Chlorophyll-Carotenoid Excitation Energy Transfer in High-Light-Exposed Thylakoid Membranes Investigated by Snapshot Transient Absorption Spectroscopy

Abstract

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.

A proteoliposome-based system reveals how lipids control photosynthetic light harvesting

“…The presence of zea in the lipid bilayer increases membrane order and rigidity (59,60) that is known to enhance the lateral membrane pressure in hydrophobic membrane regions (13). Zea is converted from violaxanthin by the xanthophyll cycle and promotes qE formation (61,62). Thus, modulation of generic physicochemical membrane properties by free zea in the membrane could be another way to control LMP and therefore light harvesting by LHCII.…”

Chlorophyll–carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis

“…Although the mechanisms of NPQ in N. oceanica remain to be determined, it is well known that electronic interactions between carotenoids (Car) and Chl play a major role in NPQ, and that coupled Chl-Car pairs can provide quenching sites (36)(37)(38). As shown in Scheme 1, two possible mechanisms of Chl-Car energy transfer and subsequent de-excitation have been reported to explain the role of Car as a direct quencher (23,39). In the first, excitation energy transfer (EET) quenching can be achieved by energy transfer from the Chl Q y state to the Car S 1 state (9, 10, 23, 40).…”

“…Unfortunately, strong scattering makes fully intact systems (e.g., leaves, live cells) difficult to study using TA. However, isolated crude thylakoid membranes, which exhibit moderate quenching capabilities and less scattering, have been a useful sample for studying NPQ in the near-native state (22,23). Nevertheless, there are significant differences between thylakoid membranes and fully intact systems in terms of the kinetics of qE induction/relaxation and the overall extent of quenching.…”

Observation of dissipative chlorophyll-to-carotenoid energy transfer in light-harvesting complex II in membrane nanodiscs

“…The in vitro environments, which employ detergent or crystallization, may introduce additional, non-native conformational changes that could alter or even denature the functional structure of membrane proteins [30][31][32] . In contrast, in vivo spectroscopy on whole leaves provides physiological information [33][34][35] . However, identifying the photophysical pathways in each of the homologous antenna complexes is not possible.…”

“…However, identifying the photophysical pathways in each of the homologous antenna complexes is not possible. Furthermore, in vivo transient absorption measurements have been shown to inevitably lead to laser-induced artifacts, such as singlet-singlet annihilation in the measured photophysics due to the large absorption cross-section of the intact protein network 34,36 . Due to these challenges and limitations, a simple, yet physiological environment has been lacking, leaving the photophysical pathways of individual antenna complexes undetermined.…”