High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.

Comparison of PsbQ and Psb27 in photosystem II provides insight into their roles.

Photosystem II (PSII) catalyzes the oxidation of water at its active site that harbors a high-valent inorganic Mn4CaOx cluster called the oxygen-evolving complex (OEC). Extrinsic subunits generally serve to protect the OEC from reductants and stabilize the structure, but diversity in the extrinsic subunits exists between phototrophs. Recent cryo-electron microscopy experiments have provided new molecular structures of PSII with varied extrinsic subunits. We focus on the extrinsic subunit PsbQ, that binds to the mature PSII complex, and on Psb27, an extrinsic subunit involved in PSII biogenesis. PsbQ and Psb27 share a similar binding site and have a four-helix bundle tertiary structure, suggesting they are related. Here, we use sequence alignments, structural analyses, and binding simulations to compare PsbQ and Psb27 from different organisms. We find no evidence that PsbQ and Psb27 are related despite their similar structures and binding sites. Evolutionary divergence within PsbQ homologs from different lineages is high, probably due to their interactions with other extrinsic subunits that themselves exhibit vast diversity between lineages. This may result in functional variation as exemplified by large differences in their calculated binding energies. Psb27 homologs generally exhibit less divergence, which may be due to stronger evolutionary selection for certain residues that maintain its function during PSII biogenesis and this is consistent with their more similar calculated binding energies between organisms. Previous experimental inconsistencies, low confidence binding simulations, and recent structural data suggest that Psb27 is likely to exhibit flexibility that may be an important characteristic of its activity. The analysis provides insight into the functions and evolution of PsbQ and Psb27, and an unusual example of proteins with similar tertiary structures and binding sites that probably serve different roles.

Binding and functions of the two chloride ions in the oxygen-evolving center of photosystem II.

Light-driven water oxidation in photosynthesis occurs at the oxygen-evolving center (OEC) of photosystem II (PSII). Chloride ions (Cl-) are essential for oxygen evolution by PSII, and two Cl- ions have been found to specifically bind near the Mn4CaO5 cluster in the OEC. The retention of these Cl- ions within the OEC is critically supported by some of the membrane-extrinsic subunits of PSII. The functions of these two Cl- ions and the mechanisms of their retention both remain to be fully elucidated. However, intensive studies performed recently have advanced our understanding of the functions of these Cl- ions, and PSII structures from various species have been reported, aiding the interpretation of previous findings regarding Cl- retention by extrinsic subunits. In this review, we summarize the findings to date on the roles of the two Cl- ions bound within the OEC. Additionally, together with a short summary of the functions of PSII membrane-extrinsic subunits, we discuss the mechanisms of Cl- retention by these extrinsic subunits.

Deletion of psbQ' gene in Cyanidioschyzon merolae reveals the function of extrinsic PsbQ' in PSII.

KEY MESSAGE: We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ' subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ' subunit in the PSII complex. We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ' extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ' protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ' is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.

Structurally conserved channels in cyanobacterial and plant photosystem II.

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn4Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn4Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn4Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn4Ca cluster.

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II.

Protein cross-linking and radiolytic footprinting coupled with high-resolution mass spectrometry were used to examine the structure of PsbP and PsbQ when they are bound to Photosystem II. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function. These interactions place strong structural constraints on the organization of PsbP when associated with the Photosystem II complex. Additionally, amino acid residues in the structurally unresolved loop 3A domain of PsbP ((90)K-(107)V), (93)Y and (96)K, are in close proximity (≤ 11.4 Å) to the N-terminal (1)E residue of PsbQ. These findings are the first, to our knowledge, to identify a putative region of interaction between these two components. Cross-linked domains within PsbQ were also identified, indicating that two PsbQ molecules can interact in higher plants in a manner similar to that observed by Liu et al. [(2014) Proc Natl Acad Sci 111(12):4638-4643] in cyanobacterial Photosystem II. This interaction is consistent with either intra-Photosystem II dimer or inter-Photosystem II dimer models in higher plants. Finally, OH(•) produced by synchrotron radiolysis of water was used to oxidatively modify surface residues on PsbP and PsbQ. Domains on the surface of both protein subunits were resistant to modification, indicating that they were shielded from water and appear to define buried regions that are in contact with other Photosystem II components.

A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).

Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.

The extrinsic proteins of photosystem II: update.

MAIN CONCLUSION: Recent investigations have provided important new insights into the structures and functions of the extrinsic proteins of Photosystem II. This review is an update of the last major review on the extrinsic proteins of Photosystem II (Bricker et al., Biochemistry 31:4623-4628 2012). In this report, we will examine advances in our understanding of the structure and function of these components. These proteins include PsbO, which is uniformly present in all oxygenic organisms, the PsbU, PsbV, CyanoQ, and CyanoP proteins, found in the cyanobacteria, and the PsbP, PsbQ and PsbR proteins, found in the green plant lineage. These proteins serve to stabilize the Mn4CaO5 cluster and optimize oxygen evolution at physiological calcium and chloride concentrations. The mechanisms used to perform these functions, however, remain poorly understood. Recently, important new findings have significantly advanced our understanding of the structures, locations and functions of these important subunits. We will discuss the biochemical, structural and genetic studies that have been used to elucidate the roles played by these proteins within the photosystem and their locations within the photosynthetic complex. Additionally, we will examine open questions needing to be addressed to provide a coherent picture of the role of these components within the photosystem.

LtGAPR1 Is a Novel Secreted Effector from Lasiodiplodia theobromae That Interacts with NbPsQ2 to Negatively Regulate Infection.

The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. Lasiodiplodia theobromae secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in Nicotiana benthamiana reduced susceptibility to L. theobromae, and the silencing of NbPsbQ2 enhanced L. theobromae infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in N. benthamiana leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection.

Determination of chemical identity and occupancy from experimental density maps.

Three basic electronic properties of molecules, electron density (ED), charge density (CD), and electrostatic potentials (ESP), are dependent on both atomic mobility and occupancy of components in the molecules. Small protein subunits may bind large macromolecular complexes with a reduced occupancy or an increased atomic mobility or both due to affinity-based functional regulation, and so may substrates, products, cofactors, ions or solvent molecule to the active sites of enzymes. A quantitative theory is presented in this study that describes the dependence of atomic functions on atomic B-factor in Fourier transforms of the corresponding maps. An application of this theory is described to an experimental ED map at 1.73-Å resolution, and to an experimental CD map at 2.2-Å resolution. All the three density functions are linearly proportional to occupancy when the structure factor F(000) term of Fourier transforms of experimental density maps is included. Upon application of this theory to both experimental CD and ESP maps recently reported for photosystem II-light harvesting complex II supercomplex at 3.2-Å resolution, the occupancy of two extrinsic protein subunits PsbQ and PsbP is determined to be 20.4 ± 0.2%, and the negative mean ESP value of vitreous ice displaced by the supercomplex on electron scattering path is estimated to be 3% of the mean ESP value of protein α-helices.

The PsbP and PsbQ family proteins in the photosynthetic machinery of chloroplasts.

The PsbP and PsbQ proteins are extrinsic subunits of the photosystem II in eukaryotic photosynthetic organisms including higher plants, green algae and euglena. It has been suggested that PsbP and PsbQ have evolved from their cyanobacterial homologs, while considerable genetic and functional modifications have occurred to generate the eukaryote-type proteins. In addition, number of PsbP and PsbQ homologs exist in the thylakoid lumen of chloroplasts. These homologs are nuclear-encoded and likely diverged by gene duplication, and recent studies have elucidated their various functions in the photosynthetic machinery. In this short review, recent findings and new idea about these components will be discussed.

Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1.