and Are Similar to Acidocalcisomes[END_REF].

C. reinhardtii can grow photoautotrophically in the presence of light and CO2, heterotrophically in the dark when acetate is available as an organic carbon source or mixotrophically with acetate, CO2 and light [START_REF] Johnson | Central Carbon Metabolism and Electron Transport in Chlamydomonas reinhardtii: Metabolic Constraints for Carbon Partitioning between Oil and Starch[END_REF]. In the latter case, CO2 is photosynthetically fixed by RuBisCO [START_REF] Johnson | Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii[END_REF], 2013), while nonphotosynthetic acetate assimilation begins by its incorporation into acetyl coenzyme A (acetylCoA) [START_REF] Johnson | Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii[END_REF], 2013). Then, acetylCoA is metabolized in the tricarboxylic acid (TCA) cycle in mitochondria or in the glyoxylate cycle in cytosol [START_REF] Johnson | Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii[END_REF], 2013;[START_REF] Singh | Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study[END_REF]. Both acetate assimilation and photosynthesis lead to starch formation [START_REF] Ball | Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii[END_REF][START_REF] Heifetz | Effects of Acetate on Facultative Autotrophy inChlamydomonas reinhardtii Assessed by Photosynthetic Measurements and Stable Isotope Analyses[END_REF][START_REF] Johnson | Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii[END_REF], 2013;[START_REF] Park | The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis[END_REF]. In spite of an abundant literature about carbon assimilation under mixotrophic conditions in C.

reinhardtii, the impact of toxic metals on the carbon metabolism has not been investigated yet.

Ion beam and X-ray beam imaging techniques are powerful tools to study the isotopic and elemental composition of single-cells [START_REF] Ortega | Bio-metals imaging and speciation in cells using proton and synchrotron radiation X-ray microspectroscopy[END_REF][START_REF] Penen | Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS[END_REF][START_REF] Roschzttardtz | Plant Cell Nucleolus as a Hot Spot for Iron[END_REF][START_REF] Sarret | Chapter One -Use of Synchrotron-Based Techniques to Elucidate Metal Uptake and Metabolism in Plants[END_REF][START_REF] Schaumlöffel | Novel Methods for Bioimaging Including LA-ICP-MS, NanoSIMS, TEM/X-EDS, and SXRF[END_REF]. Nanoscale secondary ion mass spectrometry (NanoSIMS) allows elemental and isotopic mapping with a resolution of about 50 nm. In biology, it is used to localize and to determine at a subcellular level the turnover of proteins and metabolites using stable isotopic tracers ( 13 C and 15 N) [START_REF] Gao | A critical review of NanoSIMS in analysis of microbial metabolic activities at single-cell level[END_REF][START_REF] Lechene | High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry[END_REF]. Moreover, when NanoSIMS is combined with transmission electron microscopy (TEM) in correlative imaging, isotopic tracers can be localized in the cell structure [START_REF] Clode | Subcellular imaging of isotopically labeled carbon compounds in a biological sample by ion microprobe (NanoSIMS)[END_REF][START_REF] Hoppe | NanoSIMS: Technical Aspects and Applications in Cosmochemistry and Biological Geochemistry[END_REF][START_REF] Kopp | Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis[END_REF][START_REF] Penen | Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS[END_REF]. NanoSIMS mapping has been also used to determine metal distributions in plants and microalgae [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Moore | NanoSIMS analysis of arsenic and selenium in cereal grain[END_REF][START_REF] Moore | High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots[END_REF][START_REF] Moore | Localisation of iron in wheat grain using high resolution secondary ion mass spectrometry[END_REF]. 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 [START_REF] Adams | Copper Uptake, Intracellular Localization, and Speciation in Marine Microalgae Measured by Synchrotron Radiation X-ray Fluorescence and Absorption Microspectroscopy[END_REF][START_REF] Leonardo | Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniquessubcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis[END_REF][START_REF] Leonardo | Silver Accumulation in the Green Microalga Coccomyxa actinabiotis: Toxicity, in Situ Speciation, and Localization Investigated Using Synchrotron XAS, XRD, and TEM[END_REF][START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF][START_REF] Wang | Cellular internalization and intracellular biotransformation of silver nanoparticles in Chlamydomonas reinhardtii[END_REF].

Here, we investigated the impact of cadmium on carbon assimilation in C. reinhardtii grown under mixotrophic conditions. The objective was to relate subcellular cadmium localization and speciation with acetate and CO2 assimilation. Microalgae were thus exposed up to 70 µM Cd and cultivated in the presence of labeled 13 C-[1,2] acetate to distinguish carbon originating from acetate assimilation or from photosynthesis. A multimodal approach at subcellular level allowed the localization of cadmium in the algal structure by TEM/X-EDS (X-ray energy dispersive spectroscopy) and the determination of carbon origins by measuring the 13 C/ 12 C ratio in the pyrenoid and starch using NanoSIMS. In addition, Cd was localized by µXRF, which was combined to µXANES (micro X-ray absorption near edge structure spectroscopy) at the Cd LIII-edge to identify Cd speciation. Our study showed that in C. reinhardtii cadmium sequestration in the pyrenoid impaired CO2 fixation to the benefit of acetate assimilation.

MATERIALS AND METHODS

Biological material and growth conditions

Chlamydomonas reinhardtii wild type strain (SAG 11/32b, Experimental Phycology and Culture Collection of Algae at Goettingen University (EPSAG), Germany) was grown heterotrophically in a tris-acetate-phosphate medium [START_REF] Harris | The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use[END_REF] where ethylenediaminetetraacetic acid (EDTA) and FeSO4 As the main aim of this study was the investigation of the cadmium stress response at cellular level during the exponential growth phase, cells were exposed to Cd after 24h. Therefore changes in the lag phase, the delay before the start of exponential growth, were not assayed since this delay allowed the adaptation required for cells to begin the experience of new environmental conditions, i.e. cadmium exposure in this study. The cell cycle of C. reinhardtii is about 24 h which is confirmed with the start of the exponential phase at this time point. This means that under these conditions the cells of the second (and third) cell cycle are exposed.

