Photoinhibition in optically thick samples: Effects of light attenuation on chlorophyll fluorescence-based parameters

Oxygenic photoautotrophs are, paradoxically, subject to photoinhibition of their photosynthetic apparatus, in particular one of its major components, the Photosystem II (PSII). Photoinhibition is generalized across species, light conditions and habitats, imposing substantial metabolic costs that lower photosynthetic productivity and constrain the niches of photoautotrophy. As a process driven by light reaching PSII, light attenuation in optically thick samples influences both the actual extent, and the detection, of photoinhibition. Chlorophyll fluorescence is widely used to measure photoinhibition, but fluorescence-based parameters are affected by light attenuation of both downwelling incident radiation traversing the sample to reach PSII, and emitted fluorescence upwelling through the sample. We used modelling, experimental manipulation of within-sample light attenuation, and meta-analysis of published data, to show substantial, differential effects of light attenuation and depth-integration of emitted fluorescence upon measurements of photoinhibition. Numerical simulations and experimental manipulation of light attenuation indicated that PSII photoinactivation tracked using chlorophyll fluorescence can appear to be over three times lower than the inherent cellular susceptibility to photoinactivation, in optically-dense samples such as leaves or biofilms. The meta-analysis of published data showed that this general trend was unknowingly present in the literature, revealing an overall difference of more than five times between optically thick leaves and optically thin cell suspensions. Although fluorescence-based parameters may provide ecophysiologically relevant information for characterizing the sample as a whole, light attenuation and depth integration can vary between samples independently of their intrinsic physiology. They should be used with caution when aiming to quantify in absolute terms inherent photoinhibition-related parameters in optically thick samples.