A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

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.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

Spectral properties of the CP43-deletion mutant of Synechocystis sp. PCC 6803

Spectral properties, particularly fluorescence spectra and their time-dependent behavior, were investigated for a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the 43 kDa chlorophyll-protein (CP43, PsbC). Lack of CP43 was confirmed by a size shift of the corresponding gene and by Western blotting. The CP43-deletion mutant grown under heterotrophic conditions accumulated a small amount of photosystem (PS) II, but virtually no PS II fluorescence was observed. A 686-nm fluorescence band was clearly observed by phycocyanin excitation, coming from the terminal pigments of phycobilisomes. In contrast, no PS I fluorescence was detected by phycocyanin excitation when accumulation of PS II components was not proved by a fluorescence excitation spectrum, indicating that energy transfer to PS I chlorophyll a was mediated by PS II chlorophyll a. Direct connection of phycobilisomes with PS I was not suggested. Based on these fluorescence properties, the energy flow in the CP43-deletion mutant cells is discussed.     

Isolation of an intrinsic antenna chlorophyll a-protein from the photosystem I reaction center complex of the thermophilic cyanobacterium Synechococcus sp

A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CP1-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CP1-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60-kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with 1-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll alpha molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77 degrees K. The temperature dependence of the far red band of CP60 was essentially the same as that of CP1-e between 77 and 273 degrees K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CP1-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60-kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Function of the polypeptides of the photosystem II reaction center in Chlamydomonas reinhardtii. Localization of the primary reactants

The distribution of the primary quinone and of the pheophytin acceptors has been studied in PS II particles isolated from Chlamydomonas reinhardtii, with respect to the distribution of the apoproteins of the two chlorophyll-protein complexes associated with the PS II core. We show that photoreduction of the primary quinone requires the presence of the 50 and 47 kDa polypeptides. On the contrary, charge separation between P-680 and the pheophytin acceptor molecules can occur within the chlorophyll-protein complex of which the 50 kDa polypeptide is the apoprotein. Functional analysis of the PS II fractions shows that an active PS II center contains one photoreducible quinone and one photoreducible pheophytin per 45 chlorophyll molecules. Stoichiometric analysis of the PS II fractions shows that a PS II reaction center contains 45 chlorophyll molecules associated with most likely one copy of the 50 kDa and the 47 kDa polypeptides.     

Lipid and pigment composition of a chlorophyll b-deficient mutant of Chlamydomonas reinhardii

Lipids and pigments of the chlorophyll b -deficient mutant pg-113 and the parent strain (ps) of Chlamydomonas were analysed and compared. Monogalactosyldiglyceride, digalactosyldiglyceride, diacylglyceryl(N, N, N-trimethyl)homoserine, sulfoquinovosyldiglyceride, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol were found as major lipid components. While the lipid patterns were qualitatively and quantitatively almost the same in the two strains, the C₁₆/C₁₈ fatty acid ratios were different, 0.85 in the mutant and 1.11 in the parent strain. Furthermore, the relative amounts of C₁₆- and C₁₈-monoene fatty acids were slightly enhanced and the C₁₈-trienes slightly reduced in the mutant. In the parent strain, chlorophylls a and b , α- and β-carotene, lutein, violaxanthin, neoxanthin and loroxanthin were detected by HPLC. In the mutant, similar pigments were found, except that only traces of chlorophyll b and a reduced amount of neoxanthin were present. Since no chlorophyll-protein complex CP II could be detected in the mutant by electrophoresis, the possible interrelationships between pigment deficiency and alteration of chlorophyllprotein complexes are discussed.     

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Biochemical characterization of a highly active O2-evolving Photosystem II preparation from maize

An O₂-evolving Photosystem (PS) II preparation was isolated from maize by a Triton X-100 procedure (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539). A highly active O₂-evolving preparation was obtained which evolved O₂ at 76% the rate of fresh chloroplasts (H₂O → 2,6-dichloro-p-benzoquinone) and was very sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. There was no detectable PS I activity in the preparation (2,3,5,6-tetramethyl-p-phenylenediamine → methyl viologen). When analyzed by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis the O₂-evolving preparation was shown to be highly depleted in CP I, CF₁, and devoid of cytochromes f and b-563 (the absence of which was confirmed by difference spectroscopy). The preparation was enriched in the PS II reaction center polypeptides I and II, the 34 kDa polypeptide (Metz, J., Wong, J. and Bishop, N.I. (1980) FEBS Lett. 114, 61–66), the Coomassie blue-stainable 32 kDa polypeptide (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys. Acta 581, 228–236), LHCP-associated polypeptides and cytochrome b-559. Polypeptides of unknown function at 40.5, 25, 24, 22, 16.6 and 14 kDa were also present in the O₂-evolving preparation. Triton X-114 phase partitioning (Bricker, T.M. and Sherman, L.A. (1982) FEBS Lett. 149, 197–202) indicated that the majority of these polypeptides were intrinsic. Only the polypeptides at 32, 25, 24 and 14 kDa were extrinsic. When examined by the octylglucoside procedure of Camm and Green (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) the PS II O₂-evolving preparation was shown to contain the chlorophyll-proteins CP 27, CP 29, CP II1, D, and CP a-1 and CP a-2. Chlorophyll-proteins associated with PS I were highly depleted. The visible absorption spectra indicated an enrichment of chlorophyll b and carotenoids in the preparation. The 77 K fluorescence emission spectrum (excitation wavelength = 435 nm) exhibits a strong F-686 with little F-695 shoulder and a broad, low-intensity F-735 emission.     

Structural-functional state of thylakoid membranes of wheat genotypes under water stress

Plants were grown in field conditions in the wide area under normal water supply and severe water deficit. Two wheat (Triticum aestivum L.) genotypes contrasting by architectonics and differing in drought-resistance were used: Giymatli-2/17, short stature, with broad and drooping leaves, drought-sensitive, and Azamatli-95, short stature, with vertically oriented small leaves, drought-tolerant). It was found out that Giymatli-2/17 was characterized by relatively low content of Chl a-protein of PS I (CP I) and β-subunit of ATP-synthase complex, the high content of proteins in the 33-30.5 kDa region and LHC polypeptides (28-24.5 kDa), the intensive fluorescence at 740 nm and more high photochemical activity of PS II under normal irrigation compared with Azamatli-95. However, the content of CP I (M_r 115 kDa) and apoprotein of P700 with M_r 63 kDa insignificantly increases in the drought-resistant genotype Azamatli-95 under extreme water supply condition while their content decreases in drought-sensitive cv Giymatli-2/17. Intensity of synthesis α- and β-subunits of CF₁ (55 and 53.5 kDa) also decreases in Giymatli-2/17. The levels of the core antenna polypeptides of FS II with M_r 46 and 44.5 kDa (CP47 and CP43) remains stable both in normal, and stressful conditions. At the same time the significant reduction is observed in the content of polypeptides in the 33-30.5 kDa region in the more sensitive genotype Giymatli-2/17. There is an increase in the LHC II polypeptides level in tolerant genotype Azamatli-95 in contrast to Giymatli-2/17 (where the content of these subunits is observed decreasing). The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77 K) of chloroplasts from sensitive genotype Giymatli-2/17 under water deficiency and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv Azamatli-95.