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Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria
通过测定蓝藻叶绿素荧光评估呼吸和光合作用的条件   

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Journal of Photochemistry and Photobiology B: Biology
Mar 2015

Abstract

Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis. Furthermore, chlorophyll fluorescence from cyanobacteria reflects the condition of respiration, since cyanobacterial photosynthesis shares several components of electron transport chain with respiration. This protocol presents the method to monitor the condition of both photosynthesis and respiration in cyanobacteria simply by measuring chlorophyll fluorescence in the dark and in the light with pulse amplitude modulation (PAM) chlorophyll fluorometer.

Keywords: Cyanobacteria (蓝藻), Chlorophyll fluorescence (叶绿素荧光), Non-photochemical quenching (非光化学淬灭), Photosynthesis (光合作用), Respiration (呼吸)

Background

Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis in many photosynthetic organisms (Krause and Weis, 1991; Govindjee, 1995). In the case of cyanobacteria, the photosynthetic prokaryotes, chlorophyll fluorescence can be affected not only by the condition of photosynthesis but also by that of other metabolic pathways due to possible interactions among metabolic pathways within the cells. In particular, photosynthesis and respiration share several components of electron transport chain, such as plastoquinone (PQ) in cyanobacteria (Aoki and Katoh, 1982; Peschek and Schmetterer, 1982). The redox state of the PQ pool influences the yield of chlorophyll fluorescence through the regulation of state transition (Mullineaux and Allen, 1986; Mullineaux et al., 1997), which is the main component of the non-photochemical quenching in cyanobacteria (Campbell and Öquist, 1996). Thus, the cyanobacterial respiratory chain directly affects chlorophyll fluorescence especially in the dark, where photosynthesis is not active.

Due to the influence of respiration, chlorophyll fluorescence should be measured with caution in order to estimate photosynthesis precisely in cyanobacteria (Ogawa et al., 2013). On the other hand, the involvement of both photosynthesis and respiration in cyanobacterial chlorophyll fluorescence allows the estimation of not only photosynthesis but also respiration. In this protocol, we provide the method to monitor the condition of respiration and photosynthesis in cyanobacteria through the analysis of NPQ, the chlorophyll fluorescence parameter reflecting the level of non-photochemical quenching, measured in the dark (NPQDark) and under low light (NPQLL).

Materials and Reagents

  1. Cyanobacteria
    Note: The cyanobacterium Synechocystis sp. PCC 6803 is grown at 30 °C in BG11 medium (Rippka et al., 1979), buffered with 20 mM TES-KOH (pH 8.0) or 20 mM CHES-KOH (pH 9.0) and bubbled with air for 24 h under continuous illumination using fluorescent lamps.
  2. Ferric ammonium citrate (Wako Pure Chemical Industries, catalog number: 092-00802 )
  3. Na2EDTA·2H2O (Wako Pure Chemical Industries, catalog number: 345-01865 )
  4. Sodium nitrate (NaNO3) (Wako Pure Chemical Industries, catalog number: 192-02555 )
  5. Dipotassium hydrogenphosphate (K2HPO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
  6. Magnesium sulfate (MgSO4) (anhydrous) (Wako Pure Chemical Industries, catalog number: 132-00435 )
  7. Calcium chloride (CaCl2) (Wako Pure Chemical Industries, catalog number: 038-24985 )
  8. Sodium carbonate (Na2CO3) (Wako Pure Chemical Industries, catalog number: 199-01585 )
  9. Boric acid (H3BO4) (Wako Pure Chemical Industries, catalog number: 029-02191 )
  10. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
  11. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
  12. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
  13. Disodium molybdate(VI) dihydrate (Na2MoO4·2H2O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
  14. Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
  15. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Wako Pure Chemical Industries, catalog number: 031-03752 )
  16. TES (Wako Pure Chemical Industries,, catalog number: 340-02655 )
  17. Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
  18. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (TCI, catalog number: D1328 )
    Note: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is an inhibitor of electron transport from QA to QB in photosystem II (PSII), thus oxidizing PQ pool under illumination. DCMU is used to determine maximum chlorophyll fluorescence level (Fm). We dissolve DCMU in ethanol (see below), and the stock solution at the concentration of 10 mM is added to the sample (equivalent to the final concentration of 20 μM) to determine the Fm level. DCMU solution can be stored for months in a freezer at -20 °C.
  19. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
  20. BG11 stock solutions (see Recipes)
  21. BG11 medium (see Recipes)
  22. 10 mM DCMU solution in ethanol (see Recipes)

Equipment

  1. Test tubes (IWAKI, catalog number: TEST30NP )
  2. Spherical micro quantum sensor (Heinz Walz, model: US-SQS/L )
    Note: The light meter and the spherical micro-quantum sensor are used for measuring photon flux density of growth light and actinic light of WATER-PAM. It is advisable to use spherical micro-sensor for monitoring the photon flux density of actinic light of WATER-PAM, since the light illuminate samples from multiple directions.
  3. Light meter (LI-COR, model: LI-250A )
  4. Fluorometer (Heinz Walz, model: WATER-PAM )
    Note: WATER-PAM (Heinz Walz, http://www.walz.com/products/chl_p700/water-pam/introduction.html) is a pulse amplitude modulation (PAM) fluorometer designed for measuring aquatic samples with low chlorophyll content. We use the Red LED type of the fluorometer with the emitter-detector unit of CUVETTE Version. The Red LED type of WATER-PAM is equipped with 3 LEDs peaking at 650 nm for the measuring light, 12 LEDs peaking at 660 nm for the actinic light as well as for the saturating pulse, and 3 LEDs peaking at 460 nm for blue light, which preferentially excite photosystem I (PSI) in cyanobacteria.
  5. Quartz cuvette for WATER-PAM (Heinz Walz, model: WATER-K )
  6. Spectrophotometer (JASCO, model: V-650 )
    Note: The optical density of cell cultures at 750 nm is determined by the spectrophotometer. Any other common spectrophotometer can be used for this purpose.

