Grana Formation Research Articles

Glucose Induces Thylakoid Formation and Upregulates Green Pigment Contents in Complete Dark Culture of the Angiosperm Pachiramacrocarpa

In addition to angiosperms, most plants are able to synthesize chlorophyll (Chl)-generating green tissues in total darkness. In this study, 140 plants of the angiosperm Pachira macrocarpa were divided into five groups. Among them, one group was grown for 2 weeks under natural light conditions, whereas the others were grown in complete darkness (0 μmol m−2 s−1). Dark-grown plants were then treated with 0~6% glucose for another 8 weeks. The budding and greening ratios, ultrastructure of chloroplasts (ChlPs) of newly developed leaves, and green pigment contents of pre-illuminated mature and young leaves, and totally dark-grown newly developed leaves were measured. Results showed that glucose inhibited the budding and promoted the greening of newly developed leaves. Pre-illuminated mature and young leaves were able to synthesize green pigments during the 2 weeks of dark adaption. Dark-grown newly developed leaves contained high levels of green pigments at 2 and 3 weeks after budding. Green pigments of glucose-fed newly developed leaves had increased, whereas they had decreased in control leaves. In addition, ChlPs of dark-grown glucose-fed newly developed leaves contained both giant grana and prolamellar bodies (PLBs), usually found in shade plants and etiolated seedlings, respectively. The higher the glucose concentration was, the greater the numbers of grana, thylakoids, and PLBs. Glucose increased the green pigment contents and grana formation in newly developed leaves in a dark condition, and the mechanisms are discussed.

Role of Protein-Water Interface in the Stacking Interactions of Granum Thylakoid Membranes-As Revealed by the Effects of Hofmeister Salts.

The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I—light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects—in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular—pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.

Light-induced ultrastructure changes of amyloplasts and effect of nitrogen fertilization on greening in potato tubers (Solanum tuberosum L.)

Abstract Tuber greening causes potato quality decline and economic loss. We investigated physiological mechanisms of greening by fluorescence and transmission electron microscopy. We then explored the effect of nitrogen level (0, 210, and 315 kg N ha−1) on starch granule size and tuber greening of three commercial cultivars. Results showed that chlorophyll content of tuber skin increased (5–8 times) in a quadratic manner with increasing duration of light exposure (0–7 d) in all cultivars. Light-induced greening occurred in cortical parenchyma 0–1.5 mm below the periderm, where chloroplast auto-fluorescence was evident under ultraviolet light. The greening process involved membrane lose, starch granule dissolution, and grana formation in amyloplasts, along with chloroplast development. Formation of grana lamellae was observed around amyloplasts with a diameter of 9–30 μm. Nitrogen application increased the percentage of small granule starch (

Functional Aspects of Silencing and Transient Expression of <i>psb</i>S in <i>Nicotiana benthamiana</i>

MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco rattle virus (TRV) vector to silence expression of the nuclear psbS gene in Nicotiana benthamiana. The 22-kiloDalton psbS protein is essential for xanthophyll- and H+-dependent thermal dissipation of excitation in higher plants widely known as nonphotochemical quenching (NPQ). Controls treated with the TRV-VIGS vector containing a bacterial chloramphenicol resistance gene as the silencing target were included to test for non-silencing effects of the viral vector system. PsbS protein was undetectable and both psbS mRNA transcript levels and NPQ capacity were dramatically reduced in new leaf tissue of VIGS-psbS plants only. Photosynthetic performance in TRV-VIGS-treated and uninfiltrated plants was assessed by application of CO2 exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a mild stress based on pigment content and light absorption characteristics in some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the transit sequence in the TRV vector followed by Agrobacterium-mediated transient expression of a modified gene consisting of an altered transit sequence fused to the native mature protein sequence. Nevertheless, NPQ in infused fully expanded leaves that expressed this re-introduced form was not fully restored indicating the possible importance of psbS incorporation prior to formation of grana stacks.

Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea.

