- Email: [email protected]
Evaluation of Oxygen Response Involving Differential Gene Expression in Chlamydomonas reinhardtii
604
[39]
unicellular systems
[39] Evaluation of Oxygen Response Involving Differential Gene Expression in Chlamydomonas reinhardtii By Jose´ A. Del Campo, Jeanette M. Quinn, and Sabeeha Merchant Chlamydomonas as a Model Organism
The unicellular green alga Chlamydomonas has, for decades, offered experimental advantages for the study of chloroplast function and biogenesis, photosynthesis, flagellar motility and assembly, photoreceptor biochemistry, and sexual mating.1–6 Among these are (1) the ability to manipulate the nuclear and also both organellar genomes,1,7,8 (2) facultative photosynthetic growth because of the ability of Chlamydomonas to use acetate for heterotrophic growth, (3) heterothallic mating types, which permit classical genetic approaches for the dissection of important biological problems,9 and (4) considerable genomic information through EST10 and shotgun genome sequencing projects (http://genome.jgi-psf.org/chlre1/ chlre1.home.html). Chlamydomonas has long been used as a model (see Fig. 1) for the study of nutrient-responsive signal transduction, especially in the context of the function of photosynthetic apparatus. The classical areas of interest have included inorganic (sulfur, nitrogen, phosphorus) nutrient utilization,11,12
1
J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant (eds.), in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas.’’ Kluwer Academic Publishers, 1998. 2 A. R. Grossman, Curr. Opin. Plant Biol. 3, 132 (2000). 3 E. H. Harris, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 363 (2001). 4 R. M. Dent, M. Han, and K. K. Niyogi, Trends Plant Sci. 6, 364 (2001). 5 C. D. Silflow and P. A. Lefebvre, Plant Physiol. 127, 1500 (2001). 6 O. A. Sineshchekov and E. V. Govorunova, Trends Plant Sci. 4, 201 (1999). 7 B. L. Randolph-Anderson, J. E. Boynton, N. W. Gillham, E. H. Harris, A. M. Johnson, M. P. Dorthu, and R. F. Matagne, Mol. Gen. Genet. 126, 357 (1993). 8 J. E. Boynton and N. W. Gillham, Methods Enzymol. 264, 279 (1996). 9 H. Harris, in ‘‘The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use.’’ Academic Press, San Diego, 1989. 10 J. Shrager, C. Hauser, C. W. Chang, E. H. Harris, J. Davies, J. McDermott, R. Tamse, Z. Zhang, and A. R. Grossman, Plant Physiol. 131, 401 (2003). 11 J. P. Davies and A. R. Grossman, in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas’’ (J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant, eds.), p. 613. Kluwer Academic Publishers, 1998. 12 A. Grossman and H. Takahashi, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 163 (2001).
METHODS IN ENZYMOLOGY, VOL. 381
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00
[39]
low O2 response in Chlamydomonas
605
Fig. 1. A model showing known elements involved in copper deficiency and hypoxia responses. The putative DNA-binding protein Crr1 is required for the activation of target genes when cells are facing copper deficiency and/or hypoxic conditions. The core of the CuRE elements (see text) is the sequence GTAC.
as well as trace metal requirements and mechanisms of homeostasis.13–15 Metabolism and gene expression involving CO2 and O2 have also been long-standing topics of research activity,16–18 with recently renewed emphasis from the perspective of energy metabolism19 and the identification of regulatory mutants.20–23 Chlamydomonas reinhardtii, like other organisms, responds to changes in oxygen supply through the alteration of metabolism. Some species of this genus are found in naturally oxygen-deficient habitats such as peat bogs and sewage lagoons,9 where a hypoxic response is probably critical for survival. Even laboratory cultures become oxygen depleted quite 13
S. Merchant, in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas’’ (J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant, eds.), p. 597. Kluwer Academic Publishers, 1998. 14 S. La Fontaine, J. M. Quinn, S. S. Nakamoto, M. D. Page, V. Gohre, J. L. Moseley, J. Kropat, and S. Merchant, Eukaryot. Cell 1, 736 (2002). 15 P. Rubinelli, S. Siripornadulsil, F. Gao-Rubinelli, and R. T. Sayre, Planta 215, 1 (2002). 16 P. E. Bryant, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 17, 533 (1970). 17 M. R. Badger, A. Kaplan, and J. A. Berry, Plant Physiol. 66, 407 (1980). 18 A. M. Geraghty and M. H. Spalding, Plant Physiol. 111, 1339 (1996). 19 M. L. Ghirardi, L. Zhang, J. W. Lee, T. Flynn, M. Seibert, E. Greenbaum, and A. Melis, Trends Biotechnol. 18, 506 (2000). 20 J. M. Quinn, M. Eriksson, J. L. Moseley, and S. Merchant, Plant Physiol. 128, 463 (2002). 21 H. Fukuzawa, K. Miura, K. Ishizaki, K. Kucho, T. Saito, T. Kohinata, and K. Ohyama, Proc. Natl. Acad. Sci. USA 98, 5347 (2001). 22 K. Van, Y. Wang, Y. Nakamura, and M. H. Spalding, Plant Physiol. 127, 607 (2001). 23 Y. Xiang, J. Zhang, and D. P. Weeks, Proc. Natl. Acad. Sci. USA 98, 5341 (2001).
606
unicellular systems
[39]
rapidly as a consequence of respiration if they are not sufficiently agitated.20,24 Oxygen-responsive gene expression is, therefore, probably an integral aspect of everyday metabolism for Chlamydomonas, as well as for other organisms. In addition, there are also special metabolic pathways occurring only in anaerobic Chlamydomonas cells; the most publicized of which is photosynthetic hydrogen production.25 Hydrogen Production in Low Oxygen
In the early 1930s, Gaffron and co-workers discovered the ability of unicellular green algae to produce hydrogen gas upon illumination (reviewed by Melis and Happe25). Historically, hydrogen evolution activity in green algae was induced upon prior anaerobic incubation of the cells in the dark. Under such conditions, an iron-containing hydrogenase enzyme (encoded by the HydA gene in C. reinhardtii) was induced, catalyzing light-mediated H2 evolution. Oxygen is a powerful inhibitor of hydrogen evolution at the enzyme level.19,26 It has also been shown that regulation of the hydrogenase gene takes place at the transcriptional level.27 The idea of using H2 gas from green algae as an alternative fuel source has been promoted recently.28 The pathway involves two stages, photosynthesis and H2 production, dependent on sulfur availability, with sulfur deprivation serving as a metabolic switch. When sulfur is available for cells, green algae perform normal photosynthesis (water oxidation, oxygen evolution, and biomass accumulation). In the absence of sulfur, photosynthesis in C. reinhardtii slips into a hydrogen production mode if oxygen is simultaneously removed. Electrons for H2 production may originate either at photosystem II (PSII) upon photooxidation of water or at the plastoquinone pool upon oxidation of the cellular endogenous substrate (e.g., starch degradation). Electrons are transported via photosystem I (PSI) to ferredoxin, which serves as the physiological electron donor to the hydrogenase. The signal transduction components involved in metabolic switching in this pathway are completely unknown. The ability to undertake combined molecular and classical genetic studies in this model system should facilitate the discovery of the relevant regulatory factors.
