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 S₁ 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 high-light exposure.

Protection of photosystem II against damage from excess light by nonphotochemical quenching (NPQ) includes responses on a wide range of timescales. The onset of the various phases of NPQ overlap in time making it difficult to discern if they influence each other or involve different photophysical mechanisms. To unravel the complex relationship of the known actors in NPQ, we perform fluorescence lifetime snapshot measurements throughout multiple cycles of alternating 2 min periods of high light and darkness. By comparing the data with an empirically based mathematical model that describes both fast and slow quenching responses, we suggest that the rapidly reversible quenching response depends on the state of the slower response. By studying a series of Arabidopsis thaliana mutants, we find that removing zeaxanthin (Zea) or enhancing PsbS concentration, for example, influences the amplitudes of the slow quenching induction and recovery, but not the timescales. The plants' immediate response to high light appears independent of the illumination history, while PsbS and Zea have distinct roles in both quenching and recovery. We further identify two parameters in our model that predominately influence the recovery amplitude and propose that our approach may prove useful for screening new mutants or overexpressors with enhanced biomass yields under field conditions.

Photosynthetic organisms use sunlight as the primary energy source to fix CO 2 . However, in the environment, light energy fluctuates rapidly and often exceeds saturating levels for periods ranging from seconds to hours, which can lead to detrimental effects for cells. Safe dissipation of excess light energy occurs primarily by non-photochemical quenching (NPQ) processes. In the model green microalga Chlamydomonas reinhardtii , photoprotective NPQ is mostly mediated by pH-sensing light-harvesting complex stress-related (LHCSR) proteins and the redistribution of light-harvesting antenna proteins between the photosystems (state transition). Although each component underlying NPQ has been documented, their relative contributions to the dynamic functioning of NPQ under fluctuating light conditions remains unknown. Here, by monitoring NPQ throughout multiple high light-dark cycles with fluctuation periods ranging from 1 to 10 minutes, we show that the dynamics of NPQ depend on the frequency of light fluctuations. Mutants impaired in the accumulation of LHCSRs ( npq4, lhcsr1 , and npq4lhcsr1 ) showed significantly less quenching during illumination, demonstrating that LHCSR proteins are responsible for the majority of NPQ during repetitive exposure to high light fluctuations. Activation of NPQ was also observed during the dark phases of light fluctuations, and this was exacerbated in mutants lacking LHCSRs. By analyzing 77K chlorophyll fluorescence spectra and chlorophyll fluorescence lifetimes and yields in a mutant impaired in state transition, we show that this phenomenon arises from state transition. Finally, we quantified the contributions of LHCSRs and state transition to the overall NPQ amplitude and dynamics for all light periods tested and show that both processes interact to facilitate NPQ during light fluctuations. We further assess the role of LHCSRs in cell growth under various periods of fluctuating light. These results highlight the dynamic functioning of photoprotection under light fluctuations and open a new way to systematically characterize the photosynthetic response to an ever-changing light environment.

One sentence summary

The roles of LHCSR and STT7 in NPQ vary with the light fluctuation period and duration of light fluctuation.

Photosynthetic organisms use sunlight as the primary energy source to fix CO₂ . However, in nature, light energy is highly variable, reaching levels of saturation for periods ranging from milliseconds to hours. In the green microalga Chlamydomonas reinhardtii, safe dissipation of excess light energy by nonphotochemical quenching (NPQ) is mediated by light-harvesting complex stress-related (LHCSR) proteins and redistribution of light-harvesting antennae between the photosystems (state transition). Although each component underlying NPQ has been documented, their relative contributions to NPQ under fluctuating light conditions remain unknown. Here, by monitoring NPQ in intact cells throughout high light/dark cycles of various illumination periods, we find that the dynamics of NPQ depend on the timescales of light fluctuations. We show that LHCSRs play a major role during the light phases of light fluctuations and describe their role in growth under rapid light fluctuations. We further reveal an activation of NPQ during the dark phases of all high light/dark cycles and show that this phenomenon arises from state transition. Finally, we show that LHCSRs and state transition synergistically cooperate to enable NPQ response during light fluctuations. These results highlight the dynamic functioning of photoprotection under light fluctuations and open a new way to systematically characterize the photosynthetic response to an ever-changing light environment.

The rapid response of photosynthetic organisms to fluctuations in ambient light intensity is incompletely understood at both the molecular and membrane levels. In this review, we describe research from our group over a 10-year period aimed at identifying the photophysical mechanisms used by plants, algae and mosses to control the efficiency of light harvesting by photosystem II on the seconds-to-minutes time scale. To complement the spectroscopic data, we describe three models capable of describing the measured response at a quantitative level. The review attempts to provide an integrated view that has emerged from our work, and briefly looks forward to future experimental and modelling efforts that will refine and expand our understanding of a process that significantly influences crop yields.

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.

A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (¹O₂*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of ¹O₂* in photodamage and cell signaling, few studies directly correlate ¹O₂* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of ¹O₂* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of ¹O₂* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of ¹O₂* in chlorophyll-containing photosynthetic membranes. We find that the apparent ¹O₂* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of ¹O₂* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of ¹O₂* during high light. Upon saturation of NPQ, the concentration of ¹O₂* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).

We explore the photoprotection dynamics of Nannochloropsis oceanica using time-correlated single photon counting under regular and irregular actinic light sequences. The varying light sequences mimic natural conditions, allowing us to probe the real-time response of non-photochemical quenching (NPQ) pathways. Durations of fluctuating light exposure during a fixed total experimental time and prior light exposure of the algae are both found to have a profound effect on NPQ. These observations are rationalized with a quantitative model based on the xanthophyll cycle and the protonation of LHCX1. The model is able to accurately describe the dynamics of non-photochemical quenching across a variety of light sequences. The combined model and observations suggest that the accumulation of a quenching complex, likely zeaxanthin bound to a protonated LHCX1, is responsible for the gradual rise in NPQ. Additionally, the model makes specific predictions for the light sequence dependence of xanthophyll concentrations that are in reasonable agreement with independent chromatography measurements taken during a specific light/dark sequence.

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 _y state and the Car S₁ state in high-light-exposed spinach thylakoid membranes. The kinetic and spectral analyses using transient absorption (TA) spectroscopy revealed that the Car S₁ 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 S₁ ESA signal as the thylakoid membranes were exposed to various light conditions. The obtained snapshots indicate that maximum Car S₁ 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 S₁ 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.