PsbO Protein Research Articles

The Effect of Removal of External Proteins PsbO, PsbP and PsbQ on Flash-Induced Molecular Oxygen Evolution and Its Biphasicity in Tobacco PSII.

The oxygen evolution within photosystem II (PSII) is one of the most enigmatic processes occurring in nature. It is suggested that external proteins surrounding the oxygen-evolving complex (OEC) not only stabilize it and provide an appropriate ionic environment but also create water channels, which could be involved in triggering the ingress of water and the removal of O2 and protons outside the system. To investigate the influence of these proteins on the rate of oxygen release and the efficiency of OEC function, we developed a measurement protocol for the direct measurement of the kinetics of oxygen release from PSII using a Joliot-type electrode. PSII-enriched tobacco thylakoids were used in the experiments. The results revealed the existence of slow and fast modes of oxygen evolution. This observation is model-independent and requires no specific assumptions about the initial distribution of the OEC states. The gradual removal of exogenous proteins resulted in a slowdown of the rapid phase (~ms) of O2 release and its gradual disappearance while the slow phase (~tens of ms) accelerated. The role of external proteins in regulating the biphasicity and efficiency of oxygen release is discussed based on observed phenomena and current knowledge.

Structural basis for an early stage of the photosystem II repair cycle in Chlamydomonas reinhardtii

Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins—PsbO, PsbP, and PsbQ—integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.

Single-cell microfluidics in combination with chlorophyll a fluorescence measurements to assess the lifetime of the Chlamydomonas PSBO protein

Single-cell microfluidics in combination with chlorophyll a fluorescence measurements to assess the lifetime of the Chlamydomonas PSBO protein

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Comparison of photosynthetic responses between haptophyte Phaeocystis globosa and diatom Skeletonema costatum under phosphorus limitation.

The diatom Skeletonema costatum and the haptophyte Phaeocystis globosa often form blooms in the coastal waters of the South China Sea. Skeletonema costatum commonly dominates in nutrient enrichment coastal waters, whereas P. globosa starts flourishing after the diatom blooms when phosphorus (P) is limited. Therefore, P limitation was proposed to be a critical factor affecting diatom-haptophyte transition. To elucidate the tolerance to P limitation in P. globosa compared with S. costatum, the effect of P limitation on their photosystem II (PSII) performance was investigated and their photosynthesis acclimation strategies in response to P limitation were evaluated. P limitation did not affect the growth of P. globosa over 7 days but decreased it for S. costatum. Correspondingly, the PSII activity of S. costatum was significantly inhibited by P limitation. The decline in PSII activity in S. costatum under P limitation was associated with the impairment of the oxygen-evolving complex (the donor side of PSII), the hindrance of electron transport from QA - to QB (the acceptor side of PSII), and the inhibition of electron transport to photosystem I (PSI). The 100% decrease in D1 protein level of S. costatum after P limitation for 6 days and PsbO protein level after 2 days of P limitation were attributed to its enhanced photoinhibition. In contrast, P. globosa maintained its photosynthetic activity with minor impairment of the function of PSII. With accelerated PSII repair and highly increased non-photochemical quenching (NPQ), P. globosa can avoid serious PSII damage under P limitation. On the contrary, S. costatum decreased its D1 restoration under P limitation, and the maximum NPQ value in S. costatum was only one-sixth of that in P. globosa. The present work provides extensive evidence that a close interaction exists between the tolerance to P limitation and photosynthetic responses of S. costatum and P. globosa.

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts of CaCl2 that were required for dissociation of the PsbORC complex. It was first demonstrated that the protein exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and promote its participation in alternative pathways of electron transport under the influence of stress factors. The hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.

Dimerization of the Free and Photosystem II-Associated PsbO Protein upon Irradiation with UV Light

Dimerization of the Free and Photosystem II-Associated PsbO Protein upon Irradiation with UV Light

The lifetime of the oxygen-evolving complex subunit PSBO depends on light intensity and carbon availability in Chlamydomonas.

PSBO is essential for the assembly of the oxygen-evolving complex in plants and green algae. Despite its importance, we lack essential information on its lifetime and how it depends on the environmental conditions. We have generated nitrate-inducible PSBO amiRNA lines in the green alga Chlamydomonas reinhardtii. Transgenic strains grew normally under non-inducing conditions, and their photosynthetic performance was comparable to the control strain. Upon induction of the PSBO amiRNA constructs, cell division halted. In acetate-containing medium, cellular PSBO protein levels decreased by 60% within 24 h in the dark, by 75% in moderate light, and in high light, the protein completely degraded. Consequently, the photosynthetic apparatus became strongly damaged, probably due to 'donor-side-induced photoinhibition', and cellular ultrastructure was also severely affected. However, in the absence of acetate during induction, PSBO was remarkably stable at all light intensities and less substantial changes occurred in photosynthesis. Our results demonstrate that the lifetime of PSBO strongly depends on the light intensity and carbon availability, and thus, on the metabolic status of the cells. We also confirm that PSBO is required for photosystem II stability in C. reinhardtii and demonstrate that its specific loss also entails substantial changes in cell morphology and cell cycle.