24
P. M. Wood, Eur. J. Biochem. 87, 8 (1978). A. Melis and T. Happe, Plant Physiol. 127, 740 (2001). 26 T. Happe, A. Hemschemeier, M. Winkler, and A. Kaminski, Trends Plant Sci. 7, 246 (2002). 27 T. Happe and A. Hamiski, Eur. J. Biochem. 269, 1022 (2002). 28 A. Melis, L. Zhang, M. Forestier, M. L. Ghirardi, and M. Seibert. Plant Physiol. 122, 127 (2000). 25
[39]
low O2 response in Chlamydomonas
607
Connections between Oxygen and Copper Nutrient Homeostasis
Copper is an essential micronutrient for all organisms because of its function in enzymes that serve as catalysts of oxygen chemistry and redox reactions. Chlamydomonas has a well-characterized response to nutritional copper deficiency.13 The best characterized of these is the replacement of plastocyanin (the most abundant copper protein in photosynthetic cells) with a heme-containing c-type cytochrome.24,29 This occurs through transcriptional activation of the Cyc6 gene encoding cytochrome c6, a heme-containing functional substitute for plastocyanin,30 and induced degradation of apoplastocyanin in the thylakoid lumen,31 which occurs probably to ensure the redistribution of copper to cytochrome oxidase.32 This transcriptional response requires copper response elements (CuREs)33 containing the sequence GTAC, which forms the core of these CuREs, and Crr1, a putative DNA-binding protein (Fig. 1).34 Crr1 is also required for the transcriptional activation in copper-deficient cells of Cpx1, encoding coproporphyrinogen III oxidase,35 and also Crd1,36 encoding the aerobic oxidative cyclase in chlorophyll biosynthesis.37 In copper-replete cells, Crd1 is replaced by Cth1. The inhibition of Cth1 accumulation under copper-deficient conditions also involves Crr1.36 Wood24 noted first that hypoxic Chlamydomonas cells accumulate cytochrome c6 even in copper-replete medium. The response of the Cyc6 gene to hypoxia is mediated at the transcriptional level via its CuREs and requires Crr1 function.20,33 Subsequently, it was discovered that each of the Cu deficiency targets mentioned previously also responds to hypoxia (e.g., Fig. 2) in a pathway involving the CuRE and Crr1 function.38 The hypoxic and nutritional copper signaling pathways in Chlamydomonas therefore seem to share some common components.20 A trivial explanation for the similar output, that is, that hypoxic cells are internally copper deficient, was ruled out because oxygen-deprived cells are able to synthesize 29
S. Merchant and L. Bogorad, EMBO J. 6, 2531 (1987). J. M. Quinn and S. Merchant, Plant Cell 7, 623 (1995). 31 H. H. Li and S. Merchant, J. Biol. Chem. 270, 23504 (1995). 32 S. S. Nakamoto, in ‘‘Compartmentalized Copper and Iron Enzymes in C. reinhardtii: Venus Versus Mars.’’ Ph.D. Dissertation, University of California, Los Angeles. Department of Chemistry and Biochemistry, 2001. 33 J. M. Quinn, P. Barraco, M. Eriksson, and S. Merchant, J. Biol. Chem. 275, 6080 (2000). 34 J. Kropat and S. Merchant, unpublished results (2003). 35 M. Eriksson et al., unpublished results (2003). 36 J. L. Moseley, M. D. Page, N. P. Alder, M. Eriksson, J. Quinn, F. Soto, S. M. Theg, M. Hippler, and S. Merchant, Plant Cell 14, 673 (2002). 37 S. Tottey, M. A. Block, M. Allen, T. Westergreen, C. Albrieux, H. U. Scheller, S. Merchant, and P. E. Jensen, Proc. Natl. Acad. Sci. USA 100, 16119 (2003). 38 J. M. Quinn, S. S. Nakamoto, and S. Merchant, J. Biol. Chem. 274, 14444 (1999). 30
608
unicellular systems
[39]
Fig. 2. Hypoxia-induced gene expression in C. reinhardtii with a time course response. Wild-type strain CC125 was grown in copper-supplemented (6 M) Tris–acetate–phosphate medium under normal aeration to a concentration of 1 106 cells/ml in a shaker (300 rpm) at room temperature using normal room lighting (approximately 1–15 mol m2 s1). Twenty milliliters was removed for RNA preparation (t ¼ 0). The remaining culture was bubbled with 98% N2 þ 2% CO2 (0% O2). Samples for RNA preparation were removed at the indicated times. RNA samples were probed with Cyc6, Cpx1, Crd1, and RbcS2 (as a control) (A). Except for Cth1 (B), all samples were analyzed in duplicate cultures.
holoplastocyanin de novo, indicating that copper is available inside the cells. Quinn et al.20 tested the idea that the connection between Cu and the hypoxic response results from modification of the Cu(II)/Cu(I) ratio by the oxygen status of the environment, assuming that the copper sensor is specific for Cu(II) vs Cu(I). In this case, the hypoxic response of Cyc6, Cpx1, Crd1, and Cth1 would simply mimic the response to Cu deficiency. However, this is not the case.20 The Cyc6 gene responds strongly to Cu deficiency and more weakly to hypoxia, whereas the Crd1 and Cpx1 genes respond strongly to hypoxia and less so to Cu deficiency. Furthermore, a specific hypoxia response element was identified in the Cpx1 gene.33 This HyRE is related to the CuRE by virtue of the GTAC core sequence, but it does not function as a CuRE.20 Quinn et al.20 concluded that the hypoxic response shares signal transduction components with the Cu deficiency response. The rationale for the intersection of the regulatory pathways is that hypoxic conditions create copper deficiency in nature by driving copper ions to insoluble Cu(I) species, which precipitate and
[39]
low O2 response in Chlamydomonas
609
perhaps have limited bioavailability. The response of Cu deficiency targets to hypoxia might be a mechanism for anticipating and preparing for Cu deficiency in a microaerobic growth environment. The pattern of cytochrome c6 and coprogen oxidase accumulation parallels the transcriptional activation of the genes, indicating that the hypoxic response can also be detected and analyzed at the protein level. However, in order to monitor changes in gene expression, it is more appropriate to analyze the effects at the level of transcript abundance, as the half-life for the various mRNAs is shorter than for the corresponding proteins. The physiological relevance of the Crr1-dependent hypoxic response of one target gene, Crd1, was confirmed by the demonstration of a conditional chlorotic phenotype in hypoxic but not aerobic crd1 mutant cells.20 The crd1 mutant strain is useful for the study of the Crr1-dependent hypoxic gene expression pathway because its remarkable chlorotic phenotype in þCu TAP medium in low air can be used as a physiological marker, and expression of the gene can be used as a molecular marker (see Fig. 2). At this point, a number of questions remain about the hypoxic response in Chlamydomonas. First, for the Cpx1 gene, the copper response element appears to be necessary and sufficient for the nutritional copper response and is also necessary for the hypoxic response. A second element, with a GTAC core, found in the Cpx1 promoter is required for the oxygen deficiency response. The second element, called a HyRE, is not necessary for the copper deficiency response. What is the relationship between the CuRE and the HyRE at the mechanistic level? Second, a well-known anaerobically induced gene, Hyd1, is normally regulated in the crr1 mutant, indicating a Crr1-independent hypoxia/anoxia sensing mechanism in C. reinhardtii. What are the regulatory components required for the regulation of HydA? Given the repertoire of molecular techniques on hand today that can be applied to this experimental model, identification of the hypoxic signaling components should be achievable. With this intention in mind, this article describes the setup of experiments involving the growth of Chlamydomonas cells in liquid cultures at different oxygen concentrations. Growth Media
Chlamydomonas was first introduced into the biochemistry of photosynthesis by Levine and co-workers.39 In a previous volume of this series,40 information concerning the culturing of Chlamydomonas cells, as well as detailed descriptions about mutagenesis procedures and photosynthesis 39 40
