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Growth of Chlamydomonas reinhardtii under Circadian Conditions   

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Adam Idoine Adam Idoine
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Takuya Matsuo Takuya Matsuo
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Abstract

The green biflagellate unicellular alga Chlamydomonas reinhardtii serves as a model to study fundamental biological processes such as the structure and function of flagella or light-driven processes including photosynthesis, its behavioral responses, life cycle and circadian clock. Light-dark, as well as temperature cycles, are major Zeitgebers to entrain the algal circadian clock. In C. reinhardtii, several processes are under circadian control and many clock-controlled genes and/or proteins have been found in the past decades as well as components of the endogenous oscillator. Here, we describe a protocol for the growth of C. reinhardtii for the synchronization and analysis of its circadian clock.

Keywords: Chlamydomonas reinhardtii, Circadian clock, Free running conditions

Background

In the past years, several clock components of C. reinhardtii have been identified and their function has been studied (for reviews, see Schulze et al., 2010; Matsuo and Ishiura, 2011; Noordally and Millar, 2015; Ryo et al., 2016; Kottke et al., 2017). In this article, we will present growth conditions for studying circadian control in C. reinhardtii. Therefore, we introduce the chronobiology nomenclature used for the growth of the algal cells under diurnal and circadian conditions (Figure 1).

At first, the circadian clock is synchronized by a light-dark cycle of 12-h light and 12-h dark, known as LD 12:12 at constant temperature. At LD0, light is turned on, and at LD12 it is switched off. LD6 thus defines the middle of the day and LD18 the middle of the night. A rhythm observed under LD conditions is called diurnal. Time measurement under diurnal conditions goes from LD0 to LD24. The next day is defined in the same way (LD0 to LD24). To find out if this rhythm is controlled by the circadian clock, the cells have to be released under so-called “free running conditions” with constant light and temperature where a circadian rhythm will continue with a period of about 24 h (Wagner and Mittag, 2009; Boesger et al., 2014). The cells are released at the end of the dark period (LD24) to constant conditions (Figure 1). Therefore, dim light (LL) is often used for C. reinhardtii, but if effects of specific light pulses are necessary as for the rhythm of photoaccumulation or for phase shifting the circadian clock, constant darkness (DD) is also used. For the rhythm of photoaccumulation (also described as rhythm of phototaxis), specialized set-ups and needs are necessary that differ depending on the home-made instrumental device (Mergenhagen, 1984; Gaskill et al., 2010; Forbes-Stovall et al., 2014; Müller et al., 2017). These are not further described in the current protocol.


Figure 1. Light conditions for investigating circadian rhythms in C. reinhardtii

Under circadian conditions, time measurement starts at LL0 and continues with the number of hours under which the organism has been put under circadian conditions. For example, LL48 means that the organism was for two days under constant conditions. LL30 symbolizes the middle of subjective day and LL42 the middle of subjective night. Subjective day (or day phase) and subjective night (night phase) are commonly used terms for free running conditions in chronobiology. Since transients may occur upon transfer to constant conditions, circadian rhythms are usually measured after the organism has been exposed for at least 12 h to constant conditions, and often after exposure for 24 h.