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

Chloroplast biogenesis during rehydration of the resurrection plantXerophyta humilis: parallels to the etioplast–chloroplast transition

De-etiolation of dark-grown seedlings is a commonly used experimental system to study the mechanisms of chloroplast biogenesis, including the stacking of thylakoid membranes into grana, the response of the nuclear-chloroplast transcriptome to light, and the ordered synthesis and assembly of photosystem II (PSII). Here, we present the xeroplast to chloroplast transition during rehydration of the resurrection plant Xerophyta humilis as a novel system for studying chloroplast biogenesis, and investigate the role of light in this process. Xeroplasts are characterized by the presence of numerous large and small membrane-bound vesicles and the complete absence of thylakoid membranes. While the initial assembly of stromal thylakoid membranes occurs independently of light, the formation of grana is light dependent. Recovery of photosynthetic activity is rapid in plants rehydrated in the light and correlates with the light-dependent synthesis of the D1 protein, but does not require de novo chlorophyll biosynthesis. Light-dependent synthesis of the chlorophyll-binding protein Lhcb2 and digalactosyldiacylglycerol synthase 1 correlated with the formation of grana and with the increased PSII activity. Our results suggest that the molecular mechanisms underlying photomorphogenic development may also function in desiccation tolerance in poikilochlorophyllous resurrection plants.

EGY1 plays a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-grown Arabidopsis hypocotyls

Egy1 was isolated as an ethylene-dependent gravitropism-deficient Arabidopsis mutant. Molecular studies reveal that EGY1 gene encodes a 59-kDa plastid-targeted metalloprotease. It is actively expressed in hypocotyl tissue and targets to endodermal and cortex plastid. Its protein level is up-regulated by both ethylene and light. CAB protein accumulation and chlorophyll level is severely reduced in hypocotyls and endodermal cells, respectively. Sucrose is able to restore the severely reduced starch and lipid contents as well as the deficient endodermal plastid size found in light-grown egy1 hypocotyls yet it fails to rescue the reduced plastid number and chlorophyll level in egy1 endodermal cells. The loss-of-function egy1 mutation results in a smaller size (1.9 +/- 0.3 microm in diameter) and less number (5 +/- 1) of plastids in endodermal cells, which are nearly 50% of the wild-type. EGY1 is specially required for the development of full-size endodermal plastid in seedlings that are grown on sucrose-free media under light. It plays a direct role in controlling the light-induced chlorophyll production, grana formation and plastid replication in endodermal cell. However, it plays an indirect role in regulation of endodermal plastid size. It is likely that the ethylene-dependent gravitropism-deficient phenotype of egy1 hypocotyls may result from the smaller size and less number of endodermal plastids. Gravicurvature assays performed on ethylene-insensitive mutants, etr1-1, etr2-1, ers2-1, ein4-1 and ein2-5, have clearly demonstrated the necessary role for ethylene in vigorous gravitropism of light-grown hypocotyls. The degree of ethylene-dependent gravicurvature is positively correlated with the combined state of endodermal plastid mass and number. Neither ethylene nor EGY1-regulated full-size endodermal plastid is sufficient for promotion of vigorous hypocotyl gravitropism. Presence of 4 full-size plastids per endodermal cell together with ethylene pretreatment of hypocotyls becomes sufficient to trigger vigorous gravicurvature in light-grown seedlings. A model is therefore proposed to address the role of EGY1 in regulation of endodermal plastid size and number as well as the stimulatory effect of ethylene on hypocotyl gravitropism.

Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying the premature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs in appressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) as well as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacity for a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement of photochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimately intertwined.

Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone.

We have determined the stoichiometric composition of membrane components (lipids and proteins) in spinach thylakoids and have derived the molecular area occupied by these components. From this analysis, the lipid phase diffusion space, the fraction of lipids located in the first protein solvation shell (boundary lipids), and the plastoquinone (PQ) concentration are derived. On the basis of these stoichiometric data, we have analyzed the motion of PQ between photosystem (PS) II and cytochrome (cyt.) bf complexes in this highly protein obstructed membrane (protein area about 70%) using percolation theory. This analysis reveals an inefficient diffusion process. We propose that distinct structural features of the thylakoid membrane (grana formation, microdomains) could help to minimize these inefficiencies and ensure a non-rate limiting PQ diffusion process. A large amount of published evidence supports the idea that higher protein associations exist, especially in grana thylakoids. From the quantification of the boundary lipid fraction (about 60%), we conclude that protein complexes involved in these associations should be spaced by lipids. Lipid-spaced protein aggregations in thylakoids are qualitatively different to previously characterized associations (multisubunit complexes, supercomplexes). We derive a hierarchy of protein and lipid interactions in the thylakoid membrane.

