By dynamic changes in protein structure and function, the photosynthetic membranes of plants are able to regulate the partitioning of absorbed light energy between utilization in photosynthesis and photoprotective non-radiative dissipation of the excess energy. This process is controlled by features of the intact membrane, the transmembrane pH gradient, the organization of the photosystem II antenna proteins and the reversible binding of a specific carotenoid, zeaxanthin. Resonance Raman spectroscopy has been applied for the first time to wild type and mutant Arabidopsis leaves and to intact thylakoid membranes to investigate the nature of the absorption changes obligatorily associated with the energy dissipation process. The observed changes in the carotenoid Resonance Raman spectrum proved that zeaxanthin was involved and indicated a dramatic change in zeaxanthin environment that specifically alters the pigment configuration and red-shifts the absorption spectrum. This activation of zeaxanthin is a key event in the regulation of light harvesting.When plants are exposed to high light intensities a sustained decline in the maximum photosynthetic quantum efficiency, called photoinhibition, frequently occurs. This inevitably leads to the drop in plant productivity, endangering the yield of agricultural crops (1). However, plants have acquired a process of non-radiative dissipation of the excess light energy. This process is referred to as non-photochemical quenching (NPQ), 1 and it is recognized as a central regulatory mechanism for protecting plants from the photodamage (2). The major part of NPQ is induced as a response to the formation of a proton gradient across the thylakoid membrane and is referred to as qE. The xanthophyll cycle is the second factor that controls qE; under excess light conditions violaxanthin bound to the PSII light harvesting antenna undergoes de-epoxidation to zeaxanthin. The ⌬pH and zeaxanthin allosterically control the proportion of light harvesting antenna that is in the dissipating state (3). At physiological values of ⌬pH, the formation of qE is mostly dependent on the presence of zeaxanthin.At present the molecular mechanism of qE has not been determined. A major obstacle has been the fact that it is a relatively complex process dependent upon features of an intact chloroplast membrane (2, 3). New analytical approaches are required. qE has been shown to be associated with an absorbance change centered around 535 nm (⌬A 535 ), referred to as light scattering (4, 5). Many different experiments indicate that the ⌬A 535 is closely associated with qE: inhibition of qE by antimycin removed ⌬A 535 despite ⌬pH still being present (6); the kinetics of formation and relaxation of qE and ⌬A 535 are the same (Ref. 4 and Fig. 1, inset); close correlations between qE and ⌬A 535 were found under a wide range of conditions in leaves and chloroplasts (4 -6). Particularly clear was the fact that the loss of qE in the npq4 mutant of Arabidopsis thaliana was associated with the loss of ⌬A 535 (7). Unde...