Lijin Tian

Site, rate, and mechanism of photoprotective quenching in cyanobacteria.

In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ∼72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ∼29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (∼240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.

Picosecond Kinetics of Light Harvesting and Photoprotective Quenching in Wild-Type and Mutant Phycobilisomes Isolated from the Cyanobacterium Synechocystis PCC 6803

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.

Molecular insights into Zeaxanthin-dependent quenching in higher plants

Photosynthetic organisms protect themselves from high-light stress by dissipating excess absorbed energy as heat in a process called non-photochemical quenching (NPQ). Zeaxanthin is essential for the full development of NPQ, but its role remains debated. The main discussion revolves around two points: where does zeaxanthin bind and does it quench? To answer these questions we have followed the zeaxanthin-dependent quenching from leaves to individual complexes, including supercomplexes. We show that small amounts of zeaxanthin are associated with the complexes, but in contrast to what is generally believed, zeaxanthin binding per se does not cause conformational changes in the complexes and does not induce quenching, not even at low pH. We show that in NPQ conditions zeaxanthin does not exchange for violaxanthin in the internal binding sites of the antennas but is located at the periphery of the complexes. These results together with the observation that the zeaxanthin-dependent quenching is active in isolated membranes, but not in functional supercomplexes, suggests that zeaxanthin is acting in between the complexes, helping to create/participating in a variety of quenching sites. This can explain why none of the antennas appears to be essential for NPQ and the multiple quenching mechanisms that have been observed in plants.

LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells

Significance Too much light can be dangerous for photosynthetic organisms because the simultaneous presence of excitation energy and molecular oxygen, as it occurs in the photosynthetic membranes, may lead to the formation of reactive oxygen species and induce photodamage. To avoid these effects, photosynthetic organisms dissipate a large part of the absorbed energy as heat in a process known as nonphotochemical quenching (NPQ). This process is very complex, and the molecular understanding of its mechanisms in vivo represents a challenge. Here we have developed a “minimal NPQ cell” of the green alga Chlamydomonas reinhardtii. Using these cells, we could check a series of suggestions coming from in vitro studies and obtain new insights on the mechanism of NPQ in this alga. 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.

Probing the picosecond kinetics of the photosystem II core complex in vivo

Photosystems I (PSI) and II (PSII) are two major pigment-protein complexes of photosynthetic organisms that function in series to convert sunlight energy into chemical energy. We have studied the picosecond fluorescence behaviour of the core of both photosystems in vivo by using two Synechocystis PCC 6803 mutants: BE cells contain PSI but are lacking both PSII and the light-harvesting complexes called phycobilisomes (PBs) whereas PAL cells contain both PSI and PSII but lack the PBs. Measurements were performed at room temperature and at 77 K. The fluorescence kinetics of PSI and PSII can nicely be separated, en passant providing the PSI/PSII ratio. At room temperature, the PSI kinetics are identical to those of isolated PSI whereas the PSII kinetics can equally well be described by the in vitro trap-limited model of Y. Miloslavina, M. Szczepaniak, M. G. Muller, J. Sander, M. Nowaczyk, M. Rogner and A. R. Holzwarth, Biophys J., 2009, 96(2), 621-631, and by the transfer-to-the-trap-limited model of C. D. van der Weij-de Wit, J. P. Dekker, R. van Grondelle and I. H. M. van Stokkum, J. Phys. Chem. A, 2011, 115(16), 3947-3956, albeit that the in vivo kinetics turn out to be somewhat slower. At 77 K several low-energy pigments are observed in both photosystems which complicate the overall dynamics but the PSII kinetics can still be described by both a trap-limited and a transfer-to-the-trap-limited model.