Grana formation: entropy-assisted local order in chloroplasts?

Grana formation is a prominent feature of the ultrastructure of chlorophyll b-containing chloroplasts, serving to fine-tune photosynthetic efficiency. This paper examines the physical forces that determine the state of minimum free energy of the chloroplast as manifested by grana formation. In particular, it considers the interplay of van der Waals attraction, electrostatic repulsion and short-range hydration repulsion between thylakoid membranes. In addition to these interactions as determinants of the free energy of the system, it is also proposed that ordering of thylakoid membranes and of intramembrane protein complexes is driven by an increase in the overall entropy of the system. This local order may partly come about by an increase in entropy associated with a greater free volume for diffusion of membrane and stromal components.

The xanthophyll cycle of higher plants: influence of antenna size and membrane organization

The development of the photosynthetic apparatus of intermittent light grown pea plants under continuous illumination has been investigated. We determined the formation of antenna proteins and the synthesis of pigments at different stages of greening and compared the data with the changes in the xanthophyll cycle reactions. The limited convertibility of violaxanthin in the de-epoxidation reactions of the cycle was found to be closely related to the presence of antenna proteins and could be attributed to direct (pigment binding) and indirect (grana formation) functions of antenna proteins. The reduced epoxidation rate in intermittent light plants was found to be accelerated with increasing amounts of antenna proteins. However, the changes in the epoxidation rates were not consistent with the assignment of the epoxidase activity to LHC II, the major light harvesting complex protein of photosystem II. This interpretation was further supported by an unchanged epoxidase activity in – also LHC II depleted – bundle sheath cells of the C 4 plant Sorghum bicolor and stroma fractions of isolated spinach thylakoids. We assume that the basic function of antenna proteins in the xanthophyll cycle of higher plants is mainly related to the binding of the substrate and/or to interactions with the de-epoxidase/epoxidase. By that antenna proteins seem to be responsible for the limited violaxanthin convertibility as well as they are required for highest epoxidation rates.

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.

Cytochemical Localization of Photosystem I in Relation to Differentiation of the Thylakoid Membranes during Greening of Barley Leaves

Changes in distribution of photosystem I (PS I) in the developing thylakoid membranes of greening barley (Hordeum vulgare L. cv. Amagi-Nijo) chloroplasts were studied by a cytochemical method using 3,3'-diaminobenzidine (DAB). The activity was detected on the thylakoid of etiochloroplasts after 1 h of greening. Stacks of a few thylakoids (thought to be an initiation of grana) appeared at about 2-3 h of greening. Until 4 h, the DAB reaction was observed uniformly throughout the whole thylakoid system (stroma and grana thylakoids). After 6 h of greening, The DAB reaction was detected heterogeneously in the etiochloroplasts; more intense on the stroma thylakoid and peripheral grana thylakoid. Further illumination led to more distinct heterogeneity of the DAB reaction. Thus, redistribution of PS I proceeded differentially after onset of grana formation in the developing etiochloroplasts. Measurements of chlorophyll content, light saturation of PS I activity and fluorescence emission spectra of greening barley leaves suggest that redistribution of PS I may reflect developmental changes in assembly of the PS I complex with light-harvesting chlorophyll-protein complex.

Membrane in chloroplasts. A topological approach to grana and frets

The membrane system of chloroplasts, where photosynthesis takes place, presents a remarkable organization of flat thykaloïds in grana and frets which contributes to the efficiency of the photochemical and biochemical steps of the process. A topological analysis of this organization is proposed, which develops a relationship with screw dislocation walls in lamellar liquid crystals. This analysis makes possible a rather simple geometrical description of the organization depending upon two parameters only: the stacking distances of thykaloïds in grana and frets. This suggests that the formation of grana and frets, and their overall structure, might be viewed as a separation between two lamellar stockings of thykaloïds whose different spacings are associated with a distribution along the membrane of the molecules intervening in the photochemical process.