R. P. Levine, Science 162, 768 (1968). A. San Pietro (ed.), Methods Enzymol. 23 (1971).
610
unicellular systems
[39]
studies, can be found. For details concerning the preparation of growth media, both liquid and solid, and particularly copper-free media, see Quinn and Merchant.41 For general information concerning the cultivation of Chlamydomonas cells, including various types of growth media, see Harris.9 Culturing
Several different strains have been tested for the response to oxygen concentration in liquid cultures, including CC125, CC425, and 2137 as wild-type strains, and the mutant crr1-1, which is affected in the regulation of copper metabolism (see earlier discussion). As diagrammed in Fig. 3, to generate different oxygen concentrations for the culturing of Chlamydomonas cells, we used a mixture of three different gases: CO2, compressed air, and nitrogen. The use of CO2 in the gas mixture is important because CO2 is a nutrient and of course also buffers the medium. Variation in air content relative to nitrogen changes both CO2 and O2 levels. By providing CO2 at a fixed concentration (0.2 to 2% depending on the experiment), the key variable in the experiment is O2. CO2 concentration in air (0.036% or 350 ppm) is considered to be low CO211 conditions from a physiological point of view,42 leading to low growth rates and low biomass accumulation in algal cultures because CO2 is a substrate for photosynthesis. The ratio of CO2/O2 is also metabolically relevant43,44 because the active site of Rubisco discriminates poorly against O2. Therefore, in air, a significant fraction of ribulose-1,5-bisphosphate is oxygenated instead of carboxylated,43,44 leading to the photorespiratory pathway for salvage of phosphoglycolate. To avoid photorespiration, algae induce a carbon concentration mechanism (CCM), involving carbonic anhydrases (CA)42,45–48 and other components as well. The CCM is repressed by high CO2 (e.g., 5%).21–23 The effect of O2 on regulation of the CCM is not known, but is probably a question worth addressing. Nevertheless, 41
J. M. Quinn and S. Merchant, Methods Enzymol. 297, 263 (1998). A. Kaplan and L. Reinhold, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 539 (1999). 43 J. Berry, J. Boynton, A. Kaplan, and M. Badger, Carnegie Inst. Wash. Yearbook 75, 423 (1976). 44 W. R. King and Andersen, Arch. Microbiol. 128, 84 (1980). 45 M. Rawat and J. V. Moroney, Plant Physiol. 109, 937 (1995). 46 J. Karlsson, A. K. Clarke, Z. Y. Chen, S. Y. Hugghins, Y. I. Park, H. D. Husic, J. V. Moroney, and G. Samuelsson, EMBO J. 17, 1208 (1998). 47 R. P. Funke, J. L. Kovar, and D. P. Weeks, Plant Physiol. 114, 237 (1997). 48 M. Eriksson, J. Karlsson, Z. Ramazanov, P. Gardestrom, and G. Samuelsson, Proc. Natl. Acad. Sci. USA 93, 12031 (1996). 42
[39] low O2 response in Chlamydomonas
Fig. 3. (continued)
611
612
unicellular systems
[39]
Fig. 3. (A) Scheme for the experimental setup used to grow Chlamydomonas cells under different oxygen concentrations. The amount of each gas is monitored on a multichannel gas flowmeter. The mixture of different gases is provided to the cultures using silicone tubing. The oxygen concentration in the cultures is measured using a Clark-type oxygen electrode in a flask at the end of the series containing TAP medium without cells. Note that it is preferable to measure the O2 content in each flask individually, but there may be technical difficulties in
[39]
low O2 response in Chlamydomonas
613
because of the critical metabolic function of CO2 and the known responses to CO2 (e.g., modification of phototactic behavior49), we have fixed the CO2 concentration in experiments in our laboratory. We have not considered the effect of the CO2/O2 ratio, although the ratio is known to be critical for photosynthetic carbon metabolism. Ideally, the CO2 concentration should be fixed to correspond to air levels. However, this low level is technically difficult to achieve consistently and reproducibly when the CO2 is supplied from a pure CO2 gas tank. Therefore, for low air experiments (hypoxic conditions), we used two different mixtures: 97.8% nitrogen, 2% air, and 0.2% CO2 or 96% nitrogen, 2% air, and 2% CO2. It may be possible to provide HCO31 in the medium in place of CO2 and vary only O2 and nitrogen in the gas mixture. We have not tried this option. Other variables to consider include light intensity and acetate for heterotrophic growth because these affect photosynthesis and respiration and hence O2 content during the experiment. It is also possible to induce hypoxic conditions by growing Chlamydomonas cells without vigorous agitation, for example, in Erlenmeyer flasks fitted with cotton plugs (200 ml medium in 250-ml flasks) where the cells are kept suspended by low basal stirring using a magnetic stir bar.20,24 Note that growth under photoautotrophic conditions (illumination þ CO2) will generate O2 in the cells and in the growth medium. Therefore, continuous bubbling or flushing is necessary to maintain control of hypoxic conditions. Happe and Kaminski50 flushed the cell suspension with argon in the dark in order to promote anaerobic conditions for inducing hydrogenase activity in Chlamydomonas cultures. Bubbling and Flask Setup
In order to deliver the desired mixture of gases for bubbling the cultures, we use three different gas flowmeters (Aalborg Instruments & Controls, Inc.) in two scales (one from 0 to 25 ml of gas per minute and another from 0 to 5 liters per minute of gas) because the amount needed for one gas is quantitatively different from the other (e.g., 99.8% air vs 0.2% CO2). The output mixture has a total flow of 2.5 liters/min1 liter of culture1. All gases are driven using flexible plastic tubes (Tygon) from the tanks to the flowmeters and from these to the cultures (see Fig. 3). The diameter for 49 50
W. Nultsch, Arch. Microbiol. 112, 179 (1977). T. Happe and A. Kaminski, Eur. J. Biochem. 269, 1022 (2002).
maintaining sterile conditions for extended periods. It is useful to place the electrode at various points of exit from the gas supply in order to evaluate the effect of position of each flask. It is also preferable to mount the flasks in parallel as diagrammed rather than in series. (B) Details for connections of the different components.
