Quality control of photosystem II

Photosystem II is particularly vulnerable to excess light. When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein. Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested. We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes. The cross-linked products are then digested by a stromal protease(s). These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases. Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II. Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions.     

Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors

Irradiation of Spinach oleracea intact leaf tissue and of mesophyll protoplasts of Valerianella locusta at 20° C with strong light resulted in severe (40–80%) inhibition of photosynthesis, measured as photosystem II electron transport activity in isolated thylakoids or as fluorescence parameter F_V/F_M on intact leaf disks. No net degradation of the D1 protein of photosystem II was seen under these conditions. However, in the presence of streptomycin, an inhibitor of chloroplast protein synthesis, net D1 degradation (up to about 80%) did occur with a half-time of 4–6h, and photoinhibition was enhanced. Thylakoid ultrastructure remained stable during photoinhibition, even when substantial degradation of D1 took place in the presence of streptomycin. When leaf disks were irradiated at 2°C, streptomycin did not influence the degree of photoinhibition, and net Dl degradation did not occur. These results suggest that in excess (photoinhibitory) light at 20°C, turnover (coordinated degradation and synthesis) of D1 diminished the degree of photoinhibition. The observed photoinhibition is thought to be due to the accumulation of inactive photosystem II reaction centres still containing D1. In the presence of streptomycin, the Dl protein was degraded (probably in the previously inactivated centres), but restoration of active centres via D1 synthesis was blocked, leading to more severe photoinhibition. Low temperature (2°C), by restricting both degradation and resynthesis of D1, favoured the accumulation of inactive centres. Streptomycin and chloramphenicol (another inhibitor of chloroplast protein synthesis) were tested for side-effects on photosynthesis. Strong inhibitory effects of chloramphenicol, but much less severe effects of streptomycin were observed.     

On the molecular mechanism of light-induced D1 protein degradation in photosystem II core particles.

The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.     

Massive breakdown of the Photosystem II polypeptides in a mutant of the cyanobacterium Synechocystis sp. PCC 6803

Degradation of the D1 protein of the Photosystem II (PS II) complex was studied in the Fad6/desA::Km^r mutant of a cyanobacterium Synechocystis sp. PCC 6803. The D1 protein of the mutant was degraded during solubilization of thylakoid membranes with SDS at 0°C in darkness, giving rise to the 23 kDa amino-terminal and 10 kDa carboxy-terminal fragments. Moreover, the D2 and CP43 proteins were also degraded under such conditions of solubilization. Degradation of the D2 protein generated 24, 17 and 15.5 kDa fragments, and degradation of the CP43 protein gave rise to 28, 27.5, 26 and 16 kDa fragments. The presence of Ca²⁺ and urea protected the D1, D2 and CP43 proteins against degradation. Degradation of the D1 protein was also inhibited by the presence of a serine protease inhibitor suggesting that the putative protease involved belonged to the serine class of proteases. The protease had the optimum activity at pH 7.5; it was active at low temperature (0°C) but a brief heating (65°C) during solubilization destroyed the activity. Interestingly, the protease was active in isolated thylakoid membranes in complete darkness, suggesting that proteolysis may be a non-ATP-dependent process. Proteolytic activity present in thylakoid membranes seemed to reside outside of the PS II complex, as demonstrated by the 2-dimensional gel electrophoresis. These results represent the first (in vitro) demonstration of strong activity of a putative ATP-independent serine-type protease that causes degradation of the D1 protein in cyanobacterial thylakoid membranes without any induction by visible or UV light, by active oxygen species or by any chemical treatments.     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

Quantitative correlation of peripheral and intrinsic core polypeptides of photosystem II with photosynthetic electron-transport activity ofAcetabularia mediterranea in red and blue light

The high photosynthetic activity (O₂ production and CO₂ consumption) ofAcetabularia mediterranea Lamour. (=A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light, and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity completely within 3–5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre complex of photosystem I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity of the reaction-centre components D1 (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells (the 16-kDa component could not be found inAcetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast, the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna, comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via the core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment.     

