Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)–zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica. We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

The photosystem II (PSII) protein PsbS and the enzyme violaxanthin deepoxidase (VDE) are known to influence the dynamics of energy-dependent quenching (qE), the component of nonphotochemical quenching (NPQ) that allows plants to respond to fast fluctuations in light intensity. Although the absence of PsbS and VDE has been shown to change the amount of quenching, there have not been any measurements that can detect whether the presence of these proteins alters the type of quenching that occurs. The chlorophyll fluorescence lifetime probes the excitedstate chlorophyll relaxation dynamics and can be used to determine the amount of quenching as well as whether two different genotypes with the same amount of NPQ have similar dynamics of excited-state chlorophyll relaxation. We measured the fluorescence lifetimes on whole leaves of Arabidopsis thaliana throughout the induction and relaxation of NPQ for wild type and the qE mutants, npq4, which lacks PsbS; npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin; and npq1 npq4, which lacks both VDE and PsbS. These measurements show that although PsbS changes the amount of quenching and the rate at which quenching turns on, it does not affect the relaxation dynamics of excited chlorophyll during quenching. In addition, the data suggest that PsbS responds not only to ΔpH but also to the Δψ across the thylakoid membrane. In contrast, the presence of VDE, which is necessary for the accumulation of zeaxanthin, affects the excited-state chlorophyll relaxation dynamics.PsbS | nonphotochemical quenching | fluorescence lifetime | carotenoids | photosystem II P lants regulate light harvesting by photosystem II (PSII) in response to changes in light intensity. One way that plants are able to regulate light harvesting is through turning on and off mechanisms that dissipate excess energy. This energy dissipation is assessed via nonphotochemical quenching (NPQ) measurements of chlorophyll fluorescence. Energy-dependent quenching (qE) is the NPQ process with the fastest kinetics. It turns on and off in seconds to minutes, allowing plants to respond to rapid fluctuations in light intensity, which is thought to reduce photodamage (1, 2).Illumination causes the formation of gradients of electrical potential (Δψ) and of proton concentration (ΔpH) across the thylakoid membrane. Although it has been suggested that Δψ may play a role in qE (3), only ΔpH is thought to trigger different proteins and enzymes to induce qE (4). The major known factors involved in induction of qE are the enzyme violaxanthin deepoxidase (VDE) (5) and the PSII protein PsbS (6). The mutant npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin, has a phenotype with lower qE compared with the wild type (7). Transient absorption measurements suggest that zeaxanthin may quench excited chlorophyll (8). The npq4 mutant, which lacks PsbS, shows no rapidly reversible quenching of chlorophyll fluorescence, suggesting that PsbS is required for qE in vivo (6). PsbS is pH sensitive (9) but is not though...

Nonphotochemical quenching (NPQ) provides an essential photoprotection in plants, assuring safe dissipation of excess energy as heat under high light. Although excitation energy transfer (EET) between chlorophyll (Chl) and carotenoid (Car) molecules plays an important role in NPQ, detailed information on the EET quenching mechanism under in vivo conditions, including the triggering mechanism and activation dynamics, is very limited. Here, we observed EET between the Chl Q state and the Car S state in high-light-exposed spinach thylakoid membranes. The kinetic and spectral analyses using transient absorption (TA) spectroscopy revealed that the Car S excited state absorption (ESA) signal after Chl excitation has a maximum absorption peak around 540 nm and a lifetime of ∼8 ps. Snapshot TA spectroscopy at multiple time delays allowed us to track the Car S ESA signal as the thylakoid membranes were exposed to various light conditions. The obtained snapshots indicate that maximum Car S ESA signal quickly rose and slightly dropped during the initial high-light exposure (<3 min) and then gradually increased with a time constant of ∼5 min after prolonged light exposure. This suggests the involvement of both rapidly activated and slowly activated mechanisms for EET quenching. 1,4-Dithiothreitol (DTT) and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) chemical treatments further support that the Car S ESA signal (or the EET quenching mechanism) is primarily dependent on the accumulation of zeaxanthin and partially dependent on the reorganization of membrane proteins, perhaps due to the pH-sensing protein photosystem II subunit S.