Thylakoid Domains Research Articles

Thylakoid membrane appression in the giant chloroplast of Selaginella martensii Spring: A lycophyte challenges grana paradigms in shade-adapted species

In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.

Effect of light on the rearrangements of PSI super-and megacomplexes in the non-appressed thylakoid domains of maize mesophyll chloroplasts

We demonstrated the existence of PSI-LHCI-LHCII-Lhcb4 supercomplexes and PSI-LHCI-PSII-LHCII megacomplexes in the stroma lamellae and grana margins of maize mesophyll chloroplasts; these complexes consist of different LHCII trimers and monomer antenna proteins per PSI photocentre. These complexes are formed in both low (LL) and high (HL) light growth conditions, but with different contents. We attempted to identify the components and structure of these complexes in maize chloroplasts isolated from the leaves of low and high light-grown plants after darkness and transition to far red (FR) light of high intensity. Exposition of plants from high and low light growth condition on FR light induces different rearrangements in the composition of super- and megacomplexes. During FR light exposure, in plants from LL, the PSI-LHCI-LHCII-Lhcb4 supercomplex dissociates into free LHCII-Lhcb4 and PSI-LHCI complexes, and these complexes associate with the PSII monomer. This process occurs differently in plants from HL. Exposition to FR light causes dissociation of both PSI-LHCI-LHCII-Lhcb4 supercomplexes and PSI-PSII megacomplexes. These results suggest a different function of super- and megacomplex organization than the classic state transitions model, which assumes that the movement of LHCII trimers in the thylakoid membraneis considered as a mechanism for balancing light absorption between the two photosystems in light stress. The behavior of the complexes described in this article does not seem to be well explained by this model, i.e., it does not seem likely that the primary purpose of these megacomplexes dynamics is to balance excitation pressure. Rather, as stated in this article, it seems to indicate a role of these complexes for PSI in excitation quenching and for PSII in turnover.

The singular properties of photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum suggest new alternative functions.

Cytochrome c550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast-where it interacts with PSII-, it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.

Isolation and Characterization of a Novel ATPase-Photosystem I Reaction Center Complex in the Chloroplast Thylakoid Membrane

A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplast ATP synthase and the photosystem I (PSI) reaction-center complex has been isolated from spinach thylakoid membranes. This super-complex has been characterized by assay of ATPase activity, negative stain electron microscopy, tandem mass spectrometry, and Western assay of subunits. High-resolution Orbitrap mass spectrometry was used to quantitatively analyze the presence and relative amount of the multiple major and minor polypeptide components in the super-complex. This is the first isolation of a higher order complex involving intact ATP synthase and Photosystem I to be identified and documented by negative stain electron microscopy. The importance of the ATPase-PSI super-complex in the thylakoid domain may imply efficient channeling of substrates and cofactors utilized for localized CO2 fixation. Studies supported by NIHGMS-038323 and the Henry Koffler Distinguished Professorship (WAC), and NIHDK-063491 (JPW).

Phosphorylation-induced lateral rearrangements of thylakoid protein complexes upon light acclimation.

Understanding the mechanistic basis of balanced excitation energy distribution between photosystem II and photosystem I (PSII and PSI) requires detailed investigation of the thylakoid light‐harvesting system composed of energetically connected LHCII trimers. The exact mechanisms controlling the excitation energy distribution remain elusive, but reversible phosphorylation is known to be one important component. Here, we addressed the role of grana margins in regulation of excitation energy distribution, as these thylakoid domains host all the complexes of photosynthetic light reactions with dynamic response to environmental cues. First, the effect of detergents for the thylakoid membrane connectivity is explained. We show that a specific interaction between the separate LHCII trimers as well as between the LHCII trimers and the PSII and PSI–LHCI complexes is a prerequisite for energetically connected and functional thylakoid membrane. Second, we demonstrate that the optimization of light reactions under changing light conditions takes place in energetically connected LHCII lake and is attained by lateral rearrangements of the PSII–LHCII and PSI–LHCI–LHCII complexes depending especially on the phosphorylation status of the LHCII protein isoform LHCB2.

Loss of LHCI system affects LHCII re-distribution between thylakoid domains upon state transitions.

LHCI, the peripheral antenna system of Photosystem I, includes four light-harvesting proteins (Lhca1-Lhca4) in higher plants, all of which are devoid in the Arabidopsis thaliana knock-out mutant ΔLhca. PSI absorption cross-section was reduced in the mutant, thus affecting the redox balance of the photosynthetic electron chain and resulting in a more reduced PQ with respect to the wild type. ΔLhca plants developed compensatory response by enhancing LHCII binding to PSI. However, the amplitude of state transitions, as measured from changes of chlorophyll fluorescence in vivo, was unexpectedly low than the high level of PSI-LHCII supercomplex established. In order to elucidate the reasons for discrepancy, we further analyzed state transition in ΔLhca plants. The STN7 kinase was fully active in the mutant as judged from up-regulation of LHCII phosphorylation in state II. Instead, the lateral heterogeneity of thylakoids was affected by lack of LHCI, with LHCII being enriched in stroma membranes with respect to the wild type. Re-distribution of this complex affected the overall fluorescence yield of thylakoids already in state I and minimized changes in RT fluorescence yield when LHCII did connect to PSI reaction center. We conclude that interpretation of chlorophyll fluorescence analysis of state transitions becomes problematic when applied to mutants whose thylakoid architecture is significantly modified with respect to the wild type.

