Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors

The nuclear-encoded Chl a/b and Chl a/c antenna proteins of photosynthetic eukaryotes are part of an extended family of proteins that also includes the early light-induced proteins (ELIPs) and the 22 kDa intrinsic protein of PS II (encoded by psbS gene). All members of this family have three transmembrane helices except for the psbS protein, which has four. The amino acid sequences of these proteins are compared and related to the three-dimensional structure of pea LHC II Type I (Kühlbrandt and Wang, Nature 350: 130–134, 1991). The similarity of psbS to the three-helix members of the family suggests that the latter arose from a four-helix ancestor that lost its C-terminal helix by deletion. Strong internal similarity between the two halves of the psbS protein suggests that it in turn arose as the result of the duplication of a gene encoding a two-helix protein. Since psbS is reported to be present in at least one cyanobacterium, the ancestral four-helix protein may have been present prior to the endosymbiotic event or events that gave rise to the photosynthetic eukaryotes. The Chl a/b and Chl a/c antenna proteins, and the immunologically-related proteins in the rhodophytes may have had a common ancestor which was present in the early photosynthetic eukaryotes, and predated their division into rhodophyte, chromophyte and chlorophyte lineages. The LHC I-LHC II divergence probably occurred before the separation of higher plants from chlorophyte algae and euglenophytes, and the different Types of LHC I and LHC II proteins arose prior to the separation of angiosperms and gymnosperms.Abbreviations CAB Chl a/b-binding - ELIP early light-induced protein - FCP fucoxanthin-Chl a/c protein - PCR polymerase chain reaction - TMH trans-membrane helix     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

Identification of STN7/STN8 kinase targets reveals connections between electron transport,metabolism and gene expression

The thylakoid‐associated kinases STN7 and STN8 are involved in short‐ and long‐term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA‐binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin‐NADP reductase as targets for the thylakoid‐associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7‐dependent phosphorylation site are similar to those of phosphorylated light‐harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the ?1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long‐term adaptation processes affecting metabolite accumulation and gene expression.     

More nitrogen partition in structural proteins and decreased photosynthetic nitrogen-use efficiency of <Emphasis Type="Italic">Pinus massoniana</Emphasis> under in situ polluted stress

Masson pine (Pinus massoniana L.) trees in the Pearl River Delta have shown growth decline since late 1980s, particularly those around industrially polluted regions. As nitrogen is an important nutritional element composing functional proteins, structural proteins and photosynthetic machinery, investigation on nitrogen allocation is helpful to understand nutrient alteration and its regulation mechanism in response to pollution stress. Current year (C) and 1-year old needles (C + 1) of five mature trees were sampled in industrially polluted site and unpolluted natural reserve for bioassay. Needles of declining trees had significantly higher leaf nitrogen per unit area (N_L) but lower photosynthetic capacity (P _max), which resulted in lower photosynthetic nitrogen use efficiency (PNUE) than those of healthy trees. Nitrogen fraction to the photosynthetic apparatus in the C and C + 1 needles at polluted site was 27 and 22%, significantly lower than the corresponding healthy needles (48 and 32%). The content of structural proteins was positively correlated with N_L in C and C + 1 needles. Moreover, the C and C + 1 needles of declining trees had about 1.8 times structural protein as those of healthy trees, suggesting that more nitrogen allocation to structural protein are needed for stronger structural defenses under polluted stress. Decreases in PNUE of declining pine trees could be partially explained by increases in structural protein nitrogen.     

Chemical modification by trinitrobenzenesulfonate of a lipid and proteins of intracytoplasmic membranes isolated from Chromatium vinosum and Azotobacter vinelandii

1. 1. The structure of intracytoplasmic membranes of a photosynthetic bacterium Chromatium vinosum and a nitrogen-fixing bacterium Azotobacter vinelandii was studied by chemical modification of amino groups of phospatidylethanolamine and proteins with trinitrobenzensulfonate.

2. 2. Almost all the constituents of intracytoplasmic membranes of C. vinosum were solubilized in a mixture of chloroform, methanol and trichloroacetic acid. One-third of proteins in the intracytoplasmic membranes of C. vinosum was found solubilized in a mixture of chloroform and methanol. By using a column chromatography with Sephadex LH-20 in organic solvents, the unmodified as well as the trinitrophenylated proteins and also the trinitrophenylated phosphatidylethanolamine were separated from the other colored substances.

3. 3. In the chemical modification of the intracytoplasmic membrane preparations, 30% of phosphatidylethanolamine and 15% of protein amino groups in C. vinosum and 45% of phosphatidylethanolamine and 20% of protein amino groups in A. vinelandii were estimated to be exposed to the aqueous phase. In the single-layered liposomes composed of phosphatidylethanolamine and phospatidylglycerol with a ratio of 2:1, 40% of phosphatidylethanolamine were estimated to be exposed to the aqueous phase.

