Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ') extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.     

Metal metabolism in the red alga Cyanidium caldarium and its relationship to metal tolerance

The unicellular red alga Cyanidium caldarium is tolerant to high levels of various metal ions. Cells of this alga cultured with divalent metal ions at 5 mM contained an elevated concentration of each metal, with the highest level for Zn followed by Mn > Ni > Cu. This order is in fair agreement with the toxicity levels reported previously, with the exception of Mn, which shows a toxicity level comparable to that of Ni. Transmission electron microscopy indicated the presence of electron-dense bodies in the algal cells, and elemental analysis by energy dispersive X-ray spectrometry showed high levels of Fe and P in these bodies. Accumulation of Zn was found in these particles in Zn-treated algal cells, whereas no such deposition was found for Cu, Ni, or Mn in cells treated with the respective metals. Although trapping of Zn in the intracellular bodies may contribute to reduction of metal activity in the cells, this effect can be overcome by high intracellular levels of Zn that result in a high degree of toxicity. The correlation between intracellular concentration and toxic levels of metal ions implies that the reduced incorporation of the metals is a major detoxification mechanism in this alga.     

Structure of the Rubisco operon from the unicellular red alga Cyanidium caldarium: evidence for a polyphyletic origin of the plastids

Summary The genes for both subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) were located on the plastid DNA (ptDNA) of the unicellular red algaCyanidium caldarium. Both genes are organized together in an operon. The sequence homology of both genes to the corresponding genes from the unicellular red algaPorphyridium aerugineum is remarkably high, whereas homology to Rubisco genes from chloroplasts and two recent cyanobacteria is significantly lower. These data provide strong evidence for a polyphyletic origin of chloroplasts and rhodoplasts. In addition the genes for the small subunit of Rubisco (rbcS) from red algae show about 60% homology torbcS genes from cryptophytes and chromophytes. Thus, homologies in therbcS gene indicate a close phylogenetic relationship between rhodoplasts and the plastids of Chromophyta.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Alteration of thylakoid membrane structure in Brassica rapa ssp. oleifera during ageing in high and low light

Abstract Alterations in the composition and structure of thylakoids were studied in Brassica rapa ssp. oleifera grown under high and low irradiance (800 μmol m^?2 s^?1 and 80 μmol m^?2 s^?1). During ageing, both high and low light induced a decrease in total protein particle density and in the relative amount of 80–90 Å cytochrome b6/f and 90–100 Å ATP-synthetase. The density of PSII complexes in stacked (EFs) and unstacked (EFu) thylakoids also decreased. In high light, a shift was noted towards smaller PSII complexes in the EFs face with decreasing attached antenna complex CP29, but the relative amount of the antenna chlorophyll a-protein complexes of photosystem II (CPa) remained stable. In contrast, the proportion of peripheral LHCH on the PFs face and the density of PFs particles increased together with an increase in grana size. In low light, a shift occurred towards larger PSII complexes on the EFs face, along with a decrease in the proportion of CPa complexes and the PFs particle density (peripheral LHCH), though a marked increase was observed in the proportion of chlorophyll a/b-protein complexes in SDS-PAGE. The amount of photosystem I in green gel remained fairly stable, although the density of PFu particles (including PSI) increased in low and slightly diminished in high light. The results indicate that the organization of thylakoid components depends strongly on the light conditions and stage of development.     

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA^Ile (GAU) and tRNA^Ala (UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by small-sized rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.     

Effect of L-methionine-DL-sulphoximine, a specific inhibitor of glutamine synthetase, on ammonium and nitrate metabolism in the unicellular alga Cyanidium caldarium

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L-methionine-DL-sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell.     

Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: An overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Photosystem I: its biogenesis and function in higher plants

Photosystem I (PSI), the plastocyanin-ferredoxin oxidoreductase of the photosynthetic electron transport chain, is one of the largest bioenergetic complexes known. It is composed of subunits encoded in both the chloroplast genome and the nuclear genome and thus, its assembly requires an intricate coordination of gene expression and intensive communication between the two compartments. In this review, we first briefly describe PSI structure and then focus on recent findings on the role of the two small chloroplast genome-encoded subunits PsaI and PsaJ in the stability and function of PSI in higher plants. We then address the sequence of PSI biogenesis, discuss the role of auxiliary proteins involved in cofactor insertion into the PSI apoproteins and in the establishment of protein-protein interactions during subunit assembly. Finally, we consider potential limiting steps of PSI biogenesis, and how they may contribute to the control of PSI accumulation.     