Light acclimation involves dynamic re-organization of the pigment-protein megacomplexes in non-appressed thylakoid domains.

Thylakoid energy metabolism is crucial for plant growth, development and acclimation. Non-appressed thylakoids harbor several high molecular mass pigment-protein megacomplexes that have flexible compositions depending upon the environmental cues. This composition is important for dynamic energy balancing in photosystems (PS) I and II. We analysed the megacomplexes of Arabidopsis wild type (WT) plants and of several thylakoid regulatory mutants. The stn7 mutant, which is defective in phosphorylation of the light-harvesting complex (LHC) II, possessed a megacomplex composition that was strikingly different from that of the WT. Of the nine megacomplexes in total for the non-appressed thylakoids, the largest megacomplex in particular was less abundant in the stn7 mutant under standard growth conditions. This megacomplex contains both PSI and PSII and was recently shown to allow energy spillover between PSII and PSI (Nat. Commun., 6, 2015, 6675). The dynamics of the megacomplex composition was addressed by exposing plants to different light conditions prior to thylakoid isolation. The megacomplex pattern in the WT was highly dynamic. Under darkness or far red light it showed low levels of LHCII phosphorylation and resembled the stn7 pattern; under low light, which triggers LHCII phosphorylation, it resembled that of the tap38/pph1 phosphatase mutant. In contrast, solubilization of the entire thylakoid network with dodecyl maltoside, which efficiently solubilizes pigment-protein complexes from all thylakoid compartments, revealed that the pigment-protein composition remained stable despite the changing light conditions or mutations that affected LHCII (de)phosphorylation. We conclude that the composition of pigment-protein megacomplexes specifically in non-appressed thylakoids undergoes redox-dependent changes, thus facilitating maintenance of the excitation balance between the two photosystems upon changes in light conditions.

Light-harvesting II antenna trimers connect energetically the entire photosynthetic machinery — including both photosystems II and I

In plant chloroplasts, the two photosystems (PSII and PSI) are enriched in different thylakoid domains and, according to the established view, are regarded as energetically segregated from each other. A specific fraction of the light harvesting complex II (LHCII) has been postulated to get phosphorylated by the STN7 kinase and subsequently to migrate from PSII to PSI as part of a process called ‘state transition’. Nevertheless, the thylakoid membrane incorporates a large excess of LHCII not present in the isolatable PSII–LHCII and PSI–LHCII complexes. Moreover, LHCII phosphorylation is not limited to a specific LHCII pool and “state 2” condition, but is found in all thylakoid domains in any constant light condition. Here, using a targeted solubilization of pigment–protein complexes from different thylakoid domains, we demonstrate that even a minor detachment of LHCII leads to markedly increased fluorescence emission from LHCII and PSII both in grana core and non-appressed thylakoid membranes and the effect of the detergent to detach LHCII is enhanced in the absence of LHCII phosphorylation. These findings provide evidence that PSII and PSI are energy traps embedded in the same energetically connected LHCII lake. In the lake, PSI and LHCII are energetically connected even in the absence of LHCII phosphorylation, yet the phosphorylation enhances the interaction required for efficient energy transfer to PSI in the grana margin regions.

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.

Structural and functional dynamics of plant photosystem II.

Given the unique problem of the extremely high potential of the oxidant P(+)(680) that is required to oxidize water to oxygen, the photoinactivation of photosystem II in vivo is inevitable, despite many photoprotective strategies. There is, however, a robustness of photosystem II, which depends partly on the highly dynamic compositional and structural heterogeneity of the cycle between functional and non-functional photosystem II complexes in response to light level. This coordinated regulation involves photon usage (energy utilization in photochemistry) and excess energy dissipation as heat, photoprotection by many molecular strategies, photoinactivation followed by photon damage and ultimately the D1 protein dynamics involved in the photosystem II repair cycle. Compelling, though indirect evidence suggests that the radical pair P(+)(680)Pheo(-) in functional PSII should be protected from oxygen. By analogy to the tentative oxygen channel of cytochrome c oxidase, oxygen may be liberated from the two water molecules bound to the catalytic site of the Mn cluster, via a specific pathway to the membrane surface. The function of the proposed oxygen pathway is to prevent O(2) from having direct access to P(+)(680)Pheo(-) and prevent the generation of singlet oxygen via the triplet-P(680) state in functional photosytem IIs. Only when the, as yet unidentified, potential trigger with a fateful first oxidative step destroys oxygen evolution, will the ensuing cascade of structural perturbations of photosystem II destroy the proposed oxygen, water and proton pathways. Then oxygen has direct access to P(+)(680)Pheo(-), singlet oxygen will be produced and may successively oxidize specific amino acids of the phosphorylated D1 protein of photosystem II dimers that are confined to appressed granal domains, thereby targeting D1 protein for eventual degradation and replacement in non-appressed thylakoid domains.

Counter-current distribution of spinach chloroplasts in an aqueous two-phase system

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.