In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
The monomeric photosystem I-light-harvesting antenna complex I (PSI-LHCI) supercomplex from the extremophilic red alga Cyanidioschyzon merolae represents an intermediate evolutionary link between the cyanobacterial PSI reaction center and its green algal/higher plant counterpart. We show that the C. merolae PSI-LHCI supercomplex is characterized by robustness in various extreme conditions. By a combination of biochemical, spectroscopic, mass spectrometry, and electron microscopy/single particle analyses, we dissected three molecular mechanisms underlying the inherent robustness of the C. merolae PSI-LHCI supercomplex: (1) the accumulation of photoprotective zeaxanthin in the LHCI antenna and the PSI reaction center; (2) structural remodeling of the LHCI antenna and adjustment of the effective absorption cross section; and (3) dynamic readjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK core subunit. We show that the largest low light-treated C. merolae PSI-LHCI supercomplex can bind up to eight Lhcr antenna subunits, which are organized as two rows on the PsaF/PsaJ side of the core complex. Under our experimental conditions, we found no evidence of functional coupling of the phycobilisomes with the PSI-LHCI supercomplex purified from various light conditions, suggesting that the putative association of this antenna with the PSI supercomplex is absent or may be lost during the purification procedure.
- MeSH
- biologická adaptace MeSH
- chlorofyl metabolismus MeSH
- cirkulární dichroismus MeSH
- fluorescenční spektrometrie MeSH
- fotosystém I - proteinový komplex chemie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- molekulární evoluce MeSH
- Rhodophyta chemie fyziologie MeSH
- sinice chemie fyziologie MeSH
- světlo MeSH
- světlosběrné proteinové komplexy chemie metabolismus MeSH
- teplota MeSH
- zeaxanthiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In nature, plants are continuously exposed to varying environmental conditions. They have developed a wide range of adaptive mechanisms, which ensure their survival and maintenance of stable photosynthetic performance. Photosynthesis is delicately regulated at the level of the thylakoid membrane of chloroplasts and the regulatory mechanisms include a reversible formation of a large variety of specific protein-protein complexes, supercomplexes or even larger assemblies known as megacomplexes. Revealing their structures is crucial for better understanding of their function and relevance in photosynthesis. Here we focus our attention on the isolation and a structural characterization of various large protein supercomplexes and megacomplexes, which involve Photosystem II and Photosystem I, the key constituents of photosynthetic apparatus. The photosystems are often attached to other protein complexes in thylakoid membranes such as light harvesting complexes, cytochrome b 6 f complex, and NAD(P)H dehydrogenase. Structural models of individual supercomplexes and megacomplexes provide essential details of their architecture, which allow us to discuss their function as well as physiological significance.
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.
- MeSH
- Arabidopsis metabolismus MeSH
- elektronová mikroskopie MeSH
- fotosystém II - proteinový komplex chemie metabolismus ultrastruktura MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- proteiny huseníčku metabolismus ultrastruktura MeSH
- světlosběrné proteinové komplexy chemie metabolismus ultrastruktura MeSH
- tylakoidy metabolismus ultrastruktura MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b6f (Cytb6f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1-3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains.
- MeSH
- Arabidopsis metabolismus MeSH
- chlorofyl metabolismus MeSH
- chloroplasty metabolismus MeSH
- fotosyntéza fyziologie MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- komplex cytochromů b6f metabolismus MeSH
- NADH-dehydrogenasa metabolismus MeSH
- oxidace-redukce MeSH
- světlo MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- transport elektronů fyziologie MeSH
- tylakoidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Photo-reduction of O2to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.
