Functional Organization of the Chlorophyll-Containing Complexes

The stepwise synthesis and assembly of photosynthetic membrane components in the y-l mutant of Chiamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or Ught harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae lnk the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylis and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent. The greening process of the y-l mutant of Chlamydomonas reinhardi as well as, e.g., of Euglena (21, 22, 25), Scendesmus (9, 11), and higher plants (3), forms a valuable tool for the study of the connection and interrelation of the various parts of the photosynthetic apparatus, notably the antennae pigments, reaction centers, and electron transfer components (5, 15, 30). Dark-grown Chlamydomonas y-l cells contain only small amounts of Chl and photosynthetic membrane remnants (30). Residual activities of both PSI and PSII reaction centers can still be detected (15, 30) as well as the presence of relatively large amounts of Cyt, plastocyanin, ferredoxin, NADP reductase, and the CO2 fixation enzymes (30). However, the residual reaction centers are not properly integrated within the electron carrier chain (15), and thus electron transfer activities are barely detect'Supported by Stiftung Volkswagenwerke, Grant No. AZ-I 1-2882. able. The organization of the residual Chl present in the membrane of dark-grown cells differs from that of normal cells. Chlprotein complexes cannot be detected by the usual techniques (23); the quantum yield of fluorescence at room temperature is several times higher than that of normal light-grown cells (26), and at 77 K only one fluorescence band appears which peaks at 680 nm (12, 23) instead of the usual three peaks at 685, 695, and 714 nm, characteristic of fully greened Chlamydomonas cells. Upon reexposure ofdark grown cells to light, Chl is synthesized, and active photosynthetic membranes are rapidly formed before any cell division occurs (greening) (30). The greening process involves synthesis of both chloroplastic and cytoplasmic translation products. It can be carried out in steps when first CAP2 is introduced to inhibit the synthesis of chloroplastic translated polypeptides required for the formation of both PSI and PSII reaction centers (30). The newly formed membranes contain relatively large amounts of Chl but are photosynthetically inactive (30). The high fluorescence yield at room temperature is maintained (26). The fluorescence emission spectrum at 77 K now consists of two bands with peaks at 686 and 705 to 708 nm, the latter being predominant (12, 23). Among the Chl-protein complexes only the Chl a/b protein complex (LHC or CPII) can now be detected by SDS-PAGE (5). To complete the developmental process, cells are washed free of CAP, allowing restoration of the synthesis of chloroplastic translated polypeptides (repair). There are three variations of the repair process which can take place either in the light (when additional Chl is synthesized), in the dark (no additional Chl synthesis) or in the light in the presence of cycloheximide (CHI) which inhibits cytoplasmic protein synthesis and reduces Chl synthesis severely (30). Photosynthetic activities are reestablished to a large extent within a few hours in all the above conditions in the absence of cell division. However, only in the light-incubated cells, which synthesize large additional amounts of Chl, is the CPI complex detected together with a substantial reduction of the fluorescence yield at room temperature. The difference between these cells and those repaired in the dark probably arises from differences in the organization of the newly synthesized Chl in the photosynthetic membranes. The results presented here, part of which have been published as a preliminary communication (23), indicate that photosynthetic 2 Abbreviations: CAP, chloramphenicol; CHI, cycloheximide; CPI, Chl a-protein complex I; CPII, Chl a,b-protein complex; DCIP, 2,6-dichlorophenolindophenol; LDS, lithium dodecyl sulfate; LHC, light harvesting complex; MV, methylviologen; PAGE, polyacrylamide gel electrophoresis; RCII, RCI, reaction centers of photosystems II and I, respectively. 637 www.plantphysiol.org on July 19, 2018 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 70, 1982 membranes formed in presence of CAP contain, in addition to the LHC, a component of the PSI antenna responsible for the 77 K fluorescence at 705 to 708 nm. When the polypeptides required for the formation of reaction centers are synthesized and integrated in the membrane in absence of additional Chl synthesis (repair in the dark), energy transfer between preexisting antennae and the newly formed active reaction centers is impaired. The difference between the 'light' and 'dark' repair is the synthesis of new Chl protein complexes, whose function is probably to link between the main antennae and the reaction centers and to optimize excitation energy transfer. The simplest assumption is that each photosystem consists of at least three components, which can be formed and integrated stepwise: a reaction center, a connecting antenna close to the reaction center, and a major light harvesting antenna. MATERIALS AND METHODS Synthesis of Inactive Membranes and Repair of Photosynthetic Activity. Chlamydomonas reinhardi y-l cells were grown on a mineral medium with acetate (5 mM) as the sole carbon source (30). Cells grown in the dark for four to five generations, depleted of photosynthetic membranes, were harvested by centrifugation (4,000g, 4 min), washed, and resuspended in fresh growth medium at a final concentration of I to 2 x I07 cells/ml. CAP (200 ,ug/ml) was added to the cell suspension and the cells were then exposed to white fluorescent light (10 w m-2). After 6 h of incubation ('CAP greening'), the cells were washed free of CAP, resuspended in fresh growth medium, and further incubated in the dark or light with or without the addition of CHI (2 ,ug/ml) (repair). At the end of this incubation, the cells were washed in 30 mm Tris-HCl, pH 8.0, containing 10 mm KCI (TK buffer), and stored in ice until use. For radioactive labeling of proteins, [14C]acetate was added during the repair process (1 to 2 ,uCi/,Imol, I ,Lmol/ml). Preparation of Membrane Fractions. Chloroplast membranes were prepared by passing a cell suspension in TK buffer (5 x 108 cells/ml) through a French press (7,000 p.s.i. O° C). The homogenate was layered on a discontinuous sucrose gradient (15%; 30%o; 60%1o; w/v, in TK buffer) and centrifuged in a SW27 Beckman rotor at 25,000 rpm for 2 h. The green layer of the interphase between 30%o and 60%o sucrose was collected, and the chloroplast membranes were washed in TK buffer free of sucrose. Membranes prepared in this way were used for electrophoretic analysis of radioactively labeled proteins and detection of the Chl-protein complexes CPI and CPII. For measurements of photosynthetic activity, the French press homogenate was centrifuged at 2,000g for 3 min (in order to remove whole cells and large debris) and then at 27,000g for 10 min. The resulting pellet was suspended in TK buffer and stored in ice until use, without loss of activity (2-3 h). Assays of Photosynthetic Activity. Photosynthetic 02 evolution in whole cells was measured polarographically using saturating white (unfiltered) light. Various activities of electron transport were measured on isolated chloroplast membranes; electron transport from reduced DCIP to MV was assessed polarographically by the resulting 02 uptake, under white light (reaction mixture: 25 ,ug Chl/ml, 100 JM DCIP, 5 mm ascorbate, 10 JIM MV, and 10 tiM DCMU, in TK buffer). Photoreduction ofDCIP was measured spectrophotometrically using an Aminco-Chance dual wavelength spectrophotometer and recording the light minus dark absorbance differences at 580 nm (reaction mixture: 2-5 ,ug Chl/ml, 100 JIM DCIP, in TK buffer). The exciting light was filtered through a Baird-Atomic 650 nm interference fiter (half-band width, 20 nm; maximum intensity, 85 w m-2). Photoreduction of NADP with H20 as an electron donor was measured with membranes as by Berzborn and Bishop (7) and carried out in the Aminco-Chance spectrophotometer. The excitation light was filtered through a wide-band 525 to 625 nm Baird-Atomic interference filter (maximum intensity, 15 w m-2). Measurements of fluorescence yield and kinetics were carried out using a red sensitive (EMI 9558) photomultiplier and Philips PM 3234 storage oscilloscope. Illumination was at right angle to the detector and was switched on by an