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Photoprotective nonphotochemical quenching (NPQ) of absorbed solar energy is vital for survival of photosynthetic organisms, and NPQ modifications significantly improve plant productivity. However, the exact NPQ quenching mechanism is obscured by discrepancies between reported mechanisms, involving xanthophyll-chlorophyll (Xan-Chl) and Chl-Chl interactions. We present evidence of an experimental artifact that may explain the discrepancies: strong laser pulses lead to the formation of a novel electronic species in the major plant light-harvesting complex (LHCII). This species evolves from a high excited state of Chl a and is absent with weak laser pulses. It resembles an excitonically coupled heterodimer of Chl a and lutein (or other Xans at site L1) and acts as a de-excitation channel. Laser powers, and consequently amounts of artifact, vary strongly between NPQ studies, thereby explaining contradicting spectral signatures attributed to NPQ. Our results offer pathways toward unveiling NPQ mechanisms and highlight the necessity of careful attention to laser-induced artifacts.

In the first step of the photosynthetic process, light is absorbed by the pigments associated with the antenna proteins, known as light-harvesting complexes (Lhcs), which in vivo are functionally organized as hetero-oligomers. The architecture of the pigments, chlorophylls, and carotenoids bound to each LHC is responsible for the efficient excitation energy transfer resulting in photochemistry. So far, the only LHC studied in depth was LHCII, the most abundant membrane protein of plants, while less information was available for the other antennae. In particular, despite the availability of the structure of CP29 obtained at near atomic resolution in 2011 (Pan et al., 2011), the mismatch in pigment content and spectroscopic properties between CP29 in solution and in the crystal has hampered the possibility to use the structure to interpret the experimental data. In this work, we purified CP29 and its larger assembly (CP29-LHCII-CP24) from the membrane in very mild conditions using a His-tag, and we have studied their pigment binding and spectroscopic properties. In addition, we have performed mutation analysis in vivo to obtain mutants of CP29 lacking individual chlorophylls. The peculiar properties of this antenna support its role in directing the energy flow from the external antennae to the reaction center.

LHCSR3 is a member of the Light-Harvesting Complexes (LHC) family, which is mainly composed of pigment-protein complexes responsible for collecting photons during the first steps of photosynthesis. Unlike related LHCs, LHCSR3 is expressed in stress conditions and has been shown to be essential for the fast component of photoprotection, non-photochemical quenching (NPQ), in the green alga Chlamydomonas reinhardtii. In plants, which do not possess LHCSR homologs, NPQ is triggered by the PSBS protein. Both PSBS and LHCSR3 possess the ability to sense pH changes but, unlike PSBS, LHCSR3 binds multiple pigments. In this work we have analyzed the properties of the pigments bound to LHCSR3 and their excited state dynamics. The data show efficient excitation energy transfer between pigments with rates similar to those observed for the other LHCs. Application of an exciton model based on a template of LHCII, the most abundant LHC, satisfactorily explains the collected steady state and time-resolved spectroscopic data, indicating that LHCSR3 has a LHC-like molecular architecture, although it probably binds less pigments. The model suggests that most of the chlorophylls have similar energy and interactions as in LHCII. The most striking difference is the localization of the lowest energy state, which is not on the Chlorophyll a (Chl a) 610-611-612 triplet as in all the LHCB antennas, but on Chl a613, which is located close to the lumen and to the pH-sensing region of the protein.

Summary The light-harvesting complexes (LHCs) of plants can regulate the energy flux to the reaction centers in response to fluctuating light by virtue of their vast conformational landscape. They do so by switching from a light-harvesting state to a quenched state, dissipating the excess absorbed energy as heat. However, isolated LHCs are prevalently in their light-harvesting state, which makes the identification of their photoprotective mechanism extremely challenging. Here, ensemble time-resolved fluorescence experiments show that monomeric CP29, a minor LHC of plants, exists in various emissive states with identical spectra but different lifetimes. The photoprotective mechanism active in a subpopulation of strongly quenched complexes is further investigated via ultrafast transient absorption spectroscopy, kinetic modeling, and mutational analysis. We demonstrate that the observed quenching is due to excitation energy transfer from chlorophylls to a dark state of the centrally bound lutein.

International audience; Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex. T he capture and storage of light energy by photosynthetic organisms is the process that sustains virtually all life on earth, but it is also a hazardous business. If the absorbed energy exceeds the capacity of the metabolic reactions, it can result in photo-oxidation events that can ultimately result in the organism's death. 1 Plants and algae have evolved elaborate mechanisms to protect themselves against oxidative damage.

In a world undergoing rapid changes in sunlight quality and quantity, the successful design of photosynthesis must rely on the ability of plants and algae to quickly and reversibly adapt to the different conditions. A fast response to the fluctuating environment is given by the Light-Harvesting Complexes (LHCs). LHCs are large pigment-protein complexes responsible for the first steps of photosynthesis. LHCs capture light and deliver excitations to Photosystem I and II (PSI and PSII) where photosynthesis starts. Spectroscopy studies showed that LHCs can switch between states of long and short fluorescence lifetime. Quenched fluorescence states help preventing reactions with oxygen eventually leading to oxidative stress. Although postulated for long time, the role of protein conformational changes in tuning optical properties of LHCs remained unconfirmed until only recently. We here present the role of specific protein domains in tuning pigment-pigment interactions in a series of LHCs. A first example is LHCSR3, the trigger of photoprotection in the green alga Chlamydomonas reinhardtii, which possesses a peculiar C-terminus. This domain is pH-sensitive (pH is a stress-indicator in the alga) and causes tuning of fluorescence to quenched states upon acidification [Liguori, N. et al JACS 2013]. Additionally we show that this mechanism is absent in all related LHC proteins from PSII, which indeed do not present the same residue composition at this domain [Liguori, N. et al in preparation]. However LHCII, which is the most abundant LHC in both plants and algae, still undergoes significant conformational changes at a different domain: the N-terminus. It is a highly disordered domain and, as we show, a strong correlation is found between the different conformations of this domain and the different pigment-pigment interactions [Liguori, N. et al Scientific Reports in press].

To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.

Feedback mechanisms that dissipate excess photo-excitations in Light-Harvesting Complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment. We show that this ability to respond to different environments is missing in the related major Light Harvesting Complex II (LHCII) despite the high structural homology. Via mutagenesis and spectroscopy characterization we show that LHCSR uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.View Large Image | View Hi-Res Image | Download PowerPoint Slide