Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba

Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophoretic fractions. On the basis of these data the in vivo ratios of xanthophylls per chlorophyll a could be estimated. The results show that the light-harvesting complexes from Chlorella and from Sinapis exhibit identical ratios of total xanthophylls per chlorophyll a. By contrast, in the prasinophycean alga Mantoniella, the light-harvesting complex markedly differs from the other chlorophyll b containing proteins. It contains, in addition to neoxanthin and violaxanthin, high amounts of prasinoxanthin and its epoxide, which contribute significantly to light absorption. The concentration of chlorophyll b in the complex is very much higher in the antenna of Mantoniella than in those of Chlorella and Sinapis. Furthermore, it must be emphasized that in addition to chlorophyll b, a third chlorophyll species acts in the energy transfer to chlorophyll a. This chlorophyll c-like pigment is found to be present in a concentration which improves very efficiently the absorption in blue light. In light of these results it can be concluded that the absorption cross section in Mantoniella is higher not only because of an enhanced number of light-harvesting particles in the membrane, but also because of a higher ratio of accessory pigments to chlorophyll a.Abbreviations Chl Chlorophyll - FP Free Pigments - HPLC High Performance Liquid Chromatography - LHC Light-harvesting Chlorophyll protein complex - PAGE Polyacrylamide Gel Electrophoresis - PS Photosystem     

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

We have adapted the procedure for the isolation of PSII membranes from higher plants (D.A. Berthold et al., 1981, FEBS Lett. 134, 231–234) to the green algae Chlamydomonas reinhardtii. The chlorophyll (Chl)-binding proteins from this PSII preparation have been further separated into single Chl-binding polypeptides and characterized spectroscopically. Seven single polypeptides were shown to bind Chl a and Chl b. In particular, we demonstrate that polypeptides p9, p10 and p22, which had not been previously shown to bind Chl a and b, have characteristics similar to those of CP29, CP26 and CP24 from higher plants. We note, however, that p9 and p10 are phosphorylatable in C. reinhardtii, at variance with CP29 and CP26 from higher plants. Our data support the notion that the PSII antenna systems in C. reinhardtii and in higher plants are very similar. Therefore, studies on the organization and regulation of light-harvesting processes in C. reinhardtii may provide information of general relevance for both green algae and higher plants.Abbreviations Chl chlorophyll - IEF isoelectrofocusing - LHC light harvesting complex - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PS photosystem - RC reaction centre - SDS sodium dodecylsulfate We thank Dr. J. Olive (Institut Jacques Monod, Paris, France) for the electron-microscopy analysis, C. de Vitry (Institut de Biologie Physico-Chimique, Paris, France) for the kind gift of a PSII RC preparation and P. Dainese and M.L. Di Paolo (Universitá di Padova, Padova, Italy) for helpfull discussions. Professor Strasser and Elizbeth Scwartz (Université de Genova, Genova, Switzerland) are thanked for assistance in taking low-temperature fluorescence emission spectra. Roberto Bassi was recipient of a short-term fellowship from the European Molecular Biology Organization fellowship, during the early phases of the work.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Characterization of the thylakoid membranes of the tobacco aurea mutant Su/su and of three green revertant plants

Phenotypic characterizations of the semidominant aurea tobacco (Nicotiana tabacum L.) mutant Su/su, the homozygous mutant Su/Su and three green revertants (R1, R2, and R3) are presented. The leaf color of Su/su plants varies from yellow to light-green when grown under high and low energy fluence rates (33.0 and 3.3 W m^–2), respectively. The change in visual phenotype under high-light conditions is correlated with decreased content of chlorophyll per leaf area, agranal chloroplast ultrastructure, changes in the number of chlorophyll-protein complexes, and absence of two or more of the light harvesting chlorophyll-polypeptides of 25,000–29,000 dalton. The homozygous mutant grown under low light was shown to be completely lacking in grana stacks and to be deficient in chlorophyll-protein complexes. Revertant R1 was found to be identical to wild-type plants in all parameters examined (leaf color, chloroplast ultrastructure, chlorophyll-protein complexes, chlorophyll-protein complex polypeptides) except in chlorophyll content. It did not show an increased chlorophyll and carotenoid content as did the wild-type plants when exposed to high light. Revertants R2 and R3 were similar to the heterozygous mutant Su/su in most of the parameters examined. They yellowed because of a loss of chlorophyll and an increase in the amount of carotenoids, had agranal chloroplasts, and had variant chlorophyll-protein complexes when grown under high light intensities. However, each appeared to contain some of the light-harvesting pigment-protein complex polypeptides found to be absent in Su/su when grown under high-light conditions.Abbreviations HL high light - LL low light - SDS sodium dodecyl sulfate This paper is part of a Ph.D. thesis by P.J.K. in the Program in Genetics, Michigan State University     

On the location of the carotenoids in the light-harvesting pigment-protein complexes of the photosynthetic bacterium Rhodopseudomonas capsulata

Measurements of pronase-induced shifts of the absorption spectrum and of the isobestic point of the light-induced difference spectrum of the carotenoids show that the pool responsible for the light-induced absorption changes in Rhodopseudomonas capsulata wild type is more sensitive to pronase treatment than the bulk carotenoids. The most likely explanation for this, in the context of the work of Kakitani et al. (Kakitani, T., Honig, B. and Crofts, A.R. (1982) Biophys. J. 39, 57–63), is that the field indicating carotenoids, or at least that part of the molecules which determines their spectral characteristics, are imbedded in the LHC II pigment-protein complexes, close to the membrane surface. The importance of the location of the carotenoids for the measurement of the electrical potential differences is briefly discussed.     