614
[39]
unicellular systems
the tubes may vary according to the regulator output, the flowmeter input and output, the type of filters, and so on. Once the gas mixture exits the multichannel flowmeter, the outlet pipe is split to the same number of branches to correspond to the number of flasks that are needed (see Fig. 3). Ideally, an individual flowmeter for each culture flask should be used, which allows bubbling of all flasks using exactly the same flow rate. A trouble commonly encountered with bubbled cultures is bacterial contamination. To avoid this, gas to the cultures is filtered through 0.45-m filters (Pall Gelman Laboratory) and supplied through a sterile disposable pipette. An additional 125-ml flask, containing 50 ml of rich medium (e.g., LB), is aerated in the same way as the Chlamydomonas cultures. This is included for monitoring contamination during the course of the experiments. Normally, we grow Chlamydomonas in 250-ml Erlenmeyer flasks containing 100 ml of liquid TAP medium. The culture flasks are placed on a shaker at 100 rpm for additional agitation, as bubbling alone is not enough to keep the cells from settling. In our experience, cultures can be kept in axenic conditions for at least 5 days of growth. They multiply from 4 105 cells/ ml at inoculation to 2 107 cell/ml after day 5 for wild-type CC125 in 99.8% air þ 0.2% CO2 corresponding to a doubling time in the log phase of 17 h. The doubling time for Chlamydomonas grown in optimal conditions in TAP medium, using a shaker at 220 rpm, 24 , and a light intensity of about 60 mole m2 s1, ranges from 6 to 8 h. Other Culture Conditions
Cultures are maintained at room temperature (22–24 ) with ambient illumination (10–15 mole m2 s1) provided by overhead standard ceiling fluorescent lights. Due to the ability of Chlamydomonas cells to grow heterotrophically, wild-type cultures in TAP medium9 reach a cell density of 1 107 cells/ml in 2% air þ 0.2% CO2 in 4 days (stationary phase) under these conditions. For standard culture conditions in our laboratory (with out bubbling, at 24 in a shaker incubator, 220 rpm, 60 mole m2 s1), the cell density reached for the same wild-type Chlamydomonas strain in the same time is 2.1 107 cell/ml. A higher light intensity can be used by placing lamps (fluorescent type, to avoid unwanted heating) close to the cultures, which is important for species that are incapable of growing heterotrophically (like some diatoms).51
51
L. A. Zaslavskaia, J. C. Lippmeier, C. Shih, D. Ehrhardt, A. R. Grossman, and K. E. Apt, Science 292, 2073 (2001).
[39]
low O2 response in Chlamydomonas
615
Monitoring Oxygen Concentration
The oxygen content of the cultures is measured with a standardized oxygen electrode (Orion Research, Inc.). To avoid contamination of the experimental cultures, the oxygen concentration is measured in a parallel flask containing TAP medium to verify that the correct amount of oxygen is delivered to the cultures. The electrode can be calibrated by bubbling air in an aqueous solution (21% oxygen or 100% air). The second point for calibration (0% oxygen) can be achieved by bubbling nitrogen gas or by adding dithionite, which consumes oxygen. Different Ways for Bubbling
It is possible to set up different systems for bubbling the cultures. As shown in Fig. 4, we use a sterile pipette and a cotton plug placed in the flask. This system needs to be assembled in a laminar flow hood to keep the culture sterile once the medium has been sterilized and cells inoculated. Alternatively, a rubber stopper with two holes can also be used. Through one hole, a glass tube (or a pipette) is added for bubbling the culture. In the other one, a short curved glass tube serves as exhaust for the air. We recommend using a sterile cotton plug on the exhaust tube, which minimizes the chance of contamination when cultures are removed for analysis. Sample Preparation
Total RNA is prepared and analyzed by hybridization as described.41 It is important to remember for the preparation of RNA from hypoxic cells that the t1/2 of the mRNA encoding Cyc6 (about 45 min) can be comparable to the RNA preparation time and for Cpx1 appears to be shorter. Five micrograms of total RNA is loaded per lane. Probes for Cpx1, Cyc6, and RbcS2 (encoding the small subunit of Rubisco) are prepared as described.38 For the detection of Crd1 transcripts, the 55 102-bp XhoI/PstI fragment from pCrd1-552 is used. A gene-specific probe for Cth1 is made from a 345-bp XhoI fragment corresponding to 4305 to 4650 of the Cth1 genome sequence.36 Specific activities of probes ranged from 3 to 6 108 cpm g1 DNA for the experiments shown here. The blots are exposed at 80 to film (XRP-1; Eastman-Kodak, Rochester, NY)
52
J. Moseley, J. Quinn, M. Eriksson, and S. Merchant, EMBO J. 19, 2139 (2000).
616
unicellular systems
[39]
Fig. 4. Alternative options for bubbling cultures. (A) A cotton plug with an inserted pipette is used. (B) A rubber stopper where two holes have been made: one for the tube that reaches the cell suspension and the second one for a exhaust tube in order to avoid high pressure inside the flask.
with two intensifying screens and are typically developed after overnight exposure. Concluding Remarks: Linking Hypoxia and Copper-Deficient Responses through the crr1 Mutant
The copper- and oxygen-responsive expression of Cyc6, Cpx1, and Crd1 is mediated via a common pathway. Evidence for this common pathway is (1) regulation of all three genes by copper or oxygen requires the trans-acting locus CRRI,20,36 (2) the induction of Cyc6 and Cpx1 expression by copper or oxygen deficiency can be blocked by the same inhibitor, that is, mercuric ions,20,53,54 and (3) the cis elements mediating copper- and oxygen-responsive expression have the same core sequence. The core of 53 54
K. L. Hill, H. H. Li, J. Singer, and S. Merchant, J. Biol. Chem. 266, 15060 (1991). K. L. Hill and S. Merchant, EMBO J. 14, 857 (1995).
[39]
low O2 response in Chlamydomonas
617
the two CuREs of Cyc6 was identified initially by deletion analysis of the Cyc6 promoter30 and extensive site-directed mutagenesis to be the tetranucleotide sequence GTAC.33 Each Cyc6 CuRE is independently capable of conferring copper-responsive expression.30,33 The same GTAC core was found to define a single CuRE in the Cpx1 promoter required for copper-responsive expression of this gene.33,38 Mutation of a second downstream GTAC sequence did not affect copper-responsive expression. When the oxygen-responsive expression of Cpx1 was analyzed, it was found that mutation of either the GTAC core of the Cpx1 CuRE or the downstream GTAC sequence would eliminate oxygen-responsive expression of Cpx1.20 This second GTAC thus defines the core of a cis element specific for oxygen-responsive expression and is therefore designated a hypoxiaresponsive element. This difference in the cis element requirement for copper- vs oxygen-responsive expression of Cpx1 correlates with the increased accumulation of Cpx1 transcripts relative to Cyc6 transcripts in oxygen-deficient cells compared to copper-deficient cells.20 Therefore, it is clear that while copper- and oxygen-responsive expression of these genes is mediated by a common pathway, mechanistic details do differ, and these details potentially relate to the function of these gene products under the different growth conditions. This difference between the responses to copper vs oxygen deficiency highlights the need for the study of oxygen-responsive expression in C. reinhardtii. Acknowledgments We thank the members of the group, especially Janette Kropat and Stephen Tottey, for their comments on the manuscript. This work was supported by the National Institutes of Health (GM42143) (SM) and a postdoctoral fellowship from the Spanish Ministry for Education (JC).
