placed in a Clark-type oxygen electrode assay chamber main- tained at 25 ��C and containing 2.25 ml of de-oxygenated MOPS buffer (50 mM, pH 6.8). The algal suspension was kept in the dark for 2 min, exposed to 700 mol m 2 s 1 of actinic light filtered through a solution of 1% CuSO4 for 3 min, and then returned to the dark for 1.5 min. Clark-type oxygen electrodes were used simultaneously to measure H2 and O2 production rates. Chlorophyll Measurements���Chlorophyll a and b content was determined spectrophotometrically in 95% ethanol (5). Metabolite Analysis���Organic acid analysis was performed by liquid chromatography (Hewlett Packard Series 1050 HPLC) using an Aminex HPX-87H (300 7.8 mm) ion exchange col- umn. Anaerobically adapted cells were collected at the indi- cated times and centrifuged, and the supernatant was trans- ferred to a new vial and frozen in liquid N2 for subsequent analysis. Samples were thawed, centrifuged, and filtered prior to HPLC injection. Twenty l of cell culture supernatant was injected onto the column and eluted with 8 mM filtered sulfuric acid (J. T. Baker Inc.) at a flow rate of 0.5 ml min 1 at 45 ��C. Retention peaks were recorded using Agilent ChemStation software, and quantification was performed by comparisons with known amounts of standards for each of the organic acids. Ethanol was measured using the YSI 2700 SELECT electro- chemical probe (YSI Inc.). This system allows direct reading of ethanol in solution at the enzyme sensor. The enzyme alcohol oxidase, which converts ethanol to acetaldehyde, was immobi- lized on the enzyme membrane. Identical supernatants were used for metabolite and ethanol analysis 10 l of the superna- tant was required for the measurement. Extraction of RNA for Real Time PCR and Microarray Analysis���Total RNA was isolated using the plant RNA reagent protocol, as described by the manufacturer (Invitrogen). Approximately 40 g of isolated RNA was treated with 5 units of RNase-free DNase (Ambion) for 30 min at room tempera- ture. The Qiagen RNeasy MinElute kit (Qiagen) was used to purify DNase-treated total RNA from degraded DNA, tRNA, 5.5 rRNA, DNase, contaminating proteins, and potential inhib- itors of the reverse transcriptase reaction. The A260 of the eluted RNA was measured, and 4 g of purified RNA was reserved to prepare labeled samples for microarray analysis. Reverse Transcription Reactions���First strand cDNA synthe- sis was primed from purified, total RNA template using specific primers for each of the Chlamydomonas genes of interest (shown as the reverse primers in supplemental Table 1), or with (dT)12���18 for the ferredoxin genes (the genes are designated FDX, with PETF as the exception). The reverse transcription reaction was done using the reverse transcriptase Superscript III kit (Invitrogen), as described by the manufacturer. The spe- cific primers were annealed to 250 ng of total RNA and extended for 1 h at 55 ��C using 200 units of reverse transcriptase Superscript III. (dT)12���18 primers were annealed to 250 ng of total RNA and extended for 1 h at 50 ��C using 200 units of reverse transcriptase Superscript III. Quantitative Real Time PCR���Levels of specific transcripts in total mRNA from each sample were quantified by real time PCR using the Engine Opticon system (Bio-Rad). Four l of single-stranded cDNA from the reverse transcriptase reaction (see above) was used as template for the real time PCR experi- ments. The real time PCR amplifications were performed using reagents from the DyNAmo HS SYBR green real time PCR kit (Finnzymes). Specific primers were designed to amplify gene regions consisting of 100���200 nucleotides. Amplifications were performed using the following cycling parameters: an ini- tial single step at 95 ��C for 10 min (denaturation) was followed by 40 cycles of the following: (a) 94 ��C for 10 s (denaturation), (b) 56 ��C for 15 s (primer annealing for the RACK1, HYD1, HYD2, HYDEF, HYDG, PFL1, PFR1, PAT1, ACK2, PETF, FDX2, FDX3, FDX4, FDX5, FDX6, AMYB1, AMYB3, ADH1, CAT1, ppGpp synthetase/degradase, TAB2, HCP4, PDC1, and NADTH genes) or 54 ��C for 15 s (primer annealing for the PAT2, ACK1, and PFLA genes), and (c) 72 ��C for 15 s (elonga- tion). Annotations associated with these genes are described elsewhere in the text and figure legends. A final single step at 72 ��C for 10 min followed these 40 cycles. Melting curve analy- ses were performed on all PCRs to ensure that single DNA species were amplified, and the product sizes were verified by agarose gel electrophoresis. The relative expression ratio of a target gene was calculated based on the 2 CT method (46), using the average cycle threshold (CT) calculated from triplicate measurements. Relative expression ratios from three independ- ent experiments are reported. The RACK1 gene, previously named CBLP, was used as a constitutive control gene for nor- malization. The primers used for reverse transcription and real time PCR are described in supplemental Table 1 and were designed using Primer3 software. Microarray Fabrication���Microarrays were fabricated at the Stanford Functional Genomics Facility at Stanford University. Oligonucleotides to be printed were suspended in 8 l of 3 SSC on a Beckman Coulter BioMek FX liquid handling robot to a con- centration of 50 M. The print material was deposited onto Corning GAPS II or UltraGAPS slides using a custom-built microarray robot equipped with Majer Precision MicroQuill 2000 array pins. Replicate spots were created by printing the entire plate set twice in succession. The ���.gal��� file, which reports the specific genes (and gene models when available) and their loca- tion on the array as well as gene annotation information, is available on line. Printed slides were maintained in a desiccator. Prior to use, slides were hydrated in a humidity chamber (100% humidity) for 5 min followed by immediate snap drying on a 100 ��C hot plate ( 3 s, array side up), and the array elements were then UV cross-linked to the aminosilane surface of the slide at 600 mJ using a Stratalinker (Stratagene). Labeling and Purification of Reverse-transcribed cDNAs��� Labeling and purification of reverse-transcribed cDNAs were performed as described previously (47). Four micrograms of purified RNA was adjusted to 4 l with sterile milliQ-treated water. One microliter of oligo-dT(V) (2 g l 1), consisting of 23 consecutive T residues followed at the 3 end by an A, T, G, or C, was added to the solution prior to heating the reaction mixture for 10 min at 70 ��C and then quickly chilled the mixture on ice. The following reagents were then added to the reaction mixture: 2 l of 5 superscript buffer 1 l of 0.1 M dithiothre- itol, 0.2 l of 50 dNTPs (5 mM dATP, dCTP, dGTP, and 10 mM dTTP), 1 l of Cy3- or Cy5-dUTP, and 0.8 l of Superscript III (200 units l 1) the final reaction volume was 10 l. After Anaerobic Acclimation in C. reinhardtii AUGUST 31, 2007��� VOLUME 282���NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 25477 by guest, on November 30, 2010 www.jbc.org Downloaded from

allowing the reaction to proceed at 42 ��C for 2 h, an additional aliquot of 0.5 l Superscript III was added, and the reaction was continued for an additional 1 h at 50 ��C. The reaction was stopped by the addition of 0.5 l of 500 mM EDTA and 0.5 l of 500 mM NaOH, and the solution was incubated at 70 ��C for 10 min to degrade RNA. Neutralization of the reaction mixture was achieved by adding 0.5 l of 500 mM HCl. The QIAquick PCR purification kit (Qiagen) was used to purify labeled cDNA. Cy3- and Cy5-labeled cDNAs were mixed with 90 l of DNase-free water and 500 l of Buffer PB (from kit), and the solution was immediately placed onto a QIAquick column. The column was washed with 750 l of kit Buffer PE by centrifugation at maximum speed for 1 min, and the flow- through was discarded. The wash procedure was repeated, and the column was centrifuged at maximum speed in an Eppen- dorf microcentrifuge for 2.5 min to remove the remaining buffer. The column was then transferred to a new 1.5-ml Eppendorf tube, and 50 l of 1/10 kit Buffer EB (v/v), preheated to 40 ��C, were applied to the resin bed. After 1 min of incuba- tion, the column was centrifuged at maximum speed for 4 min to elute the labeled cDNAs. The eluate was then dried in the dark in a SpeedVac (keeping the probe in the dark prevents photobleaching). Hybridization to the Oligonucleotide Array���Hybridization to the arrays was performed as described previously (47). All solutions used in the prehybridization and hybridization pro- tocols were filtered through 0.2- m Nalgene Bottle-Top filters and, when possible, autoclaved. Prehybridization was per- formed immediately before starting the hybridization. The arrays were incubated for 1 h in the pre-warmed prehybridiza- tion solution (5 SSC, 25% formamide, 0.1% SDS, 0.1 mg/ml bovine serum albumin) at 42 ��C. Following this incubation, the slides were transferred to 0.1 SSC and gently agitated at room temperature for 5 min. The 0.1 SSC wash was repeated, and the slides were then transferred to double distilled H2O for 30 s and dried by centrifugation at 1,000 rpm for 10 min (Eppendorf Centrifuge 5810R). The dried, labeled cDNA was resuspended in 25 l of milliQ-treated water followed by the addition of 25 l of 2 hybridization buffer (6 SSC, 0.2% SDS, 0.4 g/ l poly(A), 0.4 g l 1 yeast tRNA, 40% formamide), both pre- heated to 40 ��C (preventing precipitation of SDS). Resuspended samples were boiled for 3 min and centrifuged for 2 min in an Eppendorf microcentrifuge at full speed to remove debris, and then 50 l of the probe solution was placed in the middle of the prehybridized dried array. The solution spreads over the entire surface of the array when a large coverslip (microscope cover glass, 12-544-G22X60-1.5, Fisher) is carefully placed over probe solution on the array surface. Three drops of 10 l of 3 SSC were placed on the surface of the array (not too close to the position of the coverslip), and then the array was sealed in an air-tight chamber and incubated in a 42 ��C water bath for 24 h. To wash the slides following the hybridization, containers with 350 ml of 2 SSC, 0.1% SDS were preheated to 42 ��C, and 350 l of freshly prepared 0.1 M dithiothreitol was added to each just before use. Hybridization chambers were removed from the water bath, and individual slides were immersed in 2 SSC, 0.1% SDS (in one of the containers) until the coverslip moved away from the slides. The slides were then transferred to fresh, preheated 2 SSC, 0.2% SDS, and gently dipped up and down for 5 min. This was followed by 5-min washes in 0.1 SSC, 0.1% SDS and then 0.1 SSC, both at room temperature. Slides were rinsed in 0.01 SSC for 10 s and immediately dried by centrif- ugation. Detailed and updated versions of the protocols used for RNA labeling, slide prehybridization, hybridization, and washing are available on line. Scanning, Quantification, and Analysis of the Slides���Mi- croarray slides were scanned for Cy5 and Cy3 fluorescent sig- nals using a GenePix 4000B scanner (Molecular Devices). The images, representing spot intensities from scanned slides, were imported into SpotReader (version 1.3.0.5, Niles Scientific) and quantified. Spot positions were defined according to a pre- defined microarray layout that was subsequently adjusted by eye to help optimize spot recognition. Spot signals that were distorted by dust, locally high backgrounds, or printing flaws were not included in subsequent analyses. Analyses of the data were performed using GeneSpring 6.1 (Agilent Technologies). Images of the fluorescence at 532 nm for Cy3 and 635 nm for Cy5 were recorded and analyzed from the complete array sets (three biological replicates for the time point 0.5 h, four biolog- ical replicates for the time points 2 and 4 h, and three slides per time point for each biological replicate, with two copies of each cDNA per slide). RNA samples used to synthesize the probes that were hybridized to the slides were from independent experiments. Error models were computed based on replicates. Signal ratios were considered to meet threshold criteria if they passed Student���s t test for significance with a p value of 0.05. RESULTS Hydrogenase activity, photosynthesis, cellular respiration, and organic acid accumulation were all recorded over the period in which the cells were acclimating to anoxic conditions. As presented in Table 1, the maximum rate of photosynthetic O2 evolution rapidly declines under dark, anaerobic conditions (within 30 min there is a 3���4-fold decline), and the rate of respiration remains approximately constant whereas the rate of H2 evolution increases high rates of H2 production occur between 2 and 4 h following the initiation of anaerobic acclima- tion. After 24 h under dark, anaerobic conditions, H2 produc- tion by the cells decreases to 50 mol of H2/mg of chloro- phyll 1 h 1 (not shown). These rates of H2 production were at levels consistent with previous reports (21, 26). Congruent with elevated H2 production following exposure TABLE 1 Rates of H2 photoevolution, O2 photoevolution and dark O2 consumption ( mol gas mg Chl 1 h 1) during acclimation to anoxia Aliquots of acclimated cells were anaerobically transferred with a gas-tight syringe to an anaerobic cell containing deoxygenated buffer as described under ���Experi- mental Procedures.��� Samples were illuminated and then returned to darkness. The rates of H2 and O2 photoproduction were measured concomitantly with two Clark- type electrodes (one poised to measure H2, the other O2). The rates of O2 consump- tion were measured in the dark after the illumination period. Time Rate of O2 production (light) Rate of O2 consumption (dark) Rate of H2 production (light) h 0 139.3 21.5 40.1 7.5 0 0.5 41.3 5.5 36 8.3 72.2 12.9 2 37.7 5.8 36.7 7.4 123.3 25.8 4 37.0 9.8 39.6 7.6 135.9 19.9 Anaerobic Acclimation in C. reinhardtii 25478 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282���NUMBER 35��� AUGUST 31, 2007 by guest, on November 30, 2010 www.jbc.org Downloaded from