Culture and exposure media

C. reinhardtii (wild type C137) was cultured to late log phase (~2x10⁶ cells/mL) in a (4 times) diluted Tris-acetate-phosphate (TAP) medium (Harris, 1989), under a 12:12 hours light: dark regime of 50 μmol photons m^-2 s^-1 of fluorescent white light and rotary shaking (100 rpm) at 20º C. Cultures were prepared for cadmium exposure by centrifuging at 3,300 x g for 7 min.

prior to resuspending the cells in a (4 times) diluted TAP media without trace metals.

Following the third separation by centrifugation, the cell pellet was resuspended in a (4 times) diluted TAP without trace metals at a cell density of approximately 6.4x10⁵ cells/mL.

Cadmium concentrations employed in the present study were shown previously to have slight, but measurable, effects on the growth rate of C. reinhardtii in culture and to be more than two orders of magnitude below lethal concentrations (Macfie et al., 1994; Macfie and Welbourn, 2000). In order to avoid artifacts due to metal contamination, acid-washed, polycarbonate vessels were used. To ensure a constant concentration of free metal (i.e., to avoid metal depletion by uptake or metal complexation by exudates production (Kola, 2004)) exposure solutions were buffered with 5x10^-4 M citrate. Free metal concentrations were estimated from measured dissolved concentrations and thermodynamic calculations (MINTEQA2, Ver 1.50, (Allison et al., 1999)). For the conditions employed here, the concentration of free Cd (Cd²⁺) never exceeded 10% of the total cadmium. The addition of Cd and citrate to the exposure media did not significantly (<10%) change the speciation of the other components in the TAP media.

5.2.2. Differential display

The cell suspension prepared as previously described was divided into six aliquots: a non metal control; 0.03 µM Cd²⁺; 0.34 µM Cd²⁺; 3.40 µM Cd²⁺; 0.50 µM Ni²⁺;and 0.17 µM Cu²⁺

in a diluted (4 times) TAP medium that included 5x10^-4 M citrate but not added trace metals.

Cells were exposed to the experimental media for 2 hours under light after which 15 mL of the experimental media were centrifuged at 1,000 x g for 10 min. The pellet was washed (1 min. with diluted TAP medium containing 10^-3 M ethylenediaminetetraacetic acid (EDTA) then centrifuged at 12,280 x g for 5 min. Pellets were frozen on dry ice and stored at -80 °C

until extraction of the RNA. Total RNA was isolated by vortexing the cells in the presence of glass beads followed by purification with a Qiagen RNeasy kit (Qiagen, Valencia, CA, USA).

The RNA quality and quantity were estimated from the measured absorbance at 280 and 260 nm and by analysis of the intactness of rRNA species with an Agilent 2100 BioAnalyzer (Agilent Technologies, Foster City, CA, USA). The RNA quality was considered acceptable when the 28S rRNA/18S rRNA ratio was ≥2.

The differential display technique was carried out as described previously (Kornmann et al., 2001) with a few modifications. A master stock solution of double stranded 3'-terminal cDNA Mbo1 restriction fragments was obtained by PCR amplification. The PCR reactions were performed in a cocktail (pH 9.2) containing 50 mM Tris-HCl, 16 mM (NH₄)₂SO₄, 1.75 mM MgCl₂, 5% dimethylsulfoxide, 0.2 mM deoxyribonucleoside triphosphates, 2 units of Thermus aquaticus polymerase (TAQ)/50 µL reaction mixture and 1 μM of each of the two primers (5'GGTCCATCCAACCGATC-3' and 5'-ATTGGCGCGCCTAAGCTT-3').

Amplification (Biometra Thermocycler, Biometra, Goettingen, Germany) was performed using the following temperature cycles: 4 min. at 94 °C; 23 cycles (1 min. (94 °C), 50 s (52

°C), 2 min. 30 s (72 °C)), and 10 min. at 72 °C. After the large-scale amplification (pooled results of eight amplifications), the product was purified by extraction in a (25:24:1) phenol:

chloroform: isoamyl alcohol mixture then precipitated with isopropanol. The precipitated cDNA was resuspended at a concentration of 100 ng/µl in 10 mM Tris-HCl and 1 mM EDTA at pH 8.1 measured by spectrophotometry at 260 nm using an extinction coefficient of 6600/cm M (Sambrook et al., 1989). Gel electrophoresis was performed on 10 μL of each suspension to visually confirm that each sample contained the same cDNA concentration.

Aliquots of cDNA (3 ng) from the master stock solution were added to 50 mM Tris-HCl, 16 mM (NH4)2SO4, 1.75 mM MgCl2, 5% dimethylsulfoxide, 0.2 mM deoxyribonucleotide triphosphates, 0.6 μCi [α^-32P] deoxycytidine triphosphate, two units of TAQ and 1 μM of each of the two primers then amplified by touchdown PCR. The following program was used: 5 min. 95° C, 2 times (1 min. 95° C, 1 min. 50 s 54° C, 2 min. 30 s 72° C), 2 times (1 min. 95°

C, 1 min. 50 s 52° C, 2 min. 30 s 72° C), 2 times (1 min. 95° C, 1 min. 50 s 50° C, 2 min. 30 s 72° C), 2 times (1 min. 95° C, 1 min. 50 s 48° C, 2 min. 30 s 72° C), 2 times (1 min. 95° C, 1 min. 50 s 46° C, 2 min. 30 s 72° C), 14 times (1 min. 95° C, 1 min. 50 s 44° C, 2 min. 30 s 72° C). The radiolabeled PCR fragments were displayed on a denaturing 5% acrylamide gel

and visualized by autoradiography. The procedures for fragment isolation, cloning and sequencing have been described previously (Kornmann et al., 2001).

Basic local alignment search tool (BLASTn) analysis (Altschul et al., 1997) against the C.

reinhardtii genome was performed to identify the genes corresponding to the cloned cDNA fragments. Sequenced fragments were compared with the Joint Genome Institute (JGI) C.

reinhardtii genome database (Ver 3) (http://www.chlamy.org/) and the GenBank database.

For all annotations found in the JGI C. reinhardtii database, protein sequences were searched using a position specific iterative (PSI)-BLAST.

5.2.3. Microarray analysis

Cell suspensions were exposed to 0.34 µM Cd²⁺ (free metal) in a diluted (4 times) TAP for 2 hours, while control treatments received no metal. Microarray analysis was performed on cDNA isolated from four identical treatments. The RNA extraction, purification, quantification and quality assessment were performed as described above for the differential display experiments. Samples were concentrated to 1 µg/µl with Microcon filters (Millipore, Billerica MA, USA). Reverse transcription was performed on 4 μg of total RNA. Labeling, hybridization, and washing procedures were performed as described previously (Eberhard et al., 2006) with minor modifications. Specifically, Cy5 and Cy3 labeled deoxycytidine triphosphate were used instead of labeled deoxythimidine triphosphate. In addition, because the wash procedure was performed in a room equipped with ozone removal, no dithiothreitol (DTT) was added to the wash solution. A dye swap labeling reaction was performed where controls and samples were labeled with Cy5 and Cy3 and vice versa and used in the separate trial experiments. Samples were hybridized to microarray slides (Chip ver 2.0, Carnegie Institution, Stanford, CA, USA) containing 10⁴ oligos, each spotted twice per slide that represented unique genes and covered approximately 80% of the genome (Eberhard et al., 2006). The slides were scanned using a ScanArray instrument (Agilent Technologies, Santa Clara, CA, USA), and the red and green channels were quantified with GenePix Pro 5.1 (Molecular Devices, Sunnyvale, CA, USA). GeneSpring software (Agilent Technologies, Santa Clara, CA, USA) was used for the statistical analysis of mean intensities obtained from the four biological replicates. A Locally Weighted Linear Regression (Lowess) normalization

(Quackenbush, 2002) was used to adjust the control value for each measurement. The criteria used for selection of the up regulated genes were based on a 1.4-fold increase in induction and a p value < 0.05.