Cells were harvested after 72 h for different analyses, i.e. at the end of the third cell cycle when these cells experienced similar exposure conditions. It is a good approximation although cultivation was not synchronized. This procedure also applied in our previous studies [START_REF] Bräutigam | Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii[END_REF][START_REF] Bräutigam | Analytical approach for characterization of cadmium-induced thiol peptides-a case study using Chlamydomonas reinhardtii[END_REF][START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF].

The concentrations of free metals in the medium were simulated (Table 1) with the freeware Visual MINTEQ 3.0 (vminteq.lwr.kth.se/visual-minteq-ver-3-0/) (Gustafsson, 2013) using Lindsay's database containing thermodynamic equilibrium constants compiled by [START_REF] Lindsay | Chemical equilibria in soils[END_REF]. Before use, glassware was washed with 5% HNO3. All solutions were prepared with ultrapure water (18 MΩ cm at 25 °C) obtained from a Milli-Q system (Millipore, Bedford, MA).

Growth rate determination

Optical density at 730 nm (OD730nm) was measured every 12 h in each control and Cd-exposed algal cultures, grown in triplicate, using a spectrophotometer. Growth rates µ were calculated as follows: µ = (ln Nf -ln Ni)/Δt. Nf was the OD730nm of the culture measured at the end of the exponential phase. Ni was the OD730nm of the culture at the beginning of the exponential phase and Δt was the duration of the exponential phase (in days). Relative growth rate was expressed as the ratio µ/µ0 determined during the exponential phase of growth (µ: growth rate during cadmium exposure, µ0: growth rate in control conditions). The Excel macro REGTOX (http://www.normalesup.org/~vindimian/en_index.html) was used to calculate the half maximal efficient concentrations (EC50) applying the Hill model.

Chlorophyll concentration measurement

The chlorophyll concentration assay was adapted from Arnon et al. [START_REF] Arnon | Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta Vulgaris[END_REF]. After 72 h cultivation, 1 ml of each control and Cd-exposed algal cultures (grown in triplicate) was centrifuged at 13000 × g for 1 min. Each algal pellet was resuspended in 1 mL of 80% acetone and vortexed for 2 min. Then, solubilized chlorophyll was separated from cell debris by a centrifugation at 13000 × g for 4 min. After that, the concentration (C) of chlorophyll a and b in the culture medium was determined spectrophotometrically at 652 nm and calculated as follows: C = Abs652nm × 1000 / 34.5 with C in mg mL -1 . Finally, in order to relate the chlorophyll content to the biomass, the determined chlorophyll concentration was normalized to the optical density at 730 nm whose value is proportional to the number of cells in the medium (unit: µg mL -1 OD730nm -1 ).

Starch concentration measurement

After 72 h cultivation, microalgae were harvested from in triplicate grown control and Cdexposed (70 µM) algal cultures by centrifugation at 3000 × g for 3 min and each pellet was resuspended in 1.5 mL water. In order to estimate the dry biomass, a 0.5 mL aliquot from each sample was dried at 60 °C for 24 h and the resulting pellet was weighted. The remaining 1 mL samples were washed once with water and once with 80% ethanol, and centrifuged (3000 × g for 3 min). Then, each pellet was boiled in 2 mL water for 10 min. Finally, starch concentrations were determined in these extracts, using the enzymatic starch assay kit HK (Sigma Aldrich) described in [START_REF] Delrue | Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin[END_REF].

Cadmium uptake

After 48 h cadmium exposure (70 µM), microalgae were harvested from in triplicate grown cultures by centrifugation at 3000 × g for 3 min. In order to obtain the intracellular Cd concentration, each algal pellet was subjected to the following washing protocol according to [START_REF] Macfie | Effects of cadmium, cobalt, copper, and nickel on growth of the green alga Chlamydomonas reinhardtii: The influences of the cell wall and pH[END_REF]: a first time in water, then twice in a mix of Na2-EDTA (1 mM) / CaCl2

(3.77 mM) and finally in water with centrifugation between in (3000 × g for 3 min). The remaining pellets were dried at 60°C for 24 h and weighted. This dry algal biomass was digested in a mixture of 1 mL 70% (v/v) HNO3 and 1 mL 30% (v/v) H2O2 at 80 °C for 3 h.

Samples were then diluted to reach a 2% HNO3 concentration. The cadmium concentration was determined by ICP-MS (Inductively Coupled Plasma Mass Spectrometry, 7500 model, Agilent Technologies).

Sample preparation for TEM/X-EDS and NanoSIMS

Microalgae from a control and a Cd-exposed (70 µM) culture containing 13 C-labeled acetate in the medium were harvested after 72 h of inoculation by centrifugation at 3,000 × g for 3 min.

A protocol including high pressure freezing, and freeze substitution followed by resin inclusion was used to preserve cell integrity. For high-pressure freezing, each microalgae pellet was resuspended and incubated for 1 h in 150 mM mannitol. After a centrifugation at 3000 × g for 3 min, the harvested cells were included in agarose 2%. Then, the inclusion was plunged in cryo-protectant 1-hexadecene and high-pressure freezing was performed immediately (EM HPM 100, Leica Microsystems, Vienna, Austria). Samples were maintained in liquid nitrogen until the freeze-substitution steps.

Freeze-substitution was adapted from O'Toole's protocol (O'Toole, 2010). Under liquid nitrogen, algal samples were post-fixed in 1% osmium tetroxide, 0.1% uranyl acetate and 0.25% glutaraldehyde in anhydrous acetone and transferred into the Automatic Freeze Substitution System AFS2 (Leica microsystems). Temperature was maintained at -90 °C for 3 d and then warmed to -30 °C for 8 h. Samples were removed from freeze-substitution system, kept at 0 °C for 1 h and washed with water-free acetone.

Resin inclusion was carried out at room temperature. Samples were embedded in epoxy resin (EPON 812, Delta Microscopies, Mauressac, France) using a graded resin and acetone series.

