We describe a technique to measure the fluorescence decay profiles of intact leaves during adaptation to high light and subsequent relaxation to dark conditions. We show how to ensure that photosystem II reaction centers are closed and compare data for wild type Arabidopsis thaliana with conventional pulse-amplitude modulated (PAM) fluorescence measurements. Unlike PAM measurements, the lifetime measurements are not sensitive to photobleaching or chloroplast shielding, and the form of the fluorescence decay provides additional information to test quantitative models of excitation dynamics in intact leaves.

Photosynthetic organisms use various photoprotective mechanisms to dissipate excess photoexcitation as heat in a process called nonphotochemical quenching (NPQ). Regulation of NPQ allows for a rapid response to changes in light intensity and in vascular plants, is primarily triggered by a pH gradient across the thylakoid membrane (∆pH). The response is mediated by the PsbS protein and various xanthophylls. Time-correlated single-photon counting (TCSPC) measurements were performed on to quantify the dependence of the response of NPQ to changes in light intensity on the presence and accumulation of zeaxanthin and lutein. Measurements were performed on WT and mutant plants deficient in one or both of the xanthophylls as well as a transgenic line that accumulates lutein via an engineered lutein epoxide cycle. Changes in the response of NPQ to light acclimation in WT and mutant plants were observed between two successive light acclimation cycles, suggesting that the character of the rapid and reversible response of NPQ in fully dark-acclimated plants is substantially different from in conditions plants are likely to experience caused by changes in light intensity during daylight. Mathematical models of the response of zeaxanthin- and lutein-dependent reversible NPQ were constructed that accurately describe the observed differences between the light acclimation periods. Finally, the WT response of NPQ was reconstructed from isolated components present in mutant plants with a single common scaling factor, which enabled deconvolution of the relative contributions of zeaxanthin- and lutein-dependent NPQ.