Photosynthesis Research 35: 171-178, 1993. O 1993 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Composition of photosynthetic pigments in thylakoid membrane vesicles from spinach Ren6 K. Juhler 1, Eva Andreasson 2, Shi-Gui Yu 2 & Per-Ake Albertsson 2 ~Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark; 2Department of Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 O0 Lund, Sweden Received 16 March 1992; accepted in revised form 9 September 1992

Key words:

carotenoids, chlorophylls, HPLC, phase partition, xanthophylls

Abstract

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 /3-carotene was linearly related to chlorophyll a. Introduction

In higher plants the first events of photosynthesis, the light-dependent reactions, take place in the thylakoid membrane. The thylakoid membrane is continuous and forms stacked grana regions as well as unstacked stroma lamella regions. Thus, the two major domains of the photosynthetic membrane are grana and stroma lamella regions, respectively. One can also distinguish between the partition region (the appressed part of grana stacks) and the stroma exposed end membranes of the grana region. Furthermore, margins of both grana and stroma lamella membranes and frets, the interconnection between grana and stroma region, must be taken into consideration when investigating the domains of the thylakoid membrane. Disintegration followed by subfractionation has, over the years, been a successful approach to investigating the domains of this photosyn-

thetic membrane. Long ago, it was shown that the components of the photosystems could be partially separated by detergents (Anderson and Boardman 1966) or by mechanical disintegration (Jacobi and Lehmann 1968, Sane et al. 1970) followed by centrifugation. Photosystem I was enriched in a light fraction while Photosystem II was enriched in a heavier fraction. Separation of press-treated thylakoid membranes in an aqueous two-phase system (Albertsson 1986) yielded inside-out vesicles highly enriched in Photosystem II (Akerlund et al. 1976, Andersson et al. 1977, Andersson and Akerlund 1978). The inside-out vesicles were supposed to be formed when neighboring membrane parts of the grana seal upon disintegration. Thus, it was concluded that the appressed grana region of the thylakoid membrane is enriched in Photosystem II (Andersson et al. 1980). From these findings and from electron microscopy studies, a model implying almost complete

172 lateral heterogeneity in the thylakoid membrane could be suggested, nearly all Photosystem II (PS II~) being situated in the appressed granal domain of the thylakoid membrane and Photosystem I (PSI) being restricted to the stromal exposed part of the thylakoid (stroma lamella, margins and end membranes) together with a minor part of Photosystem II (PS 118). In addition, Photosystem I is also heterogenous. PS I~ is located in the grana periphery and PS 18 in the stroma lamellae (Svensson et al. 1991). For further references see Andersson and Anderson (1980), Andersson et al. (1985) and Albertsson et al. (1990a). In higher plants the main pigments of the photosynthetic apparatus are chlorophyll a, chlorophyll b and the carotenoids. Carotenoids can be divided into two groups; the carotenes which consist of carbon and hydrogen only and the xanthophylls which also contain oxygen. In higher plants, the major carotenoids are the carotene /3-carotene and the xanthophylls neoxanthin, violaxanthin and lutein. The photosynthetic pigments are not uniformly distributed among the components of the photosynthetic apparatus. Chlorophyll b and the xanthophylls are, for example, enriched in the light-harvesting complex of PS II, whereas chlorophyll a and /3-carotene are typical pigments of the reaction center complexes PSI and PS II (Siefermann-Harms 1985, Lichtenthaler 1987, Melis 1991). We have fractionated the thylakoid membrane into two well-separated vesicle populations originating from the grana and stroma lamellae, respectively. This was achieved by sonication followed by aqueous two-phase partitioning (Andreasson et al. 1988). The grana vesicles were further fractionated by sonication and aqueous two-phase partitioning (Svensson et al. 1989). In this study we put the question of how the different pigments of the photosynthetic apparatus are distributed among the different vesicle populations.

Materials and methods

Chemicals Tetrahydrofuran was obtained from Rathburn (Walkerburn, Scotland), Dextran 500 from Phar-

macia (Uppsala, Sweden) and Poly(ethylene glycol) 4000 (Carbovax PEG 3350) was supplied by Union Carbide (New York, NY).

Thylakoid membrane vesicle isolations Nomenclature. The naming of the different vesicles depends on the mode of preparation. Grana derived vesicles are either called alpha vesicles or B3 depending on whether they are prepared by counter current distribution or a batch procedure. Membrane vesicles originating from stroma lamella membrane vesicles are called beta vesicles if they are prepared by counter current distribution, T3 if they are prepared by a batch partition procedure or Y100 if isolated by differential centrifugation. Spinach (Spinacia oleracea L.) was grown at 20°C under cool white fluorescent light (400/xmol m -2 s -1) with a light period of 12h. Chloroplasts were isolated and osmotically broken and thylakoid membranes were obtained as described previously (Andreasson et al. 1988). Grana-originating vesicles (alpha vesicles, B3) and vesicles originating from the stroma lamella membrane region (beta vesicles, T3) were obtained after sonication of stacked thylakoid membranes present in an aqueous two-phase system followed by either two-phase partitioning by counter current distribution, yielding alpha and beta vesicles, respectively, or by a batch procedure in three steps yielding B3 and T3 as described earlier (Andreasson et al. 1988). The supernatant remaining after each centrifugation after the separation procedure was colorless indicating minimal losses of pigment during the vesicle preparation. The grana originating vesicles were further fractionated by sonication and aqueous twophase partitioning essentially as described by Svensson and Albertsson (1989) with the modifications described by Albertsson et al. (1991). The top phases obtained after each sonicationphase-partitioning step were named after the total time of ultrasonic exposure in seconds (180s, 360s, 540s and 720s). The remaining bottom phase after the last step was named BS. The two-phase system used to obtain both the alpha and beta vesicles as well as the further fractionated alpha vesicles (180 s, 360 s, 540 s, 720s and BS) comprised 5.7% (w/w) Dextran

173 500, 5.7% (w/w) poly (ethylene glycol) 4000, 10 mM sodium phosphate buffer (pH 7.4), 5 mM NaCI and 20 mM sucrose with the temperature maintained at 4 °C. Another fraction, named Y100, representing the stroma lamellae was prepared as described in Henrysson and Sundby (1990) by passing stacked thylakoid membranes twice through a Yeda press. After centrifugation at 40000 x g for 30 min the vesicles in the supernatant were collected by centrifugation at 100 000 x g for 90 min. HPLC: 50/zl of thylakoid membrane vesicles at a chlorophyll concentration of about 1400/xM was extracted three times with 0.8 ml 80 acetone in the presence of an equimolar mixture of solid N a : H P O 4 with KH2PO 4. Pigment content was determined by gradient reverse phase HPLC using a 120 x 4 mm column with octadecyl silica (Shandon Hypersil 5/zm spherical particles). Pigments were separated using a gradient of tetrahydrofuran in water and quantified as described by Juhler and Cox (1990).

high in the grana fraction and low in the stroma lamellae when compared with unfractionated thylakoid membranes (Fig. 1). When comparing the content of the individual carotenoids in the grana fraction, stroma lamellae and whole thylakoids (Fig. 2), it can be seen that the violaxanthin content is around 18% of total carotenoid content, whereas the lutein and neoxanthin content is low in the stroma lamellae fraction and high in the grana fraction. Also, it was found that the /3-carotene contribution to the total carotenoid in stroma lamellae fractions is high, while it is rather low in grana fractions when compared to unfractionated thylakoid membranes (Fig. 2). These results essentially agree with those of Henry et al. (1983). The chlorophyll a to b ratio for the vesicles originating from the grana (alpha vesicles) is 2.2 and that of stroma lamella membranes (beta 5

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Results

Comparison between grana and stroma lamella membranes

The HPLC method used allows for the determination of the major pigments of higher plants: Neoxanthin, violaxanthin, zeaxanthin, lutein, /3carotene, chlorophyll a and chlorophyll b. The zeaxanthin content is not considered as it contributed with less than 1% of the total carotenoid content in all fractions. No unresolved peaks occurred in the chromatograms, indicating that only very small amounts if any, of additional carotenoids like antheraxanthin could be present. Other pigments, like pheophytins and chlorophyll epimers that occurs in minor amounts within thylakoid membranes of higher plants has not been detected (Watanabe et al. 1985). The HPLC analysis of the pigment composition of the two membrane fractions originating from the grana (alpha vesicles, B3) and stroma lamellae (beta vesicles, T3, Y100) displays a clear pattern both when comparing carotenoid composition alone and pigment composition as a whole. The ratio of xanthophylls to carotenes is

l

chl a / b

2

(chl a + b } / ( e + x )

x/c

Fig. 1. Pigment composition in ( and (>

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 (Andreass...
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