The following steps were applied for infiltration: 3:1 acetone:resin, 1:1 acetone:resin, 1:3 acetone:resin, and 2 baths with 100% resin. Finally, resin embedded samples were polymerized for 48 h at 60 °C.

Samples were cut in 70 nm and 300 nm sections for TEM and NanoSIMS, respectively, using a diamond knife (Diatome, Biel-Bienne, Switzerland) on an ultra-microtome [EM Ultracut-UC7, Leica Microsystems) and placed on the respective sample holder. In order to carry out correlative imaging by TEM and NanoSIMS, a first 70 nm section was placed on a copper grid for TEM and then, an adjacent 300 nm section was placed on a silicon wafer (Wafer Solution, Le Bourget du lac, France) for NanoSIMS as previously described [START_REF] Penen | Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS[END_REF]. Cells were localized and observed first by TEM and then, relocalized and analyzed by NanoSIMS.

TEM/X-EDS analysis

A transmission electron microscope FEI TECNAI 12 (Eindhoven, The Netherlands) using an accelerating voltage 120 kV and equipped with an X-Energy Dispersive Spectroscopy (X-Flash 6T 60 BRUKER-SYNERGIE 4, Evry, France) was used. The parallel electron beam of the nanoprobe was focused on 0.25-1.2 µm areas. Under these conditions X-EDS spectra of 1-2k counts s -1 , a deadtime of 15-20% and analytical time of 120 -240 s per spectrum were generated.

NanoSIMS analysis

A nanoscale secondary ion mass spectrometer NanoSIMS 50L (CAMECA, Gennevilliers, France) was used to perform SIMS analysis. This device was equipped with a primary Cs + ion source (lateral resolution down to 50 nm) for the mapping of electronegative elements, and with a novel O -RF plasma primary ion source enabling a high sensitivity for electropositive elements combined to a high lateral resolution (40 nm lateral resolution) [START_REF] Malherbe | A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level[END_REF]. The seven parallel electron multiplier detectors equipped on the NanoSIMS 50L instrument allowed detection of seven elements at the same time. In order to remove major interferences on the chosen element masses, the mass resolution (M/ΔM) was tuned to be about 5000. Initially, ion maps (256×256 pixels, 64-144 µm²) of 12 C -, 13 C -, 12 C 14 N -(for N detection)

and 32 S -were carried out using the primary Cs + ion source. Mapping of 31 P + and 40 Ca + was then performed using the O -RF plasma primary ion source.

NanoSIMS data processing

Image processing was performed using ImageJ software (v.1.48, Wayne Rasband, National

Institutes of Health (NIH), Bethesda, MD, USA). Processing of NanoSIMS images required additionally the openMIMS plugin developed at the National Resource for Imaging Mass Spectrometry (NRIMS, Cambridge, MA, USA). Ion maps were corrected for detector dead time (44 ns) with the openMIMS plugin and the 13 C/ 12 C ratio maps were corrected for the mass bias as a function of the 13 C/ 12 C ratio measured on microalgae not enriched in 13 C. The 13 C/ 12 C ratio was measured in regions of interest (ROI) defined on pyrenoid and starch granules of six control cells and six Cd-exposed cells as detailed in Figure S1. The isotopic enrichment of 13 C was expressed as its relative abundance δ 13 C compared to the 13 C natural isotopic abundance and calculated using the following formula:

𝛿 13 C = Rsample Rnat -1 (1)
where Rsample is the measured 13 C/ 12 C ratio in the ROI of the cells and Rnat is the natural carbon isotope ratio.

The origin of carbon in starch granules from either acetate or CO2 as carbon source was determined by data processing adapted from [START_REF] Terrado | Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis[END_REF]. First, the fractional abundance Fstarch of the carbon isotope 13 C in starch granules of the sum of both carbon isotopes is given as (in percentage):

Fstarch = 13 C 13 C + 12 C ×100 (2)
which can also be expressed as

Fstarch = Rstarch 1 + Rstarch ×100 ( 3
)
where Rstarch is the 13 C/ 12 C ratio measured in the ROI within starch granules. Then, the origin of starch carbon was determined using the following formulas:

Fstarch = Facetate × facetate + FCO2 × fCO2 (4) with: facetate + fCO2 = 1 (5) facetate = Fstarch -FCO2 Facetate -FCO2 × 100 and fCO2 = Fstarch -Facetate FCO2 -Facetate × 100 (6)
The formula (6) results from ( 4) and ( 5) where Facetate and FCO2 are the fractional abundance of 13 C in the carbon sources acetate (99%) and CO2 (1.09%), respectively, and facetate and fCO2

represent the percentage of the origin of carbon within the ROI (starch granule) from the respective source.

Samples preparation for synchrotron-based techniques

Microalgae from a control and a Cd-exposed (70 µM) culture were harvested after 72 h of inoculation by centrifugation at 3000 × g for 3 min. They were rapidly washed in ultrapure water three times to remove metals and organic compounds weakly adsorbed on the cell wall.

Two sample preparations were used.

For µXRF and Cd LIII-edge µXANES, droplets of microalgae suspension were deposited on 4 μm-thick Ultralene film (SPEX sample prep Metuche, NJ). After decantation for 10 min, microalgae were frozen into liquid nitrogen-chilled isopentane (-160 °C).

For Cd LIII-edge XANES bulk analysis, the algae pellet was frozen in liquid nitrogen, homogenized and pressed as a 5 mm frozen pellet. All the samples were kept frozen at -80 °C until measurements and transferred to the sample stage in their hydrated frozen state.

