On the analysis of non-photochemical chlorophyll fluorescence quenching curves: I. Theoretical considerations.

Non-photochemical quenching (NPQ) protects photosynthetic organisms against photodamage by high light. One of the key measuring parameters for characterizing NPQ is the high-light induced decrease in chlorophyll fluorescence. The originally measured data are maximal fluorescence (Fm') signals as a function of actinic illumination time (Fm'(t)). Usually these original data are converted into the so-called Stern-Volmer quenching function, NPQSV(t), which is then analyzed and interpreted in terms of various NPQ mechanisms and kinetics. However, the interpretation of this analysis essentially depends on the assumption that NPQ follows indeed a Stern-Volmer relationship. Here, we question this commonly assumed relationship, which surprisingly has never been proven. We demonstrate by simulation of quenching data that particularly the conversion of time-dependent quenching curves like Fm'(t) into NPQSV(t) is (mathematically) not "innocent" in terms of its effects. It distorts the kinetic quenching information contained in the originally measured function Fm'(t), leading to a severe (often sigmoidal) distortion of the time-dependence of quenching and has negative impact on the ability to uncover the underlying quenching mechanisms and their contribution to the quenching kinetics. We conclude that the commonly applied analysis of time-dependent NPQ in NPQSV(t) space should be reconsidered. First, there exists no sound theoretical basis for this common practice. Second, there occurs no loss of information whatsoever when analyzing and interpreting the originally measured Fm'(t) data directly. Consequently, the analysis of Fm'(t) data has a much higher potential to provide correct mechanistic answers when trying to correlate quenching data with other biochemical information related to quenching.