Materials and Reagents

  1. Petri dishes
  2. Aluminum foil
  3. Autoclave tape
  4. Whatman® Prepleated Qualitative Filter Paper (GE Healthcare, catalog number: 1201-320 )
  5. Sterile tooth picks
  6. Indicator paper pH-Fix 0-14 (Machery-Nagel, catalog number: 92110 )
  7. Cotton plug
  8. Nunc®-flasks (NuncTM EasYFlaskTM 25cm2 with filter cap, gamma irradiated) (Thermo Fisher Scientific, catalog number: 156367 )
  9. Chlamydomonas reinhardtii cells
  10. Wild-type strain SAG 73.72 cells
  11. Double-distilled water (ddH2O; conductivity ≤ 0.1 μS/cm)
  12. NH4Cl
  13. CaCl2•2H2O
  14. MgSO4•7H2O
  15. K2HPO4
  16. KH2PO4
  17. Na2EDTA
  18. H3BO3
  19. FeSO4•7H2O
  20. 20% KOH
  21. ZnSO4•7H2O
  22. MnCl2•4H2O
  23. CoCl2•6H2O
  24. CuSO4•5H2O
  25. (NH4)6Mo7O24•4H2O
  26. Tris [Tris(hydroxymethyl)-aminomethane]
  27. Lugol's solution (iodine-potassium iodide solution; Merck, catalog number: 1092611000 )
  28. Liquid nitrogen
    Caution: Extremely cold liquid (-196 °C); it may cause cryogenic burns or injury and displaces oxygen, which could lead to rapid suffocation in closed rooms; transport and store it always in containers designed for cryogenic liquids; handle it with special devices using protective clothing, cold insulating gloves and a face shield.
  29. TAP salt solution (see Recipes)
  30. 1 M potassium phosphate buffer (see Recipes)
  31. Hutner's trace elements (see Recipes)
  32. Tris-acetate-phosphate (TAP) medium (see Recipes)

Equipment

  1. 1 L beaker
  2. Heater
  3. Erlenmeyer-flasks
  4. Magnetic stirrer
  5. Autoclave
  6. Sterile bench
  7. Culture room
  8. Laboratory sample shaker
  9. pH-electrode
  10. Centrifuge
  11. Neubauer improved counting chamber (Marienfeld, catalog number: 0640010 )

Procedure

  1. Take C. reinhardtii cells from a one to two weeks inoculated TAP-agar Petri dish with a sterile toothpick and suspend them in up to 50 ml sterile (see Notes) TAP medium in a 100 ml Erlenmeyer-flask with a magnetic stirring bar under a sterile bench. As an alternative, you can also inoculate the cells in 10 ml sterile TAP-medium in Nunc®-flasks (25 cm2) under constant shaking (70 rpm) on a laboratory sample shaker. We routinely use wild-type strain SAG 73.72 obtained by the Culture Collection of Algae at the University of Göttingen, Germany (Sammlung von Algenkulturen der Universität Göttingen: SAG). 
  2. Grow this preculture in a 12-h light/12-h dark cycle (LD 12:12) with a light intensity of 75 μE m−2 sec−1 (1 E = 1 mol of photons) and “cool white” neon lights (Lumilux) at 23 °C-24 °C until a cell density of about 4-5 x 106 cells per ml is reached.
  3. For determination of the cell density, mix 90 μl of the cell solution with 10 μl Lugol's solution to immobilize the cells. Pipette 10 μl each (duplicate) in the upper and the lower count field of a Neubauer improved counting chamber and count the cells therein by microscopy (400x magnification). Determine the average cell number (ACN) from the cell numbers of five squares for each count field and calculate the cell concentration considering the dilution using the following equation: algal cells/ml = CAN x 5 x 104 x (10/9) (Guillard and Sieracki, 2005). 
  4. Use this preculture to start the main culture. Take an autoclaved Erlenmeyer-flask with TAP medium (e.g., 100 ml) and add 1/100 of the desired volume of the main culture from the preculture (e.g., 1 ml) under a sterile bench.
  5. Inoculate and cultivate the main culture under the same conditions mentioned above till a cell density of 2-3 x 106 cells per ml is reached. 
  6. Transfer the cells to conditions of constant dim light (LL-conditions) with a light intensity of 15-20 μE m−2 sec−1 at 23-24 °C. Thereby, the beginning of the constant dim light period is defined as time zero (LL0). 
  7. After 24 h at LL-conditions, harvest the cells by centrifugation (3,000 x g for 5 min at 4 °C) according to the experimental design at different circadian times corresponding to subjective day (LL25 to LL36) and subjective night (LL37 to LL48). After discarding the supernatant, cells can be stored at -80 °C after being frozen in liquid nitrogen (see Notes) or directly used for further purposes.