Software

  1. PC software ‘WinControl’ (WALZ, ver.3.22)
    Note: WATER-PAM is operated from the PC software‘WinControl’.

Procedure

  1. The goal of this protocol
    To estimate the condition of both photosynthesis and respiration, we determine NPQ, a parameter representing non-photochemical quenching of chlorophyll fluorescence, which predominantly reflects the redox state of PQ pool. NPQ is calculated as (Fm - Fm’)/Fm’, where Fm is the maximum fluorescence level determined under fully oxidized PQ pool (i.e., State 1 condition), while Fm’ is that under reduced PQ pool conditions where State 2 is partly induced.
    In this protocol, Fm’ is determined both in the dark (Fm’Dark) and under illumination with low light (Fm’LL), and each value is used for calculation of NPQ in the dark (NPQDark = (Fm - Fm’Dark)/Fm’Dark) or that under illumination with low light (NPQLL = (Fm - Fm’LL)/Fm’LL), respectively. From these two parameters, we can collect information about the condition of photosynthesis and respiration.

  2. Culture of cyanobacteria
    The cyanobacterium Synechocystis sp. PCC 6803 is cultured at 30 °C in BG11 medium in a test tube (see in the “Recipes” section), bubbled with air for 24 h under continuous light (120 μmol m-2 sec-1) and served for the measurement. Photon flux density of growth light is determined by a spherical micro-sensor (WALZ, US-SQS/L) with a light meter (LI-250A, LI-COR Biosciences).

  3. Setting WATER-PAM for the measurement
    Prior to the measurement by WATER-PAM, set the levels of the measuring light, actinic light and saturating pulse in the <Settings> panel of the WinControl software. The levels should be set as follows:
    1. Measuring light frequency (<Freq.> setting) at the level 2 of 12 steps.
    2. Measuring light intensity (<Int.> setting) at the level 2 of 12 steps.
      Note: By these settings, measuring light is weak enough to avoid excitation of photosystems (see also the note below).
    3. Saturating pulse intensity (<Int.> setting) at the level 12 of 12 steps and saturating pulse width at 0.8 sec.
    4. Actinic light amplitude (<Ampl.> setting) at the level 10 of 12 steps and actinic light intensity (<Int.> setting) at the level that gives the necessary photon flux density of the actinic light.
      Note: We determine photon flux density of actinic light in a cuvette (WALZ, WATER-K) filled with MilliQ water by using a spherical micro-quantum sensor (WALZ, US-SQS/L) with a light meter (LI-COR Biosciences, LI-250A). Photon flux density of measuring light, saturating pulse and actinic light by the settings described above is < 0.01, > 4,000 or 100 μmol m-2 sec-1, respectively.

  4. The procedure for recording chlorophyll fluorescence in cyanobacteria by WATER-PAM
    1. Preparation of a sample for the measurement
      Cell cultures are adjusted to the optical density of 0.2 at 750 nm, which is approximately equivalent to 1 μg/ml of chlorophyll in Synechocystis sp. PCC 6803, when cells are grown under the condition described in the “Materials and Reagents” section.
      Notes:
      1. Prepare > 4 ml of the sample. 2 ml of the sample will be used for the measurement of Fm’, and another 2 ml for the measurement of Fm.
      2. In the case of Synechocystis sp. PCC 6803, the sample is not necessary to be stirred during the measurement within about 15 min. If you deal with samples necessary to be stirred, the stirring device for CUVETTE Version of WATER-PAM (WALZ, WATER-S) is available for stirring cell culture during the measurement.
    2. Measurements of Fm’, the quenched fluorescence level, either in the dark (Fm’Dark) or under illumination with low light (Fm’LL)
      1. The sample (2 ml) is dark-acclimated for 10 min prior to the measurement in the sample chamber of the WATER-PAM.
        Note: Please make sure that checkboxes in the <Status> panel (circled by a yellow square in Figure 1, a screenshot of the software) at the bottom left of the window of the WinControl software are all cleared except for the <PM active> checkbox during the dark-acclimation.
      2. After the dark-acclimation, time course change in the level of chlorophyll fluorescence is determined in the <Chart> panel (circled by a red square in Figure 1) of the WinControl software as follows: 
        1. First, start the recording of chlorophyll fluorescence by clicking <Start Onl. Rec.> button at the bottom right of the <Chart> panel (or checking <Rec. Online> checkbox at the top right of the <Chart> panel). Then, start illumination with measuring light by checking <Meas. Light> checkbox in the <Status> panel at the bottom left of the window. The level of chlorophyll fluorescence rises from zero to the Fo’ level (This is NOT the Fo level in the case of cyanobacteria. See Note described below), the minimum fluorescence level with reduced PQ pool already in the dark.
          Note: Please keep in mind that, unlike in the case of land plants, the chlorophyll fluorescence level is already quenched from original Fo level to Fo' level in the dark-acclimated cyanobacterial cells, due to the reduction of PQ pool by respiratory electron transport in the dark.
        2. Subsequent application of 0.8 sec pulse of saturating light by checking the <SAT-Pulse> checkbox in the <Status> panel at the bottom left of the window induces the increase of fluorescence level to Fm’ level in the dark (Fm’Dark). The value of this Fm’ level in the dark, which is used for calculating NPQDark = (Fm - Fm’Dark)/Fm’Dark, is displayed in the <Result> panel (circled by a blue square in Figure 1) at the right side of the window and can be always checked in the <Report> panel.
          Note: Please note that the peak height of the fluorescence increase triggered by the application of saturating light may not reach the level of Fm' (indicated by 'x' in Figure 1) because of the problem in pixel resolution of the screen.
        3. Subsequently, start illumination with actinic light by checking <Act. Light> checkbox in the <Status> panel. After the fluorescence level reaches the stable level under illumination, apply a saturating pulse to the sample to determine the Fm’ level under illumination with low light (the Fm’ level used for calculating NPQLL, which is calculated as (Fm - Fm’LL)/Fm’LL).