Photosynthesis in plants and algae relies on the coordinated function of photosystems (PS) I and II. Their efficiency is augmented by finely-tuned light-harvesting proteins (Lhcs) connected to them. The most recent Lhcs (in evolutionary terms), Lhcb6 and Lhcb3, evolved during the transition of plants from water to land and have so far been considered to be an essential characteristic of land plants. We used single particle electron microscopy and sequence analysis to study architecture and composition of PSII supercomplex from Norway spruce and related species. We have found that there are major land plant families that lack functional lhcb6 and lhcb3 genes, which notably changes the organization of PSII supercomplexes. The Lhcb6 and Lhcb3 proteins have been lost in the gymnosperm genera Picea and Pinus (family Pinaceae) and Gnetum (Gnetales). We also revealed that the absence of these proteins in Norway spruce modifies the PSII supercomplex in such a way that it resembles its counterpart in the alga Chlamydomonas reinhardtii, an evolutionarily older organism. Our results break a deep-rooted concept of Lhcb6 and Lhcb3 proteins being the essential characteristic of land plants, and beg the question of what the evolutionary benefit of their loss could be.
- MeSH
- biologická evoluce * MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- fylogeneze MeSH
- podjednotky proteinů chemie metabolismus MeSH
- rostlinné geny MeSH
- rostlinné proteiny metabolismus MeSH
- sekvenční homologie aminokyselin MeSH
- světlosběrné proteinové komplexy metabolismus ultrastruktura MeSH
- vyšší rostliny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Gymnosperms, unlike angiosperms, are able to synthesize chlorophyll and form photosystems in complete darkness. Photosystem I (PSI) formed under such conditions is fully active, but photosystem II (PSII) is present in its latent form with inactive oxygen evolving complex (OEC). In this work we have studied light-induced gradual changes in PSII function in dark-grown cotyledons of Norway spruce (Picea abies) via the measurement of chlorophyll a fluorescence rise, absorption changes at 830 nm, thermoluminescence glow curves (TL) and protein analysis. The results indicate that in dark-grown cotyledons, alternative reductants were able to act as electron donors to PSII with inactive OEC. Illumination of cotyledons for 5 min led to partial activation of PSII, which was accompanied by detectable oxygen evolution, but still a substantial number of PSII centers remained in the so called PSII-Q(B)-non-reducing form. Interestingly, even 24 h long illumination was not sufficient for the full activation of PSII centers. This was evidenced by a weak attachment of PsbP protein and the absence of PsbQ protein in PSII particles, the absence of PSII supercomplexes, the suboptimal maximum yield of PSII photochemistry, the presence of C band in TL curve and also the presence of up-shifted Q band in TL in DCMU-treated cotyledons. This slow light-induced activation of PSII in dark-grown cotyledons could contribute to the prevention of PSII overexcitation before the light-induced increase in PSI/PSII ratio allows effective operation of linear electron flow.
- MeSH
- chlorofyl chemie metabolismus MeSH
- fotosyntéza účinky záření MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- kotyledon růst a vývoj metabolismus účinky záření MeSH
- kyslík metabolismus MeSH
- luminiscenční měření metody MeSH
- rostlinné proteiny metabolismus MeSH
- semenáček růst a vývoj metabolismus účinky záření MeSH
- smrk růst a vývoj metabolismus účinky záření MeSH
- světlo * MeSH
- teplota MeSH
- tma * MeSH
- transport elektronů účinky záření MeSH
- tylakoidy metabolismus účinky záření MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.
- MeSH
- elektronová mikroskopie MeSH
- ferredoxiny metabolismus MeSH
- fotosystém I - proteinový komplex chemie izolace a purifikace metabolismus MeSH
- ječmen (rod) chemie enzymologie účinky záření MeSH
- listy rostlin chemie enzymologie účinky záření MeSH
- molekulární modely MeSH
- NAD metabolismus MeSH
- NADPH-dehydrogenasa chemie izolace a purifikace metabolismus MeSH
- nativní elektroforéza na polyakrylamidovém gelu MeSH
- oxidace-redukce MeSH
- světlo MeSH
- světlosběrné proteinové komplexy chemie izolace a purifikace metabolismus MeSH
- transport elektronů MeSH
- tylakoidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