µXRF imaging

Measurements were performed on the ID21 beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) equipped with a fixed exit Si(111) two-crystal monochromator [START_REF] Cotte | The ID21 X-ray and infrared microscopy beamline at the ESRF: status and recent applications to artistic materials[END_REF]. The X-ray photons were focused by a KB mirror system providing a sub-micron resolution of 0.6 μm (H) × 0.3 μm (V) FWHM on the sample. The Xray fluorescence signal was recorded with a large 100 mm 2 SDD detector (Bruker) while the samples were scanned to obtain elemental maps. All the measurements were carried out under cryogenic conditions at -160 °C using a liquid N2 cryostat to limit beam radiation damage. The fluorescence signal was normalized by the incident photon intensity (I0) measured with a photodiode. Phosphorus, sulfur, chloride, and cadmium maps were recorded using an incident energy of 3570 eV, below the absorption edge of potassium, while potassium and calcium maps were recorded at 4100 eV as described in [START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy[END_REF]. The fluorescence signal was deconvoluted from fluorescence background and fluorescence elemental overlapping to obtain elemental maps using the PYMCA software [START_REF] Solé | A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra[END_REF].

Cd LIII-edge µXANES and XANES. µXANES spectra were recorded with the same lateral resolution as for µXRF in fluorescence mode on points of interest visualized on the µXRF maps. For Cd model compounds and bulk samples, XANES spectra were recorded in fluorescence mode with a defocused beam (100 µm × 100 µm). Measurements were also done under cryogenic conditions to limit radiation damage and speciation change. µXANES and XANES spectra were collected in the energy range 3520-3590 eV. Approximately 30 scans were calibrated with a metallic Cd foil, averaged, and normalized according to standard methods using ATHENA software [START_REF] Ravel | ATHENA , ARTEMIS , HEPHAESTUS : data analysis for X-ray absorption spectroscopy using IFEFFIT[END_REF]. Then, experimental normalized spectra were compared to a library of standard spectra previously collected [START_REF] Huguet | Cd speciation and localization in the hyperaccumulator Arabidopsis halleri[END_REF][START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy[END_REF][START_REF] Isaure | Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the nonaccumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques[END_REF][START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF]. A fingerprint approach was used to simulate the unknown spectra by linear combination fits (LCFs) of Cd model compounds as described by [START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy[END_REF]. The quality of the fits was estimated by the normalized sum-squares residuals NSS = Σ(Xanesexperimental-Xanesfit)²/Σ(Xanesexperimental)²×100, in the 3520-3580 for µXANES spectra and 3520-3590 eV range for XANES spectra. Linear combination fits with one, two and three components were tested and the combination with n+1 components was retained if the NSS parameter decreased more that 20% in comparison to the fit with n components (Figure S2). The uncertainty of the proportion of each compound was estimated to 10% [START_REF] Isaure | Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the nonaccumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques[END_REF].

Statistics

One-way ANOVA followed by a post-hoc Tuckey test Statistical analyses were performed on growth rate, chlorophyll concentration, starch concentration and 13 C/ 12 C ratio measured on NanoSIMS maps to identify significant differences among the different conditions. Results

were considered significant for a p < 0.05. Kruskal-Wallis one-way analysis of variance on ranks (p<0.05) was performed when the condition of homogeneity of variances was not respected.

RESULTS

Availability of cadmium and essential trace elements in TAPEDDHA medium

In a first approach, cadmium availability in TAPEDDHA medium, i.e. free Cd 2+ concentration, was simulated using Visual Minteq software (Table 1). Concentrations of 10, 20, 30, 40, 50 and 70 µM of CdCl2 added to the medium correspond to free ionic Cd concentrations of 1, 4, 7, 10, 13 and 20 µM. Cd availability was thus increased by 10 compared to the classic TAP medium for the higher Cd concentration.

The free ion concentrations of several essential trace elements in the TAPEDDHA medium were markedly different from that in the original TAP medium (Table 1). Most important, the concentration of free Zn 2+ increased by 10000 and of free Cu 2+ by 100 whereas Co 2+ availability decreased by 10000 compared to the classic TAP medium.

Cadmium exposure leads to starch accumulation

C. reinhardtii was exposed to a range of 10-70 µM CdCl2 for 48 h in TAPEDDHA medium and relative growth rates were measured in order to determine Cd efficient concentrations (ECx)

with the Hill model (Figure 1A). Up to 20 µM Cd, no significant effect on the algae growth was observed (EC5 = 22 µM Cd [15-31 µM Cd CI95%]) but increasing Cd concentrations induced a strong decrease of the relative growth rate (EC50 = 74 µM Cd [67-86 µM Cd CI95%]).

In order to investigate the impact of cadmium on C. reinhardtii in stress conditions, the Cd exposure concentration of 70 µM (i.e. 20 µM free Cd 2+ ) was applied in this study since it was the nearest to EC50 value. These concentrations can seem quite high compared to other studies focused on Cd concentrations in the 10-100 nM range [START_REF] Stoiber | Relationships between surface-bound and internalized copper and cadmium and toxicity in Chlamydomonas reinhardtii[END_REF][START_REF] Lavoie | Influence of essential elements on cadmium uptake and toxicity in a unicellular green alga: The protective effect of trace zinc and cobalt concentrations[END_REF] . However, contrary to these studies, the present experiments were carried out in mixotrophic conditions, which likely modified the cell response. Our observations support that under mixotrophic conditions C. reinhardtii was more resistant against Cd stress.

When exposed to 70 µM Cd, the intracellular cadmium concentration was 1530 ± 132 µg g -1

(dry weight). The global impact of this cadmium stress on C. reinhardtii vitality was examined by the determination of growth rate, chlorophyll concentration and starch concentration (Figure 1B, C and D). Compared to the control condition, Cd induced a decrease of the growth rate (from 0.76 ± 0.05 to 0.39 ± 0.06 d -1 ) and the chlorophyll concentration (from 8.3 ± 0.4 to 6.4 ± 0.3 µg mL -1 OD730nm -1 ) while starch concentration was increased (from 47.6 ± 4.7 to 87.9 ± 13.2 mg g -1 ). These results showed an important stress in C. reinhardtii when it exposed to 70 µM Cd leading to an impaired photosynthesis and an accumulation of starch. Carbon metabolism in C. reinhardtii was thus strongly impacted by Cd toxicity.