      Figure 1. The window of the PC software “WinControl”, displaying time course change in chlorophyll fluorescence in <Chart> panel. Black arrowheads represent the time of illumination with saturating pulse. In the measurement shown in Figure 1, effect of high light (HL) was examined following the routine determination of Fm’ in the dark and under low light.

    3. Measurement of Fm, the maximum fluorescence level
      1. To measure the Fm level, add 20 μM DCMU (final concentration) to 2 ml of the fresh sample prepared in Step D1.
        Note: Since the chlorophyll fluorescence in cyanobacteria is already quenched in the dark, the maximum fluorescence level (Fm) must be determined in the presence of DCMU in the light, which oxidizes PQ pool and thus brings the cells to State 1 to eliminate quenching of the fluorescence. It is possible to add DCMU to the sample after determining Fm’ levels as described above, but sometimes actinic illumination may change the condition of the sample to interfere the precise determination of Fm, especially in the case of gene-disruptants. Thus, it is advisable to determine Fm level in a separate fresh sample.
      2. Set the sample in the sample chamber of WATER-PAM, and then start recording with measuring light. To determine the Fm level, apply saturating pulse to the sample under different levels of actinic light. Actinic light should be changed from low level to high level by clicking the upward arrow at the right side of <Act. Int> value in <Basic> panel at the bottom of the window, and find the maximum level of fluorescence signal by confirming the value of Fm’ in the <Result> panel.
        Note: There is no need to dark-acclimate the sample prior to the measurement of Fm in this procedure. And note that the value of Fm is displayed in the <Fm’> Box but not in the <Fm> Box in the <Result> panel in this procedure, because the <Fo, Fm> button is not used (see the “Notes” section).

Data analysis

  1. In cyanobacteria, it has been reported that the level of non-photochemical quenching is high both under dark condition and under high light condition, while low under light intensity near to growth light condition, and the light-dependency of non-photochemical quenching describes a concave curve (Campbell and Öquist, 1996; Sonoike et al., 2001). The high level of non-photochemical quenching in the dark reflects the reduction of the PQ pool by the respiratory chain, while the low level of that under growth light condition reflects the oxidation of PQ pool through the activity of PSI. The situation is different in the disruptant of ndhF1 gene which encodes a subunit of type 1 NAD(P)H dehydrogenase (NDH-1) complex donating electron to the PQ pool in the respiratory chain (Mi et al., 1992; Battchikova et al., 2011), of the cyanobacterium Synechocystis sp. PCC 6803. The difference between NPQdark and NPQLL was diminished and the concave shape of the light-dependency curve was hardly observed (Ogawa and Sonoike, 2015). This indicates that the light-dependency of non-photochemical quenching reflects the balance between respiration and photosynthesis in cyanobacteria, through the redox state of the PQ pool. In other words, the level of non-photochemical quenching reflects the condition of both respiration and photosynthesis.
  2. The value of NPQLL could be affected by the condition of respiration as well as by that of photosynthesis. We have reported that the disruption of ndhF1 gene or ndhD1/D2 genes, which encode subunits of the NDH-1 complex serving in the respiratory electron transport, brought about the decrease of NPQLL as well as of NPQDark through the oxidation of the PQ pool under illumination due to the poor electron supply to the PQ pool from the respiratory chain (Ogawa and Sonoike, 2015). To assess whether the change in the value of NPQLL is caused by respiration or photosynthesis, it is also necessary to determine NPQDark.
  3. The value of fluorescence level necessary to calculate NPQ can be checked in the panel at the right side of the window (circled by a blue square in Figure 1) during the measurement or any time in the panel. In the panel, the measured fluorescence level is displayed in the order of time course. The value of Fm’Dark or that of Fm’LL, in the measurement demonstrated in Figure 1, is 481 or 508, respectively, and that of Fm measured with a fresh sample is 638. In this case, NPQDark and NPQLL are calculated as;
    NPQDark = (Fm - Fm’Dark)/Fm’Dark = (638 - 481)/481 = 0.326
    NPQLL = (Fm - Fm’LL)/Fm’LL= (638 - 508)/508 = 0.256
    In this particular case, the value of NPQDark (0.362) is higher than that of NPQLL (0.256). This is a sure sign of the reduced PQ pool in the dark usually brought about by the electron flow from respiratory chain. In the gene-disruptant of Synechocystis sp. PCC 6803 deficient in electron supply to PQ pool from respiratory NDH-1 complex, NPQDark is much smaller than that in wild type strain (Ogawa and Sonoike, 2015). Furthermore, in some cyanobacterial species that adapted to low light environment, the level of NPQDark was reported to be also very low (Misumi et al., 2016). NPQ decreases upon light illumination from NPQDark to NPQLL, presumably due to the oxidation of PQ pool by the action of PSI. Thus, the difference between the level of NPQDark and that of NPQLL could be used for the evaluation of the relative content of PSI. At least in certain cases, the positive correlation between relative difference of NPQ (i.e., (NPQDark - NPQLL)/NPQLL) and the photosystem stoichiometry (i.e., the ratio of PSI/PSII) was observed (Ogawa and Sonoike, 2015). Interestingly, these characteristics were observed not only in cyanobacteria but also in eukaryotic algae. It has been reported that in the glaucophyte Cyanophora paradoxa, PQ pool in the dark is reduced by chlororespiration, and it exhibits high NPQDark in the dark-acclimated cells (Misumi and Sonoike, 2017). Thus, this protocol can be applied to the evaluation of interaction between photosynthesis and chlororespiration at least in some eukaryotic algae, in addition to the measurements in cyanobacteria. For the interpretation of the results, however, one must pay attention to the points described below.
  4. It must be noted that, not only fluorescence from chlorophyll of PSII but also that from phycobilisome, which does not quench as PSII fluorescence does, contributes to the fluorescence signal in cyanobacteria (Campbell et al., 1998). Contribution of the phycobilisome fluorescence to the fluorescence signal causes underestimation of the chlorophyll fluorescence parameters, including NPQ (Ogawa and Sonoike, 2016). Therefore, the values of NPQ cannot be compared between samples with different phycobilisome content. Correction of the fluorescence signal through the subtraction of the contribution of phycobilisome fluorescence is necessary to determine the actual level of chlorophyll fluorescence (Ogawa and Snoike, 2016).
  5. The value of NPQ can be affected by the action of orange carotenoid protein (OCP) under strong blue light (Kirilovsky, 2007). In this protocol, however, action of OCP is negligible, since red LEDs are used as the actinic light for the measurements.