[39]
unicellular systems
[39] Evaluation of Oxygen Response Involving Differential Gene Expression in Chlamydomonas reinhardtii By Jose´ A. Del Campo, Jeanette M. Quinn, and Sabeeha Merchant Chlamydomonas as a Model Organism
The unicellular green alga Chlamydomonas has, for decades, offered experimental advantages for the study of chloroplast function and biogenesis, photosynthesis, flagellar motility and assembly, photoreceptor biochemistry, and sexual mating.1–6 Among these are (1) the ability to manipulate the nuclear and also both organellar genomes,1,7,8 (2) facultative photosynthetic growth because of the ability of Chlamydomonas to use acetate for heterotrophic growth, (3) heterothallic mating types, which permit classical genetic approaches for the dissection of important biological problems,9 and (4) considerable genomic information through EST10 and shotgun genome sequencing projects (http://genome.jgi-psf.org/chlre1/ chlre1.home.html). Chlamydomonas has long been used as a model (see Fig. 1) for the study of nutrient-responsive signal transduction, especially in the context of the function of photosynthetic apparatus. The classical areas of interest have included inorganic (sulfur, nitrogen, phosphorus) nutrient utilization,11,12
1
J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant (eds.), in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas.’’ Kluwer Academic Publishers, 1998. 2 A. R. Grossman, Curr. Opin. Plant Biol. 3, 132 (2000). 3 E. H. Harris, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 363 (2001). 4 R. M. Dent, M. Han, and K. K. Niyogi, Trends Plant Sci. 6, 364 (2001). 5 C. D. Silflow and P. A. Lefebvre, Plant Physiol. 127, 1500 (2001). 6 O. A. Sineshchekov and E. V. Govorunova, Trends Plant Sci. 4, 201 (1999). 7 B. L. Randolph-Anderson, J. E. Boynton, N. W. Gillham, E. H. Harris, A. M. Johnson, M. P. Dorthu, and R. F. Matagne, Mol. Gen. Genet. 126, 357 (1993). 8 J. E. Boynton and N. W. Gillham, Methods Enzymol. 264, 279 (1996). 9 H. Harris, in ‘‘The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use.’’ Academic Press, San Diego, 1989. 10 J. Shrager, C. Hauser, C. W. Chang, E. H. Harris, J. Davies, J. McDermott, R. Tamse, Z. Zhang, and A. R. Grossman, Plant Physiol. 131, 401 (2003). 11 J. P. Davies and A. R. Grossman, in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas’’ (J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant, eds.), p. 613. Kluwer Academic Publishers, 1998. 12 A. Grossman and H. Takahashi, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 163 (2001).
METHODS IN ENZYMOLOGY, VOL. 381
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00
[39]
low O2 response in Chlamydomonas
605
Fig. 1. A model showing known elements involved in copper deficiency and hypoxia responses. The putative DNA-binding protein Crr1 is required for the activation of target genes when cells are facing copper deficiency and/or hypoxic conditions. The core of the CuRE elements (see text) is the sequence GTAC.
as well as trace metal requirements and mechanisms of homeostasis.13–15 Metabolism and gene expression involving CO2 and O2 have also been long-standing topics of research activity,16–18 with recently renewed emphasis from the perspective of energy metabolism19 and the identification of regulatory mutants.20–23 Chlamydomonas reinhardtii, like other organisms, responds to changes in oxygen supply through the alteration of metabolism. Some species of this genus are found in naturally oxygen-deficient habitats such as peat bogs and sewage lagoons,9 where a hypoxic response is probably critical for survival. Even laboratory cultures become oxygen depleted quite 13
S. Merchant, in ‘‘The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas’’ (J. D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant, eds.), p. 597. Kluwer Academic Publishers, 1998. 14 S. La Fontaine, J. M. Quinn, S. S. Nakamoto, M. D. Page, V. Gohre, J. L. Moseley, J. Kropat, and S. Merchant, Eukaryot. Cell 1, 736 (2002). 15 P. Rubinelli, S. Siripornadulsil, F. Gao-Rubinelli, and R. T. Sayre, Planta 215, 1 (2002). 16 P. E. Bryant, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 17, 533 (1970). 17 M. R. Badger, A. Kaplan, and J. A. Berry, Plant Physiol. 66, 407 (1980). 18 A. M. Geraghty and M. H. Spalding, Plant Physiol. 111, 1339 (1996). 19 M. L. Ghirardi, L. Zhang, J. W. Lee, T. Flynn, M. Seibert, E. Greenbaum, and A. Melis, Trends Biotechnol. 18, 506 (2000). 20 J. M. Quinn, M. Eriksson, J. L. Moseley, and S. Merchant, Plant Physiol. 128, 463 (2002). 21 H. Fukuzawa, K. Miura, K. Ishizaki, K. Kucho, T. Saito, T. Kohinata, and K. Ohyama, Proc. Natl. Acad. Sci. USA 98, 5347 (2001). 22 K. Van, Y. Wang, Y. Nakamura, and M. H. Spalding, Plant Physiol. 127, 607 (2001). 23 Y. Xiang, J. Zhang, and D. P. Weeks, Proc. Natl. Acad. Sci. USA 98, 5341 (2001).
606
unicellular systems
[39]
rapidly as a consequence of respiration if they are not sufficiently agitated.20,24 Oxygen-responsive gene expression is, therefore, probably an integral aspect of everyday metabolism for Chlamydomonas, as well as for other organisms. In addition, there are also special metabolic pathways occurring only in anaerobic Chlamydomonas cells; the most publicized of which is photosynthetic hydrogen production.25 Hydrogen Production in Low Oxygen
In the early 1930s, Gaffron and co-workers discovered the ability of unicellular green algae to produce hydrogen gas upon illumination (reviewed by Melis and Happe25). Historically, hydrogen evolution activity in green algae was induced upon prior anaerobic incubation of the cells in the dark. Under such conditions, an iron-containing hydrogenase enzyme (encoded by the HydA gene in C. reinhardtii) was induced, catalyzing light-mediated H2 evolution. Oxygen is a powerful inhibitor of hydrogen evolution at the enzyme level.19,26 It has also been shown that regulation of the hydrogenase gene takes place at the transcriptional level.27 The idea of using H2 gas from green algae as an alternative fuel source has been promoted recently.28 The pathway involves two stages, photosynthesis and H2 production, dependent on sulfur availability, with sulfur deprivation serving as a metabolic switch. When sulfur is available for cells, green algae perform normal photosynthesis (water oxidation, oxygen evolution, and biomass accumulation). In the absence of sulfur, photosynthesis in C. reinhardtii slips into a hydrogen production mode if oxygen is simultaneously removed. Electrons for H2 production may originate either at photosystem II (PSII) upon photooxidation of water or at the plastoquinone pool upon oxidation of the cellular endogenous substrate (e.g., starch degradation). Electrons are transported via photosystem I (PSI) to ferredoxin, which serves as the physiological electron donor to the hydrogenase. The signal transduction components involved in metabolic switching in this pathway are completely unknown. The ability to undertake combined molecular and classical genetic studies in this model system should facilitate the discovery of the relevant regulatory factors.