Kinetic relationships between structural changes in the thylakoid membranes and photosystems organization

Events involved in cation-induced aggregation of thylakoid membranes and grana formation were studied in broken chloroplasts. Membrane stacking was evaluated through changes in light scattering while lateral interactions and movements of pigment-protein complexes were monitored through changes in steady-state, maximum and minimum chlorophyll fluorescence ( F s , F m and F o resp.). Changes in the imbalance between the photoactivities of PS I and PS II were derived from these fluorescence parameters and the rates of these changes were compared at room temperature and at 5°C. Particularly, a distinction was made between altered protein-protein interactions within the membrane (microstructural changes) and altered membrane-membrane interactions (macrostructural changes). Following addition of MgCl 2 (5 mM), the increase in F s and in the photosystems' imbalance lagged behind the increase in F m , all of which lagged behind the increase in light scattering. This effect was mostly pronounced at the low temperature. One may conclude that membrane stacking (reflected mainly by the scattering increase) does not require and is not simultaneously coordinated with the lateral movements of pigement complexes (reflected by an increase in the imbalance) and the lateral organization of the photosystems (reflected by an increase in F m ) but is rather a prerequisite for it. Such photosystem organization occurs in later stages of the cation-induced overall process. On the other hand, in the reverse processes, which were followed by depleting Mg 2+ with EDTA, membrane destacking and grana disintegration lagged behind the decrease in light excitation imbalance in favor of PS II and thus are probably limited by lateral rearrangements of pigment complexes. The decrease in the imbalance was much faster than the decrease in F m , the last one presumably reflecting an increased excition interaction between PS I and PS II (i.e., quenching of PS II excitation by energy transfer to PS I—‘spillover’—when the two photosystems are sufficiently close, as the diffusional movement progresses). The difference between the kinetics of F m and the imbalance when they increase upon cation addition or decrease upon cation depletion implies that the changes in the imbalance of the photosystems and spillover are largely independent processes.

The greening process in cress seedlings. I. Pigment accumulation and ultrastructure after application of 5‐aminolevulinate and complexing agents

A reduced rate of greening after continuous illumination was observed in dark‐grown cress seedlings (Lepidium sativum L.) incubated with 5‐aminolevulinate (ALA) or the complexing agents 2,2′‐bipyridyl, 8‐hydroxyquinoline or 1,10‐phenanthroline. This effect cannot be explained merely by photodynamic damage caused by chlorophyll precursors which are accumulated in the dark under these conditions. Flash light experiments revealed that photoconversion of protochlorophyll(ide) to chlorophyllide was not influenced by chelator treatment. The next step in the chlorophyll pathway, the esterification of chlorophyllide, however, was inhibited. Simultaneously applicated ALA and complexing agents did not result in a synergistic reponse; on the contrary, ALA seemed to render cress plants less susceptible to the treatment with complexing agents upon subsequent irradiation. Ultrastructural studies demonstrated that grana formation in light was inhibited after pretreatment with ALA or complexing agents.

Immunogold localization of LHC II proteins in chloroplasts with different degrees of grana stacking induced by SAN‐9789.

The amount and distribution of proteins of the light‐harvesting complex associated with photosystem II (PS II) were investigated using immunogold labelling of chloroplasts of wheat (Triticum aestivum L. cv. Walde). The seedlings were grown in weak red light (16 mW m−2) after imbibition of grains with SAN‐9789 (Norflurazon, 0.028 to 28 mg I−1). Chloroplasts of these plants exhibited thylakoids with different degrees of stacking. Thylakoids of untreated plants grown in a greenhouse had most gold particles per unit membrane length in both appressed and non‐appressed regions compared to red light grown plants. The ratios of labelling between appressed and non‐appressed membranes were fairly constant in red light‐ and greenhouse‐grown plants. The labelling densities were 2.5–3 times higher in the appressed thylakoids compared to the non‐appressed thylakoids. However, at a SAN concentration of 2.8 mg I−1 there was a sharp decrease in thylakoid appressions and in labelling density of both appressed and non‐appressed membranes. The total amount of particles per chloroplast was also much lower as compared to that at lower SAN concentrations. Plants treated with the highest concentration of SAN (28 mg I−1) contained chloroplasts devoid of normal grana structures. In these plastids, the thylakoids were elongated and single. The labelling density in these membranes was ca 50% of that observed at 2.8 mg I−1. This paper thus supports earlier observations that proteins of the light‐harvesting complex of PS II (LHC II) are mainly localized in the appressed regions of the grana membranes, and may be involved in the formation of grana.