Cadmium sequestration in pyrenoid and vacuoles

C. reinhardtii cells exposed to 70 µM Cd for 48 h were examined by TEM/X-EDS and NanoSIMS (Figure 2). Figure 2 A and B show that cell structure was altered by Cd exposure, particularly the chloroplast where the pyrenoid and its starch shell were destructured. The starch plaques were also more abundant in the Cd-exposed cells corroborating the increase of starch measured by enzymatic digestion.

Elemental composition of organelles observed on TEM micrographs (Figure 2C, F) was determined on their corresponding NanoSIMS elemental maps (Figure 2E,H). Vacuoles were rich in calcium, phosphorus and sulfur. Pyrenoids were characterized by a composition rich in sulfur and nitrogen, which could be attributed to the high concentration in proteins (RuBisCO in particular) inside this organelle. Moreover, pyrenoids were surrounded by a halo of phosphorus which did not necessarily correspond to the starch shell. Finally, starch shells and starch plates were rich in nitrogen.

Additional X-EDS analysis (Figure 2D, G) revealed that cadmium concentration was high in the pyrenoid. 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 [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF]. It is important to note that the elemental composition of vacuoles and pyrenoid determined by NanoSIMS was confirmed by the X-EDS measurements.

Cadmium localized in pyrenoid is preferentially bound to sulfur ligands

Elemental µXRF maps (Figure 3A) of C. reinhardtii cells exposed to 70 µM Cd for 48 h corroborated the elemental distribution obtained by TEM/X-EDS and NanoSIMS analysis (Figure 3A). A sulfur spot was present in each cell which corresponded to the pyrenoid.

Although cadmium was detected at low level in the whole cells, it was highly concentrated in one spot in each cell co-localized with sulfur, confirming its pyrenoidal sequestration. In addition, phosphorus, calcium, and to a lesser extent cadmium were co-localized in spots of 1 µm size representing Ca polyphosphate granules in vacuoles.

In combination with µXRF, µXANES gave information about Cd binding ligands in areas of interest evidenced by µXRF. In contrast, XANES collected in bulk samples allowed to determine the average in situ cadmium binding in the microalgae. Cd LIII-edge XANES was demonstrated powerful to distinguish sulfur from oxygen ligands due to absence of a peak at 3539 eV in sulfur binding atoms [START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy[END_REF]. Moreover, as indicated by arrows in Cd reference XANES spectra in figures 3B and 3C, the second oscillation was shifted to higher energy values for Cd-acetate and Cd-malate where Cd is bound to carboxyl groups compared to Cd bound to phosphate groups in Cd-phosphate and Cd-phytate [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF]. This enabled a differentiation between the oxygen donor ligands in Cd-carboxyl and Cd-phosphate.

In contrast, Cd-phytochelatin 2 (Cd-PC2), Cd-glutathione (Cd-GSH) and Cd-cysteine spectra had similar pattern, thus hampering the exact determination of the nature of sulfur donor ligands; however, they can be encompassed as thiol groups. Therefore, the distribution of Cd ligands, as results of linear combination fits (LCFs) of Cd LIII-edge XANES and µXANES spectra, was described as ratios of Cd-S, Cd-O-P, and Cd-O-C compounds (Figure 3D).

In the S and Cd-enriched areas attributed to the pyrenoid, µXANES analysis revealed that Cd was mainly coordinated by thiol ligands (67  10% and 56  10% for the two presented pyrenoids), but also by carboxyl groups (33  10% and 44  10%). No phosphate ligand was identified in these spots. A pyrenoid consists principally of proteins, mainly RuBisCO, thus µXANES results could indicate cadmium binding to protein thiol groups. Finally, Cd bound to carboxyl groups could be attributed to Cd sequestered to the starch shell around the pyrenoid since µXANES here probed a three-dimensional volume.

XANES measurements of bulk samples showed that Cd-thiol ligands in algae accounted for 31  10% only while Cd-carboxyl groups amounted to 25  10%. 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 [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF].

Cadmium promotes carbon incorporation from acetate in starch

In order to investigate the impact of Cd on carbon fixation and to distinguish inorganic and organic carbon sources of C. reinhardtii, i.e. CO2 and acetate, 13 C labeled acetate was employed for tracer experiments. C. reinhardtii control cells and cells exposed to 70 µM Cd for 48 h in TAPEDDHA medium containing 13 C labeled acetate were examined by NanoSIMS (Figure 4).

13

C/ 12 C ratio maps highlight intracellular areas of carbon resulting from acetate assimilation (high 13 C/ 12 C ratio) (Figure 4A andB).

Two different subcellular regions of interest (ROI) were selected in 13 C/ 12 C ratio maps (Figure S1) to investigate carbon assimilation: the pyrenoid as organelle of CO2 fixation and the starch plates as a final storage form of carbon in C. reinhardtii. Based on data from ROI in 6 individual control cells and 6 exposed cells (Figure S1), the 13 C isotopic enrichment δ 13 C was calculated as represented in Figure 4C. Hence, 13 C enrichment was found in the pyrenoid, but it did not change significantly under Cd exposure. In contrast, starch plates showed a 5-fold higher 13 C enrichment under Cd exposure than in unexposed cells. In particular, the study of carbon isotopic distribution in starch as the final carbon storage form allowed the quantification of carbon assimilation from CO2 and acetate sources.

The determination of facetate and fCO2 in starch granules revealed that, under control conditions, 73% of starch carbon came from CO2 ( 12 C) and 27% from acetate ( 13 C) (RSD ± 9%). In contrast, Cd stress seemed to disturb CO2 ( 12 C) assimilation which decreased to 39% while acetate ( 13 C) assimilation contributed for 61% of starch carbon (Figure 4D) (RSD ± 13%).

Moreover, when the isotopic distribution of starch carbon was combined with the starch concentration previously analyzed (Figure 1D), it appeared that overall CO2 carbon ( 12 C) assimilation remained limited (1.28 ± 0.18 mmol ( 12 C) g -1 in control condition; 1.26 ± 0.43 mmol ( 12 C) g -1 under Cd exposure) (Figure 4E) relative to the highly increased starch biosynthesis under Cd exposure. In contrast, acetate carbon ( 13 C) assimilation was about 4-fold higher during Cd exposure (0.48 ± 0.15 mmol ( 13 C) g -1 under control conditions; 1.99 ± 0.47 mmol ( 13 C) g -1 under Cd exposure). These results suggest that Cd exposure interfered with CO2 assimilation due to the Cd fixation in the pyrenoid possibly due to the Cd binding to protein thiol groups.

