Per Svensson

Separation and characterization of stroma and grana membranes: evidence for heterogeneity in antenna size of both photosystem I and photosystem II

A rapid procedure to fractionate the thylakoid membrane into two well-separated vesicle populations, one originating from the grana and the other from the stroma-membrane region, has been developed. This was achieved by sonication of thylakoids present in an aqueous two-phase system followed by partitioning either by countercurrent distribution or by a batch procedure in three steps. The membrane populations were analysed according to their composition and photochemical activities. The grana membranes comprise, on chlorophyll basis, about 60% of the thylakoid material and are enriched in PS II, but also contain some PS I, while the stroma membranes comprise about 40% and are enriched in PS I, but also contain some PS II. Cytochrome f was slightly enriched in the grana-derived vesicle fraction. The properties of both PS I and PS II differ between the two populations. The PS I of the grana fraction (PS Iα) reached half-saturation at about half the light intensity of the PS I in the stroma-membrane fraction (PS Iβ). The rate of P-700 photooxidation under low light illumination was higher for PS Iα than for PS Iβ (30% larger rate constant), showing that PS Iα has a larger antenna. The PS II of the grana fraction (PS IIα) reached half-saturation at half the light intensity compared to the PS II of the stroma-membrane fraction (PS IIβ). The results show that the grana-derived membranes contain PS Iα and PS IIα which have larger functional antenna sizes than the corresponding PS Iβ and PS IIβ of the stroma membranes. The results suggest that the photosystems of the grana are designed to allow effective electron transport both at low and high light intensities, while the stroma-membrane photosystems mainly work at high light intensities as a supplement to the grana systems.

The domain organization of the plant thylakoid membrane

A model of the photosynthetic membrane from higher plants is presented. The different photosystems, PSIα, PSIβ, PSIIα and PSIIβ, are located in separate domains. The photosystems with the largest antenna systems, the alpha systems, are in the grana and the other in the stroma lamellae. In each grana disc PSIα is located in a flat annulus surrounding a circular PSIIα domain. In this the PSIIα units with the largest antennae are found in the center. The model is consistent with results from recent membrane fractionation experiments.

Heterogeneity among photosystem I

Thylakoids from spinach were sonicated and separated by aqueous two-phase partitioning into two vesicle populations originating from grana (α vesicles) and stroma lamellae (β vesicles), respectively, according to a procedure described earlier (Andreasson et al. (1988) Biochim. Biophys. Acta 936, 339–350). The functional antenna sizes of Photosystem I for these two vesicle populations were determined by measuring the kinetics of the photooxidation of P700. Under green light illumination, the antenna size of Photosystem I of the α vesicles (PSI α ) was 40% larger (40% larger rate constant of photooxidation) than that of Photosystem I of the β vesicles (PSI β ). By using actinic light, which is preferentially absorbed by chlorophyll b , this difference was accentuated (60%) indicating that the antenna of PSIα contains relatively more chlorophyll b . The experiments did not support the possibility that this difference in functional antenna size was due to ‘spill over’ from Photosystem II. A model is presented which shows that PSIα is located in the periphery of the grana and PSI β in the stroma lamellae.

Localization of cytochrome f in the thylakoid membrane: evidence for multiple domains

Abstract Thylakoids from spinach were fragmented by sonication and the viscles so obtained were separated into different populations by aqueous two-phase partitioning using the dextran-poly(ethylene glycol) system. The different vesicle populations were analyzed with respect to the concentrations of P700 and cytochrome. The P700 content varied between 0.93 (PS-II-enriched grana vesicles named BS) and 4.85 (stroma membrane vesicles named Y100) mmol per mol chlorophyll. The cytochrome f content varied between 1.92 (vesicles originating from the grana periphery, named 120S vesicles) and 3.12 (PS-II-enriched grana vesicles named BS) mmol/mol chlorophyll. A plot of the P700 content against the cytochrome f content of the different vesicle populations was compared with hypothetical models of membrane vesicles. The results show that the thylakoid membrane consists of at least three different domains with respect to cytochrome f. These are suggested to be: (1) the stroma lamellae; (2) the core of the partition region of the grana; and (3) a peripheral annulus of the grana discs including the margins (and perhaps also the end membranes).