The light-harvesting system of Euglena gracilis during the cell cycle

The apoproteins of the light-harvesting chlorophyll-protein complexes LHCI and CP29 (apparent molecular weights of 27 kDa and 29 kDa, respectively) of Euglena gracilis were identified immunologically. Both complexes are present in the thylakoids of autotrophically cultured Euglena cells during the whole cell cycle. The relative amount of each apoprotein tends to increase towards the end of the cell cycle. The light-harvesting chlorophyll-protein complex of photosystem II, LHCII, of E. gracilis contains chlorophyll a, chlorophyll b, neoxanthin, diadinoxanthin and beta-carotene. Its chlorophyll a/b ratio is about 1.7 during the whole cell cycle. About 9 h after cell division the ratio of diadinoxanthin to chlorophyll a is doubled for a time of 3–4 h. The relevance of this increase during one developmental stage is discussed in relation to the insertion and-or assembly of newly synthesized LHCII.Abbreviations LHCP light-harvesting chlorophyll-protein complex - PS photosystem This research was partly supported by the Deutsche Forschungsge meinschaft.     

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria

Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.     

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Summary Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c ₂ light-harvesting complex, phycoerythrin and the -subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c ₂ LHC in both appressed and unappressed thylakoid membranes.Abbreviations PE phycoerythrin - chl chlorophyll - LHC light-har-vesting complex     

Orientation of the pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. III. A quantitative comparison of the low-temperature linear dichroism spectra of thylakoids and isolated pigment-protein complexes

The low-temperature linear dichroism spectrum of thylakoids oriented in polyacrylamide gel can be adequately described by a linear combination of the corresponding spectra of particles of light-harvesting complex, Photosystem I and Photosystem II, isolated by Triton X-100 extraction. The main conclusions which can be derived from this observation are: (1) The in vivo orientation of the pigments within each of the three complexes is not significantly affected by the extraction and purification procedures. (2) The various photosynthetic pigments are oriented roughly to the same extent in each of the three main biochemical constituents of the thylakoid. (3) All the complexes investigated behave like ellipsoids, the largest dimensions of which are lying in the plane of the photosynthetic membrane.     

Molecular assembly of Zn porphyrin complexes using synthetic light-harvesting model polypeptides

Synthetic single α-helix hydrophobic polypeptides, which have similar amino acid sequences to the hydrophobic core in the native light-harvesting 1-β polypeptide from Rhodobacter sphaeroides, formed Zn porphyrin complexes on a gold electrode, as well as in n-octyl-β-glucoside micelles: this process is dependent on the structure of the pigments and the polypeptides. Interestingly, an enhanced photoelectric current was observed when Zn mesoporphyrin monomer complexed with the synthetic light-harvesting model polypeptide in an α-helical configuration was assembled with a defined orientation onto the electrode. Analog of these light-harvesting model complexes are also useful in providing insights into the effect of polypeptide structure on the formation of light-harvesting complexes on and off electrodes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts

Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.     

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound.

The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex.

In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10–20% of the total Photosystem II reaction centre complex and the light-harvesting complex.

The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex.

The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.     

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 s for the B800-850 complex, 14±2 s for the B850 complex incorporated with spheroidene, and 19±2 s for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 s. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.     

The novel light-harvesting pigment-protein complex ofMantoniella squamata (Chlorophyta): Phylogenetic implications

Summary A light-harvesting pigment-protein complex has been isolated fromMantoniella squamata (Micromonadophyceae, Chlorophyta) by nondenaturing polyacrylamide-gel electrophoresis. The complex runs as two bands of molecular weights 54,000 and 55,000. There are two constituent polypeptides of molecular weights 20,500 and 22,000. Antibodies were raised to the 20,500-dalton polypeptides from this complex and to the 24,500-dalton polypeptide from the analogous complex ofPedinomonas minor (Micromonadophyceae). The antibodies to theM. squamata polypeptide are specific for both polypeptides of theM. squamata light-harvesting complex, as well as for a 27,000-dalton polypeptide of undetermined function. The antibodies to theP. minor polypeptide are specific for polypeptide components of the light-harvesting complex of that alga. The antibodies specific for theM. squamata light-harvesting complex polypeptides do not cross react with any polypeptides ofP. minor thylakoid membranes, as demonstrated by crossed immunoelectrophoresis. Similarly, no polypeptides ofM. squamata thylakoids cross react with the antibodies specific forP. minor light-harvesting complex polypeptides. These results indicate that the light-harvesting complex ofM. squamata is structurally very different from that ofP. minor. In a survey of several land plants and green algae, including representatives of all classes of green algae, a light-harvesting complex homologous to that ofM. squamata was found only inMicromonas pusilla. All other organisms tested possessed a lightharvesting complex homologous to that ofP. minor. The evolutionary and taxonomic implications of the novelM. squamata light-harvesting complex are discussed.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.