Alexander V. Ruban

Identification of a mechanism of photoprotective energy dissipation in higher plants

Under conditions of excess sunlight the efficient light-harvesting antenna found in the chloroplast membranes of plants is rapidly and reversibly switched into a photoprotected quenched state in which potentially harmful absorbed energy is dissipated as heat, a process measured as the non-photochemical quenching of chlorophyll fluorescence or qE. Although the biological significance of qE is established, the molecular mechanisms involved are not. LHCII, the main light-harvesting complex, has an inbuilt capability to undergo transformation into a dissipative state by conformational change and it was suggested that this provides a molecular basis for qE, but it is not known if such events occur in vivo or how energy is dissipated in this state. The transition into the dissipative state is associated with a twist in the configuration of the LHCII-bound carotenoid neoxanthin, identified using resonance Raman spectroscopy. Applying this technique to study isolated chloroplasts and whole leaves, we show here that the same change in neoxanthin configuration occurs in vivo, to an extent consistent with the magnitude of energy dissipation. Femtosecond transient absorption spectroscopy, performed on purified LHCII in the dissipative state, shows that energy is transferred from chlorophyll a to a low-lying carotenoid excited state, identified as one of the two luteins (lutein 1) in LHCII. Hence, it is experimentally demonstrated that a change in conformation of LHCII occurs in vivo, which opens a channel for energy dissipation by transfer to a bound carotenoid. We suggest that this is the principal mechanism of photoprotection.

Molecular basis of photoprotection and control of photosynthetic light-harvesting

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

The photoprotective molecular switch in the photosystem II antenna.

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.

Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll-protein complex

A new hypothesis is presented to explain the major molecular process that regulates the efficiency of light harvesting by chloroplast membranes. It is proposed that in excess light the decrease in the thylakoid lumen pH causes an increase in aggregation of the light harvesting complexes of photosystem II resulting in formation of an efficient pathway for non‐radiative dissipation of excitation energy. The aggregation is potentiated by the conversion of violaxanthin to zeaxanthin. This hypothesis is based upon (i) similarity between the spectroscopic changes associated with energy dissipation and those observed upon aggregation of isolated light harvesting complex; and (ii) the link between changes in light scattering and increased energy dissipation.

Control of the light harvesting function of chloroplast membranes: The LHCII‐aggregation model for non‐photochemical quenching

Dissipation of excess excitation energy within the photosystem II light‐harvesting antenna (LHCII) by non‐photochemical quenching (NPQ) is an important photoprotective process in plants. An update to a hypothesis for the mechanism of NPQ [FEBS Letters 292, 1991] is presented. The impact of recent advances in understanding the structure, organisation and photophysics of LHCII is assessed. We show possible locations of the predicted regulatory and quenching pigment‐binding sites in the structural model of the major LHCII. We suggest that NPQ is a highly regulated concerted response of the organised thylakoid macrostructure, which can include different mechanisms and sites at different times.

Photoprotective Energy Dissipation Involves the Reorganization of Photosystem II Light-Harvesting Complexes in the Grana Membranes of Spinach Chloroplasts

The rapidly reversible macrostructural changes in higher-plant chloroplast thylakoid membrane organization accompanying photoprotective energy dissipation (qE) are studied using freeze-fracture electron and laser confocal microscopy. qE is shown to involve the aggregation of light-harvesting complexes and their segregation from photosystem II. Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes.

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.

