Excess absorbed light energy can damage the photosynthetic apparatus of photoautotrophs and lead to the photoinhibition. Most photoautotrophs possess a xanthophyll cycle mediated regulatory mechanism that dissipates the excess absorbed light energy through harmless non-radiative pathways to prevent photodamage, known as the photoprotective mechanism. The photoprotective capacity of phytoplankton has attracted much attention in recent marine ecological studies in view of its significant influence on the distribution of phytoplankton in the water column. The photoprotective mechanism will lead to a depression of the chlorophyll fluorescence yield, known as the non-photochemical quenching (NPQ), hence it can be measured by in vivo chlorophyll fluorescence detection. However, the commonly used fluorescence parameter for evaluating the photoprotective capacity of phytoplankton, NPQ_m (maximum NPQ), is not an ideal parameter for practical applications in field ecological studies. In this study, we propose that the initial slope of the NPQ induction curves (denoted as NPQ/t), which indicates the change rate of NPQ at the beginning of induction (< 1 min), can be used as a fine indicator of the photoprotective capacity. Compared to NPQ_m, NPQ/t is easier for standardization, and it can be measured faster, and it provides more comprehensive information on the photoprotective capacity.
Experiments were conducted to validate the reasonability and effectiveness of using NPQ/t as an indicator of the photoprotective capacity of phytoplankton. Three phytoplankton species (Tetraselmis chui, Chaetoceros mulleri, and Dicrateria inornata) were used in the experiments. Culture samples of each species were incubated under different light intensities (150, 50, and 10 μE m^-2 s^-1, respectively) and adapted for 72 h. NPQ/t, NPQ_m and photoprotective pigment content were then measured for each culture sample using a Water-PAM fluorometer and high-performance liquid chromatography (HPLC), respectively.
Our results showed the following: (1) The NPQ/t value was linearly correlated with time at the beginning of fluorescence induction (averaged linear regression R² = 0.786); hence, NPQ/t was proved to be a constant for a given sample. The NPQ/t values measured in this study ranged from 0.00012 s^-1 to 0.016 s^-1, depending on the culture conditions. (2) NPQ/t was significantly correlated with NPQ_m and the photoprotective pigment content for all algal species tested, indicating that NPQ/t is equivalent to the traditional indicators used for evaluating the photoprotective capacity of phytoplankton. (3) High-light adapted cultures showed larger NPQ/t than low-light adapted cultures for all species tested; NPQ/t values of the different species were in the order: T. chui > C. mulleri > D. inornata, which was independent of light intensity. These results showed that NPQ/t is an effective indicator for evaluating the photoprotective capacity of phytoplankton. A theoretical analysis based on a dynamic model of the xanthophyll cycle was conducted to demonstrate the intrinsic biological characteristics of NPQ/t. It revealed that NPQ/t is equal to the product of NPQ_m and k_NPQ (rate constant of NPQ induction), which confirmed that NPQ/t is a constant for a given sample. The new method developed in this study is more efficient and applicable in ecological studies of photoprotective capacity of phytoplankton than the traditional methods. In addition, we believe that this method can also be useful in studies of the photoprotective mechanism in higher plants.