PSI–LHCI of Chlamydomonas reinhardtii: Increasing the absorption cross section without losing efficiency

Photosystem I (PSI) is an essential component of photosynthetic membranes. Despite the high sequence and structural homologies, its absorption properties differ substantially in algae, plants and cyanobacteria. In particular it is characterized by the presence of low-energy chlorophylls (red forms), the number and the energy of which vary in different organisms. The PSI–LHCI (PSI–light harvesting complex I) complex of the green alga Chlamydomonas reinhardtii (C.r.) is significantly larger than that of plants, containing five additional light-harvesting complexes (together binding ≈ 65 chlorophylls), and contains red forms with higher energy than plants. To understand how these differences influence excitation energy transfer and trapping in the system, we studied two PSI–LHCI C.r. particles, differing in antenna size and red-form content, using time-resolved fluorescence and compared them to plant PSI–LHCI. The excited state kinetics in C.r. shows the same average lifetime (50 ps) as in plants suggesting that the effect of antenna enlargement is compensated by higher energy red forms. The system equilibrates very fast, indicating that all Lhcas are well-connected, despite their long distance to the core. The differences between C.r. PSI–LHCI with and without Lhca2 and Lhca9 show that these Lhcas bind red forms, although not the red-most. The red-most forms are in (or functionally close to) other Lhcas and slow down the trapping, but hardly affect the quantum efficiency, which remains as high as 97% even in a complex that contains 235 chlorophylls.

LHCII Populations in Different Quenching States Are Present in the Thylakoid Membranes in a Ratio that Depends on the Light Conditions.

LHCII is the major antenna complex of plants and algae, where it is involved in light harvesting and photoprotection. Its properties have been extensively studied in vitro, after isolation of the pigment-protein complex from the membranes, but are these properties representative for LHCII in the thylakoid membrane? In this work, we have studied LHCII in the cells of the green alga C. reinhardtii acclimated to different light conditions in the absence of the other components of the photosynthetic apparatus. We show that LHCII exists in the membranes in different fluorescence quenching states, all having a shorter excited-state lifetime than isolated LHCII in detergent. The ratio between these populations depends on the light conditions, indicating that the light is able to regulate the properties of the complexes in the membrane.

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I

Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment.

Light Harvesting and Blue-Green Light Induced Non-Photochemical Quenching in Two Different C-Phycocyanin Mutants of Synechocystis PCC 6803

Cyanobacteria are oxygen-evolving photosynthetic organisms that harvest sunlight and convert excitation energy into chemical energy. Most of the light is absorbed by large light harvesting complexes called phycobilisomes (PBs). In high-light conditions, cyanobacteria switch on a photoprotective mechanism called non-photochemical quenching (NPQ): During this process, absorption of blue-green light transforms the inactive orange form of the orange carotenoid protein OCP (OCP(o)) into the red active form OCP(r) that subsequently binds to the PB, resulting in a substantial loss of excitation energy and corresponding decrease of the fluorescence. In wild-type cells, the quenching site is a bilin chomophore that fluoresces at 660 nm and which is called APC(Q)(660). In the present work, we studied NPQ in two different types of mutant cells (CB and CK) that possess significantly truncated PBs, using spectrally resolved picosecond fluorescence spectroscopy. The results are in very good agreement with earlier in vitro experiments on quenched and unquenched PBs, although the fraction of quenched PBs is far lower in vivo. It is also lower than the fraction of PBs that is quenched in wild-type cells, but the site, rate, and location of quenching appear to be very similar.

Emission enhancement and lifetime modification of phosphorescence on silver nanoparticle aggregates

Silver nanoparticle aggregates have been shown to support very large enhancements of fluorescence intensity from organic dye molecules coupled with an extreme reduction in observed fluorescence lifetimes. Here we show that for the same type of aggregates, similar enhancement factors (~75× in intensity and ~3400× in lifetime compared to the native radiative lifetime) are observed for a ruthenium-based phosphorescent dye (when taking into account the effect of charge and the excitation/emission wavelengths). Additionally, the inherently long native phosphorescence lifetimes practically enable more detailed analyses of the distribution of lifetimes (compared with the case with fluorescence decays). It was thus possible to unambiguously observe the deviation from mono-exponential decay which we attribute to emission from a distribution of fluorophores with different lifetimes, as we could expect from a random aggregation process. We believe that combining phosphorescent dyes with plasmonic structures, even down to the single dye level, will offer a convenient approach to better characterize plasmonic systems in detail.