Characterizing non-photochemical quenching in leaves through fluorescence lifetime snapshots

Abstract

We describe a technique to measure the fluorescence decay profiles of intact leaves during adaptation to high light and subsequent relaxation to dark conditions. We show how to ensure that photosystem II reaction centers are closed and compare data for wild type Arabidopsis thaliana with conventional pulse-amplitude modulated (PAM) fluorescence measurements. Unlike PAM measurements, the lifetime measurements are not sensitive to photobleaching or chloroplast shielding, and the form of the fluorescence decay provides additional information to test quantitative models of excitation dynamics in intact leaves.

Dissecting and modeling zeaxanthin- and lutein-dependent nonphotochemical quenching in Arabidopsis thaliana

“…Lastly, HPLC data confirmed the presence of lutein epoxide, which is conclusive evidence of the functionality of the nonnative zeaxanthin epoxidase from N. oceanica in A. thaliana plants. All plants were between 6 and 9 weeks of age at the time of experiments, and all measurements were completed before the stage of bolting as described previously (41).…”

“…However, the change from 745 to 620 μmol photons m −2 s −1 should not significantly impact the analysis reported in this work. In contrast to PAM traces, data from TCPSC measurements are relatively insensitive to changes in high light intensity, because time resolving the fluorescence eliminates sensitivity to nonquenching processes, including chloroplast avoidance (41). The remaining differences that could have implications for the reconstruction of wt from components were subsequently accounted for by normalization of the average lifetimes and the resulting zeaxanthin concentration in lut2 compared with wt.…”

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

“…18−21 Figure 2a presents amplitudeweighted average lifetimes (τ avg ) and corresponding NPQ parameters (NPQτ) extracted from 68 fluorescence decays at 680 nm ( Figure S2). NPQτ is a lifetime-based parameter analogous to the conventional NPQ value (= (F m − F m ′ )/F m ′ ), 18 and details of the calculation of NPQτ are presented in Experimental Methods section. When the actinic light was turned on, thylakoid samples showed a rapid decrease in Chl lifetime from 1.3 to 0.34 ns, eventually plateauing after 5 min, and 90% of maximum NPQτ was achieved within 3 min.…”

Studying fast dynamics in biological complexes : from photosynthesis in vivo to single DNA molecules in vitro

“…To avoid back reflections, the sample rotator was placed at an angle of ~80 degrees with respect to the excitation beam. The upper surface of the leave was facing the excitation light, on the rear of the leaves a piece of wet tissue was placed to prevent them from dehydration and overheating (for detached leaves without access to water qE is significantly reduced (Sylak-Glassman et al 2016)). Laser powers of 100nW or 1mW were used to keep the reaction centers in either the open or closed state, respectively.…”

“…Furthermore, the method does not give information about the rates at of photosynthetic charge separation (kCS) and NPQ (kNPQ). To overcome these shortcomings we have studied NPQ with time-resolved picosecond/nanosecond fluorescence measurements , Lambrev et al 2012, Lambrev et al 2010, Sylak-Glassman et al 2016. Such measurements are not sensitive to photobleaching and chloroplast movement, and in addition, can be used to reveal the rates of qE both in the presence of open and closed RCs.…”