24
P. M. Wood, Eur. J. Biochem. 87, 8 (1978). A. Melis and T. Happe, Plant Physiol. 127, 740 (2001). 26 T. Happe, A. Hemschemeier, M. Winkler, and A. Kaminski, Trends Plant Sci. 7, 246 (2002). 27 T. Happe and A. Hamiski, Eur. J. Biochem. 269, 1022 (2002). 28 A. Melis, L. Zhang, M. Forestier, M. L. Ghirardi, and M. Seibert. Plant Physiol. 122, 127 (2000). 25
[39]
low O2 response in Chlamydomonas
607
Connections between Oxygen and Copper Nutrient Homeostasis
Copper is an essential micronutrient for all organisms because of its function in enzymes that serve as catalysts of oxygen chemistry and redox reactions. Chlamydomonas has a well-characterized response to nutritional copper deficiency.13 The best characterized of these is the replacement of plastocyanin (the most abundant copper protein in photosynthetic cells) with a heme-containing c-type cytochrome.24,29 This occurs through transcriptional activation of the Cyc6 gene encoding cytochrome c6, a heme-containing functional substitute for plastocyanin,30 and induced degradation of apoplastocyanin in the thylakoid lumen,31 which occurs probably to ensure the redistribution of copper to cytochrome oxidase.32 This transcriptional response requires copper response elements (CuREs)33 containing the sequence GTAC, which forms the core of these CuREs, and Crr1, a putative DNA-binding protein (Fig. 1).34 Crr1 is also required for the transcriptional activation in copper-deficient cells of Cpx1, encoding coproporphyrinogen III oxidase,35 and also Crd1,36 encoding the aerobic oxidative cyclase in chlorophyll biosynthesis.37 In copper-replete cells, Crd1 is replaced by Cth1. The inhibition of Cth1 accumulation under copper-deficient conditions also involves Crr1.36 Wood24 noted first that hypoxic Chlamydomonas cells accumulate cytochrome c6 even in copper-replete medium. The response of the Cyc6 gene to hypoxia is mediated at the transcriptional level via its CuREs and requires Crr1 function.20,33 Subsequently, it was discovered that each of the Cu deficiency targets mentioned previously also responds to hypoxia (e.g., Fig. 2) in a pathway involving the CuRE and Crr1 function.38 The hypoxic and nutritional copper signaling pathways in Chlamydomonas therefore seem to share some common components.20 A trivial explanation for the similar output, that is, that hypoxic cells are internally copper deficient, was ruled out because oxygen-deprived cells are able to synthesize 29
S. Merchant and L. Bogorad, EMBO J. 6, 2531 (1987). J. M. Quinn and S. Merchant, Plant Cell 7, 623 (1995). 31 H. H. Li and S. Merchant, J. Biol. Chem. 270, 23504 (1995). 32 S. S. Nakamoto, in ‘‘Compartmentalized Copper and Iron Enzymes in C. reinhardtii: Venus Versus Mars.’’ Ph.D. Dissertation, University of California, Los Angeles. Department of Chemistry and Biochemistry, 2001. 33 J. M. Quinn, P. Barraco, M. Eriksson, and S. Merchant, J. Biol. Chem. 275, 6080 (2000). 34 J. Kropat and S. Merchant, unpublished results (2003). 35 M. Eriksson et al., unpublished results (2003). 36 J. L. Moseley, M. D. Page, N. P. Alder, M. Eriksson, J. Quinn, F. Soto, S. M. Theg, M. Hippler, and S. Merchant, Plant Cell 14, 673 (2002). 37 S. Tottey, M. A. Block, M. Allen, T. Westergreen, C. Albrieux, H. U. Scheller, S. Merchant, and P. E. Jensen, Proc. Natl. Acad. Sci. USA 100, 16119 (2003). 38 J. M. Quinn, S. S. Nakamoto, and S. Merchant, J. Biol. Chem. 274, 14444 (1999). 30
608
unicellular systems
[39]
Fig. 2. Hypoxia-induced gene expression in C. reinhardtii with a time course response. Wild-type strain CC125 was grown in copper-supplemented (6 M) Tris–acetate–phosphate medium under normal aeration to a concentration of 1 106 cells/ml in a shaker (300 rpm) at room temperature using normal room lighting (approximately 1–15 mol m2 s1). Twenty milliliters was removed for RNA preparation (t ¼ 0). The remaining culture was bubbled with 98% N2 þ 2% CO2 (0% O2). Samples for RNA preparation were removed at the indicated times. RNA samples were probed with Cyc6, Cpx1, Crd1, and RbcS2 (as a control) (A). Except for Cth1 (B), all samples were analyzed in duplicate cultures.
holoplastocyanin de novo, indicating that copper is available inside the cells. Quinn et al.20 tested the idea that the connection between Cu and the hypoxic response results from modification of the Cu(II)/Cu(I) ratio by the oxygen status of the environment, assuming that the copper sensor is specific for Cu(II) vs Cu(I). In this case, the hypoxic response of Cyc6, Cpx1, Crd1, and Cth1 would simply mimic the response to Cu deficiency. However, this is not the case.20 The Cyc6 gene responds strongly to Cu deficiency and more weakly to hypoxia, whereas the Crd1 and Cpx1 genes respond strongly to hypoxia and less so to Cu deficiency. Furthermore, a specific hypoxia response element was identified in the Cpx1 gene.33 This HyRE is related to the CuRE by virtue of the GTAC core sequence, but it does not function as a CuRE.20 Quinn et al.20 concluded that the hypoxic response shares signal transduction components with the Cu deficiency response. The rationale for the intersection of the regulatory pathways is that hypoxic conditions create copper deficiency in nature by driving copper ions to insoluble Cu(I) species, which precipitate and
[39]
low O2 response in Chlamydomonas
609
perhaps have limited bioavailability. The response of Cu deficiency targets to hypoxia might be a mechanism for anticipating and preparing for Cu deficiency in a microaerobic growth environment. The pattern of cytochrome c6 and coprogen oxidase accumulation parallels the transcriptional activation of the genes, indicating that the hypoxic response can also be detected and analyzed at the protein level. However, in order to monitor changes in gene expression, it is more appropriate to analyze the effects at the level of transcript abundance, as the half-life for the various mRNAs is shorter than for the corresponding proteins. The physiological relevance of the Crr1-dependent hypoxic response of one target gene, Crd1, was confirmed by the demonstration of a conditional chlorotic phenotype in hypoxic but not aerobic crd1 mutant cells.20 The crd1 mutant strain is useful for the study of the Crr1-dependent hypoxic gene expression pathway because its remarkable chlorotic phenotype in þCu TAP medium in low air can be used as a physiological marker, and expression of the gene can be used as a molecular marker (see Fig. 2). At this point, a number of questions remain about the hypoxic response in Chlamydomonas. First, for the Cpx1 gene, the copper response element appears to be necessary and sufficient for the nutritional copper response and is also necessary for the hypoxic response. A second element, with a GTAC core, found in the Cpx1 promoter is required for the oxygen deficiency response. The second element, called a HyRE, is not necessary for the copper deficiency response. What is the relationship between the CuRE and the HyRE at the mechanistic level? Second, a well-known anaerobically induced gene, Hyd1, is normally regulated in the crr1 mutant, indicating a Crr1-independent hypoxia/anoxia sensing mechanism in C. reinhardtii. What are the regulatory components required for the regulation of HydA? Given the repertoire of molecular techniques on hand today that can be applied to this experimental model, identification of the hypoxic signaling components should be achievable. With this intention in mind, this article describes the setup of experiments involving the growth of Chlamydomonas cells in liquid cultures at different oxygen concentrations. Growth Media
Chlamydomonas was first introduced into the biochemistry of photosynthesis by Levine and co-workers.39 In a previous volume of this series,40 information concerning the culturing of Chlamydomonas cells, as well as detailed descriptions about mutagenesis procedures and photosynthesis 39 40
