Abstract
Chlorophyll fluorescence measurement is a widely used technique to determine photosynthetic performance. Light energy absorbed by a chlorophyll molecule can be dissipated by driving photochemical energy conversion, as heat in non-photochemical quenching processes, or it is re-emitted as fluorescence. The loss of light energy as chlorophyll fluorescence is primarily derived from photosystem II. Photosystem II is a thylakoid-embedded multiprotein complex which provides the high redox potential needed to oxidize water. Within photosystem II photons of light are captured and used to energize electrons. Energized electrons are fed into the linear electron transport chain and photosystem II replenishes lost electrons with electrons from splitting of water. Chlorophyll fluorescence yield can be quantified using a modulated fluorometer device. In such a device, a modulated measuring light beam (switched on and off at a high frequency) and the parallel detection of fluorescence exclusively excited by the measuring light allows chlorophyll fluorescence measurements in the presence of photosynthetic (actinic) light. In addition, high intensity, but short duration light flashes (saturating pulses) are used to determine maximum fluorescence yields in dark and light adapted leaves. In this protocol the procedure to receive a typical fluorescence graph of Arabidopsis wild-type leaves is given. Furthermore, this procedure can be used to identify Arabidopsis mutant plants affecting photosystem II, on the basis of the respective fluorescence graphs and values.
Keywords: Arabidopsis, Phtotosynthesis, Photosystem II, Chlorophyll fluorescence, Pulse Amplitude Modulation PAM
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 2. Fluorescence graphs of a wild-type (A) and a reference PSII mutant plant (B), see Note 3. Application of measuring light (ML) and saturated light pulses (SP) are indicated. Exposure to actinic light is shown and fluorescence parameters F0, Fm, Ft and Fm’ are denoted.
Notes
Acknowledgments
This protocol was adapted or modified from previous work by Meurer et al. (1995). We thank Thilo Rühle for discussion and comments on the protocol. This work was carried out in the laboratory of Prof. Dario Leister (Biozentrum der LMU München, Department Biologie I, Munich, Germany) and supported by funds from the Deutsche Forschungsgemeinschaft (LE 1265/20-1) to D.L.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.