Notes

  1. The procedure of measuring chlorophyll fluorescence by PAM fluorometer in cyanobacteria is different in many ways from that generally used for land plants. Fm level can be determined in land plants by illumination with saturating pulse in the dark, where photosynthesis is not active and the PQ pool is oxidized. However, PQ pool is reduced in the dark by respiratory chain in many cyanobacterial species (Misumi et al., 2016). Thus, Fm level should be determined either in the presence of DCMU in the light (Campbell et al., 1998) or under weak blue light which preferentially excites PSI (El Bissati et al., 2000) by oxidizing PQ pool in cyanobacteria. In this protocol, the procedure of measuring Fm in the presence of DCMU is introduced.
  2. Since PC software of WATER-PAM, WinControl, is not developed for cyanobacteria, there are two problems of this software to use in cyanobacterial measurements. First, the Fm level cannot be determined by clicking the button in the dark, since Fm could not be attained by dark acclimation in cyanobacteria as described above. Secondary, the values of the chlorophyll fluorescence parameters calculated automatically by the software are meaningless, since Fo and Fm levels cannot be determined by the action of the button. If you want to use the button, it is necessary to use it under illumination with weak blue light, which preferentially excites PSI and oxidizes the PQ pool.

Recipes

Note: The solutions are prepared with distilled water unless otherwise specified.

  1. BG11 stock solutions
    1. Stock solution #1
      0.3 g of ferric ammonium citrate
      0.05 g of Na2EDTA·2H2O
      Per 100 ml of H2O
    2. Stock solution #2
      30 g of NaNO3
      0.78 g of K2HPO4
      0.73 g of MgSO4 (anhydrous)
      Per 1,000 ml of H2O
    3. Stock solution #3
      1.43 g of CaCl2 (anhydrous)
      Per 100 ml of H2O
    4. Stock solution #4
      2 g of NaNO3
      Per 100 ml of H2O
    5. Stock solution #5
      2.86 g of H3BO4
      1.81 g of MnCl2·4H2O
      0.22 g of ZnSO4·7H2O
      0.08 g of CuSO4·5H2O
      0.021 g of Na2MoO4·2H2O
      10 μl of sulfuric acid
      0.049 g of Co (NO3)2·6H2O
      Per 1,000 ml of H2O
    6. Stock solution #6
      1 M TES adjusted to pH 8.0 by 1 M KOH
    Excepting the stock solution #1, the other BG11 stock solutions are autoclaved at 121 °C for 20 min. All the BG11 stock solutions are stored at 4 °C 
  2. BG11 medium
    1. For 1 L BG11 medium, add
      2 ml of Stock solution #1
      50 ml of Stock solution #2
      2 ml of Stock solution #3
      1 ml of Stock solution #4
      1 ml of Stock solution #5
      20 ml of Stock solution #6
      Adjust the final volume to 1,000 ml with H2O
    2. Distribute BG11 medium into test tubes each with 30 ml
    3. Autoclave the distributed BG11 medium at 121 °C for 20 min and then store at room temperature
  3. 10 mM DCMU solution in ethanol
    Dissolve DCMU in ethanol, store the solution at -20 °C

Acknowledgments

This work was supported by JSPS Grant-in-Aid for Scientific Research on Innovative Areas (No. 16H06552 and No. 16H06553 to K.S.) and Grant-in-Aid for Scientific Research (B) (No. 16H04809 to K.S.). The authors declare no conflicts of interest.