DISCUSSION

Effect of the culture medium on cell vitality C. reinhardtii was cultivated in a modified version of TAP medium where EDTA was replaced with EDDHA (TAPEDDHA) in order to increase Cd availability and, at the same time, Cd toxicity. However, under control conditions, this modification induced already a decrease of cell vitality. Indeed, in our previous work [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF], C. reinhardtii grown in classic TAP medium showed a higher growth rate (1.15 ± 0.03 d -1 ) and a higher production of chlorophyll (10.78 ± 0.38 µg ml -1 OD730nm -1 ) compared to the present study where microalgae have been cultivated in TAPEDDHA medium (Figure 1B,C). These effects of TAPEDDHA medium on cell vitality in control conditions could be explained by changes in the availability of some trace elements compared to the classic TAP medium (Table 1). Indeed, higher availability mainly of zinc but also, to a lesser extent, of copper, and in addition deficiency of cobalt was found in the control TAPEDDHA medium.

Cell response to cadmium stress

Cell vitality results showed that a 70 µM Cd exposure for 48 h induced a decrease of the chlorophyll concentration, which is a well-known sign of photosynthesis impairment [START_REF] Nagel | In Vitro Evolution and Preliminary Characterization of a Cadmium-Resistant Population of Chlamydomonas reinhardtii[END_REF][START_REF] Collard | Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium[END_REF][START_REF] Nagel | Impaired photosynthesis in a cadmium-tolerant Chlamydomonas mutant strain[END_REF][START_REF] Prasad | Toxicity of Cadmium and Copper in Chlamydomonas reinhardtii Wild-Type (WT 2137) and Cell Wall Deficient Mutant Strain (CW 15)[END_REF]. The degradation of the chlorophyll pool was concomitant with a growth decrease and an increase of carbon storage in the form of starch. Usually, this stress reaction has been described in C. reinhardtii during nitrogen [START_REF] Ball | Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii[END_REF][START_REF] Cakmak | Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii[END_REF], phosphorus [START_REF] Ball | Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii[END_REF] and sulfur [START_REF] Ball | Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii[END_REF][START_REF] Cakmak | Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii[END_REF] starvation.

Moreover, [START_REF] Juergens | The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Juergens | The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows during Nutrient Deprivation in Chlamydomonas reinhardtii[END_REF] showed that starch was accumulated to maintain a carbon pool during N-starvation instead of delaying photoinhibition and oxidative stress due to energetic overflows from photosynthesis. In case of metallic stress, starch storage was reported in Chlamydomonas acidophila [START_REF] Nishikawa | Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism[END_REF] exposed to 20 µM Cd where the size of starch plates observed microscopically increased 2.43fold. Metal stress could thus induce, in an indirect way, symptoms similar to those of nutrient starvation. Working with C. reinhardtii, [START_REF] Bräutigam | Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii[END_REF] showed that Cd stress induced the synthesis of thiol polypeptides like phytochelatins, resulting in a decrease in the intracellular concentration of its precursors like glutathione, γ-EC and cysteine leading presumably to a sulfur starvation. Although, sulfur supply by the medium should be sufficient [START_REF] Bräutigam | Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii[END_REF], an intracellular sulfur deficiency is conceivable. Furthermore, a proteomic study [START_REF] Gillet | Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach[END_REF] revealed that enzymes involved in sulfur metabolism were modified under Cd stress in order to promote the synthesis of phytochelatins.

Cadmium is bound to RuBisCO in pyrenoid and to phosphate in acidocalcisome

For the first time, a pyrenoidal sequestration of Cd and its binding to sulfur ligands were clearly observed in C. reinhardtii. In addition, high sulfur and nitrogen content indicated a high protein concentration in the pyrenoid. A model of the impact of Cd sequestration on carbon fixation in C. reinhardtii is proposed in Figure 5. Pyrenoids are essentially composed of the protein RuBisCO [START_REF] Borkhsenious | The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii[END_REF]. They are the place of the carbon fixation from CO2 via the Calvin cycle [START_REF] Harris | The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use[END_REF]. Beside RuBisCO, the enzyme RuBisCO-activase is involved in this process [START_REF] Mckay | RuBisCo activase is present in the pyrenoid of green algae[END_REF]. Thus, it can be hypothesized that Cd binding to protein thiol groups interferes with CO2 fixation in C. reinhardtii, for example due to conformational changes caused by cadmium-thiol bonds. Indeed, it has been reported that modifications at cysteines 449 and 459 of the RuBisCO large subunit induce a conformational change and a decrease of its carboxylase activity [START_REF] Marín-Navarro | Cysteines 449 and 459 modulate the reductionoxidation conformational changes of ribulose 1•5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress[END_REF]. Moreover, the RuBisCO large subunit protein is down-regulated under Cd exposure [START_REF] Gillet | Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach[END_REF]. For higher plants, it has been shown that Cd impairs the CO2 assimilation rate by RuBisCO activity decrease, e.g. in the crop Lactuca sativa [START_REF] Dias | Cadmium toxicity affects photosynthesis and plant growth at different levels[END_REF] and in

Nicotiana rustica [START_REF] Afef | Relationship between physiological and biochemical effects of cadmium toxicity in Nicotiana rustica[END_REF].

