Grana Thylakoid Membranes Research Articles

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023Å−1, corresponding to spatially and statistically averaged repeat distance values of about 275–330Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.

Isolation and characterization of lamellar aggregates of LHCII and LHCII-lipid macro-assemblies with light-inducible structural transitions.

We describe the method of isolation of loosely stacked lamellar aggregates of LHCII that are capable of undergoing light-induced reversible structural changes, similar to those in granal thylakoid membranes (LHCII, the main chlorophyll a/b light-harvesting antenna complex of photosystem II). This unexpected structural flexibility of the antenna complexes depends largely on the lipid content that is retained during the isolation. As revealed by circular dichroism, in lipid-LHCII aggregates, the pigment-pigment interactions are very similar to those in the thylakoid membranes, while they differ significantly from those in solubilized trimers. The essence of the procedure is to adjust--for the plant material used--the proper conditions of detergent solubilization and purification that are mild enough for the associated lipids but provide sufficient purity. Microcrystals and most other LHCII preparations, which are more delipidated, are not capable of similar changes. The light-induced structural reorganizations can be enhanced by the addition of different thylakoid lipids, which--depending on the lipid species--also lead to the transformation of the lamellar structure. The preparation of different LHCII-lipid macro-assemblies is also described. Both in structurally flexible LHCII preparations and in thylakoids, the changes originate from a thermo-optic effect: fast local thermal transients, T-jumps, due to the dissipation of the (excess) excitation energy, which lead to elementary structural transitions in the close vicinity of the dissipating centers. This can occur because thylakoids and structurally flexible LHCII assemblies, but, e.g., not the microcrystals, exhibit a thermal instability below the denaturation temperature, and thus (local) heating leads to reorganizations without the loss of the molecular architecture of the constituents. We also list the main biochemical and biophysical techniques that can be used for testing the structural flexibility of LHCII, and discuss the potential physiological significance of the structural changes in light adaptation and photoprotection of plants.

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.

Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene.

Etiolation symptoms and the greening process are usually studied on dark-germinated seedlings and this raises the question--can these results be generalized for plants growing under field conditions? This work examines various aspects of the plastid differentiation under the covering of the achene wall, which often remains attached to the cotyledons of sunflower (Helianthus annuus) seedlings grown under light. Cotyledons of 7- to 10-d-old sunflower seedlings grown in the dark and on light were examined. The partially covered cotyledons were sectioned into light-exposed, covered and transition zones. Pigment contents, 77 K fluorescence spectroscopy, electron microscopy and fluorescence imaging, along with fluorescence kinetic methods, were used. The light-exposed zone of the partially covered cotyledons was similar to cotyledons developed without achene covering. However, some of the plastids had prolamellar bodies among the granal thylakoid membranes; despite this no protochlorophyllide was detected. The fully covered, yellowish sections contained protochlorophyllide forms emitting at 633 and 655 nm and well-developed prolamellar bodies, similar to those of etiolated cotyledons. In addition, reduced amounts of chlorophyll a, chlorophyll b and stacked thylakoid membrane pairs were found in this region. The transitional sections showed a mixture of the characteristics of the covered and exposed sections. Various, but significantly different values of the photosynthetic activity parameters were found in each sector of the partially covered cotyledons. The partial covering of the achene wall shades the cotyledon tissues effectively, enough to provoke the appearance of etiolation phenomena, i.e. the permanent presence of flash-photoactive protochlorophyllide complexes and prolamellar bodies (with or without protochlorophyllide), which proves that these phenomena may appear under natural illumination conditions.

Thermo-optically induced reorganizations in the main light harvesting antenna of plants. I. Non-Arrhenius type of temperature dependence and linear light-intensity dependencies.

Thermo-optically induced structural reorganizations have earlier been identified in isolated LHCII, the main chlorophyll a/b light harvesting complexes of Photosystem II, and in granal thylakoid membranes [Cseh et al. (2000) Biochemistry 39: 15250-15257; Garab et al. (2002) Biochemistry 41: 15121-15129]. According to the thermo-optic mechanism, structural changes can be induced by fast, local thermal transients due to the dissipation of excess excitation energy. In this paper, we analyze the temperature and light-intensity dependencies of thermo-optically induced reversible and irreversible reorganizations in the chiral macrodomains of lamellar aggregates of isolated LHCII and of granal thylakoid membranes. We show that these structural changes exhibit non-Arrhenius type of temperature dependencies, which originate from the 'combination' of the ambient temperature and the local thermal transient. The experimental data can satisfactorily be simulated with the aid of a simple mathematical model based on the thermo-optic effect. The model also predicts, in good accordance with experimental data published earlier and presented in this paper, that the reorganizations depend linearly on the intensity of the excess light, a unique property that is probably important in light adaptation and photoprotection of plants.

Isolation and characterization of lamellar aggregates of LHCII and LHCII-lipid macro-assemblies with light-inducible structural transitions.

