A major application of PAM-2500 fluorometers in ecophysiology consists in 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).
In the given example, the intensity of red actinic light is increased stepwise every 3 minutes. At the end of each intensity step, fluorescence ratio parameters including Y(II) are assessed with the help of a saturation pulse. While Y(II) decreases with increasing PAR, ETR first rises and then levels off at high PAR values. Fitting a theoretical function (see black line) to the data points yields estimates for the alpha and ETRmax. All saturation pulse data are stored in a report file from where they can be exported to spread-sheet programs, like Excel, e.g. for graphical display of light saturation curves.
The fast acquisition mode of the PAM-2500 enables recordings of rapid fluorescence kinetics with 10 µs time resolution. It may be emphasized that 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 Fo-I1 (or O-J) transient directly reflects the closure of PS II reaction centers by charge separation (QA-reduction). The rate of this transient is proportional to the applied light intensity (photochemical phase). At a given high light intensity, the rate provides a relative measure of the optical absorption cross-section of PS II. The I1-I2-Fm (or J-I-P) transients reflect the reduction of secondary acceptor pools (mainly plastoquinone), the rate of which is limited by dark reactions (thermal phases). 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 by defined far-red preillumination.