Genes encoding the photosystem II proteins are under purifying selection: an insight into the early evolution of oxygenic photosynthesis

The molecular evolution concerns coding sequences (CDSs) of genes and may affect the structure and function of proteins. Non-uniform use of synonymous codons during translation, known as codon usage bias (CUB), depends on the balance between mutations bias and natural selection. We estimated different CUB indices, i.e. the effective number of codons (ENC), G + C content in the 3rd codon positions (GC3), and codon adaptation index for CDSs of intrinsic proteins of photosystem II (PSII), such as psbA (D1), psbD (D2), psbB (CP47), psbC (CP43),and CDSs of the extrinsic protein psbO (PsbO). These genes occur in all organisms that perform oxygenic photosynthesis (OP) on Earth: cyanobacteria, algae and plants. Comparatively, a similar analysis of codon bias for CDSs of L and M subunits that constitute the core proteins of the type II reaction centre (RCII) in anoxygenic bacteria was performed. Analysis of CUB indices and determination of the number of synonymous (dS) and nonsynonymous substitutions (dN) in all analysed CDSs indicated that the crucial PSII and RCII proteins were under strong purifying (negative) selection in course of evolution. Purifying selection was also estimated for CDSs of atpA, the α subunit of ATP synthase, an enzyme that was most likely already present in the last universal common ancestor(LUCA). The data obtained point to an ancient origin of OP, even in the earliest stages of the evolution of life on Earth.

Transcriptome sequencing reveals that photoinduced gene IP-L affects the expression of PsbO to response to virus infection in Nicotiana benthamiana

CP-interacting Protein-L (IP-L) is a host protein interacting with tomato mosaic virus (ToMV) coat protein (CP) in Nicotiana benthamiana plant. In this study, we analyzed the relationship between IP-L, photosynthesis and virus infection. Our results showed that the promoter of IP-L contained many light-responsive elements, and its expression was regulated by light rhythms, light quality and light intensity. We tried to construct IP-L overexpression transgenic N. benthamiana, but the plant shows homologous co-inhibition to IP-L, which resulted in the endogenous IP-L silencing. RNA-Seq of IP-L silenced N. benthamiana revealed that IP-L was mainly involved in photosynthesis, light harvesting, photosystem II and chlorophyll binding. Strikingly, IP-L exerted a marked influence on multiple light-dependent pathways such as light stimulus, photosynthesis and chloroplast. IP-L silencing increased the expression of photosynthesis-related proteins PsbO, PsaD, PsaF and PetE, while silencing these 4 proteins increased the expression of IP-L. However, only silencing PsbO had a certain regulatory effect on the infection of TMV-GFP. Silencing PsbO could significantly reduce the content of chlorophyll b and cause the leaves to fade green and slightly yellow. All in all, for the first time we proved the relationship between IP-L and photosynthesis, clarified the coordination between virus, IP-L and photosynthesis, and provides a new clue for understanding the molecular mechanism of virus infection affecting photosynthesis.

Similarities and Differences in the Effects of Toxic Concentrations of Cadmium and Chromium on the Structure and Functions of Thylakoid Membranes in Chlorella variabilis.

Trace metal contaminations in natural waters, wetlands, and wastewaters pose serious threats to aquatic ecosystems—mainly via targeting microalgae. In this work, we investigated the effects of toxic amounts of chromium and cadmium ions on the structure and function of the photosynthetic machinery of Chlorella variabilis cells. To halt the propagation of cells, we used high concentrations of Cd and Cr, 50–50 mg L−1, in the forms of CdCl2 x 2.5 H2O and K2Cr2O7, respectively. Both treatments led to similar, about 50% gradual diminishment of the chlorophyll contents of the cells in 48 h, which was, however, accompanied by a small (~10%) but statistically significant enrichment (Cd) and loss (Cr) of ß-carotene. Both Cd and Cr inhibited the activity of photosystem II (PSII)—but with more severe inhibitions with Cr. On the contrary, the PsbA (D1) protein of PSII and the PsbO protein of the oxygen-evolving complex were retained more in Cr-treated cells than in the presence of Cd. These data and the higher susceptibility of P700 redox transients in Cr-treated cells suggest that, unlike with Cd, PSII is not the main target in the photochemical apparatus. These differences at the level of photochemistry also brought about dissimilarities at higher levels of the structural complexity of the photosynthetic apparatus. Circular dichroism (CD) spectroscopy measurements revealed moderate perturbations in the macro-organization of the protein complexes—with more pronounced decline in Cd-treated cells than in the cells with Cr. Also, as reflected by transmission electron microscopy and small-angle neutron scattering, the thylakoid membranes suffered shrinking and were largely fragmented in Cd-treated cells, whereas no changes could be discerned with Cr. The preservation of integrity of membranes in Cr-treated cells was most probably aided by high proportion of the de-epoxidized xanthophylls, which were absent with Cd. It can thus be concluded that beside strong similarities of the toxic effects of Cr and Cd, the response of the photosynthetic machinery of C. variabilis to these two trace metal ions substantially differ from each other—strongly suggesting different inhibitory and protective mechanisms following the primary toxic events.

