Instantaneous switching between different modes of non-photochemical quenching in plants. Consequences for increasing biomass production

Photosynthetic productivity usually saturates far below the maximum solar light intensity, meaning that in those conditions many absorbed photons and the resulting electronic excitations of the pigment molecules can no longer be utilized for photosynthesis. To avoid photodamage, various protection mechanisms are induced that dissipate excess excitations, which otherwise could lead to the formation of harmful molecular species like singlet oxygen. This Non-Photochemical Quenching (NPQ) of excitations can be monitored via a decrease of the chlorophyll fluorescence. There is consensus that in plants 1) there are at least two major NPQ (sub)processes and 2) NPQ (de)activation occurs on various time scales, ranging from (tens of) seconds to minutes. This relatively slow switching has a negative effect on photosynthetic efficiency, and Kromdijk et al. demonstrated in 2016 (Science 354, 857) that faster switching rates can lead to increased crop productivity. Very recently, we were involved in the discovery of a new NPQ process that switches off well within a millisecond (Farooq et al. (2018) Nat. Plants 4, 225). Here we describe the current level of knowledge regarding this process and discuss its implications.</p

Singlet-triplet annihilation in single LHCII complexes

In light harvesting complex II (LHCII) of higher plants and green algae, carotenoids (Cars) have an important function to quench chlorophyll (Chl) triplet states and therefore avoid the production of harmful singlet oxygen. The resulting Car triplet states lead to a non-linear self-quenching mechanism called singlet–triplet (S–T) annihilation that strongly depends on the excitation density. In this work we investigated the fluorescence decay kinetics of single immobilized LHCIIs at room temperature and found a two-exponential decay with a slow (3.5 ns) and a fast (35 ps) component. The relative amplitude fraction of the fast component increases with increasing excitation intensity, and the resulting decrease in the fluorescence quantum yield suggests annihilation effects. Modulation of the excitation pattern by means of an acousto-optic modulator (AOM) furthermore allowed us to resolve the time-dependent accumulation and decay rate (B7 ms) of the quenching species. Inspired by singlet–singlet (S–S) annihilation studies, we developed a stochastic model and then successfully applied it to describe and explain all the experimentally observed steady-state and time-dependent kinetics. That allowed us to distinctively identify the quenching mechanism as S–T annihilation. Quantitative fitting resulted in a conclusive set of parameters validating our interpretation of the experimental results. The obtained stochastic model can be generalized to describe S–T annihilation in small molecular aggregates where the equilibration time of excitations is much faster than the annihilation-free singlet excited state lifetime.VU University and by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT).Nederlandse Organisatie voor Wetenschappelijk Onderzoek, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305), and by the EU FP7 project PAPETS (GA 323901).Academy Professor grant from the Netherlands Royal Academy of Sciences (KNAW). University of Pretoria's Research Development Programme (Grant No.A0W679) Research Council of Lithuania (LMT grant no. MIP-080/2015).http://www.rsc.orgpccp2016-08-31hb201

Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the singleliposome level and for the first time apply it to study photosynthetic pigment protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment

Instantaneous switching between different modes of non-photochemical quenching in plants. Consequences for increasing biomass production

Photosynthetic productivity usually saturates far below the maximum solar light intensity, meaning that in those conditions many absorbed photons and the resulting electronic excitations of the pigment molecules can no longer be utilized for photosynthesis. To avoid photodamage, various protection mechanisms are induced that dissipate excess excitations, which otherwise could lead to the formation of harmful molecular species like singlet oxygen. This Non-Photochemical Quenching (NPQ) of excitations can be monitored via a decrease of the chlorophyll fluorescence. There is consensus that in plants 1) there are at least two major NPQ (sub)processes and 2) NPQ (de)activation occurs on various time scales, ranging from (tens of) seconds to minutes. This relatively slow switching has a negative effect on photosynthetic efficiency, and Kromdijk et al. demonstrated in 2016 (Science 354, 857) that faster switching rates can lead to increased crop productivity. Very recently, we were involved in the discovery of a new NPQ process that switches off well within a millisecond (Farooq et al. (2018) Nat. Plants 4, 225). Here we describe the current level of knowledge regarding this process and discuss its implications