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.     

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.     

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.     

Identification of Lhcb gene family encoding the light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

The Lhcb gene family in green plants encodes several light-harvesting Chl a/b-binding (LHC) proteins that collect and transfer light energy to the reaction centers of PSII. We comprehensively characterized the Lhcb gene family in the unicellular green alga, Chlamydomonas reinhardtii, using the expressed sequence tag (EST) databases. A total of 699 among over 15,000 ESTs related to the Lhcb genes were assigned to eight, including four new, genes that we isolated and sequenced here. A sequence comparison revealed that six of the Lhcb genes from C. reinhardtii correspond to the major LHC (LHCII) proteins from higher plants, and that the other two genes (Lhcb4 and Lhcb5) correspond to the minor LHC proteins (CP29 and CP26). No ESTs corresponding to another minor LHC protein (CP24) were found. The six LHCII proteins in C. reinhardtii cannot be assigned to any of the three types proposed for higher plants (Lhcb1-Lhcb3), but were classified as follows: Type I is encoded by LhcII-1.1, LhcII-1.2 and LhcII-1.3, and Types II, III and IV are encoded by LhcII-2, LhcII-3 and LhcII-4, respectively. These findings suggest that the ancestral LHC protein diverged into LHCII, CP29 and CP26 before, and that LHCII diverged into multiple types after the phylogenetic separation of green algae and higher plants.     

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.     

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.     

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.     

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.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

Arabidopsis mutants deleted in the light-harvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in photoprotection

The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C₂S₂ particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection.     

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis

Light‐harvesting complex II (LHCII) contains three highly homologous chlorophyll‐a/b‐binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo‐ and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so‐called state‐transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of ‘state 2 light’ results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non‐phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super‐ and mega‐complexes that comprised both photosystem (PS)I and PSII, and the state 2‐specific PSI–LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.     

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.     

Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal That Light-Harvesting Complex II Loosely Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.     

The immunophilin CYCLOPHILIN28 affects PSII-LHCII supercomplex assembly and accumulation in Arabidopsis thaliana

In plant chloroplasts, photosystem II (PSII) complexes, together with light-harvesting complex II (LHCII), form various PSII-LHCII supercomplexes (SCs). This process likely involves immunophilins, but the underlying regulatory mechanisms are unclear. Here, by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen-localized immunophilin CYCLOPHILIN28 (CYP28) to wild-type and transgenic complemented lines, we determined that CYP28 regulates the assembly and accumulation of PSII-LHCII SCs. Compared to the wild type, cyp28 plants showed accelerated leaf growth, earlier flowering time, and enhanced accumulation of high molecular weight PSII-LHCII SCs under normal light conditions. The lack of CYP28 also significantly affected the electron transport rate. Blue native-polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M-LHCII-Lhcb4-Lhcb6 complexes in cyp28 versus wild-type plants. Peptidyl-prolyl cis/trans isomerase (PPIase) activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity. Mutant analysis suggested that CYP28 may regulate PSII-LHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity. Furthermore, the Lhcb6-P139 residue is critical for PSII-LHCII SC assembly and accumulation. Therefore, our findings suggest that CYP28 likely regulates PSII-LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity.     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Investigation of the lateral light-induced migration of photosystem II light-harvesting proteins by nano-high performance liquid chromatography electrospray ionization mass spectrometry

This study reports a detailed analysis of the light-induced lateral migration of the photosystem II (PSII) antennae between appressed and non-appressed thylakoid membranes. The relative PSII antennae that migrated to stroma lamellae were readily established on the basis of peak areas of the separated stroma proteins in the ultraviolet chromatograms. Phosphorylation was predicted by intact molecular mass measurements, and this was confirmed by immunoblotting. When thylakoid membrane and chloroplasts were illuminated at 100 microE m(-2)s(-1), light-harvesting complex type II (Lhcb2) was the first PSII antenna to migrate, preferentially in phosphorylated form. However, the amount of Lhcb2 that migrated decreased after the first 20 min when the total amount of the three different Lhcb1 isoforms (1.1, 1.2, and 1.3) reached maximum. Lhcb1.1 was always found in the unphosphorylated form and migrated later than the other two isoforms, although the latter were also found to have low levels of phosphorylation. At the same time, major antennae on the grana were not found to be phosphorylated, whereas Lhcb4 showed a significant increase in molecular mass. At higher light intensity Lhcb2 migration was negligible, whereas migration of Lhcb1 isoforms was little changed, increasing in irradiated chloroplasts. Because there was no significant phosphorylation at high light intensity, and yet pigments were found to have significantly increased on the stroma lamellae, it may be that pigments play a role in migration and that, in fact, there is no direct correlation between phosphorylation and migration. We hypothesize that the Lhcb1 isoforms expressed by the multigene families play a role in plant adaptation.     

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.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.