In addition, TEM/X-EDS, µXRF and Cd LIII-edge XANES measurements have shown that Cd was also coordinated by phosphate ligands and localized in 0.5-1 µm sized vacuoles containing phosphorus and calcium. In our previous work [START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF], cadmium was shown to be localized in vacuoles which were compared to acidocalcisomes due to their specific elemental composition in phosphorus and calcium [START_REF] Goodenough | Acidocalcisomes: Ultrastructure, Biogenesis, and Distribution in Microbial Eukaryotes[END_REF][START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Komine | Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components[END_REF][START_REF] Komine | Secretory granules in the cytoplasm of a wall-less mutant of Chlamydomonas reinhardtii contain processed lightharvesting complex apoproteins and HSP70[END_REF][START_REF] Ruiz | The Polyphosphate Bodies of Chlamydomonas reinhardtii Possess a Proton-pumping Pyrophosphatase and Are Similar to Acidocalcisomes[END_REF].

CO2 fixation is impaired by cadmium stress

Cadmium has been shown to disturb the light-dependent part of the photosynthesis [START_REF] Faller | Mechanism of Cd2+ toxicity: Cd2+ inhibits photoactivation of Photosystem II by competitive binding to the essential Ca2+ site[END_REF] but no study has previously focused on the impact of Cd on carbon assimilation. C.

reinhardtii is able to grow photoautotrophically using CO2, heterotrophically using acetate in the dark and mixotrophically using both carbon sources in the light. In the present study, mixotrophic growth conditions under constant illumination induced carbon storage as starch (Figure 1D). Moreover, NanoSIMS analysis revealed that, carbon from acetate was accumulated in the pyrenoid and in starch granules (Figure 4B). The pathways involved in acetate assimilation as well as the impact of cadmium on these pathways are discussed in the next paragraphs and are represented in Figure 5.

The accumulation of carbon from acetate in the pyrenoid can be explained by the assimilation of acetate through the tricarboxylic acid cycle (TCA). Indeed, acetate assimilation starts with its incorporation into acetyl coenzyme A (acetyl-CoA), either directly converted by acetyl-CoA synthetase, or in two steps with the successive action of acetate kinase and phosphate acetyltransferase. Acetyl-CoA is used as substrate in the TCA cycle in mitochondria. The TCA cycle produces ATP as well as NADH and releases the carbon of acetyl-CoA as CO2 [START_REF] Johnson | Central Carbon Metabolism and Electron Transport in Chlamydomonas reinhardtii: Metabolic Constraints for Carbon Partitioning between Oil and Starch[END_REF]. Then, the mitochondrial carbonic anhydrase CaH4/5 [START_REF] Raven | A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii[END_REF] converts CO2 in bicarbonate which is carried to the pyrenoid by the CO2 concentrating mechanism (CCM) [START_REF] Meyer | Origins and diversity of eukaryotic CO2-concentrating mechanisms: lessons for the future[END_REF]. This latter is a combination of active carbon transporters [START_REF] Burow | Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2[END_REF][START_REF] Im | Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii[END_REF][START_REF] Miura | Expression Profiling-Based Identification of CO2-Responsive Genes Regulated by CCM1 Controlling a Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii[END_REF][START_REF] Pollock | The Chlamydomonas reinhardtii proteins Ccp1 and Ccp2 are required for long-term growth, but are not necessary for efficient photosynthesis, in a low-CO2 environment[END_REF], carbonic anhydrases [START_REF] Moroney | The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles[END_REF] specific to cellular compartments and the soluble proteins (LCIB/LCIC) [START_REF] Yamano | Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii[END_REF] allowing CO2 trapping inside the pyrenoid. In the present work, the isotopic ratio of carbon ( 13 C enrichment) inside the pyrenoid was similar under control and exposure conditions (Figure 4C) showing that cadmium did not seem to disturb TCA cycle and CCM.

Starch biosynthesis involves the successive actions of ADP-glucose pyrophosphorylase (AGPP) and starch synthase (SS) on the final product of the gluconeogenesis pathway (glucose-1-phosphate (G1P)). Carbon from acetate enters gluconeogenesis in two different ways, once incorporated into AcetylCoA. On the one hand, as mentioned in the previous paragraph, AcetylCoA feeds the TCA cycle releasing CO2. In the chloroplast, the two carbon sources thus share the CO2 fixation step by RuBisCO into the Calvin cycle pathway. The final product of the Calvin cycle (glyceraldehyde-3-phosphate (G3P)) can enter the gluconeogenesis [START_REF] Johnson | Central Carbon Metabolism and Electron Transport in Chlamydomonas reinhardtii: Metabolic Constraints for Carbon Partitioning between Oil and Starch[END_REF]. On the other hand, Acetyl-CoA feeds the glyoxylate cycle which is a TCA cycle where CO2 releasing steps are by-passed [START_REF] Johnson | Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii[END_REF], 2013) in the cytosol.

Indeed, using AcetylCoA and isocitrate as substrates, isocitrate lyase produces glyoxylate and succinate. This latter is successively converted in mitochondria to fumarate, malate, oxaloacetate then phosphoenolpyruvate (PEP) which is exported to cytosol and enters the gluconeogenesis pathway.

C. reinhardtii is able to assimilate inorganic carbon via PEP carboxylation by the two PEP carboxylases CrPpc1/CrPpc2, leading to the production of oxaloacetate [START_REF] Mamedov | Identification and expression analysis of two inorganic C-and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas reinhardtii[END_REF]. However, the algal PEP carboxylase activity is mainly anaplerotic and not photosynthetic [START_REF] Giordano | An Anaplerotic Role for Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii[END_REF]. Although the impact of cadmium on PEPC activity is not established in C. reinhardtii, it has been shown that Cd inhibits PEPC activity in higher plants like Miscanthus species [START_REF] Guo | Different Growth and Physiological Responses to Cadmium of the Three Miscanthus Species[END_REF] and Zea mays [START_REF] Wang | Changes of photosynthetic activities of maize (Zea mays L.) seedlings in response to cadmium stress[END_REF].

Despite 13 C-[1,2] labeling of acetate, our experiments do not allow us to distinguish the pathways taken by acetate carbons to be finally incorporated in starch. However, unlabeled carbon assimilation from CO2 can only pass by the Calvin cycle. In consequence, after a 48 h Cd exposure, the relative decrease in 12 C from 73 to 39% of starch carbon (Figure 4D) shows that carbon fixation through Calvin cycle pathway is limited by Cd stress but not completely inhibited. Hence, the high increase of starch biosynthesis under Cd stress (Figure 1D) is achieved in mixotrophic conditions by an about 4-fold higher carbon assimilation from acetate.

