One pulse, one light curve: Fast characterization of the light response of microphytobenthos biofilms using chlorophyll fluorescence

Abstract

Research on microphytobenthos (MPB) photosynthesis has increasingly relied on pulse amplitude modulation fluorometry, often through the generation of light response curves of the relative electron transport of PSII (rETR), generated through the sequential exposure to a range of actinic irradiances. However, fast, vertical phototactic responses by the motile diatoms that dominate these biofilms occur during the generation of sequential light curves. Light‐induced vertical migration is known to confound the characterization of the inherent physiological response of the cells forming the biofilm. The alternative approach of shortening the duration of the light curve protocol prevents the reaching of a physiological steady state and results in light curves heavily dependent on initial light conditions. This study tested the application to MPB of a recently introduced method allowing for the fast generation of non‐sequential light curves, based on steady state and temporally independent measurements (“single‐pulse light curves,” SPLC). This method was compared to the commonly used sequential protocols regarding its ability to (1) measure a steady‐state light response, (2) minimize the effects of vertical migration, and (3) minimize the effects of photoacclimation state induced by initial conditions. The results indicate that the SPLC method can be optimized by applying one light exposure period of just 60 s prior to the generation of the light curve, resulting in a clearly advantageous alternative to the sequential protocols. It effectively allows characterizing the steady‐state light response of MPB samples while avoiding the effects of light‐induced vertical migration and of the initial light conditions on light curve parameters.

Rapid light curves (RLC) or non-sequential steady-state light curves (N-SSLC): which fluorescence-based light response curve methodology robustly characterizes phytoplankton photosynthetic activity and acclimation status?

“…As a consequence, at daily scale, ETR m_RLC and E k_RLC followed more closely rapid light variations than ETR m_N-SSLC and E k_N-SSLC did and some rapid changes in phytoplankton photosynthetic activity were not detected when the measurements were made with N-SSLC. These observations are in accordance with studies made on microphytobenthos showing that patterns of variation and absolute values of photosynthetic parameters are influenced by the duration of light steps (Frankenbach and Serôdio 2017;Herlory et al 2007;Lefebvre et al 2011;Perkins et al 2006). Differences between photosynthetic parameters measured on microphytobenthic algae grown under experimental high light or low light conditions have thus been shown to decrease as a function of increasing duration of light steps.…”

“…As a consequence, lower values of α _RLC are obtained in comparison to α _SSLC because SSLC allow NPQ to completely relax during the first light steps Lefebvre et al 2011;Serôdio et al 2006). In our study, discrepancies between α _RLC and α _N-SSLC cannot be explained by the incomplete dissipation of NPQ during the first light steps because at the beginning of RLC and N-SSLC, the level of NPQ was always close to zero and NPQ processes only start to be activated during the curves building (by light dose accumulation Frankenbach and Serôdio (2017) showed that photosynthetic parameters extracted from non sequential light response curves were less affected by light conditions to which algae were exposed 15 min before the curve measurement than photosynthetic parameters extracted from RLC. As highlighted by multiple linear regressions, temperature and phytoplankton communities composition have also played a role in deviations between RLC and N-SSLC photosynthetic parameters.…”

“…Differences between photosynthetic parameters measured on microphytobenthic algae grown under experimental high light or low light conditions have thus been shown to decrease as a function of increasing duration of light steps. As a result, SSLC can reduce or negate the real level of the previous 1 hour photoacclimation while RLC with short light steps keep this difference (Frankenbach and Serôdio 2017;Perkins et al 2006). These results have important implications for the design of future phytoplankton photosynthesis studies at daily scale because they show that N-SSLC may not be appropriate to accurately measure phytoplankton responses to short-term environmental variations.…”

“…The use of longer RLC light steps would reduce differences between the photosynthetic parameters extracted from RLC and N-SSLC because RLC ability to capture acclimation to recent light history is inversely proportional to light steps duration. Indeed, the longer the light steps, the more phytoplankton acclimate during the curve building and the more it is difficult to characterize differences in short-term acclimation status of algae exposed to contrasted environmental conditions just before the RLC measurement (Frankenbach and Serôdio 2017;Lefebvre et al 2011;Perkins et al 2006).…”

“…Measurements lost their independency and the photosynthetic activity measured at each light step depends not only on the irradiance to which the sample is exposed at the moment of measurement but also on the irradiance experienced (and accumulated) during the previous step(s) (recent light history) (Frankenbach and Serôdio 2017;Herlory et al 2007). Both protocols are time-consuming as they require several minutes to a few hours to be completed.…”