Data analysis

The design and nomenclature of LD (see Procedure, Step 2) and/or LL conditions (see Procedure, Step 6) is shown in Figure 1 and explained under “Background”. Experimental examples for such growth conditions include the diurnal rhythm of protein abundance for Casein Kinase 1 and the circadian controlled complex formation of the eyespot protein SOUL3 (both in Figure 2; Schulze et al., 2013), the circadian rhythm of nitrite reductase activities (Figure 7; Iliev et al., 2006), growth and bioluminescence circadian rhythms of algal strains (Figure 3; Matsuo et al., 2008) or circadian rhythms of mRNA abundance of clock-relevant Rhythm of Chloroplast (ROC) genes (Figure 8; Matsuo et al., 2008).

Notes

  1. Sterilize the TAP salt solution and the phosphate stock solution by autoclaving (110 °C, 20 min).
  2. Do not store the harvested cells longer than 3 months at -80 °C.
  3. For the trace elements (Hutner et al., 1950), the following procedure should be followed:
    1. At first, all salts except EDTA should be dissolved, each in 75 ml ddH2O. The H3BO3 and FeSO4•7H2O solutions must be heated up for dissolving. Add the disodium EDTA to 250 ml ddH2O and heat the solution to dissolve the EDTA. Combine the hot FeSO4•7H2O solution with the hot EDTA solution. 
    2. Mix all other solutions in a 1 L beaker and heat them up to 70 °C, then add the hot FeSO4-EDTA solution. Keep the temperature at 70 °C and add ~85 ml hot 20% KOH dropwise to obtain a pH of 6.5-6.8 (check with indicator paper). Bring the solution to a final volume of 1 L and let the solution cool down to room temperature. 
    3. Seal the flask with a cotton plug to allow air exchange. Stir the solution with a magnetic stirring bar for several days until the initial clear green color turns to purple. 
    4. Filter the solution through a Whatman® Prepleated filter paper until the solution is clear. Aliquots can be stored at -20 °C.

Recipes

Note: Recipes are adapted from Harris et al. (1989).

  1. TAP salt solution
    16 g NH4Cl
    2 g CaCl2•2H2O
    1 g MgSO4•7H2O
    Add ddH2O to 1 L
  2. 1 M potassium phosphate buffer (pH 7.0)
    43.55 g K2HPO4 in 250 ml
    34.02 g KH2PO4 in 250 ml
    Prepare both solutions separately and add gradually the KH2PO4 solution to the 250 ml K2HPO4 solution until the pH is adjusted to 7.0 (pH electrode)
  3. Hutner’s trace elements (1 L)
    Note: According to Hutner et al. (1950), and Wagner and Mittag (2009).
    For 1 L final mix, the following amounts of salts are needed. The procedure for preparing the trace solution is described above (see Notes).
    22 g ZnSO4•7H2O
    11.4 g H3BO3
    5.06 g MnCl2•4H2O
    1.61 g CoCl2•6H2O
    1.57 g CuSO4•5H2O
    1.1 g (NH4)6Mo7O24•4H2O
    50 g Na2EDTA
    4.99 g FeSO4•7H2
  4. Tris-acetate-phosphate (TAP) medium
    For 1 L TAP medium add:
    2.42 g Tris
    1 ml phosphate buffer (1 M)
    25 ml TAP salt solution (see above)
    1 ml Hutner's trace elements (see above)
    Adjust the pH to 7.0 with acetic acid
    Fill up to 1 L with ddH2O
    Autoclave for 20 min at 110 °C
    For TAP agar plates, add 20 g agar to the solution before autoclaving

Acknowledgments

The work was supported by grants from the German Research Foundation (DFG) to M. Mittag and V. Wagner. The authors declare that they have no conflicts of interest or competing interests.

