Eva Andreasson

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.

Heterogeneity in photosystem I — the larger antenna of photosystem Iα is due to functional connection to a special pool of LHCII

Abstract Thylakoids from spinach were fragmented by sonication and separated by aqueous two-phase partitioning into grana originating vesicles (α vesicles) and stroma membrane originating vesicles (β vesicles) (Andreasson et al. (1988) Biochim. Biophys. Acta 936, 339–350). PS Iα and PS Iβ were isolated from these two vesicle populations respectively, and their properties were compared. PS Iα contains more of light harvesting complex II (LHCII) than PS Iβ. The LHCII is functionally connected to PS I, a connection which is abolished by addition of octyl glucoside. The grana-derived α vesicles were further fragmented by sonication and separated by aqueous two-phase partitioning into vesicles enriched in margins and vesicles enriched in PS IIα from the center of the grana. By non-denaturating isoelectric focusing it was found that the LHCII pattern of PS Iα was almost identical to that of LHCII of the margin enriched vesicles but very different from that of LHCII associated with PS IIα. It is concluded that: (1) PS Iα (originating from grana membranes) has a larger functional antenna size than PS Iβ (originating from stroma membranes) because of association with LHCII; (2) PS Iα is located in the periphery of the grana (margins and end membranes) and associated with a different pool of LHCII compared to that associated with PS IIα of the more central core of the grana.

Composition of photosynthetic pigments in thylakoid membrane vesicles from spinach

Thylakoid membranes from spinach were fragmented mechanically and separated into vesicles originating from grana and stroma-exposed lamellae (Andreasson et al. (1988) Biochim Biophys Acta 936: 339–350). The grana vesicles were further fragmented and separated into smaller vesicles originating from different parts of the grana (Svensson and Albertsson (1989) Photosynth Res 20: 249–259). All vesicles so obtained were analyzed with respect to chlorophyll and carotenoid composition by reverse phase HPLC. For all fractions the following relations (mole/mole) were found: 1 carotenoid per 4 chlorophyll (a+b), 2 lutein per 5 chlorophyll b and 5 violaxanthin per 100 chlorophyll (a + b). The contents of lutein and neoxanthin were each linearly related to chlorophyll b and β-carotene was linearly related to chlorophyll a.

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).

Fractionation of the thylakoid membrane from Dunaliella salina – heterogeneity is found in Photosystem I over a broad range of growth irradiance

Thylakoids from the green alga, Dunaliella salina, were fragmented by sonication and the appressed grasna membranes separated from stroma lamellae by partitioning in aqueous two-phase systems. The concentration and antenna size of Photosystem I in the two membrane domains were determined for cultures grown at three different light intensities. Although the antenna size of both PS is decreased with increasing growth irradiance, the antenna size of Photosystem I in the grana was approximately 25-30% greater than the antenna size of Photosystem I in the stroma lamellae. Counter-current distribution analysis of sonicated thylakoids revealed that the amount of stroma lamellae increased whereas the amount of the stacked membranes decreased at higher growth irradiance. The overall decrease in the antenna size of PS I, in D. salina, at higher light intensities can therefore be explained by the combined effect of a decrease in the antenna size of Photosystem I, both in the grana and the stroma lamellae, and a relative increase in the amount of stroma lamellae which has smaller Photosystem I antennae than the Photosystem I centers found in the grana. Light-induced protein phosphorylation increased the relative amount of the stroma lamella fraction. This is interpreted as a result of partial unstacking of the grana. It is suggested that this may be a mechanism for increasing the cyclic electron transport around Photosystem I. (Less)

Heterogeneity of the Functional Antenna Size of Photosystem I from Spinach Thylakoids

We have recently fractionated the thylakoid membrane from spinach chloroplasts into two well separated vesicle populations (1). This was achieved by sonication and aqueous two-phase partitioning. A counter-current distribution diagram after such a fractionation is shown in Fig.1. The left peak comprises the grana derived α-vesicles while the right peak comprises the stroma derived β-vesicles. In this study we focused on examining the light harvesting properties of PSI of the two vesicle populations (PSIα and PSIβ respectively). We find different functional antenna sizes of PSI. PSIα has an antenna size about 30% larger than PSIβ. Our results imply that this heterogeneity in antenna size reflects the situation in the thylakoid membrane in vivo and is not due to any spill-over from PSII.

Photosynthetic Characterization of Thylakoid Membrane Preparations Isolated from Spinach Grown under Different Light Conditions

The quality and quantity of light during plant growth determine photosynthetic structural and functional properties of higher plants. Light environment varies not only on a large scale, such as, seasonally, diurnally, and spatially, but also at the level of micro-circumstances of the chloroplasts. Our previous experiments have shown that in vivo there are different domains in the thylakoid membranes, which have different Chla/b ratios and different average antenna sizes of photosystem IIα (PSIIα), the dominating form of PSII(1). For optimal photosynthesis a battery of photosynthetic units with different antenna sizes and pigment contents might be beneficial to the plant to adapt to changes in its light environment and to efficiently utilize the light quanta.

Organization of the Thylakoid Membrane with Respect to the Four Photosystems, PSIα, PSIβ, PSIIα and PSIIβ

The thylakoid membrane is an extremely complicated structure with several different domains having specialized functions. There is a heterogeneity among the photosystems. Of the two classes of PSII the main, PSIIα, with its larger antenna is localized in the grana, while the other, PSIIβ, with its smaller antenna is localized in the stroma membrane. Recently we presented evidences that showed that also among PSI there is heterogeneity (1). One form, PSIα, is localized in the grana and has a larger antenna than the other form, PSIβ, which is localized in the stroma membrane. In this paper we calculate how much chlorophyll is associated with the four photosystems. Two alternative and partly independent approaches are used.