Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts

The chloroplast thylakoid membrane of green plants is organized in stacked grana membranes and unstacked stroma membranes. We investigated the structural organization of Photosystem II (PSII) in paired grana membrane fragments by transmission electron microscopy. The membrane fragments were obtained by a short treatment of thylakoid membranes with the mild detergent n-dodecyl-alpha, d-maltoside and are thought to reflect the grana membranes in a native state. The membranes frequently show crystalline macrodomains in which PSII is organized in rows spaced by either 26.3 nm (large-spaced crystals) or 23 nm (small-spaced crystals). The small-spaced crystals are less common but better ordered. Image analysis of the crystals by an aperiodic approach revealed the precise positions of the core parts of PSII in the lattices, as well as features of the peripheral light-harvesting antenna. Together, they indicate that the so-called C(2)S(2) and C(2)S(2)M supercomplexes form the basic motifs of the small-spaced and large-spaced crystals, respectively. An analysis of a pair of membranes with a well-ordered large-spaced crystal reveals that many PSII complexes in one layer face only light-harvesting complexes (LHCII) in the other layer. The implications of this type of organization for the efficient transfer of excitation energy from LHCII to PSII and for the stacking of grana membranes are discussed.     

"Respirasome"-like supercomplexes in green leaf mitochondria of spinach

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.     

Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)-PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone Q_A in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S₂/Q_A⁻. These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.     

Structural variability of plant photosystem II megacomplexes in thylakoid membranes

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane‐bound light‐harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear‐native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non‐parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non‐parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non‐parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.     

Light harvesting in photosystem II

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some “extra” Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.     

High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes

A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PS II arrays and PS II supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.     

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.     

Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.     

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 ? resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C(2)S(2)M(2) supercomplexes, to avoid overlap.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

Native architecture and acclimation of photosynthetic membranes in a fast-growing cyanobacterium

Efficient solar energy conversion is ensured by the organization, physical association, and physiological coordination of various protein complexes in photosynthetic membranes. Here, we visualize the native architecture and interactions of photosynthetic complexes within the thylakoid membranes from a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) using high-resolution atomic force microscopy. In the Syn2973 thylakoid membranes, both photosystem I (PSI)-enriched domains and crystalline photosystem II (PSII) dimer arrays were observed, providing favorable membrane environments for photosynthetic electron transport. The high light (HL)-adapted thylakoid membranes accommodated a large amount of PSI complexes, without the incorporation of iron-stress-induced protein A (IsiA) assemblies and formation of IsiA–PSI supercomplexes. In the iron deficiency (Fe⁻)-treated thylakoid membranes, in contrast, IsiA proteins densely associated with PSI, forming the IsiA–PSI supercomplexes with varying assembly structures. Moreover, type-I NADH dehydrogenase-like complexes (NDH-1) were upregulated under the HL and Fe⁻ conditions and established close association with PSI complexes to facilitate cyclic electron transport. Our study provides insight into the structural heterogeneity and plasticity of the photosynthetic apparatus in the context of their native membranes in Syn2973 under environmental stress. Advanced understanding of the photosynthetic membrane organization and adaptation will provide a framework for uncovering the molecular mechanisms of efficient light harvesting and energy conversion.     

Dynamic flexibility in the structure and function of photosystem II in higher plant thylakoid membranes: the grana enigma

Grana are not essential for photosynthesis, yet they are ubiquitous in higher plants and in the recently evolved Charaphyta algae; hence grana role and its need is still an intriguing enigma. This article discusses how the grana provide integrated and multifaceted functional advantages, by facilitating mechanisms that fine-tune the dynamics of the photosynthetic apparatus, with particular implications for photosystem II (PSII). This dynamic flexibility of photosynthetic membranes is advantageous in plants responding to ever-changing environmental conditions, from darkness or limiting light to saturating light and sustained or intermittent high light. The thylakoid dynamics are brought about by structural and organizational changes at the level of the overall height and number of granal stacks per chloroplast, molecular dynamics within the membrane itself, the partition gap between appressed membranes within stacks, the aqueous lumen encased by the continuous thylakoid membrane network, and even the stroma bathing the thylakoids. The structural and organizational changes of grana stacks in turn are driven by physicochemical forces, including entropy, at work in the chloroplast. In response to light, attractive van der Waals interactions and screening of electrostatic repulsion between appressed grana thylakoids across the partition gap and most probably direct protein interactions across the granal lumen (PSII extrinsic proteins OEEp-OEEp, particularly PsbQ-PsbQ) contribute to the integrity of grana stacks. We propose that both the light-induced contraction of the partition gap and the granal lumen elicit maximisation of entropy in the chloroplast stroma, thereby enhancing carbon fixation and chloroplast protein synthesizing capacity. This spatiotemporal dynamic flexibility in the structure and function of active and inactive PSIIs within grana stacks in higher plant chloroplasts is vital for the optimization of photosynthesis under a wide range of environmental and developmental conditions.     

Revealing the architecture of the photosynthetic apparatus in the diatom Thalassiosira pseudonana

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure.

The antenna organization of photosystem II in the diatom Thalassiosira pseudonana strongly differs from Chaetoceros gracilis, while the architecture of the photosystem I antenna remains the same.     

Photosystem II assembly and repair are differentially localized in Chlamydomonas

Many proteins of the photosynthesis complexes are encoded by the genome of the chloroplast and synthesized by bacterium-like ribosomes within this organelle. To determine where proteins are synthesized for the de novo assembly and repair of photosystem II (PSII) in the chloroplast of Chlamydomonas reinhardtii, we used fluorescence in situ hybridization, immunofluorescence staining, and confocal microscopy. These locations were defined as having colocalized chloroplast mRNAs encoding PSII subunits and proteins of the chloroplast translation machinery specifically under conditions of PSII subunit synthesis. The results revealed that the synthesis of the D1 subunit for the repair of photodamaged PSII complexes occurs in regions of the chloroplast with thylakoids, consistent with the current model. However, for de novo PSII assembly, PSII subunit synthesis was detected in discrete regions near the pyrenoid, termed T zones (for translation zones). In two PSII assembly mutants, unassembled D1 subunits and incompletely assembled PSII complexes localized around the pyrenoid, where we propose that they mark an intermediate compartment of PSII assembly. These results reveal a novel chloroplast compartment that houses de novo PSII biogenesis and the regulated transport of newly assembled PSII complexes to thylakoid membranes throughout the chloroplast.