Correlation between pigment composition and apoproteins of the light‐harvesting complex II (LHC II) in wheat (Triticum aestivum)

Seedlings of wheat (Triticum aestivum L. cv. Walde, Weibull) grown in continuous weak red light (16 mW m−2) with or without SAN‐9789, contained significantly lower amounts of chlorophylls and carotenoids compared to untreated plants grown in a greenhouse. The Chl alb ratios were 3.6 in the greenhouse‐grown plants, 5.1 in untreated and ca 16 in SAN‐treated plants grown in weak red light, respectively. The main difference in polypeptide composition of thylakoids isolated from red light‐grown plants, compared to those grown in the greenhouse, was a lower amount of proteins of the light‐harvesting complex (LHC) II. The amount of apo‐LHC and LHC were correlated to the xanthophyll to β‐carotene ratios in these plants. The absence of grana and the absence of proteins of the light‐harvesting complex 11 in SAN‐treated plants, support the general dogma that these proteins are involved in the formation of grana. Since the amount of apo‐LHC and LHC could be correlated to the presence of carotenoids as well as the chlorophylls, it is concluded that the carotenoids are necessary for the correct assembly and stabilization of the apoproteins of LHC II in the thylakoid membranes.

Variations in the relative content of the peripheral and inner light-harvesting chlorophyll [formula omitted] complex (LHC II) subpopulations during thylakoid light adaptation and development

The light-harvesting chlorophyll a b- protein complex of Photosystem II (LHC II) is composed of two subpopulations. One population is tightly bound to the Photosystem II core and contains predominantly a 27 kDa polypeptide. The other population, enriched in a 25 kDa polypeptide, is more peripheral bound and is able to undergo reversible detachment from the core due to protein phosphorylation or moderate heat. This pool of LHC II is therefore thought to be responsible for short-term adaptation of the Photosystem II antenna. In this study we show that the peripheral subpopulation is also responsible for long-term adaptation to variations in light regime. By two-dimensional gel electrophoresis it was found that spinach or pea grown under low irradiance contained an increased amount of total LHC II and also showed an increased proportion of the 25 kDa polypeptide relative to the 27 kDa polypeptide. The additional LHC II incorporated during the low-light adaptation was calculated to possess a 27 25 kDa polypeptide ratio around 2, a ratio close to what has previously been found for the peripheral pool of LHC II. This demonstrates a specific variation in the level of the peripheral LHC II in response to long-term light adaptation. By analyzing the LHC II polypeptide composition of developing pea etioplasts, we also demonstrate that in the early stage of membrane development the Photosystem II core and its tightly bound LHC II antenna is first inserted, while the peripheral LHC II is inserted at a later stage concomitant with grana formation and segregation of the two photosystems.

Effect of trivalent lanthanide cations on chlorophyll fluorescence and thylakoid membrane stacking

We have used trivalent lanthanide metal cations in the buffering media of pea chloroplasts to probe the stacking arrangement of thylakoid membranes and the spatial distribution of chlorophyll-protein complexes of Photosystems I and II. Measurements of steady-state chlorophyll fluorescence emission spectra of pea chloroplasts at room temperature demonstrate that, within this tripositive valency group, the extent of membrane appression is a function of hydrated metal ionic radius. These results are in agreement with a recent investigation using monovalent and divalent metal cations (Karukstis, K.K. and Sauer, K. (1985) Biochim. Biophys. Acta 806, 374–389). In addition, the lanthanide cation concentration effective in producing the maximum chlorophyll fluorescence intensity upon grana formation is dependent on hydrated ionic size. The current investigation supports the proposed hypothesis that cation screening ability defines the extent of intermembrane separation as well as the extent of lateral distribution of chlorophyll-protein complexes.