Regulation of Light Harvesting in Green Plants (Indication by Nonphotochemical Quenching of Chlorophyll Fluorescence)

ce has become one of the most powerful methods for assessing photosynthetic performance in plant physiological experiments (Horton and Bowyer, 1990; Krause and Weis, 1991). This has resulted almost entirely from the development of methods to distinguish photochemical and nonphotochemical quenching of fluorescence. Moreover, it is now clear that the process of nonphotochemical quenching itself indicates important regulatory adjustments in the photosynthetic membrane in response to altered external and internal conditions (Demmig-Adams and Adams, 1992; Horton and Ruban, 1992). In particular, the dissipation of excess absorbed excitation that is monitored by the main component of nonphotochemical quenching is a process that is necessary if plants are to avoid photoinhibition and photodestruction under conditions of light stress. When light is absorbed by the Chl molecules in the thylakoid membrane, the excited state has several alternative and competing fates: a small proportion is emitted as fluorescence, but, under light-limiting conditions, the major pathway of de-excitation is through photosynthetic electron transfer. The effect of photochemical utilization of energy is to quench fluorescence, and it is well known that when photosynthetic electron flow is saturated the yield of fluorescence rises. This photochemical quenching has been termed qP and, using the light-doubling principle as applied with modulated fluorimetry, it is easy to calculate it in leaves, chloroplasts, and cells (Schreiber et al., 1986; Horton and Bowyer, 1990; van Kooten and Snel, 1990). However, qP does not account for all of the quenching observed. Indeed, in light saturating for electron transport, qP tends to zero, yet there can be large amounts of quenching. Such quenching is therefore called nonphotochemical quenching and refers to the difference between the initial, dark-adapted maximum level of fluorescence and that recorded after a period of illumination. This quenching can be calculated in a number of ways, leading to it being termed variously as qN (Schreiber et al., 1986; van Kooten and Snel, 1990), NPQ (Bilger and Bjorkman, 1994), or SV, (Gilmore and Bjorkman, 1994); these all refer to the

The Effects of Illumination on the Xanthophyll Composition of the Photosystem II Light-Harvesting Complexes of Spinach Thylakoid Membranes

The xanthophyll composition of the light-harvesting chlorophyll a/b proteins of photosystem II (LHCII) has been determined for spinach (Spinacia oleracea L.) leaves after dark adaptation and following illumination under conditions optimized for conversion of violaxanthin into zeaxanthin. Each of the four LHCII components was found to have a unique xanthophyll composition. The major carotenoid was lutein, comprising 60% of carotenoid in the bulk LHCIIb and 35 to 50% in the minor LHCII components LHCIIa, LHCIIc, and LHCIId. The percent of carotenoid found in the xanthophyll cycle pigments was approximately 10 to 15% in LHCIIb and 30 to 40% in LHCIIa, LHCIIc, and LHCIId. The xanthophyll cycle was active for the pigments bound to all of the LHCII components. The extent of deepoxidation for complexes prepared from light-treated leaves was 27, 65, 69, and 43% for LHCIIa, -b, -c, and -d, respectively. These levels of conversion of violaxanthin to zeaxanthin were found in LHCII prepared by three different isolation procedures. It was estimated that approximately 50% of the zeaxanthin associated with photosystem II is in LHCIIb and 30% is associated with the minor LHCII components.

Photosynthetic acclimation: does the dynamic structure and macro-organisation of photosystem II in higher plant grana membranes regulate light harvesting states?

The efficiency of light harvesting in higher plant photosynthesis is regulated in response to external environmental conditions. Under conditions of excess light, the normally highly efficient light‐harvesting system of photosystem II is switched into a state in which unwanted, potentially harmful, energy is dissipated as heat. This process, known as nonphotochemical quenching, occurs by the creation of energy quenchers following conformational change in the light‐harvesting complexes, which is initiated by the build up of the thylakoid pH gradient and controlled by the xanthophyll cycle. In the present study, the evidence to support the notion that this regulatory mechanism is dependent upon the organization of the different antenna subunits in the stacked grana membranes is reviewed. We postulate that nonphotochemical quenching occurs within a structural locus comprising the PsbS subunit and components of the light‐harvesting antenna, CP26, CP24, CP29 and LHCIIb (the major trimeric light‐harvesting complex), formed in response to protonation and controlled by the xanthophyll cycle carotenoids.