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.     

Incorporation of Selenomethionine into Induced Intracytoplasmic Membrane Proteins of Rhodobacter species

Efficient multiple- or single-wavelength anomalous dispersion (MAD/SAD) techniques that use tunable X-ray sources at third-generation synchrotrons exploit the anomalous scattering of certain heavy atoms for determination of experimental phases. Development of methods for the in vivo substitution of methionine by selenomethionine (SeMet) has revolutionized the process for determination of structures of soluble proteins in recent years. Herein, we report methods for biosynthetic incorporation of SeMet into induced intracytoplasmic membrane proteins of two species of the Rhodobacter genus of purple non-sulfur photosynthetic bacteria. Amino acid analysis of a membrane protein complex that was purified to homogeneity determined that the extent of SeMet incorporation was extensive and approached quantitative replacement. Diffraction-quality crystals were obtained from SeMet-labeled membrane proteins purified from 2 l of culture. These methods augment the potential utility of photosynthetic bacteria and their inducible membrane systems for the production of foreign membrane proteins for structure determination.     

Thiobencarb Herbicide Reduces Growth,Photosynthetic Activity,and Amount of Rieske Iron‐Sulfur Protein in the Diatom Thalassiosira pseudonana

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis‐related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (F_v/F_m ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron‐sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b₆f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron‐sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:437‐444, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21505     

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.Abbreviations BSA bovine serum albumin - D1 32 kDa Q_B-binding protein - EDTA ethylenediaminetetraacetic acid - LHCII light-harvesting complex II - PMSF phenylmethylsulfonyl fluoride - PS photosystem - tricine N-[tris(hydroxymethyl)methyl] glycine     

Photosynthesis or persistence: nitrogen allocation in leaves of evergreen and deciduous Quercus species

Photosynthetic nitrogen use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) is one of the most important factors for the interspecific variation in photosynthetic capacity. PNUE was analysed in two evergreen and two deciduous species of the genus Quercus. PNUE was lower in evergreen than in deciduous species, which was primarily ascribed to a smaller fraction of nitrogen allocated to the photosynthetic apparatus in evergreen species. Leaf nitrogen was further analysed into proteins in the water‐soluble, the detergent‐soluble, and the detergent‐insoluble fractions. It was assumed that the detergent‐insoluble protein represented the cell wall proteins. The fraction of nitrogen allocated to the detergent‐insoluble protein was greater in evergreen than in deciduous leaves. Thus the smaller allocation of nitrogen to the photosynthetic apparatus in evergreen species was associated with the greater allocation to cell walls. Across species, the fraction of nitrogen in detergent‐insoluble proteins was positively correlated with leaf mass per area, whereas that in the photosynthetic proteins was negatively correlated. There may be a trade‐off in nitrogen partitioning between components pertaining to productivity (photosynthetic proteins) and those pertaining to persistence (structural proteins). This trade‐off may result in the convergence of leaf traits, where species with a longer leaf life‐span have a greater leaf mass per area, lower photosynthetic capacity, and lower PNUE regardless of life form, phyllogeny, and biome.     

Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae)

The effects of nitrogen starvation on biomass composition and photosynthetic function were examined in the marine cryptophyte Rhodomonas sp. Batch-cultured cells in N-sufficient medium showed a 2.5-fold increase in total carbohydrate content, and a 33% increase in cell volume when the cultures reached the stationary growth phase. These cultures also increased the ratio of phycoerythrin (PE)/hydrosoluble proteins from 6 to 22% by the 4th and 10th day of culture, respectively. In contrast, light-saturated photosynthetic activity (P_m) progressively decreased, and the value obtained at the beginning of the stationary phase was about 45% of that obtained for cells in the late exponential growth phase. Transfer to N-lacking medium caused a 3.2-fold increase in cell volume. N starvation also triggered a rapid decline in N-containing compounds such as hydrosoluble proteins and photosynthetic pigments, causing an almost complete loss of PE. The ratio of PE/hydrosoluble proteins decreased from 6 to 1% after 6 d of N deprivation. Furthermore, the PSII fluorescence capacity declined under N-starved conditions, which caused a pronounced decrease in both the P_m (circa 90%) and the apparent photosynthetic efficiency (circa 55%). Under these conditions, photosynthetically fixed carbon was used to synthesize large amounts of carbohydrates. We suggest that, in addition to the role of phycoerythrin as a light-harvesting pigment, Rhodomonas sp. responds to N-depleted conditions by mobilizing combined nitrogen from biliproteins.     