References

  1. Aoki, M. and Katoh, S. (1982). Oxidation and reduction of plastoquinone by photosynthetic and respiratory electron transport in a cyanobacterium Synechococcus sp. Biochem Biophys Acta 682: 307-314.
  2. Battchikova, N., Eisenhut, M. and Aro, E. M. (2011). Cyanobacterial NDH-1 complexes: novel insights and remaining puzzles. Biochim Biophys Acta 1807(8): 935-944.
  3. Campbell, D., Hurry, V., Clarke, A. K., Gustafsson, P. and Oquist, G. (1998). Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiol Mol Biol Rev 62(3): 667-683.
  4. Campbell, D. and Öquist, G. (1996). Predicting light acclimation in cyanobacteria from nonphotochemical quenching of photosystem II fluorescence, which reflects state transitions in these organisms. Plant Physiol 111(4): 1293-1298.
  5. El Bissati, K., Delphin, E., Murata, N., Etienne, A. and Kirilovsky, D. (2000). Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms. Biochim Biophys Acta 1457(3): 229-242.
  6. Govindjee. (1995). Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust J Plant Physiol 22: 131-160.
  7. Kirilovsky, D. (2007). Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism. Photosynth Res 93(1-3): 7-16.
  8. Krause, G. H. and Weis, E. (1991). Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42: 313-349.
  9. Mi, H., Endo, T., Schreiber, U., Ogawa, T. and Asada, K. (1992). Electron donation from cyclic and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis PCC 6803. Plant Cell Physiol 33: 1233-1237.
  10. Misumi, M., Katoh, H., Tomo, T. and Sonoike, K. (2016). Relationship between photochemical quenching and non-photochemical quenching in six species of cyanobacteria reveals species difference in redox state and species commonality in energy dissipation. Plant Cell Physiol 57: 1510-1517.
  11. Misumi, M. and Sonoike, K. (2017). Characterization of the influence of chlororespiration on the regulation of photosynthesis in the glaucophyte Cyanophora paradoxa. Sci Rep 7: 46100.
  12. Mullineaux, C. W. and Allen, J. F. (1986). The state 2 transition in the cyanobacterium Synechococcus 6301 can be driven by respiratory electron flow into the plastoquinone pool. FEBS Lett 205: 155-160.
  13. Mullineaux, C. W., Tobin, M. J. and Jones, G. R. (1997). Mobility of photosynthetic complexes in thylakoid membranes. Nature 390: 421-424.
  14. Ogawa, T., Harada, T., Ozaki, H. and Sonoike, K. (2013). Disruption of the ndhF1 gene affects Chl fluorescence through state transition in the cyanobacterium Synechocystis sp. PCC 6803, resulting in apparent high efficiency of photosynthesis. Plant Cell Physiol 54(7): 1164-1171.
  15. Ogawa, T. and Sonoike, K. (2015). Dissection of respiration and photosynthesis in the cyanobacterium Synechocystis sp. PCC6803 by the analysis of chlorophyll fluorescence. J Photochem Photobiol B 144: 61-67.
  16. Ogawa, T. and Sonoike, K. (2016). Effects of bleaching by nitrogen deficiency on the quantum yield of photosystem II in Synechocystis sp. PCC 6803 revealed by Chl fluorescence measurements. Plant Cell Physiol 57(3): 558-567.
  17. Peschek, G. A. and Schmetterer, G. (1982). Evidence for plastoquinol-cytochrome f/b-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria. Biochem Biophys Res Commun 108(3): 1188-1195.
  18. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J General Microbiology 111: 1-61.
  19. Sonoike, K., Hihara, Y. and Ikeuchi, M. (2001). Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803. Plant Cell Physiol 42(4): 379-384.

简介

叶绿素荧光测量已被广泛用于监测光合作用的状况。 此外,蓝细菌的叶绿素荧光反映了呼吸的状况,因为蓝藻光合作用与呼吸作用共享电子传递链的几个组成部分。 该协议提供了监测蓝藻光合作用和呼吸作用的方法,只需通过测量黑暗和光照下的叶绿素荧光以及脉冲幅度调制(PAM)叶绿素荧光计。

【背景】叶绿素荧光测量已广泛用于监测许多光合生物体的光合作用条件(Krause和Weis,1991; Govindjee,1995)。在蓝细菌的情况下,光合原核生物,叶绿素荧光不仅受光合作用条件的影响,而且还受其他代谢途径的影响,这是由于细胞内代谢途径之间的可能相互作用。特别是,光合作用和呼吸作用共享电子传递链的几个组成部分,如蓝细菌中的质体醌(PQ)(Aoki and Katoh,1982; Peschek and Schmetterer,1982)。 PQ池的氧化还原状态通过调节状态转变影响叶绿素荧光的产量(Mullineaux和Allen,1986; Mullineaux等人,1997),其是非天然存在的主要组分,蓝藻中的光化学猝灭(Campbell和Öquist,1996)。因此,蓝藻呼吸链直接影响叶绿素荧光,特别是在光合作用不活跃的黑暗中。

由于呼吸的影响,应谨慎测量叶绿素荧光以精确估计蓝藻中的光合作用(Ogawa et al。,2013)。另一方面,光合作用和呼吸作用参与蓝藻叶绿素荧光,不仅可以估计光合作用,还可以估计呼吸作用。在这个协议中,我们提供了通过分析NPQ分析监测蓝藻呼吸和光合作用的方法,NPQ是反映非光化学猝灭水平的NPQ,在黑暗中测量(NPQ )和低光照下(NPQ

关键字:蓝藻, 叶绿素荧光, 非光化学淬灭, 光合作用, 呼吸

材料和试剂

  1. 蓝细菌
    注:蓝藻Synechocystis sp。 PCC 6803在30℃下在BG11培养基(Rippka等,1979)中生长,用20mM TES-KOH(pH8.0)或20mM CHES-KOH(pH9.0)缓冲并在空气中连续鼓泡24小时使用荧光灯照明。
  2. 柠檬酸铁铵(Wako Pure Chemical Industries,目录号:092-00802)
  3. Na 2 EDTA·2H 2 O(Wako Pure Chemical Industries,目录号:345-01865)
  4. 硝酸钠(NaNO 3)(Wako Pure Chemical Industries,目录号:192-02555)
  5. 磷酸氢二钾(KH 2 HPO 4)(Wako Pure Chemical Industries,目录号:164-04295)
  6. 硫酸镁(MgSO 4)(无水)(Wako Pure Chemical Industries,目录号:132-00435)
  7. 氯化钙(CaCl 2 2)(Wako Pure Chemical Industries,目录号:038-24985)
  8. 碳酸钠(Na 2 CO 3)(Wako Pure Chemical Industries,目录号:199-01585)
  9. 硼酸(H 3 BO 4)(Wako Pure Chemical Industries,目录号:029-02191)
  10. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Wako Pure Chemical Industries,目录号:139-00722)
  11. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Wako Pure Chemical Industries,目录号:264-00402)
  12. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Wako Pure Chemical Industries,目录号:039-04412)
  13. 钼酸钠(VI)二水合物(Na 2 MoO 4·2H 2 O)(Wako Pure Chemical Industries,目录号:196-02472)
  14. 硫酸(Wako Pure Chemical Industries,目录号:192-04696)
  15. 硝酸钴(II)六水合物(Co(NO 3)2·6H 2 O)(Wako Pure Chemical Industries,目录号:031- 03752)
  16. TES(Wako Pure Chemical Industries,目录号:340-02655)
  17. 氢氧化钾(KOH)(Wako Pure Chemical Industries,目录号:168-21815)
  18. 3-(3,4-二氯苯基)-1,1-二甲基脲(DCMU)(TCI,目录号:D1328)
    注意:3-(3,4-二氯苯基)-1,1-二甲基脲(DCMU)是在光系统II(PSII)中从QA到QB的电子传递抑制剂,因此在光照下氧化PQ池。 DCMU用于确定最大叶绿素荧光水平(Fm)。我们将DCMU溶解在乙醇中(见下文),并将10mM浓度的储备溶液加入样品中(相当于最终浓度为20μM)以确定Fm水平。 DCMU溶液可在-20°C的冷冻箱中储存数月。
  19. 乙醇(Wako Pure Chemical Industries,目录号:057-00456)
  20. BG11储备液(见食谱)
  21. BG11中(见食谱)
  22. 10mM DCMU乙醇溶液(见食谱)

设备

  1. 试管(IWAKI,目录号:TEST30NP)
  2. 球形微量子传感器(Heinz Walz,型号:US-SQS / L)
    注:光度计和球形微量子传感器用于测量生长光和WATER-PAM光化光的光子通量密度。建议使用球形微型传感器来监测WATER-PAM光化光的光通量密度,因为光线会照射多个方向的样品。
  3. 测光表(LI-COR,型号:LI-250A)
  4. 荧光计(Heinz Walz,型号:WATER-PAM)
    注:WATER-PAM(Heinz Walz, http:// www .walz.com / products / chl_p700 / water-pam / introduction.html )是一种脉冲幅度调制(PAM)荧光仪,用于测量叶绿素含量低的水生样品。我们使用荧光计的红色LED类型与CUVETTE版本的发射器 - 检测器单元。红色LED类型的WATER-PAM配备了3个峰值为650 nm的LED,用于测量光,12个LED在660 nm处为光化光以及饱和脉冲峰化,3个LED在460 nm处峰值为蓝色光,它优先激发蓝藻中的光系统I(PSI)。
  5. 石英比色皿用于WATER-PAM(Heinz Walz,型号:WATER-K)
  6. 分光光度计(JASCO,型号:V-650)
    注:750nm处的细胞培养物的光密度由分光光度计测定。任何其他常见的分光光度计都可以用于此目的。

软件

  1. PC软件'WinControl'(WALZ,ver.3.22)
    注意:WATER-PAM是通过PC软件'WinControl'运行的。

程序

  1. 这个协议的目标
    为了估计光合作用和呼吸作用的条件,我们确定了代表叶绿素荧光的非光化学猝灭的NPQ,其主要反映了PQ池的氧化还原状态。 NPQ计算为(Fm-Fm')/ Fm',其中Fm是在完全氧化的PQ池(即,状态1状态)下确定的最大荧光水平,而Fm'是在PQ降低其中状态2部分被诱导的池条件。
    在该协议中,Fm'既在黑暗中(Fm'Dark>)和在低光照下(Fm'LL )下被确定,并且每个值被用于计算的NPQ在黑暗中(NPQ =(Fm-Fm'Dark Dark> / Fm'Dark )或者在低光照下NPQ LL =(Fm-Fm'LL )/ Fm'LL )。从这两个参数中,我们可以收集有关光合作用和呼吸状况的信息。

  2. 蓝藻培养
    蓝细菌集胞藻 sp。将PCC 6803在30℃的BG11培养基中在试管中培养(参见“食谱”部分),在空气中连续光照(120μmolm -2 s -1 )并用于测量。生长光的光子通量密度由具有光度计(LI-250A,LI-COR Biosciences)的球形微型传感器(WALZ,US-SQS / L)确定。

  3. 为测量设置WATER-PAM
    在用WATER-PAM测量之前,在&lt;设置&gt;设置测量光,光化光和饱和脉冲的电平。 WinControl软件的面板。级别应设置如下:
    1. 在12级的2级测量光频率(&lt; Freq。&gt;设置)。
    2. 在12级的级别2测量光强度(&lt; Int。&gt;设置)。
      注意:通过这些设置,测量光线足够弱以避免激发光系统(另请参阅下面的注释)。
    3. 饱和脉冲强度(设置)在12级的12级和饱和脉冲宽度0.8秒。
    4. 在提供光化光所需的光子通量密度的水平上,在12级的级别10处的光化光幅度(&lt; Ampl。&gt;设置)和光化光强度(&lt; Int。&gt; > 注:我们通过使用带有光度计的球形微量子传感器(WALZ,US-SQS / L)来确定用MilliQ水填充的比色杯(WALZ,WATER-K)中的光化光通量密度(LI -COR Biosciences,LI-250A)。通过上述设置测量光,饱和脉冲和光化光的光子通量密度为&lt; 0.01,&gt; 4,000或100μmol -2 sec -1 ,分别。

  4. 通过WATER-PAM记录蓝细菌叶绿素荧光的程序
    1. 为测量准备样品
      将细胞培养物调整至在750nm处0.2的光密度,其大致等于集胞藻中1μg/ ml叶绿素。 PCC 6803,当细胞在“材料和试剂”部分描述的条件下生长时。
      备注:
      1. 准备&gt; 4毫升的样品。 2毫升的样品将用于测量Fm',另外2毫升的样品用于测量Fm。
      2. Synechocystis sp。 PCC 6803,在约15分钟内测量时不需要搅拌样品。如果您处理需要搅拌的样品,可使用CUVETTE版本的WATER-PAM(WALZ,WATER-S)搅拌装置在测量过程中搅拌细胞培养物。
    2. 在暗处(Fm'Dark)或在低光照(Fm'nL)下照射猝灭的荧光水平Fm'的测量值
      1. 在WATER-PAM的样品室中测量之前,将样品(2ml)暗适应10分钟。
        注意:请确保&lt; Status&gt;中的复选框WinControl软件的窗口左下方的面板(图1中的黄色方框圈出的软件的屏幕截图)除了&lt; PM active&gt;&gt;在黑暗适应期间复选框。
      2. 在黑暗驯化之后,叶绿素荧光水平的时程变化在&lt; Chart&gt; WinControl软件的面板(在图1中用红色方框圈起来)如下:&nbsp;
        1. 首先,通过点击&lt; Start Onl开始记录叶绿素荧光。 。REC&GT;按钮位于&lt; Chart&gt;右下角(或检查&lt;图表&gt;面板右上角的&lt; Rec。Online&gt;复选框)。然后,通过检查&lt; Meas,开始用测量光照明。光&GT; &lt; Status&gt;中的复选框窗口左下角的面板。叶绿素荧光水平从0上升到Fo'水平(这不是蓝藻细菌的Fo水平,参见下面的注释),最小荧光水平与PQ水池已经在黑暗中降低了。
          注意:请记住,与陆地植物不同的是,由于PQ池的减少,在黑暗驯化的蓝细菌细胞中叶绿素荧光水平已经从原始Fo水平猝灭至Fo'水平呼吸电子在黑暗中运输。
        2. 通过检查&lt; SAT-Pulse&gt;来随后施加0.8秒的饱和光脉冲。 &lt; Status&gt;中的复选框在窗口左下方的面板诱导荧光水平在黑暗中的Fm'水平增加(Fm'Dark )。这个Fm'在黑暗中的水平值,用于计算NPQ =(Fm-Fm'Dark )/ Fm'Dark >,显示在&lt; Result&gt;面板(在图1中用蓝色方框圈出),并且可以在&lt; Report&gt;面板。
          注意:请注意,由于屏幕像素分辨率的问题,由饱和光照引起的荧光增加的峰值高度可能不会达到Fm'的水平(由图1中的'x'表示) 。
        3. 随后,通过检查&lt; Act,开始用光化光照射。光&GT; &lt; Status&gt;中的复选框面板。在荧光水平达到照明下的稳定水平后,对样品施加饱和脉冲以确定低光照下的Fm'水平(用于计算NPQ 的Fm'水平,其计算如(Fm-Fm'LL )/ Fm'LL )。


      图1. PC软件“WinControl”的窗口,显示在<图表>中的叶绿素荧光的时间变化。面板。黑色箭头代表饱和脉冲照明时间。在图1所示的测量中,在黑暗和低光条件下常规测定Fm'后,检查了高光(HL)的影响。

    3. Fm的测量,最大荧光水平
      1. 为了测量Fm水平,向步骤D1中制备的2ml新鲜样品中加入20μMDCMU(终浓度)。
        注:由于蓝藻中的叶绿素荧光已在黑暗中猝灭,因此必须在DCMU存在的情况下测定最大荧光水平(Fm),从而氧化PQ池,从而使细胞处于状态1至消除荧光淬灭。在如上所述确定Fm'水平之后,可以将DCMU添加到样品中,但是有时光化照射可能改变样品的条件以干扰Fm的精确测定,尤其是在基因破坏的情况下。因此,建议在单独的新鲜样品中确定Fm水平。
      2. 将样品放置在WATER-PAM的样品室中,然后用测量光开始记录。为了确定Fm水平,在不同光化学水平下对样品施加饱和脉冲。通过点击&lt; Act右侧的向上箭头,光化光应该从低电平变为高电平。 INT&GT;值在&lt; Basic&gt;通过确认&lt;结果&gt;中的Fm'的值,求出荧光信号的最大值。面板。
        注意:在此程序中测量Fm之前,不需要对样品进行暗适应。并且注意Fm的值显示在&lt; Fm&gt;&gt; Box,但不在&lt; Fm&gt; &lt; Result&gt;中的框因为&lt; Fo,Fm&gt;按钮不使用(请参阅“注释”部分)。 <设置>