R. P. Levine, Science 162, 768 (1968). A. San Pietro (ed.), Methods Enzymol. 23 (1971).
610
unicellular systems
[39]
studies, can be found. For details concerning the preparation of growth media, both liquid and solid, and particularly copper-free media, see Quinn and Merchant.41 For general information concerning the cultivation of Chlamydomonas cells, including various types of growth media, see Harris.9 Culturing
Several different strains have been tested for the response to oxygen concentration in liquid cultures, including CC125, CC425, and 2137 as wild-type strains, and the mutant crr1-1, which is affected in the regulation of copper metabolism (see earlier discussion). As diagrammed in Fig. 3, to generate different oxygen concentrations for the culturing of Chlamydomonas cells, we used a mixture of three different gases: CO2, compressed air, and nitrogen. The use of CO2 in the gas mixture is important because CO2 is a nutrient and of course also buffers the medium. Variation in air content relative to nitrogen changes both CO2 and O2 levels. By providing CO2 at a fixed concentration (0.2 to 2% depending on the experiment), the key variable in the experiment is O2. CO2 concentration in air (0.036% or 350 ppm) is considered to be low CO211 conditions from a physiological point of view,42 leading to low growth rates and low biomass accumulation in algal cultures because CO2 is a substrate for photosynthesis. The ratio of CO2/O2 is also metabolically relevant43,44 because the active site of Rubisco discriminates poorly against O2. Therefore, in air, a significant fraction of ribulose-1,5-bisphosphate is oxygenated instead of carboxylated,43,44 leading to the photorespiratory pathway for salvage of phosphoglycolate. To avoid photorespiration, algae induce a carbon concentration mechanism (CCM), involving carbonic anhydrases (CA)42,45–48 and other components as well. The CCM is repressed by high CO2 (e.g., 5%).21–23 The effect of O2 on regulation of the CCM is not known, but is probably a question worth addressing. Nevertheless, 41
J. M. Quinn and S. Merchant, Methods Enzymol. 297, 263 (1998). A. Kaplan and L. Reinhold, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 539 (1999). 43 J. Berry, J. Boynton, A. Kaplan, and M. Badger, Carnegie Inst. Wash. Yearbook 75, 423 (1976). 44 W. R. King and Andersen, Arch. Microbiol. 128, 84 (1980). 45 M. Rawat and J. V. Moroney, Plant Physiol. 109, 937 (1995). 46 J. Karlsson, A. K. Clarke, Z. Y. Chen, S. Y. Hugghins, Y. I. Park, H. D. Husic, J. V. Moroney, and G. Samuelsson, EMBO J. 17, 1208 (1998). 47 R. P. Funke, J. L. Kovar, and D. P. Weeks, Plant Physiol. 114, 237 (1997). 48 M. Eriksson, J. Karlsson, Z. Ramazanov, P. Gardestrom, and G. Samuelsson, Proc. Natl. Acad. Sci. USA 93, 12031 (1996). 42
[39] low O2 response in Chlamydomonas
Fig. 3. (continued)
611
612
unicellular systems
[39]
Fig. 3. (A) Scheme for the experimental setup used to grow Chlamydomonas cells under different oxygen concentrations. The amount of each gas is monitored on a multichannel gas flowmeter. The mixture of different gases is provided to the cultures using silicone tubing. The oxygen concentration in the cultures is measured using a Clark-type oxygen electrode in a flask at the end of the series containing TAP medium without cells. Note that it is preferable to measure the O2 content in each flask individually, but there may be technical difficulties in
[39]
low O2 response in Chlamydomonas
613
because of the critical metabolic function of CO2 and the known responses to CO2 (e.g., modification of phototactic behavior49), we have fixed the CO2 concentration in experiments in our laboratory. We have not considered the effect of the CO2/O2 ratio, although the ratio is known to be critical for photosynthetic carbon metabolism. Ideally, the CO2 concentration should be fixed to correspond to air levels. However, this low level is technically difficult to achieve consistently and reproducibly when the CO2 is supplied from a pure CO2 gas tank. Therefore, for low air experiments (hypoxic conditions), we used two different mixtures: 97.8% nitrogen, 2% air, and 0.2% CO2 or 96% nitrogen, 2% air, and 2% CO2. It may be possible to provide HCO31 in the medium in place of CO2 and vary only O2 and nitrogen in the gas mixture. We have not tried this option. Other variables to consider include light intensity and acetate for heterotrophic growth because these affect photosynthesis and respiration and hence O2 content during the experiment. It is also possible to induce hypoxic conditions by growing Chlamydomonas cells without vigorous agitation, for example, in Erlenmeyer flasks fitted with cotton plugs (200 ml medium in 250-ml flasks) where the cells are kept suspended by low basal stirring using a magnetic stir bar.20,24 Note that growth under photoautotrophic conditions (illumination þ CO2) will generate O2 in the cells and in the growth medium. Therefore, continuous bubbling or flushing is necessary to maintain control of hypoxic conditions. Happe and Kaminski50 flushed the cell suspension with argon in the dark in order to promote anaerobic conditions for inducing hydrogenase activity in Chlamydomonas cultures. Bubbling and Flask Setup
In order to deliver the desired mixture of gases for bubbling the cultures, we use three different gas flowmeters (Aalborg Instruments & Controls, Inc.) in two scales (one from 0 to 25 ml of gas per minute and another from 0 to 5 liters per minute of gas) because the amount needed for one gas is quantitatively different from the other (e.g., 99.8% air vs 0.2% CO2). The output mixture has a total flow of 2.5 liters/min1 liter of culture1. All gases are driven using flexible plastic tubes (Tygon) from the tanks to the flowmeters and from these to the cultures (see Fig. 3). The diameter for 49 50
W. Nultsch, Arch. Microbiol. 112, 179 (1977). T. Happe and A. Kaminski, Eur. J. Biochem. 269, 1022 (2002).
maintaining sterile conditions for extended periods. It is useful to place the electrode at various points of exit from the gas supply in order to evaluate the effect of position of each flask. It is also preferable to mount the flasks in parallel as diagrammed rather than in series. (B) Details for connections of the different components.