Growth, photosynthesis, respiration, and intracellular free amino acid profiles in the unicellular alga Cyanidium caldarium. Effect of nutrient limitation and resupply

Growth, intrácellular free amino acid pools and photosynthetic and respiratory activities in nutrient sufficient cells and in N- K- and P-limited cells of Cyanidium caldarium (Tilden) Geitler, and responses to nutrient resupply were investigated. Addition of ammonium to N-limited cells and of phosphate to P-limited cells resulted in a stimulation of dark respiration and in a decrease in photosynthetic oxygen evolution. Addition of K to K-limited cells had no effect on rates of photosynthesis and respiration. Nutrient limited cells and sufficient cells exhibited different free amino acid profiles. Upon resupply of ammonium to N-limited cells levels of glutamine, citrulline, arginine, alanine, and serine increased. Also the levels of δ-aminolevulinic acid (δ-ALA) and putrescine increased notably. On adding phosphate to P-limited cells the level of glutamate decreased significantly whereas the level of alanine increased and the concentrations of other amino acids remained unaffected. On adding potassium to K-limited cells there was an increase in glutamate and citrulline concentrations, and a decrease in putrescine concentration, whereas concentrations of arginine and alanine remained at the very high levels observed already before addition. Resuspension of N- and K-limited cells in a complete growth only after 25-30h. In P-limited cells resumption of growth in complete medium occurred progressively and reached the maximum rate 30h later. P-, K- and N- limited cells resuspended into sufficient media showed different rates of ammonium and phosphate assimilation. The pattern of recovery from nutrient limitation is discussed according to the cellular role fulfilled by the nutrient which was growth rate-limiting.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Arrhenius plots and the involvement of thermotropic phase transitions of the thylakoid membrane in chilling impairment of photosynthesis in thermophilic higher plants

Abstract Breaks and discontinuities in Arrhenius plots of physiological and physical properties of thylakoids are not diagnostic of thermotropic lipid phase transitions of the membrane. Bulk lipid transitions, as first inferred by the membrane phase transition hypothesis, do not occur in any higher plant at chilling temperatures. Solidification of some varying, but always minor, fraction of the total membrane lipid does take place. However, the presence of minor domains of solid thylakoid membrane lipid at chilling temperatures is not unique to chilling sensitive plants but is also found in tolerant species. Minor solidification may in some plants, or groups of plants, be controlled by the specific molecular species of phosphatidylglycerol only recently investigated. In plants containing little, or no, phosphatidylglycerol with this positional distribution of fatty acids, other yet unknown constituents of the membrane must fill a similar function, since DSC thermograms indicate minor solidification also in isolated, unperturbed thylakoids from chilling tolerant species. However, chilling induced phase transitions, or other perturbations, of the thylakoid membrane are not the reason for the chilling lability of net photosynthesis in the intact plant. This conclusion follows from detailed comparison between photosynthetic membranes isolated from prechilled plants and the effects of chilling exposure on CO₂ fixation of the whole plant. Damage at the level of the thylakoid membrane does occur, although not to the extent where it can account for the proportionally much larger damage to CO₂ fixation.     

Degreening of the unicellular alga Euglena gracilis: thylakoid composition, room temperature fluorescence spectra and chloroplast morphology

Thylakoid dismantling is one of the most relevant processes occurring when chloroplasts are converted to non-photosynthetically active plastids. The process is well characterised in senescing leaves, but other systems could present different features. In this study, thylakoid dismantling has been analysed in dividing cells of the unicellular alga, Euglena gracilis , cultured in darkness. Changes in photosynthetic pigments and in the abundance of LHC and PSII core proteins (D2 and CP43) showed that: (i) during the 0–24 h interval, the decline in LHCII was faster than that in the PSII core; (ii) during the 24–48 h interval, PSII and LHCII were strongly degraded to nearly the same extent; (iii) in the 48–72 h interval, the PSII core proteins declined markedly, while LHCII was maintained. These changes were accompanied by variations in room temperature fluorescence emission spectra recorded from single living cells with a microspectrofluorimeter (excitation, 436 nm; range 620–780 nm). Emission in the 700–715 nm range was proposed to derive from LHCI-II assemblages; changes in emission at 678 nm relative to PSII matched PSII core degradation phases. Overall, the results suggest that, in degreening E. gracilis , thylakoid dismantling is somewhat different from that associated with senescence, because of the early loss of LHCII. Moreover, it is proposed that, in this alga, disruption of the correct LHCI-II stoichiometry alters the energy transfer to photosystems and destabilises membrane appression leading to the thylakoid destacking observed using transmission electron microscopy.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Characterization of chloroplast thylakoid polypeptides in the 32-kDa region: polypeptide extraction and protein phosphorylation affect binding of photosystem II-directed herbicides

In order to distinguish between two photosystem II proteins with apparent molecular weights of about 32 kDa, mild extraction procedures were used to remove several thylakoid membrane components. A 32-kDa protein that stained intensely with Coomassie brilliant blue could be extracted from the thylakoid membranes without removing the 32-kDa herbicide receptor protein, which stained poorly with Coomassie brilliant blue. The nonextracted protein was readily detectable after in vivo polypeptide labeling with [35S]methionine or after in vitro covalent tagging with [14C]azidoatrazine. The procedures used to extract the intensely stained, 32-kDa polypeptide resulted in changes in herbicide-binding characteristics, presumably due to conformational changes in the herbicide-binding environment. Alterations of membrane surface charge by protein phosphorylation also influenced herbicide binding.     

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus

Summary Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli SecA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secAY may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.