数据分析

  1. 据报道,在蓝藻中,非光化学淬灭的水平在黑暗条件下和高光照条件下都很高,而在光照强度接近生长光条件下很低,并且非光化学淬灭的光依赖性描述了凹曲线(Campbell和Öquist,1996; Sonoike等人,2001)。黑暗中非光化学淬灭的高水平反映了呼吸链中PQ池的减少,而生长光下的低水平反映了PQ池通过PSI活性的氧化。编码1型NAD(P)H脱氢酶(NDH-1)复合物的亚基的emD ndhF1基因的破坏情况与呼吸链中的PQ库捐献电子的情况不同(Mi 之间的差异减小,光依赖曲线的凹形几乎未观察到(Ogawa和Sonoike,2015)。这表明非光化学猝灭的光依赖性反映了通过PQ池的氧化还原状态在蓝细菌中的呼吸和光合作用之间的平衡。换句话说,非光化学猝灭的水平反映了呼吸和光合作用的条件。
  2. NPQ LL 的值可能受呼吸作用和光合作用的影响。我们已经报道,编码在呼吸电子传递中起作用的NDH-1复合物亚基的 ndhF1 基因或 ndhD1 / D2 基因的破坏导致通过氧化PQ池在光照下由于从呼吸链到PQ池的电子供应不足而引起的NPQ LL 和NPQ 的变化是否由呼吸或光合作用引起,还需要确定NPQ
  3. 计算NPQ所需的荧光水平值可以在测量过程中或任何时候在面板中的窗口右侧(图1中用蓝色方框圈出)的面板中检查。在<报告>面板中,测量的荧光水平按时间顺序显示。在图1所示的测量中,Fm'Dark或Fm'LL的值分别是481或508,而Fm'的值是用新鲜测量的样本为638.在这种情况下,NPQ 和NPQ 计算为;
    NPQ Dark =(Fm-Fm'Dark =)/ Fm'Dark =(638-481)/481=0.332
    NPQ LL =(Fm-Fm'LL /)/ Fm'LL =(638-508)/508=0.256
    在这种特殊情况下,NPQ (0.362)的值高于NPQ (0.256)。这是通常由来自呼吸链的电子流引起的黑暗中PQ池减少的确切迹象。在 Synechocystis sp的基因破坏者中。呼吸NDH-1复合体电子供给PQ池的PCC 6803缺乏,NPQ Dark小于野生型菌株(Ogawa和Sonoike,2015)。此外,在一些适应低光照环境的蓝藻物种中,据报NPQ Dark的水平也很低(Misumi等人,2016年)。 NPQ从黑暗到NPQ LL的光照下降,可能是由于PQ池在PSI作用下的氧化。因此,NPQ 和NPQ 之间的差异可以用于评估PSI的相对含量。至少在某些情况下,NPQ(即NPQ NPQ )/ NPQ 的相对差异与NPQ LL)和光系统化学计量比(即em / ie,PSI / PSII的比例)(Ogawa和Sonoike,2015)。有趣的是,这些特征不仅在蓝细菌中观察到,而且在真核藻类中也观察到。据报道,在萤光绿藓Cyanophora paradoxa中,黑暗中的PQ池被氯化吸收减少,并且在黑暗驯化的细胞中表现出高NPQ Dark(Misumi和Misumi Sonoike,2017)。因此,除了在蓝藻中进行测量之外,该方案至少在一些真核藻类中可用于评估光合作用和绿色呼吸之间的相互作用。然而,为了解释结果,我们必须注意以下几点。
  4. 必须注意的是,不仅PSII叶绿素发出的荧光,而且来自藻胆体的荧光都不会像PSII荧光那样猝灭,这有助于蓝藻中的荧光信号(Campbell等人,1998年) 。藻胆体荧光对荧光信号的贡献导致低估了叶绿素荧光参数,包括NPQ(Ogawa和Sonoike,2016)。因此,在不同藻胆体含量的样品之间不能比较NPQ的值。通过减去藻胆体荧光的贡献来校正荧光信号对于确定叶绿素荧光的实际水平是必要的(Ogawa和Snoike,2016)。
  5. 在强蓝光下,橙黄色类胡萝卜素蛋白(OCP)的作用可影响NPQ的值(Kirilovsky,2007)。然而,在这个协议中,OCP的行为是微不足道的,因为红色LED被用作测量光化光。

笔记

  1. 通过PAM荧光计测量蓝藻中叶绿素荧光的程序在许多方面与通常用于地面植物的方法不同。 Fm水平可以在陆地植物中通过饱和脉冲在黑暗中进行照明来确定,其中光合作用不活跃并且PQ池被氧化。然而,在很多蓝藻种类中,PQ池在黑暗中被呼吸链减少(Misumi等人,2016)。因此,Fm水平应当在存在DCMU的情况下(坎贝尔等人,1998)或在优先激发PSI的弱蓝光下测定(El Bissati等人, / em>,2000)通过氧化蓝藻中的PQ池。在该协议中,介绍了在DCMU存在下测量Fm的过程。
  2. 由于Water-PAM的WinControl软件不是针对蓝细菌开发的,因此该软件在蓝藻测量中有两个问题。首先,通过点击黑暗中的按钮不能确定Fm水平,因为如上所述Fm不能通过蓝藻中的暗适应达到。次要的,由软件自动计算的叶绿素荧光参数的值是没有意义的,因为Fo和Fm水平不能通过>按钮的作用来确定。如果您想使用按钮,则需要在弱蓝光的照明下使用它,这会优先激发PSI并氧化PQ池。

食谱

注意:除非另有说明,溶液均用蒸馏水配制。

  1. BG11库存解决方案
    1. 库存解决方案#1
      0.3克柠檬酸铁铵
      0.05g的Na 2 EDTA·2H 2 O
      每100毫升H 2 O 0
    2. 库存解决方案#2
      30克NaNO 3。
      0.78g的K 2 HPO 4 4 0.73克MgSO 4(无水)
      每1000ml H 2 O
    3. 库存解决方案#3
      1.43克CaCl 2(无水)
      每100毫升H 2 O 0
    4. 库存解决方案#4
      2克的NaNO 3 /
      每100毫升H 2 O 0
    5. 库存解决方案#5
      2.86g的H 3 BO 4 4 1.81g的MnCl 2·4H 2 O·O 0.22g的ZnSO 4·7H 2 O 0.08克CuSO 4·5H 2 O
      0.021g的Na 2 MoO 4·2H 2 O:0 0 10μl硫酸
      0.049克Co(NO 3)2·6H 2 O
      每1000ml H 2 O
    6. 库存解决方案#6
      1 M TES通过1M KOH调节至pH 8.0
    除了储备溶液#1之外,其他BG11储备溶液在121℃高压灭菌20分钟。所有BG11储备溶液都储存在4°C。
  2. BG11培养基
    1. 对于1 L BG11培养基,添加
      2毫升储备溶液#1
      50毫升储备溶液#2
      2毫升储备溶液#3
      1毫升储备溶液#4
      1毫升储备溶液#5
      20毫升储备溶液#6
      用H 2 O
      调整最终体积至1,000 ml
    2. 将BG11培养基分装到每个30毫升的试管中
    3. 将分散的BG11培养基在121°C高压灭菌20分钟,然后在室温下保存
  3. 10mM DCMU的乙醇溶液
    将DCMU溶解在乙醇中,将溶液储存在-20°C。

致谢

这项工作得到了JSPS创新领域科学研究资助(No. 16H06552和No. 16H06553至K.S.)和Grant-in-Aid for Scientific Research(B)(No. 16H04809至K.S.)的支持。作者宣称没有利益冲突。

参考

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引用:Ogawa, T. and Sonoike, K. (2018). Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria. Bio-protocol 8(9): e2834. DOI: 10.21769/BioProtoc.2834.
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