614
[39]
unicellular systems
the tubes may vary according to the regulator output, the flowmeter input and output, the type of filters, and so on. Once the gas mixture exits the multichannel flowmeter, the outlet pipe is split to the same number of branches to correspond to the number of flasks that are needed (see Fig. 3). Ideally, an individual flowmeter for each culture flask should be used, which allows bubbling of all flasks using exactly the same flow rate. A trouble commonly encountered with bubbled cultures is bacterial contamination. To avoid this, gas to the cultures is filtered through 0.45-m filters (Pall Gelman Laboratory) and supplied through a sterile disposable pipette. An additional 125-ml flask, containing 50 ml of rich medium (e.g., LB), is aerated in the same way as the Chlamydomonas cultures. This is included for monitoring contamination during the course of the experiments. Normally, we grow Chlamydomonas in 250-ml Erlenmeyer flasks containing 100 ml of liquid TAP medium. The culture flasks are placed on a shaker at 100 rpm for additional agitation, as bubbling alone is not enough to keep the cells from settling. In our experience, cultures can be kept in axenic conditions for at least 5 days of growth. They multiply from 4 105 cells/ ml at inoculation to 2 107 cell/ml after day 5 for wild-type CC125 in 99.8% air þ 0.2% CO2 corresponding to a doubling time in the log phase of 17 h. The doubling time for Chlamydomonas grown in optimal conditions in TAP medium, using a shaker at 220 rpm, 24 , and a light intensity of about 60 mole m2 s1, ranges from 6 to 8 h. Other Culture Conditions
Cultures are maintained at room temperature (22–24 ) with ambient illumination (10–15 mole m2 s1) provided by overhead standard ceiling fluorescent lights. Due to the ability of Chlamydomonas cells to grow heterotrophically, wild-type cultures in TAP medium9 reach a cell density of 1 107 cells/ml in 2% air þ 0.2% CO2 in 4 days (stationary phase) under these conditions. For standard culture conditions in our laboratory (with out bubbling, at 24 in a shaker incubator, 220 rpm, 60 mole m2 s1), the cell density reached for the same wild-type Chlamydomonas strain in the same time is 2.1 107 cell/ml. A higher light intensity can be used by placing lamps (fluorescent type, to avoid unwanted heating) close to the cultures, which is important for species that are incapable of growing heterotrophically (like some diatoms).51
51
L. A. Zaslavskaia, J. C. Lippmeier, C. Shih, D. Ehrhardt, A. R. Grossman, and K. E. Apt, Science 292, 2073 (2001).
[39]
low O2 response in Chlamydomonas
615
Monitoring Oxygen Concentration
The oxygen content of the cultures is measured with a standardized oxygen electrode (Orion Research, Inc.). To avoid contamination of the experimental cultures, the oxygen concentration is measured in a parallel flask containing TAP medium to verify that the correct amount of oxygen is delivered to the cultures. The electrode can be calibrated by bubbling air in an aqueous solution (21% oxygen or 100% air). The second point for calibration (0% oxygen) can be achieved by bubbling nitrogen gas or by adding dithionite, which consumes oxygen. Different Ways for Bubbling
It is possible to set up different systems for bubbling the cultures. As shown in Fig. 4, we use a sterile pipette and a cotton plug placed in the flask. This system needs to be assembled in a laminar flow hood to keep the culture sterile once the medium has been sterilized and cells inoculated. Alternatively, a rubber stopper with two holes can also be used. Through one hole, a glass tube (or a pipette) is added for bubbling the culture. In the other one, a short curved glass tube serves as exhaust for the air. We recommend using a sterile cotton plug on the exhaust tube, which minimizes the chance of contamination when cultures are removed for analysis. Sample Preparation
Total RNA is prepared and analyzed by hybridization as described.41 It is important to remember for the preparation of RNA from hypoxic cells that the t1/2 of the mRNA encoding Cyc6 (about 45 min) can be comparable to the RNA preparation time and for Cpx1 appears to be shorter. Five micrograms of total RNA is loaded per lane. Probes for Cpx1, Cyc6, and RbcS2 (encoding the small subunit of Rubisco) are prepared as described.38 For the detection of Crd1 transcripts, the 55 102-bp XhoI/PstI fragment from pCrd1-552 is used. A gene-specific probe for Cth1 is made from a 345-bp XhoI fragment corresponding to 4305 to 4650 of the Cth1 genome sequence.36 Specific activities of probes ranged from 3 to 6 108 cpm g1 DNA for the experiments shown here. The blots are exposed at 80 to film (XRP-1; Eastman-Kodak, Rochester, NY)
52
J. Moseley, J. Quinn, M. Eriksson, and S. Merchant, EMBO J. 19, 2139 (2000).
616
unicellular systems
[39]
Fig. 4. Alternative options for bubbling cultures. (A) A cotton plug with an inserted pipette is used. (B) A rubber stopper where two holes have been made: one for the tube that reaches the cell suspension and the second one for a exhaust tube in order to avoid high pressure inside the flask.
with two intensifying screens and are typically developed after overnight exposure. Concluding Remarks: Linking Hypoxia and Copper-Deficient Responses through the crr1 Mutant
The copper- and oxygen-responsive expression of Cyc6, Cpx1, and Crd1 is mediated via a common pathway. Evidence for this common pathway is (1) regulation of all three genes by copper or oxygen requires the trans-acting locus CRRI,20,36 (2) the induction of Cyc6 and Cpx1 expression by copper or oxygen deficiency can be blocked by the same inhibitor, that is, mercuric ions,20,53,54 and (3) the cis elements mediating copper- and oxygen-responsive expression have the same core sequence. The core of 53 54
K. L. Hill, H. H. Li, J. Singer, and S. Merchant, J. Biol. Chem. 266, 15060 (1991). K. L. Hill and S. Merchant, EMBO J. 14, 857 (1995).
[39]
low O2 response in Chlamydomonas
617
the two CuREs of Cyc6 was identified initially by deletion analysis of the Cyc6 promoter30 and extensive site-directed mutagenesis to be the tetranucleotide sequence GTAC.33 Each Cyc6 CuRE is independently capable of conferring copper-responsive expression.30,33 The same GTAC core was found to define a single CuRE in the Cpx1 promoter required for copper-responsive expression of this gene.33,38 Mutation of a second downstream GTAC sequence did not affect copper-responsive expression. When the oxygen-responsive expression of Cpx1 was analyzed, it was found that mutation of either the GTAC core of the Cpx1 CuRE or the downstream GTAC sequence would eliminate oxygen-responsive expression of Cpx1.20 This second GTAC thus defines the core of a cis element specific for oxygen-responsive expression and is therefore designated a hypoxiaresponsive element. This difference in the cis element requirement for copper- vs oxygen-responsive expression of Cpx1 correlates with the increased accumulation of Cpx1 transcripts relative to Cyc6 transcripts in oxygen-deficient cells compared to copper-deficient cells.20 Therefore, it is clear that while copper- and oxygen-responsive expression of these genes is mediated by a common pathway, mechanistic details do differ, and these details potentially relate to the function of these gene products under the different growth conditions. This difference between the responses to copper vs oxygen deficiency highlights the need for the study of oxygen-responsive expression in C. reinhardtii. Acknowledgments We thank the members of the group, especially Janette Kropat and Stephen Tottey, for their comments on the manuscript. This work was supported by the National Institutes of Health (GM42143) (SM) and a postdoctoral fellowship from the Spanish Ministry for Education (JC).