Front Cover: pH‐Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes (ChemBioChem 11/2020)

Determination of surface pKa values on PSII protein PsbO: Being close to the proton release side of Photosystem II and equipped with numerous surface carboxylate groups, PsbO is an appealing model to study side-chain-protonation reactions. Using solution NMR techniques, we were able to determine the pKa values of 21 out of 22 carboxylate groups, revealing a site-specific distribution as illustrated on the cover. More information can be found in the full paper by L. Gerland, H. Oschkinat et al. Image designed and created by Barth van Rossum.

Function of PsbO-Asp158 in photosystem II: effects of mutation of this residue on the binding of PsbO and function of PSII in Thermosynechococcus vulcanus.

PsbO-D158 is a highly conserved residue of the PsbO protein in photosystem II (PSII), and participates in one of the hydrogen-bonding networks connecting the manganese cluster with the lumenal surface. In order to examine the role of PsbO-D158, we mutated it to E, N or K in Thermosynechococcus vulcanus and characterized photosynthetic properties of the mutants obtained. The growth rates of these three mutants were similar to that of the wild type, whereas the oxygen-evolving activity of the three mutant cells decreased to 60-64% of the wild type. Fluorescence kinetics showed that the mutations did not affect the electron transfer from QA to QB, but slightly affected the donor side of PSII. Moreover, all of the three mutant cells were more sensitive to high light and became slower to recover from photoinhibition. In the isolated thylakoid membranes from the three mutants, the PsbU subunit was lost and the oxygen-evolving activity was reduced to a lower level compared to that in the respective cells. PSII complexes isolated from these mutants showed no oxygen-evolving activity, which was found to be due to large or complete loss of PsbO, PsbV and PsbU during the process of purification. Moreover, PSII cores purified from the three mutants contained Psb27, an assembly co-factor of PSII. These results suggest that PsbO-D158 is required for the proper binding of the three extrinsic proteins to PSII and plays an important role in maintaining the optimal oxygen-evolving activity, and its mutation caused incomplete assembly of the PSII complex.

YR36/WKS1-Mediated Phosphorylation of PsbO, an Extrinsic Member of Photosystem II, Inhibits Photosynthesis and Confers Stripe Rust Resistance in Wheat

Wheat stripe rust, due to infection by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease that causes significant global grain yield losses. Yr36, which encodes Wheat Kinase START1 (WKS1), is an effective high-temperature adult-plant resistance gene and confers resistance to a broad spectrum of Pst races. We previously showed that WKS1 phosphorylates the thylakoid ascorbate peroxidase protein and reduces its ability to detoxify peroxides, which may contribute to the accumulation of reactive oxygen species (ROS). WKS1-mediated Pst resistance is accompanied by leaf chlorosis in Pst-infected regions, but the underlying mechanisms remain elusive. Here, we show that WKS1 interacts with and phosphorylates PsbO, an extrinsic member of photosystem II (PSII), to reduce photosynthesis, regulate leaf chlorosis, and confer Pst resistance. A point mutation in PsbO-A1 or reduction in its transcript levels by RNA interference resulted in chlorosis and reduced Pst sporulation. Biochemical analyses revealed that WKS1 phosphorylates PsbO at two conserved amino acids involved in physical interactions with PSII and reduces the binding affinity of PsbO with PSII. Presumably, phosphorylated PsbO proteins dissociate from the PSII complex and then undergo rapid degradation by cysteine and aspartic proteases. Taken together, these results demonstrate that perturbations of wheat PsbO by point mutation or phosphorylation by WKS1 reduce the rate of photosynthesis and delay the growth of Pst pathogen before the induction of ROS.

Dynamic pH-induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen.

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β-barrel domain in response to the fluctuating acidity of the thylakoid lumen.