In this chapter we describe isolation of loosely stacked lamellar aggregates of LHCII that are capable of undergoing light-induced reversible structural changes, similar to those in granal thylakoid membranes (LHCII, the main chlorophyll a/b light-harvesting antenna complex of photosystem II). This unexpected structural flexibility of the antenna complexes depends largely on the lipid content retained during the isolation. The essence of the procedure is to adjust-for the plant material used-the proper conditions of detergent solubilization and purification mild enough for the associated lipids but also provide sufficient purity. Microcrystals and most other LHCII preparations, which are more delipidated, are not capable of similar changes. The light-induced structural reorganizations can be enhanced by the addition of different thylakoid lipids, which depending on the lipid species, also lead to the transformation of the lamellar structure. The preparation of different LHCII-lipid macro-assemblies is also described. In structurally flexible LHCII preparations and in thylakoids, the changes originate from a thermo-optic effect: fast local thermal transients, T-jumps, resulting from dissipation of the (excess) excitation energy that lead to elementary structural transitions in the vicinity of the dissipating centers. This can occur because thylakoids and structurally flexible LHCII assemblies but, for example, not the microcrystals, exhibit a thermal instability below the denaturation temperature, and thus (local) heating lead to reorganizations without the loss of the molecular architecture of the constituents. We also list the main biochemical and biophysical techniques that can be used for testing the structural flexibility of LHCII, and discuss the potential physiological significance of the structural changes in light adaptation and photoprotection of plants.

Thermooptic effect in chloroplast thylakoid membranes. Thermal and light stability of pigment arrays with different levels of structural complexity.

In chloroplast thylakoid membranes, chiral macrodomains, i.e., large arrays of pigment molecules with long-range chiral order, have earlier been shown to undergo light-induced reversible and irreversible structural changes; such reorganizations did not affect the short-range, excitonic pigment-pigment interactions. These structural changes and similar changes in lamellar aggregates of the main chlorophyll a/b light-harvesting complexes exhibited a linear dependence on the intensity of light that was not utilized in photosynthesis. It has been hypothesized that the light-induced rearrangements are driven by a thermooptic effect, i.e., thermal fluctuations due to the dissipation of excess excitation energies [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. To test this hypothesis, we have utilized circular dichroism (CD) spectroscopy to investigate the structural stability of the chiral macrodomains and the constituent bulk pigment-protein complexes of granal thylakoid membranes against heat and prolonged, intense illumination. (i) In intact thylakoid membranes, the chiral macrodomains displayed high stability below 40 degrees C, but they were gradually disassembled between 50 and 60 degrees C; the thermal stability of the chiral macrodomains could be decreased substantially by suspending the membranes in reaction media that were hypotonic or had low ionic strength. (ii) The chiral macrodomains were also susceptible to high light: prolonged illumination with intense white light (25 min, 2500 microE m(-)(2) s(-)(1), 25 degrees C) induced similar, irreversible disassembly to that observed at high temperatures; in different preparations, lower thermal stability was coupled to lower light stability. (iii) The light stability depended significantly on the temperature: between about 5 and 15 degrees C, the macrodomains in the intact thylakoids were virtually not susceptible to high light; in contrast, the same preillumination at 35-40 degrees C almost completely destroyed the chiral macrodomains. (iv) As testified by the excitonic CD bands, the molecular organization of the pigment-protein complexes in all samples exhibited very high thermal stability between about 15 and 65 degrees C, and virtually total immunity against intense illumination. These data are fully consistent with the hypothesis of a thermooptic effect, and are interpreted within the frame of a simple model.

Structural flexibility of chiral macroaggregates of light-harvesting chlorophyll a/b pigment-protein complexes. Light-induced reversible structural changes associated with energy dissipation.

In this paper, we show that stacked lamellar aggregates of the purified chlorophyll a/b light-harvesting antenna complexes (LHCII) and granal thylakoid membranes are capable of undergoing light-induced reversible changes in the chiral macroorganization of the chromophores as well as in the photophysical pathways. In granal thylakoids, the light-induced reversible structural changes, detected by circular dichroism (CD) measurements, are accompanied by reversible changes in the fluorescence yield that indicate an increased dissipation of the excitation energy. These changes become gradually more significant in excess light compared to nonsaturating light intensities, and can be eliminated by suspending the membranes in hypotonic, low-salt medium in which the chiral macroaggregates are absent. In lamellar aggregates of LHCII, the light-induced reversible changes of the main, nonexcitonic CD bands are also accompanied by reversible changes in the fluorescence yield. In small aggregates and trimers, no light-induced delta CD occurs, and the fluorescence changes are largely irreversible. It is proposed that the structural changes are induced by thermal effects due to the excess light energy absorbed by the pigments. Our data strongly suggest that the structure and function of the antenna system of chloroplasts can be regulated by the absorption of excess light energy with a mechanism independent of the operation of the photochemical apparatus.

Liquid Crystal-Like Domains of the Light Harvesting Chlorophyll a/b Protein Complex in Chloroplast Thylakoid Membranes

Abstract Recent experimental results, reviewed in this paper, suggest that the light harvesting pigment protein complex in granal thylakoid membranes exists in liquid crystal like domains. These macroarrays can play an important role in the efficient photosynthetic utilization of light energy. The experimentally observed light induced reversible structural rearrangements, on the other hand, may be required for the regulatory redistribution of the excitation energy between photosystems 1 and 2.