Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.     

Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes

The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P₇₀₀) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II_β was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II_β. It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II_β is a precursor form of photosystem II_α occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation.     

The 20 kDa apoprotein of the CP24 complex of photosystem II: An alternative model to study import and intra-organellar routing of nuclear-encoded thylakoid proteins

The 20 kDa polypeptide, the apoprotein of the chlorophyll a/b antenna complex CP24 associated with photosystem II, is a remote relative of light-harvesting complex (LHC) apoproteins and thus a member of the extended cab gene family. LHC apoproteins are poly-topic integral components of the thylakoid membrane with probably three transmembrane segments which originate in nuclear genes and are made in the cytosol as precursors. They possess exclusively stroma-targeting transit peptides for import into the organelle and integrate into the thylakoid membrane via uncleaved hydrophobic domains of the mature protein. The CP24 apoprotein displays intriguing structural differences to LHC apoproteins with a potential impact on the routing and targeting processes during biogenesis. In particular, it lacks a pronounced second hydrophobic segment in the mature polypeptide chain found in LHCPs, and carries a transit peptide that is reminiscent of thylakoid-targeting transit peptides. We have used in organello assays with isolated intact chloroplasts and the authentic precursor of the 20 kDa apoprotein from spinach, or appropriate chimaeric polypeptides consisting of a transit peptide and the mature part of various nuclear-encoded thylakoid proteins of known location and targeting epitopes, in order to resolve the characteristics of its targeting properties, as well as to determine the contribution of the individual parts of the precursor molecule to its import and subsequent intra-organellar routing. Our experiments demonstrate that the transit peptide of the CP24 apoprotein is required only for the import of the protein into the organelle. All subsequent steps, such as the integration of the protein into the thylakoid membrane, binding of chlorophyll, assembly into the CP24 complex and migration to the grana lamellae, still take place if the authentic transit peptide is replaced by a targeting signal of a nuclear-encoded stromal protein.     

Loss of a single chlorophyll in CP29 triggers re-organization of the Photosystem II supramolecular assembly

In land plants, both efficient light capture and photoprotective dissipation of chlorophyll excited states in excess require proper assembly of Photosystem II supercomplexes PSII-LHCs. These include a dimeric core moiety and a peripheral antenna system made of trimeric LHCII proteins connected to the core through monomeric LHC subunits. Regulation of light harvesting involves re-organization of the PSII supercomplex, including dissociation of its LHCII-CP24-CP29 domain under excess light. The Chl a603-a609-a616 chromophore cluster within CP29 was recently identified as responsible for the fast component of Non-Photochemical Quenching of chlorophyll fluorescence. Here, we pinpointed a chlorophyll-protein domain of CP29 involved in the macro-organization of PSII-LHCs. By complementing an Arabidopsis knock-out mutant with CP29 sequences deleted in the residue binding chlorophyll b614/b3-binding, we found that the site is promiscuous for chlorophyll a and b. By plotting NPQ amplitude vs. CP29 content we observed that quenching activity was significantly reduced in mutants compared to the wild type. Analysis of pigment-binding supercomplexes showed that the missing Chl did hamper the assembly of PSII-LHCs supercomplexes, while observation by electron microscopy of grana membranes highlighted the PSII particles were organized in two-dimensional arrays in mutant grana partitions. As an effect of such array formation electron transport rate between Q_A and Q_B reduced, likely due to restricted plastoquinone diffusion. We conclude that chlorophyll b614, rather being part of pigment cluster responsible for quenching, is needed to maintain full rate of electron flow in the thylakoids by controlling protein-protein interactions between PSII units in grana partitions.     

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a-binding protein of photosystem II (psbB) and cytochrome b-563 (petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene (psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N-terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N-terminal region of the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII).     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Novel aspects of chlorophyll a/b-binding proteins

The light-harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light-harvesting function for photosystem II while the early light-induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 Å resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non-photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing conformational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light-harvesting function and are thus important in environmental stress resistance.     

Hierarchical Response of Light Harvesting Chlorophyll-Proteins in a Light-Sensitive Chlorophyll b-Deficient Mutant of Maize

The light-sensitive chlorophyll b (Chl b)-deficient oil yellow-yellow green (OY-YG) mutant of maize (Zea mays) grown under conditions of high light exhibits differential reductions in the accumulation of the three major Chl b-containing antenna complexes and characteristic changes in thylakoid architecture. When observed by freeze-fracture electron microscopy, the most notable changes in the OY-YG thylakoid structure are: (a) a major reduction in the number of 8 nanometer particles of the protoplasmic fracture face of stacked membrane regions (PFs) paralleled by a 60% reduction in the chlorophyll-proteins (CP) associated with the peripheral light harvesting complex (LHCII) for photosystem II (PSII) and which give rise to the LHCII oligomer/monomer (CPII^*/CPII) bands on mildly dissociated green gels; (b) a sizable decrease in the proportion of 11 to 13 nanometer particles of the protoplasmic fracture face of unstacked membrane regions (PFu) that parallels the loss of light harvesting complex I (LHCI) antennae from photosystem I (PSI) centers and a 40% reduction of the band containing CP1 and LHCI (CPI^*) on mildly dissociating green gels; (c) an unchanged or slightly increased average size of particles of the exoplasmic fracture face of stacked (or appressed) membrane regions (EFs) along with a relative increase in CP29, the postulated bound LHC of PSII, and of CP47 and CP43, PSII core antenna complexes. This latter result sets the OY-YG mutant apart from all other Chl b-deficient mutants studied to date, all of which possess EFs particles that are substantially reduced in size. Based on these findings, we postulate that the bound LHCII associated with EFs particles consists mostly of CP29 chlorophyll proteins and very little, if any, CPII^*/CPII chlorophyll proteins. Indeed, the CPII^*/CPII chlorophyll proteins may be exclusively associated with the `peripheral' LHCII units that give rise to 8 nanometer PF particles. The differential effect of the Chl b deficiency on the accumulation of the three main antenna complexes (CPII^*/CPII>CPI^*>CP29) suggests, furthermore, that there is a hierarchy among Chl b-binding proteins, and that this hierarchy might be an integral part of long-term photoregulation mediating Chl b partitioning in the chloroplast.     

Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach

Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids.     

The steady-state mRNA levels for thylakoid proteins exhibit coordinate diurnal regulation

Steady-state mRNA levels for thylakoid proteins were analysed in spinach cotyledons under diurnally changing light conditions. Most fluctuate considerably throughout the day, while the levels of others show only low amplitude or no oscillation. Levels of mRNAs coding for proteins that belong to the same multiprotein complex generally oscillate in parallel and exhibit maxima that are specific for that complex: mRNAs for photosystem I proteins appear prior to those for photosystem II polypeptides and these again prior to mRNAs for the three polypeptides constituting the oxygen-evolving complex. For the mRNAs that change with high amplitudes (e.g. those for LHCP or the 20 kDa apoprotein of the CP24 complex) oscillations have also been found under constant conditions, indicating that a circadian oscillator is involved. Transgenic tobacco seedlings harbouring chimeric GUS gene fusions with 5-flanking sequences from the spinach genes Lhcb, PsaF and AtpD (encoding a light-harvesting chlorophyll a/b apoprotein of photosystem II, subunit 3 of photosystem I and subunit of the plastid ATP synthase, respectively) confirm that the differences in the amplitudes as well as the timepoints of maximum mRNA accumulation are perceived via cis-regulatory elements upstream of the respective ATG codons.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.     

Chlorophyll-protein complex composition during chloroplast development: A species comparison

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - LHC the major light-harvesting complex associated with photosystem II - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.     

PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.     

The 28 kDa apoprotein of CP 26 in PS II binds copper

Photosystem II (PS II) particles isolated from spinach in the presence of 10 M CuSO₄ contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO₄ was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1–1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.Abbreviations CP 26 and CP 29 chlorophyll a/b protein complex 26 and 29 - LHC II light-harvesting chlorophyll a/b protein complex of Photosystem II - SB14 sulfobetaine 14 A preliminary report of these results was presented at the IX Int. Congress on Photosynthesis, Nagoya, Japan, 1992.     

Characterization of Chloroplasts Isolated from Triazine-Susceptible and Triazine-Resistant Biotypes of Brassica campestris L

Chloroplasts isolated from triazine-susceptible and triazine-resistant biotypes of Brassica campestris L. were analyzed for lipid composition, ultrastructure, and relative quantum requirements of photosynthesis. In general, phospholipids, but not glycolipids in chloroplasts from the triazine-resistant biotype had a higher linolenic acid concentration and lower levels of oleic and linoleic fatty acids, than chloroplasts from triazine-susceptible plants. Chloroplasts from the triazine-resistant biotype had a 1.6-fold higher concentration of t-Δ3-hexadecenoic acid with a concomitantly lower palmitic acid concentration in phosphatidylglycerol. Phosphatidylglycerol previously has been hypothesized to be a boundary lipid for photosystem II. Chloroplasts from the triazine-resistant biotype had a lower chlorophyll a/b ratio and exhibited increased grana stacking. Light-saturation curves revealed that the relative quantum requirement for whole chain electron transport at limiting light intensities was lower for the susceptible biotype than for the triazine-resistant biotype. Although the level of the chlorophyll a/b light-harvesting complex associated with photosystem II was greater in resistant biotypes, the increased levels of the light-harvesting complex did not increase the photosynthetic efficiency enough to overcome the rate limitation that is inherited concomitantly with the modification of the Striazine binding site.     

Minor antenna proteins CP24 and CP26 affect the interactions between photosystem II subunits and the electron transport rate in grana membranes of Arabidopsis

We investigated the function of chlorophyll a/b binding antenna proteins Chlorophyll Protein 26 (CP26) and CP24 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout lines that completely lacked one or both of these proteins. All three mutant lines had a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. Photosynthesis was affected in plants lacking CP24 but not in plants lacking CP26: the former mutant had decreased electron transport rates, a lower DeltapH gradient across the grana membranes, reduced capacity for nonphotochemical quenching, and limited growth. Furthermore, the PSII particles of these plants were organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, overall electron transport, nonphotochemical quenching, and growth of the double mutant were restored to wild type. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that limitation in electron transport was due to restricted electron transport between Q(A) and Q(B), which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.