Applications for the PAM-2500
Light Saturation Curves of the Apparent Electron Transport Rate
A major application of PAM-2500 fluorometers in ecophysiology is the fast and reliable analysis of the photosynthetic performance of plants.
Two important parameters for characterizing photosynthesis are the maximum quantum yield for whole chain electron transport (“alpha”, at low light intensities) and the maximum electron transport capacity (“ETRmax”, at light saturation).
To evaluate the alpha and ETRmax, apparent electron transport rates (ETR) are derived from effective quantum yields of photosystem II (ΔF/Fm' or Y(II)) according to ETR = Y(II) x PAR x 0.42.
In this equation, the PAR corresponds to the quantum flux density of photosynthetically active radiation, and the 0.42 is the product of light absorptance by an average green leaf (0.84) times the fraction of absorbed quanta available for photosystem II (0.5).
The light response experiment consisted of 6 exposure intervals of 3 minutes. At the end of each step, the photosystem II efficiency, Y(II), and light intensity, PAR, were recorded. The relative electron transport rate (ETR, plotted on Y axis versus PAR) was derived from Y(II) and PAR. Fitting a theoretical function to the data points by the PamWin-3 software (black line) yields estimates for alpha (initial slope), ETRmax (maximum ETR), and Ik (PAR above which light saturation of photosynthesis starts).
Polyphasic Fluorescence Rise Upon Onset of Saturating Light
The fast acquisition mode of the PAM-2500 enables recording of rapid fluorescence kinetics with 10 µs time resolution. This high time resolution is achieved with pulse modulated signals.
This means that the fast kinetics of fluorescence yield is measured and, consequently, that signal amplitudes from different experiments can be directly compared irrespective of light intensity and sample geometry.
The same saturating light that serves for saturation pulses can also be used for measuring the polyphasic fluorescence rise kinetics. This type of kinetics provides valuable information on the properties of PS II and the state of its primary and secondary acceptor pools.
With a dark-acclimated sample, four characteristic levels of fluorescence yield can be distinguished in a plot with logarithmic time scale: Fo, I1, I2 and Fm (alternatively also denoted O, J, I and P).
The F0-I1 (or O-J) phase of the transient directly reflects the closure of PS II reaction centers by charge separation (QA-reduction). The initial rate of increase of this phase is proportional to the applied light intensity (photochemical phase). At a given light intensity, the initial rate provides a relative measure of the optical absorption cross-section of PS II. The I1-I2-FM (or J-I-P) phases of the transient reflect the reduction of the rest of the electron transport chain defined mainly by the reduction of the plastoquinone pool and the acceptor side of PS I; the rate of which is limited by dark reactions (thermal phase). Clear-cut separation of photochemical and thermal phases (pronounced I1 plateau) is favored by the very high light intensities provided by the PAM-2500 (up to 25,000 μmol m-2 s-1). For reproducible results, defined reduction-oxidation states are essential which can be obtained (in algae) by low intensity far-red background light.