Dynamic Interaction between the D1 protein,CP43 and OEC33 at the lumenal side of photosystem II in spinach chloroplasts: evidence from light-induced cross-Linking of the proteins in the donor-side photoinhibition

During the donor-side photoinhibition of spinach photosystem II, the reaction center D1 protein cross-linked with the antenna chlorophyll binding protein CP43 of photosystem II lacking the oxygen-evolving complex (OEC) subunit proteins. The cross-linking did not occur upon illumination of photosystem II samples that retained the OEC33, nor when OEC33-depleted photosystem II samples were reconstituted with the OEC33 prior to illumination. These results suggest that the D1 protein, CP43 and the OEC33 are located in close proximity at the lumenal side of photosystem II, and that the OEC33 suppresses the unnecessary contact between the D1 protein and CP43. Previously we presented data showing the D1 protein located adjacent to CP43 on the stromal side of photosystem II [Ishikawa et al. (1999) BIOCHIM: Biophys. Acta 1413: 147]. The present data suggest that the spatial arrangement of the D1 protein and CP43 at the lumenal side of photosystem II in spinach chloroplasts is similar to that at the stromal side of photosystem II and is consistent with the assignment of these proteins recently proposed on the crystal structures of the photosystem II complexes from cyanobacteria [Zouni et al. (2001) Nature 409: 739, Kamiya and Shen 2003 PROC: Natl. Acad. Sci. USA, 100: 98]. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the photosystem II complex from higher plants may be different from those in cyanobacteria.     

Isolation of phosphorylated and dephosphorylated forms of the CP43 internal antenna of photosystem II in Hordeum vulgare L

As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll-protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.     

Too much light? How beta-carotene protects the photosystem II reaction centre.

The photosystem II reaction centre of all oxygenic organisms is subject to photodamage by high light i.e. photoinhibition. In this review I discuss the reasons for the inevitable and unpreventable oxidative damage that occurs in photosystem II and the way in which beta-carotene bound to the reaction centre significantly mitigates this damage. Recent X-ray structures of the photosystem II core complex (reaction centre plus the inner antenna complexes) have revealed the binding sites of some of the carotenoids known to be bound to the complex. In the light of these X-ray structures and their known biophysical properties it is thus possible to identify the two beta-carotenes present in the photosystem II reaction centre. The two carotenes are both bound to the D2 protein and this positioning is discussed in relation to their ability to act as quenchers of singlet oxygen, generated via the triplet state of the primary electron donor. It is proposed that their location on the D2 polypeptide means there is more oxidative damage to the D1 protein and that this underlies the fact that this latter protein is continuously re-synthesised, at a far greater rate than any other protein involved in photosynthesis. The relevance of a cycle of electrons around photosystem II, via cytochrome b(559), in order to re-reduce the beta-carotenes when they are oxidised and hence restore their ability to quench singlet oxygen, is also discussed.     

Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants

Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O₂-evolving PSII reaction center complex.     

Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.     

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium,Synechocystis PCC 6803

Despite intense research, the mechanism of Cd²⁺ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd²⁺ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO₂‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd²⁺ (50% inhibition in ～15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O₂ polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd²⁺ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.     

Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803

In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major chlorophyll-binding protein, was present in reduced but significant amounts in the thylakoid. Herbicide binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.Abbreviations CP chlorophyll-binding protein - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - ferricyanide K₃Fe(CN)₆ - HEPES N-(2-hydroxyelthyl)piperazine-N-(2-hydroxypropane sulfonic acid) - MES 2-(N-morpholino)-ethanesulfonic acid - PCC Pasteur Culture Collection - PCR polymerase chain reaction - PS photosystem - Q_A first quinone acceptor in PSII - Q_B second quinone acceptor in PSII - Z redox-active tyrosine (Y161) in D1 serving as electron carrier between the Mn cluster and P680     

Too much of a good thing: light can be bad for photosynthesis.

