Single-Molecule Exploration of the Photodynamics of LHCII Complexes in Solution

Abstract

Plants can safely dissipate excess excitation energy during light harvesting to prevent the formation of triplet chlorophyll, which can generate deleterious singlet oxygen. With this regulation, known as non-photochemical quenching (NPQ), efficient light harvesting and photosynthesis can be balanced under fluctuating sunlight intensity without damage to the photosynthetic machinery. NPQ has been extensively studied and key physiological regulatory factors have been identified. For example, it is known that the change in lumen pH or a pH gradient across thylakoid membrane can trigger an energy-dependent quenching (qE) pathway that activates on a timescale of seconds to minutes. However, understanding the molecular mechanism behind NPQ is still a challenge. One important question is whether, upon activation of qE, the number of quenched complexes increases or the degree of quenching in each complex changes. These two cases cannot be differentiated in ensemble-averaged measurements. Therefore, we use the Anti-Brownian ELectrokinetic (ABEL) trap to investigate single copies of light-harvesting complex II (LHCII), the primary antenna in higher plants. Different from other single-molecule techniques that utilize immobilization, perturbations due to surface attachment or encapsulation are avoided, and therefore the intrinsic dynamics and heterogeneity of individual complexes are revealed. We perform measurements of fluorescence intensity, excited-state lifetime, and emission spectra of single LHCII complexes in the ABEL trap. By analyzing the correlated changes in these properties over time, we observe that individual LHCII complexes are found in distinct forms with different extents of quenching. Comparing the results from different conditions known to correlate with qE activation will give a better understanding of NPQ at the molecular level.