Photosystem II protein phosphorylation follows four distinctly different regulatory patterns induced by environmental cues

Differential redox regulation of thylakoid phosphoproteins was studied in winter rye plants in vivo. The redox state of chloroplasts was modulated by growing plants under different light/temperature conditions and by transient shifts to different light/temperature regimes. Phosphorylation of PSII reaction centre proteins D1 and D2, the chlorophyll a binding protein CP43, the major chlorophyll a/b binding proteins Lhcb1 and Lhcb2 (LHCII) and the minor light‐harvesting antenna protein CP29 seem to belong to four distinct regulatory groups. Phosphorylation of D1 and D2 was directly dependent on the reduction state of the plastoquinone pool. CP43 protein phosphorylation generally followed the same pattern, but often remained phosphorylated even in darkness. Phosphorylation of CP29 occurred upon strong reduction of the plastoquinone pool, and was further enhanced by low temperatures. In vitro studies further demonstrated that CP29 phosphorylation is independent of the redox state of both the cytochrome b₆/f complex and the thiol compounds. Complete phosphorylation of Lhcb1 and 2 proteins, on the contrary, required only modest reduction of the plastoquinone pool, and was subject to inhibition upon increase in the thiol redox state of the stroma. Furthermore, the reversible phosphorylation of Lhcb1 and 2 proteins appeared to be an extremely dynamic process, being rapidly modulated by short‐term fluctuations in chloroplast redox conditions.     

Structural and functional differentiation of the light‐harvesting protein Lhcb4 during land plant diversification

About 475 million years ago, plants originated from an ancestral green alga and evolved first as non‐vascular and later as vascular plants, becoming the primary producers of biomass on lands. During that time, the light‐harvesting complex II (LHCII), responsible for sunlight absorption and excitation energy transfer to the photosystem II (PSII) core, underwent extensive differentiation. Lhcb4 is an ancestral LHCII that, in flowering plants, differentiated into up to three isoforms, Lhcb4.1, Lhcb4.2 and Lhcb4.3. The pivotal position of Lhcb4 in the PSII‐LHCII supercomplex (PSII‐LHCIIsc) allows functioning as linker for either S‐ or M‐trimers of LHCII to the PSII core. The increased accumulation of Lhcb4.3 observed in PSII‐LHCIIsc of plants acclimated to moderate and high light intensities induced us to investigate, whether this isoform has a preferential localization in a specific PSII‐LHCIIsc conformation that might explain its light‐dependent accumulation. In this work, by combining an improved method for separation of different forms of PSII‐LHCIIsc from thylakoids of Pisum sativum L. grown at increasing irradiances with quantitative proteomics, we assessed that Lhcb4.3 is abundant in PSII‐LHCIIsc of type C₂S₂, and, interestingly, similar results were found for the PsbR subunit. Phylogenetic comparative analysis on different taxa of the Viridiplantae lineage and structural modeling further pointed out to an effect of the evolution of different Lhcb4 isoforms on the light‐dependent modulation of the PSII‐LHCIIsc organization. This information provides new insight on the properties of the Lhcb4 and its isoforms and their role on the structure, function and regulation of PSII.     

Different response of photosystem II to short and long‐term drought stress in Arabidopsis thaliana

Short‐ and long‐term drought stress on photosystem II (PSII) and oxidative stress were studied in Arabidopsis thaliana. Under drought stress, chlorophyll (Chl) content, Chl fluorescence, relative water content and oxygen evolution capacity gradually decreased, and the thylakoid structure was gradually damaged. Short‐term drought stress caused a rapid disassembly of the light‐harvesting complex II (LHCII). However, PSII dimers kept stable under the short‐term drought stress and significantly decreased only after 15 days of drought stress. Immunoblotting analysis of the thylakoid membrane proteins showed that most of the photosystem proteins decreased after the stress, especially for Lhcb5, Lhcb6 and PsbQ proteins. However, surprisingly, PsbS significantly increased after the long‐term drought stress, which is consistent with the substantially increased non‐photochemical quenching (NPQ) after the stress. Our results suggest that the PSII–LHCII supercomplexes and LHCII assemblies play an important role in preventing photo‐damages to PSII under drought stress.     

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light‐harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kou?il et al. (2016) New Phytol. 210 , 808–814]. Here we present a single‐particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light‐harvesting under varying environmental conditions.     

Light‐dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light‐harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non‐photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1‐2. This protein, which showed light intensity‐dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light‐induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.     

The Light-Harvesting Chlorophyll a/b Binding Proteins Lhcb1 and Lhcb2 Play Complementary Roles during State Transitions in Arabidopsis

Photosynthetic light harvesting in plants is regulated by phosphorylation-driven state transitions: functional redistributions of the major trimeric light-harvesting complex II (LHCII) to balance the relative excitation of photosystem I and photosystem II. State transitions are driven by reversible LHCII phosphorylation by the STN7 kinase and PPH1/TAP38 phosphatase. LHCII trimers are composed of Lhcb1, Lhcb2, and Lhcb3 proteins in various trimeric configurations. Here, we show that despite their nearly identical amino acid composition, the functional roles of Lhcb1 and Lhcb2 are different but complementary. Arabidopsis thaliana plants lacking only Lhcb2 contain thylakoid protein complexes similar to wild-type plants, where Lhcb2 has been replaced by Lhcb1. However, these do not perform state transitions, so phosphorylation of Lhcb2 seems to be a critical step. In contrast, plants lacking Lhcb1 had a more profound antenna remodeling due to a decrease in the amount of LHCII trimers influencing thylakoid membrane structure and, more indirectly, state transitions. Although state transitions are also found in green algae, the detailed architecture of the extant seed plant light-harvesting antenna can now be dated back to a time after the divergence of the bryophyte and spermatophyte lineages, but before the split of the angiosperm and gymnosperm lineages more than 300 million years ago.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

Differential accumulation of Lhcb gene products in thylakoid membranes of Zea mays plants grown under contrasting light and temperature conditions

In higher plants many different genes encode Lhcb proteins that belong to a highly conserved protein family. Evolutionary conservation of this genetic redundancy suggests that individual gene products play different roles in light harvesting and photoprotection depending on environmental conditions. We have tested the hypothesis that expression/accumulation of individual light harvesting complex (Lhc) proteins depends on plant growth conditions. Zea mays plants were grown in different temperature (13 degrees C vs. 24 degrees C) and light (high vs. low) conditions. The thylakoid membranes were isolated and fractionated by sucrose gradient and the protein content of the different bands was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences were found in the accumulation of both the major light harvesting complex of photosystem II (LHCII) complexes and the minor antenna chlorophyll proteins CP29, CP26 and CP24. In particular, temperature seems to play a major role in driving the expression/accumulation of the different proteins: the LHCII/minor antenna ratio increases with decreasing temperature. The pigment composition and the spectroscopic properties of LHCII complexes isolated from low temperature grown plants are significantly different from those of LHCII purified from high temperature grown plants. Two-dimensional maps show that different LHCII proteins are accumulated at different levels depending on growth conditions. Moreover the low temperature/high light grown plants show an increased value of nonphotochemical quenching. These results suggest a specific role of different LHCII complexes in the organization of the potosystem II and photoprotection.     

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.Light capture and its conversion to chemical energy occur in a set of transmembrane protein complexes of the thylakoid membrane. PSII, the cytochrome b₆f complex, and PSI drive photosynthetic electron flow and the creation of a proton gradient across the thylakoid membrane. ATP synthase couples the dissipation of this gradient to the synthesis of ATP. The light-harvesting antennae play an important role in collecting light and transferring energy to the photosystems. Light-Harvesting Complex I (LHCI) exclusively transfers light energy to PSI, with which it is tightly associated (Croce and van Amerongen, 2014). In contrast, LHCII, which is the most abundant complex of the thylakoid membrane, can transfer energy to PSI or PSII (Grieco et al., 2015). Light is highly variable in natural environments, and plants experience continuous changes in both the spectrum and intensity of light on timescales as short as seconds. Changes in light quality may unbalance the activity of the two photosystems since their absorption spectra differ, whereas high light intensity can lead to overexcitation and induce photodamage. At low or moderate light intensities, the LHCII complex differentially associates with PSII or PSI, in a phosphorylation-dependent process known as state transitions, to rapidly respond to changes in the spectrum of light. In brief, under light quality that activates PSII more than PSI (e.g. blue light), LHCII is phosphorylated, and as a consequence, its binding to PSI is favored (state 2). Conversely, under light that preferentially excites PSI (enriched in far-red), this association can be reverted by dephosphorylation of the LHCII antenna, which favors its binding to PSII (state 1; Goldschmidt-Clermont and Bassi, 2015; Kim et al., 2015). A protein kinase, STATE TRANSITION7 (STN7), and a protein phosphatase, PROTEIN PHOSPHATASE1 (PPH1)/THYLAKOID-ASSOCIATED PHOSPHATASE38 (TAP38), are essential for the rapid phosphorylation and dephosphorylation of the LHCII antenna that regulates its differential association to PSI or PSII (Bellafiore et al., 2005; Pribil et al., 2010; Shapiguzov et al., 2010). Only a relatively small fraction of the LHCII antenna (<20%) is estimated to participate in state transitions in Arabidopsis (Arabidopsis thaliana; Allen, 1992). However, the process is conserved across the green eukaryotes and is relevant to plant fitness (Frenkel et al., 2007). Under high light, energy-dependent quenching of LHCII predominates, and furthermore, this antenna can uncouple from PSII (Wientjes et al., 2013b).The differential association of photosystems, LHCII, and other components of the thylakoid membrane gives rise to a set of supercomplexes that are central in ensuring photosynthetic efficiency and a rapid response to environmental cues (Caffarri et al., 2009; Duffy et al., 2013; Pietrzykowska et al., 2014; Fristedt et al., 2015). Fine tuning the dynamic assembly of these supercomplexes involves the association of antennae containing specific sets of Lhcb proteins. The major LHCII antenna comprises homo- and heterotrimers of Lhcb1 to Lhcb3 (Jackowski et al., 2001), whereas the minor LHCII isoforms (Lhcb4–Lhcb6) are monomeric (de Bianchi et al., 2008). Lhcb1 and Lhcb2 share a very similar primary structure and associated pigments (Formaggio et al., 2001; Zhang et al., 2008), whereas Lhcb3 appears to have slightly different features (Standfuss and Kühlbrandt, 2004). In Arabidopsis, five genes encode Lhcb1 isoforms, three genes encode Lhcb2 isoforms, and a single gene encodes Lhcb3. The principal discriminant between these classes is a short stretch of residues at the N-terminal end, which is of particular importance since it contains the Thr that is reversibly phosphorylated during light-acclimation processes (Goldschmidt-Clermont and Bassi, 2015). During evolution, land plants have maintained a major LHCII composed of different classes of Lhcb subunits. The phosphorylated N terminus of Lhcb2 was particularly well conserved (Alboresi et al., 2008; Zhang et al., 2008).PSII-LHCII supercomplexes have been isolated from Arabidopsis with up to four LHCII trimers bound to a PSII dimer, as well as the three minor monomeric antennae (Lhcb4–Lhcb6; Caffarri et al., 2009; Kouřil et al., 2012). In the LHCII trimers of these supercomplexes, different classes of Lhcb subunits are distributed differently, suggesting a specific role in light acclimation for each of them (Damkjaer et al., 2009; Pietrzykowska et al., 2014). In the stably bound S trimer, Lhcb1 and Lhcb2 are more abundant, whereas the moderately bound M trimer contains mostly Lhcb1 and Lhcb3 (Galka et al., 2012). PSII supercomplexes isolated from spinach (Spinacia oleracea) showed the presence of an extra LHCII trimer (L trimer); therefore, it is possible that, in Arabidopsis, other trimers are associated with the PSII dimer in a more labile supercomplex that cannot be isolated (Boekema et al., 1999). A single LHCII trimer, containing Lhcb1 and Lhcb2, stably associates with PSI to constitute the PSI-LHCII supercomplex, whose formation is dependent on LHCII phosphorylation by STN7 in state 2 (Kouřil et al., 2005; Galka et al., 2012).Previous reports have shown that the relative phosphorylation of Lhcb1 and Lhcb2 isoforms differs among thylakoid supercomplexes (Galka et al., 2012; Leoni et al., 2013). Here, we address the specific roles of Lhcb1 and Lhcb2 phosphorylation in photosynthetic acclimation. The improved protocol for SDS-PAGE in the presence of Phos-tag (Wako Chemicals) that we present allows quantification of the extent of phosphorylation for each class of antenna isoforms. We report that, in the PSI-LHCII supercomplex that is assembled in state 2, only the phosphorylated form of Lhcb2 is present, whereas the phosphorylated form of Lhcb1 is excluded. In contrast, both Lhcb1 and Lhcb2 are phosphorylated to different levels in other supercomplexes. This quantitative information on the level of phosphorylation of Lhcb1 and Lhcb2 offers new insights into the specific roles of the two classes of LHCII isoforms in light acclimation and supercomplex formation.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.     