Even though light is the ultimate substrate for photosynthetic energy conversion, it can also harm plants. This toxicity is targeted to the water-splitting photosystem II and leads to damage and degradation of the reaction centre D1-polypeptide. The degradation of this very important protein appears to be a direct consequence of photosystem II chemistry involving highly oxidizing radicals and toxic oxygen species. The frequency of this damage is relatively low under normal conditions but becomes a significant problem for the plant with increasing light intensity, especially when combined with other environmental stress factors. However, the plant survives this photoinhibition through an efficient repair system which involves an autoproteolytic activity of the photosystem II complex, D1-polypeptide synthesis and reassembly of active complexes.     

Quality control of photosystem II: impact of light and heat stresses

Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.     

Quantum mechanical analysis of excitation energy transfer couplings in photosystem II

We evaluated excitation energy transfer (EET) coupling (J) between all pairs of chlorophylls (Chls) and pheophytins (Pheos) in the protein environment of photosystem II based on the time-dependent density functional theory with a quantum mechanical/molecular mechanics approach. In the reaction center, the EET coupling between Chls P_D1 and P_D2 is weaker (|J(P_D1/P_D2)| = 79 cm^?1), irrespective of a short edge-to-edge distance of 3.6 Å (Mg-to-Mg distance of 8.1 Å), than the couplings between P_D1 and the accessory Chl_D1 (|J(P_D1/Chl_D2)| = 104 cm^?1) and between P_D2 and Chl_D2 (|J(P_D2/Chl_D1)| = 101 cm^?1), suggesting that P_D1 and P_D2 are two monomeric Chls rather than a “special pair”. There exist strongly coupled Chl pairs (|J| > ～100 cm^?1) in the CP47 and CP43 core antennas, which may be candidates for the red-shifted Chls observed in spectroscopic studies. In CP47 and CP43, Chls ligated to CP47-His26 and CP43-His56, which are located in the middle layer of the thylakoid membrane, play a role in the “hub” that mediates the EET from the lumenal to stromal layers. In the stromal layer, Chls ligated to CP47-His466, CP43-His441, and CP43-His444 mediate the EET from CP47 to Chl_D2/Pheo_D2 and from CP43 to Chl_D1/Pheo_D1 in the reaction center. Thus, the excitation energy from both CP47 and CP43 can always be utilized for the charge-separation reaction in the reaction center.     

Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography.

Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution.     

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.     

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.     

The action of lipases on chloroplast membranes. III. The effect of lipid hydrolysis on chlorophyll-protein complexes in thylakoid membranes

Bean thylakoid membranes treated with various lipolytic enzymes (bean galactolipase, phospholipases A₂, C, D) showed marked changes in their acyl lipid composition. As a consequence of acyl lipids hydrolysis, destruction of some chlorophyll a-protein complexes (CP1a, CP1, CPa) or monomerization of the oligomeric of light harvesting chlorophyll a/b protein complex (LHCP) was observed. It is concluded that galactolipids and phosphatidylcholine are responsible for the stability of CP1a, CP1 and CPa, respectively. Phosphatidylglycerol and to some extent monogalactosyldiacylglycerol are essential for the stabilization of oligomeric structures of light harvesting chlorophyll a/b protein complex.Abbreviations chl chlorophyll - CP1a, CP1 chl a-protein complexes, of PSI - CPa chl a-protein complex of PSII - DGDG diagalactosyldiacylglycerol - FC free chl - GL galactolipase - LHCP^1–3 light harvesting chl a/b protein complex - MGDG monogalactosyldiacylglycerol - PAGE polyacrylamide gel electrophoresis - PC phosphatidylcholine - PG phosphatidylglycerol - PLA₂ phospholipase A₂ - PL phospholipase C - PLD phospholipase D - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - SQDG sulfoquinovosyl-diacylglycerol - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan