Planta

Planta (1990)182:391-398

9 Springer-Verlag 1990

Quantitative correlation of peripheral and intrinsic core polypeptides of photosystem II with photosynthetic electron-transport activity of Acetabularia mediterranea in red and blue light Rainer Schmid*, Rainer Wennicke, and Sigrun Fleischhauer Institut ffir Pflanzenphysiologie,Zellbiologieund Mikrobiologie, Freie Universit/it Berlin, K6nigin-Luise-Strage 12-16, 1000 Berlin 33 Received2 April; accepted 11 June 1990

Abstract. The high photosynthetic activity (02 production and COz consumption) of Acetabutaria mediterranea Lamour. (= A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light, and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity completely within 3-5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre complex of photosystern I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity of the reaction-centre components DI (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells (the 16-kDa component could not be found in Acetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast, the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna, comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via * To whom correspondence should be addressed Abbreviations: Chl = chlorophyll; DI = 32-kDa QB-binding reaction-centre subunit of photosystem II; D2 = 34-kDa QA-containing subunit of the reaction centre of photosystem II; LHCP =lightharvesting chlorophyll-protein complex of photosystem II; LiDS = lithium dodccylsulfate; Mr = relative molecular mass; PSI, PSII= photosystem I, II; RuBPCase-=ribulose-l,5-bisphosphatecarboxylase

the core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment.

Key words: Acetabularia - Blue light - Chlorophyll-protein complexes - Photosystem II (antenna function) Thylakoid (polypeptide composition)

Introduction In the siphonous unicellular green alga Acetabularia mediterranea, morphogenesis and the activities of different metabolic pathways depend on the action of blue light (see Schmid, 1984, for an overview). One of these pathways is photosynthesis. After growth of the cells in white light, a transfer to red light causes a continuous and slow decrease in photosynthetic activity (Schael and Clauss 1968; Wennicke and Schmid 1987). Only about 20% of the original activity is found after three weeks of irradiation. Since the light compensation point is lowered as a result of a simultaneous decrease in respiratory 02 consumption (Wennicke and Schmid 1987), there is net photosynthesis even after long periods of exposure to red light. Under these conditions, massive accumulation of starch (Clauss 1972) is still possible. Blue or white light, after prolonged red pre-irradiation, restores the original activities of both photosynthesis and respiration within a comparatively short period (approx. 3 d) and activates degradation of the excess starch. Thus, blue light appears to be the active light quality (see also Schmid 1984). By analysis of curves of photosynthesis versus irradiance, and of partial reactions of photosynthetic electron transport, the limiting blue-light-controlled step for total photosynthesis was found to be associated with photosystem II (PSII). The component participates in the light-dependent processes of PSII (Wennicke and Schmid 1987). Recent biophysi-

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R. Schmid et al. : Acetabularia: blue-light-dependent thylakoid composition

cal analysis (Schmid et al. 1990) indicated that the quantitative changes in the function o f c o m p o n e n t s o f the P S I I reaction-centre core are only in the range o f 2 0 30% and thus are insufficient to fully explain the large changes o f total PSII activity. As we were interested in the molecular changes that are responsible for the changes o f p h o t o s y n t h e t i c activity in vivo u n d e r red and blue light, the investigations o f the present p a p e r were intended to analyze the quantity o f polypeptides in the p h o t o s y n t h e t i c m e m b r a n e u n d e r these conditions.

Results Identification o f m e m b r a n e components. After electrophoresis o f the solubilized thylakoid polypeptides, a n u m b e r o f chlorophyll-protein complexes and free pigments closely behind the f r o n t were detectable (Fig. 1) w i t h o u t staining. We use s t a n d a r d n o m e n c l a t u r e for the complexes (see T h o r n b e r 1986). Scans o f the unstained gels at 650 and at 675 n m (Fig. 1, see Wild et al. 1980) were taken to distinguish complexes according to their Chl-b content. The absorption ratio at these wavelengths o f the free-pigment p o r tion was similar to that o f the total Chl o f the thylakoids. Therefore, it did n o t indicate preferential dissociation o f either Chl a or b, at least f r o m the m a j o r complexes. The light-harvesting complexes o f P S I I a n d their oligomeric f o r m s could easily be identified, as they displayed almost identical a b s o r p t i o n at the two wavelengths owing to their high Chl-b c o n t e n t (Figs. 1, 2). Oligomeric f o r m s o f the m a j o r light-harvesting c o m p l e x o f P S I I ( L H C P ) h a d very similar a b s o r p t i o n spectra to the m o n o m e r i c complexes (not shown). In the following text and figures, they are indexed by superscript numbers, the m o n o m e r i c c o m p l e x being L H C P a. In b o t h cases, re-electrophoresis o f L H C P 1 and L H C P 3 after heating the samples p r o d u c e d two b a n d s with relative molecular masses (Mr) o f 24 and 26 k D a , indicating that L H C P 1 was the oligomer o f L H C P 3 (data n o t shown, see Apel et al. 1975). W h e n thylakoids were solubilized and sepa-

Materials and methods Culture conditions and irradiation sources. Cultures of Acetabularia mediterranea Lamouroux (=A. aeetabulum (L.) Silva) and experimental conditions were standard procedures and were as described previously (Wennicke and Schmid I987). Preparation of thylakoids. For homogenization the cells were cut into small pieces using a pair of scissors. Then, the cell fragments were squeezed out using a Potter-Elvehjem-type glass homogenizer with a teflon pestle (Braun, Melsungen, FRG). The resulting suspension was filtered through two layers of nylon gauze, the first layer having a pore diameter of 0.1 mm and the second of 0.01 ram. This suspension was used for the isolation of chloroplast membranes as described by Apel et al. (1975). Recently, we changed to a different preparation protocol (Schmid et al. 1990) which yields a higher amount of cytoplasm-free chloroplasts and eliminates bacteria that are associated with the cell walls of the Aeetabularia cultures used. Hydrophilic thylakoid-membrane polypeptides were prepared by the Triton X-114 method (Bordier 1981 ; modification of Bricker and Sherman 1982) using N-{[2-hyxdroxy-l,l-bis(hydroxymethyl) ethyl]-amino}-l-propanesulfonic acid (Tricine)-NaOH, pH 7.5, containing 150 mM NaC1 and protease inhibitors (5 mM aminocaproic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine). Samples of known chlorophyll (Chl) content were extracted by phase separation and proteins were precipitated by trichloroacetic acid (10%, w/v, final concentration). The precipitate was washed with cold 80% acetone (v/v), dried and solubilized in the final sample buffer for electrophoresis (see below).

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Electrophoresis and quantification of thylakoid polypeptides. All electrophoreses were performed at 4-8 ~ C (Delepelaire and Chua 1979) in the discontinuous buffer and gel system of Neville (1971). Sodium dodecyl sulfate was replaced by the corresponding lithium salt (LIDS). After the separations the gels were stained with Coomassie-blue G-250. Densitometric evaluations were done using a scanning photometer (M4 QII/PMQII; Zeiss, Oberkochen, FRG). Scanning wavelengths for unstained gels were at 675 and 650 nm for Chl a and b absorption, respectively (Wild et al. 1980), and in stained gels at 590 nm. For quantification we used a peak integrator (HP 3200; Hewlett-Packard, Palo Alto, Calif., USA). Immunoblotting. For immunoblotting, the polypeptides were transferred to nitrocellulose sheets (Hybond C; Amersham-Buchler, Bramaschweig) after electrophoresis and stained with peroxidaseconjugated protein A and chloronaphthol according to Towbin et al. (1979), while the blots were washed as described by Geiger et al. (1987). Site-specific antibodies against spinach DI (32-kDa QB-binding reaction-centre subunit of PSII) and D2 (34-kDa QAcontaining subunit of the reaction-centre of PSII) were a gift from Professor A. Trebst (Bochum, FRG) and are characterized elsewhere (Geiger et al. 1987; Johanningmeier 1987). Antibodies directed against the 10- and 22-kDa polypeptides of PSII and the proteins of the water-splitting domain were kindly supplied by Dr. B. Andersson (Stockholm, Sweden; see also Andersson et al. 1984; Ljungberg et al. 1984).

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