Light-harvesting complex II binds to several small subunits of photosystem I

Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.     

The Photosystem II Light-Harvesting Protein Lhcb3 Affects the Macrostructure of Photosystem II and the Rate of State Transitions in Arabidopsis

The main trimeric light-harvesting complex of higher plants (LHCII) consists of three different Lhcb proteins (Lhcb1-3). We show that Arabidopsis thaliana T-DNA knockout plants lacking Lhcb3 (koLhcb3) compensate for the lack of Lhcb3 by producing increased amounts of Lhcb1 and Lhcb2. As in wild-type plants, LHCII-photosystem II (PSII) supercomplexes were present in Lhcb3 knockout plants (koLhcb3), and preservation of the LHCII trimers (M trimers) indicates that the Lhcb3 in M trimers has been replaced by Lhcb1 and/or Lhcb2. However, the rotational position of the M LHCII trimer was altered, suggesting that the Lhcb3 subunit affects the macrostructural arrangement of the LHCII antenna. The absence of Lhcb3 did not result in any significant alteration in PSII efficiency or qE type of nonphotochemical quenching, but the rate of transition from State 1 to State 2 was increased in koLhcb3, although the final extent of state transition was unchanged. The level of phosphorylation of LHCII was increased in the koLhcb3 plants compared with wild-type plants in both State 1 and State 2. The relative increase in phosphorylation upon transition from State 1 to State 2 was also significantly higher in koLhcb3. It is suggested that the main function of Lhcb3 is to modulate the rate of state transitions.     

Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light‐driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light‐harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI‐bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.     

Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1

During state transitions, plants regulate energy distribution between photosystems I and II through reversible phosphorylation and lateral migration of the major light-harvesting complex LHCII. Dephosphorylation of LHCII and the transition from state 2 to state 1 requires a thylakoid membrane-associated phosphatase named TAP38 or PPH1. TAP38/PPH1 specifically targets LHCII but not the core subunits of photosystem II, whereas the underlying molecular mechanism of their mutual recognition is currently unclear. Here, we present the structures of Arabidopsis thaliana TAP38/PPH1 in the substrate-free and substrate-bound states. The protein contains a type 2C serine/threonine protein phosphatase (PP2C) core domain, a Mn²⁺ (or Mg²⁺) binuclear center and two additional motifs contributing to substrate recognition. A 15-mer phosphorylated N-terminal peptide of Lhcb1 binds to TAP38/PPH1 on two surface clefts enclosed by the additional motifs. The first segment of the phosphopeptide is clamped by a pair of tooth-like arginine residues at Cleft 1 site. The binding adopts the lock-and-key mechanism with slight rearrangement of the substrate binding residues on TAP38/PPH1. Meanwhile, a more evident substrate-induced fitting occurs on Cleft 2 harboring the extended part of the phosphopeptide. The results unravel the bases for the specific recognition between TAP38/PPH1 and phosphorylated Lhcb1, a crucial step in state transitions.     

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.     

A look within LHCII: differential analysis of the Lhcb1-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis

The major antenna complex of higher-plant photosynthesis, LHCII, is composed by the products of three genes, namely, Lhcb1-2-3. In this paper, the biochemical and spectroscopic properties of each of the three gene products were investigated. The three complexes were obtained by overexpression of the apoproteins in bacteria and refolding in vitro with purified pigments, thus allowing detection of differences in the structure/function of the pigment-binding gene products. The analyses showed that Lhcb1 and Lhcb2 complexes have similar pigment binding properties, although not identical, while Lhcb3 is clearly different with respect to both pigment binding and spectral properties and cannot produce homotrimers in vitro. Heterotrimers containing Lhcb3 together with Lhcb1 and/or -2 proteins were obtained upon assembly with Lhcb proteins purified from thylakoids. The major functional characteristics of Lhcb3 with respect to Lhcb1 and -2 consisted in (i) a red-shift of one specific chlorophyll a chromophore, strongly affecting the red-most region of the absorption spectrum and (ii) a different specificity for xanthophylls binding to sites L2 and N1. These properties make Lhcb3 a relative sink for excitation energy in isolated heterotrimers with Lhcb1 + Lhcb2, and potentially, a preferential site of regulation of the antenna function in excess light conditions.     

Mechanistic aspects of the xanthophyll dynamics in higher plant thylakoids

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light.     

Photoprotection in the antenna complexes of photosystem II: role of individual xanthophylls in chlorophyll triplet quenching

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.