Snapshot transient absorption spectroscopy: toward in vivo investigations of nonphotochemical quenching mechanisms

Abstract

Although the importance of nonphotochemical quenching (NPQ) on photosynthetic biomass production and crop yields is well established, the in vivo operation of the individual mechanisms contributing to overall NPQ is still a matter of controversy. In order to investigate the timescale and activation dynamics of specific quenching mechanisms, we have developed a technique called snapshot transient absorption (TA) spectroscopy, which can monitor molecular species involved in the quenching response with a time resolution of 30 s. Using intact thylakoid membrane samples, we show how conventional TA kinetic and spectral analyses enable the determination of the appropriate wavelength and time delay for snapshot TA experiments. As an example, we show how the chlorophyll-carotenoid charge transfer and excitation energy transfer mechanisms can be monitored based on signals corresponding to the carotenoid (Car) radical cation and Car S 1 excited state absorption, respectively. The use of snapshot TA spectroscopy together with the previously reported fluorescence lifetime snapshot technique (Sylak-Glassman et al. in Photosynth Res 127:69-76, 2016) provides valuable information such as the concurrent appearance of specific quenching species and overall quenching of excited Chl. Furthermore, we show that the snapshot TA technique can be successfully applied to completely intact photosynthetic organisms such as live cells of Nannochloropsis. This demonstrates that the snapshot TA technique is a valuable method for tracking the dynamics of intact samples that evolve over time, such as the photosynthetic system in response to highlight exposure.

Complex Roles of PsbS and Xanthophylls in the Regulation of Nonphotochemical Quenching in Arabidopsis thaliana under Fluctuating Light

“…Snapshot transient absorption measurements have been developed that relate observables of quenching mechanisms to regulatory timescales. 82 Further development of similar "snapshot" versions of techniques that can relate changes in protein conformation dynamics or membrane organization to these regulatory timescales will be necessary to integrate the understanding of physical phenomena with kinetic regulatory models. In addition, improved analysis methods for multiperiod data sets such as those presented here should lead to more refined insights about the complicated and overlapping response of NPQ in the presence of dynamically changing light environments.…”

Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

“…Carotenoids as essential photosynthetic pigments can contribute to light-harvesting or act as excitation quenchers depending on the structural organization of antennas and the carotenoid state 2 , resulting in excitation energy transfer (EET) to or from chlorophylls, respectively. EET can be conducted by several mechanisms: transfer to (or from) the carotenoid S 1 excited level 3 , exciton coupling 4 , 5 and charge transfer 6 , 7 . Although functional roles of carotenoids in light-harvesting antennas are clear, there are still debates considering the involvement of specific EET mechanisms in these roles.…”

An energy-dissipative state of the major antenna complex of plants

“…Method used is adapted for fluorescence lifetime snapshot from (59) and transient absorption spectroscopy snapshot from (60). For fluorescence lifetime measurements, time-correlated single photon counting (TCSPC) was performed on detached leaves, isolated thylakoids and gel filtration fractions.…”