Proteins and Metabolites Regulated by Trinexapac-ethyl in Relation to Drought Tolerance in Kentucky Bluegrass

Plants have developed various mechanisms in adaptation to water deficit stress, including growth retardant to reduce water loss. Previous studies reported that plants treated with a growth inhibitor, trinexapac-ethyl (TE), had improved drought tolerance. The objective of this study was to determine alterations in proteins and metabolite accumulation associated with drought tolerance improvement in a perennial grass species, Kentucky bluegrass (Poa pratensis), induced by TE application. Plants were treated with TE [1.95 ml l⁻¹ (v:v); a.i. TE = 0.113%] through foliar spray for 14 days, and then subjected to drought stress by withholding irrigation for 15 days in growth chambers. TE-treated plants exhibited significantly higher relative water content and photosynthetic capacity and lower membrane leakage than nontreated plants under drought stress, suggesting TE-enhanced drought tolerance in Kentucky bluegrass. Physiological improvement in drought tolerance through TE application was associated with the increased accumulation of various proteins and metabolites, including ferritin, catalase, glutathione-S-transferase, Rubisco, heat shock protein 70, and chaperonin 81, as well as fatty acids (palmitic acid, α-linolenic acid, linoleic acid, and octadecanoic acid). Our results suggest that TE may regulate metabolic processes for antioxidant defense, protective protein synthesis, photorespiration, and fatty acid synthesis, and thereby contribute to better drought tolerance in Kentucky bluegrass.     

Isolation and Characterization of Three Porin-Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl-BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl-BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl-nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin-like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin-like proteins showed that only the antiserum against 37K protein cross-reacted with the outer membrane proteins from all the strains tested.     

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui Comparison of the ribosomal protein S11 family

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45–49%) than to the eubacterial counterparts (35%)     

Purification and composition of a thermal hysteresis producing protein from the milkweed bug,Oncopeltus fasciatus

Summary A protein which produces a thermal hysteresis (a difference between the freezing and melting points) was purified from the hemolymph of the milkweed bug,Oncopeltus fasciatus. The amino acid composition of theOncopeltus thermal hysteresis protein is somewhat different from that of the larvae of the beetle,Tenebrio molitor, which is the only other insect from which such a protein has as yet been purified. The major difference between the two is the large amount of serine (30.5% of the amino acid residues) and glycine (20.0%) present in theO. fasciatus protein. Both insect proteins have a composition which consists of approximately 60% polar amino acids and lacks large amounts of alanine. In these respects they are quite different from the fish protein antifreezes. The apparent differences in structure of the thermal hysteresis proteins and glycoproteins indicates that these proteins have evolved independently and therefore offer an interesting example of convergent evolution.     

A physiological comparison of coccolith-forming and naked cells of Coccolithus huxleyi

Summary In a naked clone of the coccolithophorid Coccolithus huxleyi, growth at light saturation was 15% slower than in a coccolith-forming clone isolated from the same parent stock. The two cell types did not differ significantly with regard to cell volume and protein content. A 10–13% smaller chlorophyll a content of the naked cells was matched by a uniform lowering of photosynthetic rates at all light intensities. It was considered that the slower growth of these cells might result from a less favourable ratio between photosynthetic output and biomass.The content of deoxyribonucleic acid was the same in coccolith-forming and naked cells, suggesting that they do not represent different phases in a sexual lifecycle.     

Characterization of a T-DNA insertion mutant for the protein import receptor atToc33 from chloroplasts

In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.Communicated by R. Hagemann     

Mulberry Leaf Metabolism under High Temperature Stress

Effects of high temperature on the activity of photosynthetic enzymes and leaf proteins were studied in mulberry (Morus alba L. cv. BC2-59). A series of experiments were conducted at regular intervals (120, 240 and 360 min) to characterize changes in activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) and sucrose phosphate synthase (SPS), photosystem 2 (PS 2) activity, chlorophyll (Chl), carotenoid (Car), starch, sucrose (Suc), amino acid, free proline, protein and nucleic acid contents in leaves under high temperature (40 °C) treatments. High temperature markedly reduced the activities of RuBPC and SPS in leaf extracts. Chl content and PS 2 activity in isolated chloroplasts were also affected by high temperature, particularly over 360 min treatment. Increased leaf temperature affected sugar metabolism through reductions in leaf starch content and sucrose-starch balance. While total soluble protein content decreased under heat, total amino acid content increased. Proline accumulation (1.5-fold) was noticed in high temperature-stressed leaves. A reduction in the contents of foliar nitrogen and nucleic acids (DNA and RNA) was also noticed. SDS-PAGE protein profile showed few additional proteins (68 and 85 kDa) in mulberry plants under heat stress compared to control plants. Our results clearly suggest that mulberry plants are very sensitive to high temperature with particular reference to the photosynthetic carbon metabolism.     

Perfusion chromatography—a new procedure for very rapid isolation of integral photosynthetic membrane proteins

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.Abbreviations Chl chlorophyll - LDAO N,N dimethyldodecylamine-N-oxide - LHC light-harvesting complex - PS photosystem - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis