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• W2048917254 abstract "Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production. Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production. The unicellular green alga Chlamydomonas reinhardtii has emerged as a prototype organism for investigating processes such as photosynthesis, nutrient deprivation, flagellar function, and H2 production (1Dent R.M. Haglund C.M. Chin B.L. Kobayashi M.C. Niyogi K.K. Plant Physiol. 2005; 137: 545-556Crossref PubMed Scopus (141) Google Scholar, 2Ghirardi M.L. King P.W. Posewitz M.C. Maness P.C. Fedorov A. Kim K. Cohen J. Schulten K. Seibert M. Biochem. Soc. Trans. 2005; 33: 70-72Crossref PubMed Scopus (87) Google Scholar, 3Ghirardi M.L. Posewitz M.C. Maness P-C. Dubini A. Yu J. Seibert M. Annu. Rev. Plant Biol. 2007; 58: 71-91Crossref PubMed Scopus (305) Google Scholar, 4Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 5Harris E.H. The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego1989Google Scholar, 6Harris E.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 363-406Crossref PubMed Scopus (538) Google Scholar, 7Im C.S. Zhang Z. Shrager J. Chang C.W. Grossman A.R. Photosynth. Res. 2003; 75: 111-125Crossref PubMed Scopus (76) Google Scholar, 8Kruse O. Rupprecht J. Bader K.P. Thomas-Hall S. Schenk P.M. Finazzi G. Hankamer B. J. Biol. Chem. 2005; 280: 34170-34177Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 9Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (904) Google Scholar, 10Moseley J.L. Chang C.W. Grossman A.R. Eukaryot. Cell. 2006; 5: 26-44Crossref PubMed Scopus (135) Google Scholar, 11Rochaix J.D. FEBS Lett. 2002; 529: 34-38Crossref PubMed Scopus (74) Google Scholar, 12Snell W.J. Pan J. Wang Q. Cell. 2004; 117: 693-697Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although most studies of Chlamydomonas have been performed under oxic conditions, it is becoming increasingly apparent that photosynthetic microorganisms frequently experience periods of anoxia. In the laboratory, Chlamydomonas cultures may be grown with limited aeration, in low nutrient medium or under low light conditions. Under such conditions, respiratory O2 uptake may exceed the production of O2 by photosynthesis leading to the onset of anoxia (9Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (904) Google Scholar, 13Moseley J. Quinn J. Eriksson M. Merchant S. EMBO J. 2000; 19: 2139-2151Crossref PubMed Scopus (139) Google Scholar, 14Quinn J.M. Eriksson M. Moseley J.L. Merchant S. Plant Physiol. 2002; 128: 463-471Crossref PubMed Scopus (76) Google Scholar). In the natural environment many photosynthetic microorganisms are also exposed to anoxic conditions. For example, in situ studies have demonstrated that cyanobacteria in Octopus Springs at Yellowstone National Park experience anaerobiosis during the night, which triggers a switch from respiratory metabolism and photosynthesis during the day to fermentation metabolism and nitrogen fixation during the night (15Steunou A.S. Bhaya D. Bateson M.M. Melendrez M.C. Ward D.M. Brecht E. Peters J.W. Kuhl M. Grossman A.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2398-2403Crossref PubMed Scopus (166) Google Scholar). Based on the draft genome sequence and available literature (16Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar), it is clear that Chlamydomonas contains genes encoding proteins that participate in a diverse set of metabolic pathways. Interestingly, Chlamydomonas contains several genes encoding proteins usually associated with strict anaerobes, including two [FeFe]-hydrogenase enzymes (18Forestier M. King P. Zhang L. Posewitz M. Schwarzer S. Happe T. Ghirardi M.L. Seibert M. Eur. J. Biochem. 2003; 270: 2750-2758Crossref PubMed Scopus (213) Google Scholar, 19Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (214) Google Scholar) and the corresponding [FeFe]-hydrogenase maturation proteins (20Posewitz M.C. King P.W. Smolinski S.L. Smith R.D. Ginley A.R. Ghirardi M.L. Seibert M. Biochem. Soc. Trans. 2005; 33: 102-104Crossref PubMed Scopus (79) Google Scholar, 21Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Moreover, Chlamydomonas is a rare example of a eukaryote that has homologs to all four predominant enzymes used in the fermentative metabolism of pyruvate. These include pyruvate formate lyase (PFL1), pyruvate ferredoxin oxidoreductase (PFR1, often designated as PFOR in other organisms), lactate dehydrogenase, and pyruvate decarboxylase (PDC1). Genes encoding subunits of a pyruvate dehydrogenase complex are also present in the genome and presumably function in the aerobic metabolism of pyruvate. The Chlamydomonas genome is ∼120 Mb (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html) and is predicted to contain over 15,000 genes, with a significant number of genes encoding polypeptides involved in anaerobic metabolism (22Grossman A.R. Croft M. Gladyshev V.N. Merchant S.S. Posewitz M.C. Prochnik S. Spalding M.H. Curr. Opin. Plant Biol. 2007; 10: 190-198Crossref PubMed Scopus (133) Google Scholar). A better understanding of anaerobic metabolism in Chlamydomonas and metabolic fluxes associated with diurnal periods of light and dark will facilitate the development of physiological models able to predict metabolic fluxes under various environmental conditions. However, at this point relatively little is known regarding the following: (a) regulatory mechanisms by which phototrophic microorganisms sense and acclimate to an anoxic environment after periods of photosynthetic activity or to an oxic environment after periods of anoxia; (b) the repertoire of genes and proteins required during anaerobiosis and how changes in gene expression link to changing cellular metabolism; and (c) changes in metabolite fluxes and the importance of these fluxes in sustaining cell viability during anaerobiosis. In the laboratory, fermentation pathways become active in Chlamydomonas as the environment becomes anaerobic. This is achieved in the dark by sparging cultures with an inert gas (or exogenous reductant) to purge them of O2 (23Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 24Ghirardi M.L. Appl. Biochem. Biotechnol. 1997; 63: 141-151Crossref PubMed Google Scholar, 25Greenbaum E.R. Guillard R.L. Sunda W.G. Photochem. Photobiol. 1983; 37: 649-655Crossref Scopus (58) Google Scholar, 26Posewitz M.C. Smolinski S.L. Kanakagiri S. Melis A. Seibert M. Ghirardi M.L. Plant Cell. 2004; 16: 2151-2163Crossref PubMed Scopus (133) Google Scholar) or in the light by depriving illuminated, sealed cultures of sulfate (9Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (904) Google Scholar, 27Kosourov S. Seibert M. Ghirardi M.L. Plant Cell Physiol. 2003; 44: 146-155Crossref PubMed Scopus (222) Google Scholar, 28Kosourov S. Tsygankov A. Seibert M. Ghirardi M.L. Biotechnol. Bioeng. 2002; 78: 731-740Crossref PubMed Scopus (258) Google Scholar, 29Zhang L. Happe T. Melis A. Planta. 2002; 214: 552-561Crossref PubMed Scopus (355) Google Scholar), which results in attenuated rates of photosynthetic O2 evolution (30Wykoff D.D. Davies J.P. Melis A. Grossman A.R. Plant Physiol. 1998; 117: 129-139Crossref PubMed Scopus (408) Google Scholar). In the dark, fermentation is coupled to the degradation of starch reserves (23Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 31Kreutzberg K. Physiologia Plantarum. 1984; 61: 87-94Crossref Scopus (77) Google Scholar, 32Ohta S. Miyamoto K. Miura Y. Plant Physiol. 1987; 83: 1022-1026Crossref PubMed Google Scholar). Formate, acetate, and ethanol are formed as major fermentative products, and H2 and CO2 gases are emitted as minor products (23Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 31Kreutzberg K. Physiologia Plantarum. 1984; 61: 87-94Crossref Scopus (77) Google Scholar, 32Ohta S. Miyamoto K. Miura Y. Plant Physiol. 1987; 83: 1022-1026Crossref PubMed Google Scholar). These products are also formed in the light after exposure to sulfur deprivation (27Kosourov S. Seibert M. Ghirardi M.L. Plant Cell Physiol. 2003; 44: 146-155Crossref PubMed Scopus (222) Google Scholar). The formation of fermentation products is primarily controlled at the level of pyruvate, and the ratios of the fermentative products may change as a consequence of culture conditions and the use of different laboratory strains. The PFL1 enzyme catalyzes the cleavage of pyruvate into formate and acetyl-coenzyme A (acetyl-CoA) (33Wagner A.F. Frey M. Neugebauer F.A. Schäfer W. Knappe J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 996-1000Crossref PubMed Scopus (306) Google Scholar). The acetyl-CoA generated can be reduced to ethanol via acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Alternatively, the acetyl-CoA can be converted to acetate via phosphate acetyl-transferase (PAT) 3The abbreviations used are:PATphosphate acetyltransferaseHPLChigh pressure liquid chromatographyROSreactive oxygen speciesADHalcohol dehydrogenaseHCPhybrid cluster proteinHIFhypoxia-inducible factorPATphosphate acetyltransferaseACKacetate kinaseMOPS4-morpholinepropanesulfonic acid. and acetate kinase (ACK), yielding ATP (16Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar). Interestingly, Atteia et al. (16Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) reported that the Chlamydomonas genome contains two copies each of the PAT and ACK genes and suggested that the conversion of acetyl-CoA to acetate likely occurs in both the chloroplast and mitochondrion. It should be noted that the PAT proteins are referred to as phosphotransacetylase in several previous publications, but are annotated as PAT in the current assembly of the Chlamydomonas genome. phosphate acetyltransferase high pressure liquid chromatography reactive oxygen species alcohol dehydrogenase hybrid cluster protein hypoxia-inducible factor phosphate acetyltransferase acetate kinase 4-morpholinepropanesulfonic acid. In Chlamydomonas, pyruvate may also be oxidized to acetyl-CoA and CO2 by PFR1, which was recently identified in the Chlamydomonas genome (16Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar). In amitochondriate eukaryotes and anaerobic microbes such as species of Clostridia, PFOR reduces ferredoxin, providing electrons for H2 generation via the catalytic activity of hydrogenase. The accumulation of ethanol and formate and the acidification of the cellular environment by organic acids can be toxic to the cells (34Kennedy R.A. Rumpho M.E. Fox T.C. Plant Physiol. 1992; 100: 1-6Crossref PubMed Scopus (368) Google Scholar), which requires dynamic responses in cellular metabolism to balance ATP production while limiting the accumulation of toxic metabolites. Anaerobiosis in Chlamydomonas is currently of significant interest as it represents a potential means to generate H2, a biological source of renewable energy. This potential with respect to energy production has stimulated several studies focused on anaerobic metabolism, and the formation and assimilation of H2 in algae (32Ohta S. Miyamoto K. Miura Y. Plant Physiol. 1987; 83: 1022-1026Crossref PubMed Google Scholar, 35Ben-Amotz A. Avron M. Plant Physiol. 1972; 49: 240-243Crossref PubMed Google Scholar, 36Kessler E. Stewart W.D.P. Algal Physiology and Biochemistry: Botanical Monograph. 10. Blackwell Scientific, Oxford, UK1974: 456-473Google Scholar, 37Klein U. Betz A. Plant Physiol. 1978; 61: 953-956Crossref PubMed Google Scholar, 38Weaver P.F. Lien S. Seibert M. Solar Energy. 1980; 24: 3-45Crossref Scopus (146) Google Scholar). In Chlamydomonas, H2 evolution is catalyzed by two [FeFe]-hydrogenases (HYD1 and HYD2) that are localized in the chloroplast stroma and are coupled to electron donation from ferredoxin (18Forestier M. King P. Zhang L. Posewitz M. Schwarzer S. Happe T. Ghirardi M.L. Seibert M. Eur. J. Biochem. 2003; 270: 2750-2758Crossref PubMed Scopus (213) Google Scholar, 19Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (214) Google Scholar, 39Happe T. Mosler B. Naber J.D. Eur. J. Biochem. 1994; 222: 769-774Crossref PubMed Scopus (151) Google Scholar). The Chlamydomonas hydrogenases are typically designated as HYDA1 and HYDA2 with the “A” indicating the catalytic enzyme. However, the HYD1 and HYD2 nomenclature is used here for consistency with current Chlamydomonas annotations. The HYD1 and HYD2 enzymes are O2-sensitive, and H2 production is only observed under anaerobic conditions (24Ghirardi M.L. Appl. Biochem. Biotechnol. 1997; 63: 141-151Crossref PubMed Google Scholar, 39Happe T. Mosler B. Naber J.D. Eur. J. Biochem. 1994; 222: 769-774Crossref PubMed Scopus (151) Google Scholar, 40Happe T. Naber J.D. Eur. J. Biochem. 1993; 214: 475-481Crossref PubMed Scopus (248) Google Scholar, 41Roessler P.G. Lien S. Plant Physiol. 1984; 76: 1086-1089Crossref PubMed Scopus (48) Google Scholar). Anaerobically maintained Chlamydomonas generates H2 in the light or dark through photosynthetic and fermentative pathways, respectively. Transient H2 photoproduction is observed after illumination of dark, anaerobically acclimated cells grown in nutrient-replete medium (4Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 21Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 23Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 24Ghirardi M.L. Appl. Biochem. Biotechnol. 1997; 63: 141-151Crossref PubMed Google Scholar, 26Posewitz M.C. Smolinski S.L. Kanakagiri S. Melis A. Seibert M. Ghirardi M.L. Plant Cell. 2004; 16: 2151-2163Crossref PubMed Scopus (133) Google Scholar, 42Graves D.A. Tevault C.V. Greenbaum E. Photochem. Photobiol. 1989; 50: 571-576Crossref Scopus (17) Google Scholar). However, H2 production rates rapidly diminish as the O2, generated from photosynthesis increases to inhibitory levels. Photosynthesis-dependent generation of volumetric amounts of H2 by Chlamydomonas occurs upon sulfur deprivation (9Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (904) Google Scholar, 27Kosourov S. Seibert M. Ghirardi M.L. Plant Cell Physiol. 2003; 44: 146-155Crossref PubMed Scopus (222) Google Scholar, 28Kosourov S. Tsygankov A. Seibert M. Ghirardi M.L. Biotechnol. Bioeng. 2002; 78: 731-740Crossref PubMed Scopus (258) Google Scholar, 43Antal T.K. Krendeleva T.E. Laurinavichene T.V. Makarova V.V. Ghirardi M.L. Rubin A.B. Tsygankov A.A. Seibert M. Biochim. Biophys. Acta. 2003; 1607: 153-160Crossref PubMed Scopus (170) Google Scholar, 44Kosourov S. Makarova V. Fedorov A.S. Tsygankov A. Seibert M. Ghirardi M.L. Photosynth. Res. 2005; 85: 295-305Crossref PubMed Scopus (63) Google Scholar). This activity can be maintained in the light over a period of several days in batch cultures, whereas H2 production in chemostats can be sustained for several months (45Fedorov A. Kosourov S. Ghirardi M. Seibert M. Appl. Biochem. Biotechnol. 2005; 121: 403-412Crossref PubMed Google Scholar). Although previous physiological studies have linked fermentation and photosynthetic electron transport to H2 production in Chlamydomonas, a precise knowledge of the metabolic and regulatory context required for H2 production will be necessary to understand current limitations in H2 yields. In this study, we have combined molecular and physiological approaches to examine dark anoxic acclimation of Chlamydomonas strain CC-425. The ability to generate H2 and the accumulation of extracellular fermentation products were monitored during the acclimation of Chlamydomonas to anaerobic conditions. The levels of transcripts encoding proteins associated with the various fermentation pathways were monitored in conjunction with the formation of these fermentation products. We also used high density, oligonucleotide (70 mer)-based microarrays to obtain insights into the genome-wide responses initiated by anoxia. Although transcripts from a number of genes associated with fermentation metabolism increased, as expected, there was an unanticipated increase in the levels of several transcripts encoding proteins involved in transcriptional/translational regulation, post-translational modification, and stress responses. Insights obtained from the cellular metabolism and gene expression data are being integrated into a larger systems framework that is focused on understanding the flexibility of whole-cell metabolism under rapidly changing environmental conditions. Strains and Growth Conditions—C. reinhardtii CC-425 (cw15, sr-u-60, arg7–8, mt+) wild-type cells were grown in Tris acetate-phosphate (TAP) medium (pH 7.2) (5Harris E.H. The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego1989Google Scholar), supplemented with 200 mg/liter arginine. Algal cultures were maintained at 25 °C, stirred (105 rpm), and exposed to continuous irradiance of 80 μmol m-2 s-1 PAR. Cell suspensions contained 16–24 μg·ml-1 total chlorophyll. Anaerobic Induction of Liquid Cell Suspensions—Chlamydomonas cultures were grown on TAP medium to ∼20 μg·ml-1 total chlorophyll, centrifuged (500 ml of cells) at 2,500 × g for 1 min, and resuspended in one-tenth volume (50 ml) of anaerobic induction buffer (AIB) containing 50 mm potassium phosphate (pH 7.0), and 3 mm MgCl2 (24Ghirardi M.L. Appl. Biochem. Biotechnol. 1997; 63: 141-151Crossref PubMed Google Scholar). Hydrogenase activity was elicited by placing concentrated cells into a vial wrapped with aluminum foil to exclude light. The vial was sealed with a rubber septum, flushed with argon for 15 min, and then incubated anaerobically in the dark at room temperature. For measuring H2 and O2 production rates, aliquots of cell cultures were placed in a Clark-type oxygen electrode assay chamber maintained 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 μmol 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 (5Harris E.H. The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego1989Google Scholar). 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 column. Anaerobically adapted cells were collected at the indicated times and centrifuged, and the supernatant was transferred to a new vial and frozen in liquid N2 for subsequent analysis. Samples were thawed, centrifuged, and filtered prior to HPLC injection. Twenty μl 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 electrochemical 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 immobilized on the enzyme membrane. Identical supernatants were used for metabolite and ethanol analysis; 10 μl of the supernatant 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 μg of isolated RNA was treated with 5 units of RNase-free DNase (Ambion) for 30 min at room temperature. 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 inhibitors of the reverse transcriptase reaction. The A260 of the eluted RNA was measured, and 4 μg of purified RNA was reserved to prepare labeled samples for microarray analysis. Reverse Transcription Reactions—First strand cDNA synthesis 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 specific 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 μl of single-stranded cDNA from the reverse transcriptase reaction (see above) was used as template for the real time PCR experiments. 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 initial 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 (elongation). 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 analyses 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-ΔΔCT method (46Livak K.J. Schmittgen T.D. Methods (Amsterdam). 2001; 25: 402-408Google Scholar), using the average cycle threshold (CT) calculated from triplicate measurements. Relative expression ratios from three independent experiments are reported. The RACK1 gene, previously named CBLP, was used as a constitutive control gene for normalization. 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 μl of 3× SSC on a Beckman Coulter BioMek FX liquid handling robot to a concentration of ∼50 μm. 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 location 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 (47Eberhard S. Jain M. Im C.S. Pollock S. Shrager J. Lin Y. Peek A.S. Grossman A.R. Curr. Genet. 2006; 49: 106-124Crossref PubMed Scopus (34) Google Scholar). Four micrograms of purified RNA was adjusted to 4 μl with sterile milliQ-treated water. One microliter of oligo-dT(V) (2 μg·μl-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 μl of 5× superscript buffer; 1 μl of 0.1 m dithiothreitol, 0.2 μl of 50× dNTPs (5 mm dATP, dCTP, dGTP, and 10 mm dTTP), 1 μl of Cy3- or Cy5-dUTP, and 0.8 μl of Superscript III (200 units·μl-1); the final reaction volume was 10 μl. After allowing the reaction to proceed at 42 °C for 2 h, an additional aliquot of 0.5 μl 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 μl of 500 mm EDTA and 0.5 μl 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 μl 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 μl of DNase-free water and 500 μl of Buffer PB (from kit), and the solution was immediately placed onto a QIAquick column. The column was washed with 750 μl 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 colu" @default.
• W2048917254 created "2016-06-24" @default.
• W2048917254 creator A5003629735 @default.
• W2048917254 creator A5015052104 @default.
• W2048917254 creator A5027061768 @default.
• W2048917254 creator A5063777860 @default.
• W2048917254 creator A5073889896 @default.
• W2048917254 date "2007-08-01" @default.
• W2048917254 modified "2023-10-07" @default.
• W2048917254 title "Anaerobic Acclimation in Chlamydomonas reinhardtii" @default.
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