Salinity tolerance of quinoa (Chenopodium quinoa Willd) as assessed by chloroplast ultrastructure and photosynthetic performance

Quinoa has been widely used as a model crop for understanding salt-tolerance in halophyte plants. However, a salt tolerance mechanism regarding the photosynthetic activity is poorly evaluated. Here we report the effects of salt stress on photosynthesis of the halophyte Chenopodium quinoa cultivated under different NaCl concentrations (0, 100, and 300 mM). Our results revealed no apparent effect of moderate salinity (100 mM NaCl) on plant growth while, a reduction of plant biomass production was detected under 300 mM NaCl without any symptom of toxicity. No significant effect on the chloroplasts ultrastructure was observed under moderate salinity. Some morphological deformations of chloroplasts such as swelling of thylakoids, disruption of envelope, accumulation of starch grains and plastoglobuli were observed following prolonged exposure to severe stress. Under moderate salinity, no considerable change was detected on the rate of primary photochemistry (fluorescence O-J phase) and the reduction of the PQ pool (J-I phase), no apparent effects on the minimal (F0), the maximal fluorescence (Fm) and the maximal photochemical efficiency (Fv/Fm) showing a high stability of PSII. The high PSII efficiency was also confirmed by the donor side intactness (constant Fk/Fj ratio) and stable antenna size. The high stability of PSII efficiency was also demonstrated by enhanced communication between antenna complex and PSII reaction centre and the stability of OEC complex (PsbQ and PsbO proteins). However, high salinity (300 mM NaCl) results in a swelling of thylakoids and disappearance of grana and in a decrease of Fm and (Fv/Fm) leading to the down-regulation of PSII activity. In addition, a significant increase in F0 occurred and could be associated to the presence of some QA− in darkness in equilibrium with a partially reduced PQ pool. High salt treatment could be responsible for the thermal phase which induced an increase in chl a fluorescence (JIP rise with oxidized PQ pool).

The presence of the low molecular mass carbonic anhydrase in photosystem II of C3 higher plants

The carrier of carbonic anhydrase (CA) activity was detected in gel among low molecular mass proteins from pea, spinach and Arabidopsis, after nondenaturing electrophoresis in PAAG of the dodecyl-β-d-maltoside treated PSII membranes (the fragments of thylakoid membrane containing PSII complexes). The elimination of Mn-stabilizing protein PsbO by treatment of PSII membranes with salts, did not lead to a decrease in CA activity observed in the gel although it reduced the amount of this protein down to 25% compared to the original sample. The isolated protein PsbO did not demonstrated CA activity. The distinguished features of CA activity of PSII membranes were as follows: 1) resistance to heating, 2) high sensitivity to ethoxyzolamide, the specific inhibitor of CA, and 3) stimulation of this activity by acetazolamide, another specific inhibitor of CA at low concentration of the latter. CA activity was not stimulated by acetazolamide in the PSII membranes samples from Arabidopsis thaliana mutants with knocked out gene At4g20990 encoding αCA4 (according to the nomenclature by Fabre et al., 2007). Taking into account the above data and our previous findings that the energy-dependent part of nonphotochemical quenching of chlorophyll a fluorescence is highly suppressed in that mutant, we suppose that thylakoid membranes of higher plants contain in the vicinity of PSII complex a true CA belonging to the α family of CAs.

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt photooxidation by PSII reaction center (demonstrated at the first time), (2) Cyt photoreduction via O2−• photoproduced on the acceptor side of PSII, and (3) Cyt photoreduction by reduced electron carriers of PSII. A step-by-step removal of components of water-oxidizing complex was accompanied by the appearance of Cyt photooxidation, an increase in the superoxide dismutase (SOD)-dependent Cyt photoreduction (related to O2−• formation), and a decrease in the SOD-independent Cyt photoreduction. Re-addition of PsbO protein diminished the Cyt-induced restoration of electron transfer in PSII. Addition of diuron led to inhibition of these photoprocesses, while exogenous Mn2+ inhibited only the Cyt c photooxidation. The results can be important for correct measurements of O2−• photoproduction in PSII and for elucidation of the role of cytochrome c550 in cyanobacterial PSII.

Effect of different methods of Ca2+ extraction from PSII oxygen-evolving complex on the QA- oxidation kinetics.

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn4CaO5 of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca2+ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca2+ extraction from OEC on the kinetics of the reduced primary electron acceptor (QA-) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from QA- to the secondary quinone acceptor QB. Electron transfer from QA- to QB in photosystem II membranes with an occupied QB site was slowed down by a factor of 8. However, addition of EGTA or CaCl2 to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of QA- oxidation by QB in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca2+ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the QA- to QB electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca2+ depletion.