References

  1. Boesger, J., Wagner, V., Weisheit, W. and Mittag, M. (2014). Comparative phosphoproteomics to identify targets of the clock-relevant casein kinase 1 in C. reinhardtii Flagella. Methods Mol Biol 1158: 187-202.
  2. Forbes-Stovall, J., Howton, J., Young, M., Davis, G., Chandler, T., Kessler, B., Rinehart, C. A. and Jacobshagen, S. (2014). Chlamydomonas reinhardtii strain CC-124 is highly sensitive to blue light in addition to green and red light in resetting its circadian clock, with the blue-light photoreceptor plant cryptochrome likely acting as negative modulator. Plant Physiol Biochem 75: 14-23.
  3. Gaskill, C., Forbes-Stovall, J., Kessler, B., Young, M., Rinehart, C. A. and Jacobshagen, S. (2010). Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas. Plant Physiol Biochem 48(4): 239-246.
  4. Guillard, R. R. L. and Sieracki, M. S. (2005). Counting cells in cultures with the light microscope. In: R. A. Andersen. (Ed.). Algal culturing techniques. pp. 239-252. Elsevier. 
  5. Harris, E. H. (1989). The Chlamydomonas sourcebook. Academic Press.
  6. Hutner, S. H., Provasoli, L., Schatz, A, and Haskins, C. P. (1950). Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94(2): 152-170.
  7. Iliev, D., Voytsekh, O., Schmidt, E. M., Fiedler, M., Nykytenko, A. and Mittag M. (2006). A heteromeric RNA-binding protein is involved in maintaining acrophase and period of the circadian clock. Plant Physiol 142(2): 797-806. 
  8. Kottke, T., Oldemeyer, S., Wenzel, S., Zou, Y. and Mittag, M. (2017). Cryptochrome photoreceptors in green algae: Unexpected versatility of mechanisms and functions. J Plant Physiol 217: 4-14.
  9. Matsuo, T. and Ishiura, M. (2011). Chlamydomonas reinhardtii as a new model system for studying the molecular basis of the circadian clock. FEBS Lett 585(10): 1495-1502.
  10. Matsuo, T., Okamoto, K., Onai, K., Niwa, Y., Shimogawara, K. and Ishiura, M. (2008). A systematic forward genetic analysis identified components of the Chlamydomonas circadian system. Genes Dev 22(7): 918-930. 
  11. Mergenhagen, D. (1984). Circadian clock: genetic characterization of a short period mutant of Chlamydomonas reinhardii. Eur J Cell Biol 33(1): 13-18.
  12. Müller, N., Wenzel, S., Zou, Y., Kunzel, S., Sasso, S., Weiss, D., Prager, K., Grossman, A., Kottke, T. and Mittag, M. (2017). A plant cryptochrome controls key features of the Chlamydomonas circadian clock and its life cycle. Plant Physiol 174(1): 185-201.
  13. Noordally, Z. B. and Millar, A. J. (2015). Clocks in algae. Biochemistry 54(2): 171-183.
  14. Ryo, M., Matsuo, T., Yamashino, T., Ichinose, M., Sugita, M. and Aoki, S. (2016). Diversity of plant circadian clocks: Insights from studies of Chlamydomonas reinhardtii and Physcomitrella patens. Plant Signal Behav 11(1): e1116661.
  15. Schulze, T., Prager, K., Dathe, H., Kelm, J., Kiessling, P. and Mittag, M. (2010). How the green alga Chlamydomonas reinhardtii keeps time. Protoplasma 244(1-4): 3-14.
  16. Schulze, T., Schreiber, S., Iliev, D., Boesger, J., Trippens, J., Kreimer, G. and Mittag, M. (2013). The heme-binding protein SOUL3 of Chlamydomonas reinhardtii influences size and position of the eyespot. Mol Plant 6(3): 931-944. 
  17. Wagner, V. and Mittag, M. (2009). Probing circadian rhythms in Chlamydomonas rheinhardtii by functional proteomics. Methods Mol Biol 479: 173-188.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Wagner, V. and Mittag, M. (2018). Growth of Chlamydomonas reinhardtii under Circadian Conditions. Bio-protocol Bio101: e2982. DOI: 10.21769/BioProtoc.2982.
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