Snapshot Transient Absorption Spectroscopy of Carotenoid Radical Cations in High-Light-Acclimating Thylakoid Membranes

Abstract

Nonphotochemical quenching mechanisms regulate light harvesting in oxygenic photosynthesis. Measurement techniques for nonphotochemical quenching have typically focused on downstream effects of quenching, such as measuring reduced chlorophyll fluorescence. Here, to directly measure a species involved in quenching, we report snapshot transient absorption (TA) spectroscopy, which rapidly tracks carotenoid radical cation signals as samples acclimate to excess light. The formation of zeaxanthin radical cations, which is possible evidence of zeaxanthin–chlorophyll charge-transfer (CT) quenching, was investigated in spinach thylakoids. Together with fluorescence lifetime snapshot data and time-resolved high-performance liquid chromatography (HPLC) measurements, snapshot TA reveals that Zea•+ formation is closely related to energy-dependent quenching (qE) in nonphotochemical quenching. Quantitative and dynamic information on CT quenching discussed in this work give insight into the design principles of photoprotection in natural photosynthesis.

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

“…Cross-linking using 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) is suggested to stop PsbS-controlled conformational changes of the thylakoid membrane, resulting in behavior similar to the npq4 Arabidposis thaliana mutant. 17,28,46 As reported previously, DTSSP-treated and npq4 thylakoids do not show any Zea •+ TA signal, consistent with the idea that the interactions of active PsbS are essential for Zea •+ formation that is indicative of CT quenching. 14,17 Interestingly, DTSSPtreated thylakoids showed a 50% lower amount of Car S 1 ESA signal compared to the untreated sample (Figure 5c).…”

“…NPQτ values are closely related to the conventional NPQ parameter (see Experimental Methods). 17,33 Notably, the appearance time of the Car S 1 ESA presented in Figure 4b coincides with the Chl* quenching indicated by NPQτ (Figure 5b).…”

“…Figure 4a shows individual snapshot TA results measured at 1 and 40 ps during a time sequence of the actinic light turning on and off. Each data point in the 1 ps trace is then scaled using the data point of the corresponding light exposure time in the 40 ps trace, which gives the result shown in Figure 4b, overlaid with data from the Zea •+ snapshot TA data of Park et al 17 Much like the Zea •+ signal, the Car S 1 ESA signal appears very rapidly (<3 min) after the actinic light is turned on, well within the time scale of the qE response. Considering the fact that Zea accumulates exponentially with a rise time of 2−3 min, 17 it seems surprising that the amount of Car S 1 ESA signal indicative of Chl-Zea EET quenching is substantial in the early stages (≤3 min) of qE.…”

“…min. 17 The rapid disappearance of Car S 1 ESA signal in response to dark exposure suggests that the EET quenching mechanism plays a central role in rapidly reversible qE quenching, which is known to improve plant fitness under fluctuating light conditions. 41,42 In addition, it is conceivable that the CT and EET mechanisms involve different protein/ carotenoid conformations with one able to relax more quickly than the other.…”

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.…”