The domain organization of the chloroplast thylakoid membrane. Localization of photosystem I and of the cytochrome b6-f complex

Abstract The relative distribution of chlorophyll (Chl) a and Chl b , of Photosystem I (PS I), and of the cytochrome b 6 - f (Cyt b 6 - f ) complex in different subchloroplast membrane vesicles is presented. Spinach chloroplasts ( Chl a Chl b = 2.7 , Chl PS I = 435:1 , Chl Cyt f = 970:1 ) were fractionated by Yeda press treatment and by sonication. Counter-current distribution of the resulting thylakoid vesicles in dextran-polyethylene glycol aqueous two-phase systems yielded a laterally extended range of chloroplast thylakoid membranes without loss of chloroplast material from the initial sample. In this analysis, PS I-enriched membrane vesicles moved in the leading edge of the counter-current distribution diagram, whereas PS II-enriched vesicles remained close to the trailing edge of the counter-current distribution diagram. Vesicles of mixed composition and vesicles depleted in both PS I and PS II were isolated from intermediate positions. The Chl PS I ratio in the various fractions approximated a hyperbolic function of the Chl a Chl b ratio with limiting values of Chl PS I = 190:1 attained at a Chl a Chl b = 6 and a Chl a Chl b = 1.67 attained when the Chl PS I ratio approaches infinity ([PS I] = 0). Such hyperbolic distribution is consistent with the exclusive localization of PS I in stroma-exposed thylakoids and of PS II in the membrane of the grana partition region. A depletion of the Cyt b 6 - f complex from the membrane domain of PS I was detected ( Chl a Chl b = 5.1 , Chl PS I = 210:1 , sol Chl Cyt f = 1510:1 ). Moreover, a chloroplast thylakoid membrane was identified, distinct both from that of PS II and from that of PS I which was enriched in the Cyt b 6 - f complex ( Chl a Chl b = 2.67 , Chl PS I = 460:1 , Chl Cyt f = 590:1 ). Our results suggested that such a Cyt b 6 - f domain is localized in the region between the appressed and the non-appressed membranes, possibly in the membrane of the fret region.

Metal Iodides in Polyiodide Networks—The Structural Chemistry of Complex Gold Iodides with Excess Iodine

Theoretical calculations that compare the Ij ion and the [AuI4](-) ion show that they are closely related and have potential energy surface (PES) minima corresponding to an L-shaped structure. Th ...

Preparation of highly enriched photosystem II membrane vesicles by a non-detergent method

An improved, non-detergent, method for preparative isolation of PS II membrane vesicles from spinach chloroplasts is presented. Thylakoids (chlorophyll (Chl) a/b ratio 2.8, Chl/P700 435) were fractionated by Yeda press treatment and aqueous two-phase partition to yield inside-out vesicles (1) (chl a/b 2.2, chl/P700 700). These vesicles were subjected a sonication — phase partitioning procedure; steps of sonication of inside-out vesicles, while still present in a dextran-polyethylene glycol two-phase system were alternated by phase partition. These steps selectively removed P700-containing membrane fragments from the inside-out vesicles and yielded a membrane fraction with improved PS II purity (Chl a/b ratio 1.9, Chl/P700 1500) and retained oxygen evolving capacity (295 μmol O2 mg Chl-1 h-1).

Counter-current distribution of sonicated inside-out thylakoid vesicles.

SummaryInside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2–2.4 (original chloroplasts 2.8–3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 > 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane.A plot of P700 per chlorophyll against chlorophyll b/(a+b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.

Fractionation of thylakoid membrane components by extraction in aqueous polymer two-phase systems containing detergent

Abstract A combination of detergent extraction and aqueous polymer two-phase partition has been used to fractionate thylakoid membrane components. The membranes were initially forced to partition to the lower phase, and subsequently, on solubilization, the components were selectively extracted to the upper phase by a stepwise increase in the detergent concentration. The detergents partitioned either strongly to the upper phase (Triton X-100) or almost equally between the phases (octyl glucoside). By this simple method polypeptide complexes could be purified several-fold in just a few rapid steps.

Subfractionation of Inside-Out Thylakoid Vesicles-Preparation of Pure Photosystem II Particles without using Detergent

Previous studies from this laboratory have shown that inside-out, photosystem II enriched, thylakoid vesicles can be prepared by mechanical press treatment followed by partition in aqueous polymer twophase systems. for references, see review (1). in this work we have further fragmented the inside-out vesicles by sonication in order to study the domain organization of the thylakoid and to prepare vesicles with increased photosystem II purity by removing contaminating stroma membranes. We have used a new type of sonication procedure. Steps of sonication of the inside-out vesicles, while still present in a dextran-poly-ethylene glycol two-phase system, are alternated by phase partition which removes P700 containing membrane fragments from photosystem II vesicles. By this multistage, sonication-partition procedure we have obtained vesicles with retained oxygen evolution and extremely low, or zero, content of P700. The chlorophyll a/b ratio of these vesicles can be as low as 1.8 compared to 2.2–2.4 for the conventional inside-out vesicles (B5-B3) and 2.8–3.0 for the chloroplast. The vesicles should be useful as representatives of the thylakoid partition region.