However, the overall amount of carbon assimilation from CO2 into starch remains limited in relation to the increase of starch production (Figure 4E). Moreover, the decrease in chlorophyll concentration (figure 1C) coupled with a massive accumulation of Cd in the pyrenoid (Figures 2D, G and 3A) seems to disturb not only the light-dependent reactions [START_REF] Faller | Mechanism of Cd2+ toxicity: Cd2+ inhibits photoactivation of Photosystem II by competitive binding to the essential Ca2+ site[END_REF] but also the light-independent reactions of photosynthesis.

In unexposed C. reinhardtii grown under mixotrophic conditions, 78% of total carbon [START_REF] Heifetz | Effects of Acetate on Facultative Autotrophy inChlamydomonas reinhardtii Assessed by Photosynthetic Measurements and Stable Isotope Analyses[END_REF] has been found to be from CO2 assimilation which is similar to the 73% of starch carbon originating from CO2 found in this work. Although Cd exposure and nutrient starvation have a similar impact on starch accumulation, the limitation of CO2 fixation is specific to Cd stress, since during nitrogen starvation, 65% of starch carbon [START_REF] Juergens | The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows during Nutrient Deprivation in Chlamydomonas reinhardtii[END_REF] as well as 80% of total carbon [START_REF] Juergens | The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows during Nutrient Deprivation in Chlamydomonas reinhardtii[END_REF] are taken from an inorganic carbon source. In addition, enzymes involved in Calvin cycle have been shown to be down-regulated under Cd stress (phosphoglycerate kinase, ribose-5phosphate isomerase) while enzymes involved in acetate assimilation were up-regulated as for the isocitrate lyase [START_REF] Gillet | Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach[END_REF] involved in the glyoxylate cycle or the phosphoglycerate mutase [START_REF] Gillet | Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach[END_REF] taking part in gluconeogenesis.

Using NanoSIMS in combination with 13 C-labeled acetate it was possible to detect, localize, and quantify 13 C-labeled starch as end product within the cells. Although these are important results, the limitation of this approach is, that between the acetate substrate and the end product no other metabolites of the carbon metabolism pathway during cadmium stress were measured.

A metabolomic or a fluxomic approach would be necessary to complete our results with regard to the overall carbon fixation and metabolism pathway involved during cadmium stress in C.

reinhardtii.

In conclusion, the present work shows that cadmium clearly impairs carbon assimilation in C.

reinhardtii grown in mixotrophic conditions. Due to the pyrenoidal sequestration of Cd, CO2 fixation is limited during Cd exposure whereas acetate assimilation is favored. Mixotrophy could thus allow phytoplankton surviving toxic metal pollution events. Although mixotrophy seems to render aquatic ecosystems more resistant, the alteration of CO2 fixation by toxic metals could contribute to the unbalance of the biogeochemical carbon cycle. Zn 2+ 2.4 10 -10 3.0 10 -6 3.1 10 -6 3.1 10 -6 Cu 2+ 6.5 10 -14 6.1 10 -10 6.9 10 -12 7.5 10 -12

TABLES

Co 2+ 1.5 10 -11 2.0 10 -7 5.6 10 -15 4.7 10 -15 Mn 2+ 10.7 10 -10 8.0 10 -10 8.3 10 -10 8.4 10 -10

MoO4 2- 5.6 10 -6 1.1 10 -8 5.6 10 -6 1.1 10 -9

Cd 2+ -2.1 10 -6 -2.0 10 -5

FIGURE CAPTIONS

Figure 1 :

 1 Figure 1: Impact of cadmium exposure to C. reinardtii grown in TAPEDDHA medium. (A)

Figure 2 :Figure 3 :

 23 Figure 2: Structure and elemental composition of C. reinhardtii cells exposed to cadmium. (A)

Table 1 :

 1 Comparison of ion availability in classic TAP medium and TAPEDDHA medium.

			Free ion concentrations (M)
			TAP	TAPEDDHA
	Metal ions	Control	70 µM Cd	Control	70 µM Cd
	Ca 2+	2.5 10 -5	2.6 10 -5	2.6 10 -5	2.6 10 -5
	Mg 2+	3.1 10 -4	3.1 10 -4	3.1 10 -4	3.1 10 -4
	Fe 2+	5.6 10 -9	5.0 10 -7	-	-
	Fe 3+	-	-	1.1 10 -22	1.2 10 -22

ACKNOWLEDGEMENTS

The work was supported by funding from the French 'Ministère de l'Enseignement Supérieur et de la Recherche' via the projects ANR-11-EQPX-0027 MARSS and ANR-10-INBS-04 FranceBioImaging, and a PhD fellowship for F.P. Furthermore, this work was supported by the bilateral (German-French) DAAD -Campus France program PROCOPE funded by the German 'Bundesministerium für Bildung und Forschung (BMBF)', the French 'Ministère des Affaires étrangères et du développement International (MAEDI)' and 'Ministère de l'Education nationale, de l'Enseignement supérieur et de la Recherche (MENESR)'. We also thank ESRF (Grenoble, France) for the provision of beamtime on ID21 beamline. The microscopy was done in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging. The authors thank kindly Prof. U. Johanningmeier and Dr. I. Bertalan from the University Halle-Wittenberg (Germany) for providing C. reinhardtii wild type strain (11/32b).

CONFLICT OF INTEREST

The authors declare no conflict of interests.

Distribution of Cd ligands after normalization of the percentages to 100%. The uncertainty is estimated to ±10%.

SUPPORTING INFORMATION

Supporting information is available at the publisher's web-site. Supporting information is given to the composition of the TAPEDDHA medium (Table S1), to the definition of regions of interest (ROI) in NanoSIMS 13 C/ 12 C images for data analysis (Figure S1), and to linear combination fits (LCFs) of XANES spectra (Figure S2).