通过测定蓝藻叶绿素荧光评估呼吸和光合作用的条件

Takako  Ogawa; Kintake   Sonoike

doi:10.21769/BioProtoc.2834

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Peer-reviewed

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

TO Takako Ogawa

K Kintake Sonoike email

发布: 2018年05月05日第8卷第9期 DOI: 10.21769/BioProtoc.2834 浏览次数: 7232

评审: Alizée MalnoëShai SAroussiAnonymous reviewer(s)

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The authors used this protocol in:

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Mar 2015

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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 (NPQ_Dark) and under low light (NPQ_LL).

Materials and Reagents

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.
Ferric ammonium citrate (Wako Pure Chemical Industries, catalog number: 092-00802 )
Na₂EDTA·2H₂O (Wako Pure Chemical Industries, catalog number: 345-01865 )
Sodium nitrate (NaNO₃) (Wako Pure Chemical Industries, catalog number: 192-02555 )
Dipotassium hydrogenphosphate (K₂HPO₄) (Wako Pure Chemical Industries, catalog number: 164-04295 )
Magnesium sulfate (MgSO₄) (anhydrous) (Wako Pure Chemical Industries, catalog number: 132-00435 )
Calcium chloride (CaCl₂) (Wako Pure Chemical Industries, catalog number: 038-24985 )
Sodium carbonate (Na₂CO₃) (Wako Pure Chemical Industries, catalog number: 199-01585 )
Boric acid (H₃BO₄) (Wako Pure Chemical Industries, catalog number: 029-02191 )
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
Disodium molybdate(VI) dihydrate (Na₂MoO₄·2H₂O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
Cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O) (Wako Pure Chemical Industries, catalog number: 031-03752 )
TES (Wako Pure Chemical Industries,, catalog number: 340-02655 )
Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
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.
Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
BG11 stock solutions (see Recipes)
BG11 medium (see Recipes)
10 mM DCMU solution in ethanol (see Recipes)

Equipment

Test tubes (IWAKI, catalog number: TEST30NP )
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.
Light meter (LI-COR, model: LI-250A )
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.
Quartz cuvette for WATER-PAM (Heinz Walz, model: WATER-K )
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

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

Procedure

English

中文翻译

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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.