On the reliability of highly magnified micrographs for structural analysis in materials science.

Highly magnified micrographs are part of the majority of publications in materials science and related fields. They are often the basis for discussions and far-reaching conclusions on the nature of the specimen. In many cases, reviewers demand and researchers deliver only the bare minimum of micrographs to substantiate the research hypothesis at hand. In this work, we use heterogeneous poly(acrylonitrile) nanofiber nonwovens with embedded nanoparticles to demonstrate how an insufficient or biased micrograph selection may lead to erroneous conclusions. Different micrographs taken by transmission electron microscopy and helium ion microscopy with sometimes contradictory implications were analyzed and used as a basis for micromagnetic simulations. With this, we try to raise awareness for the possible consequences of cherry-picking for the reliability of scientific literature.

DnaK3 Is Involved in Biogenesis and/or Maintenance of Thylakoid Membrane Protein Complexes in the Cyanobacterium Synechocystis sp. PCC 6803.

DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria.

Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide ß-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

The proteome and lipidome of Synechocystis sp. PCC 6803 cells grown under light-activated heterotrophic conditions.

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes.

The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system.

Thylakoid membrane maturation and PSII activation are linked in greening Synechocystis sp. PCC 6803 cells.

Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, "greening" of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.

Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.

A chimeric cyanophycin synthetase gene composed of the cphATe coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively.

Efficient acclimation of the chloroplast antioxidant defence of Arabidopsis thaliana leaves in response to a 10- or 100-fold light increment and the possible involvement of retrograde signals.

Chloroplasts are equipped with a nuclear-encoded antioxidant defence system the components of which are usually expressed at high transcript and activity levels. To significantly challenge the chloroplast antioxidant system, Arabidopsis thaliana plants, acclimated to extremely low light slightly above the light compensation point or to normal growth chamber light, were moved to high light corresponding to a 100- and 10-fold light jump, for 6 h and 24 h in order to observe the responses of the water-water cycle at the transcript, protein, enzyme activity, and metabolite levels. The plants coped efficiently with the high light regime and the photoinhibition was fully reversible. Reactive oxygen species (ROS), glutathione and ascorbate levels as well as redox states, respectively, revealed no particular oxidative stress in low-light-acclimated plants transferred to 100-fold excess light. Strong regulation of the water-water cycle enzymes at the transcript level was only partly reflected at the protein and activity levels. In general, low light plants had higher stromal (sAPX) and thylakoid ascorbate peroxidase (tAPX), dehydroascorbate reductase (DHAR), and CuZn superoxide dismutase (CuZnSOD) protein contents than normal light-grown plants. Mutants defective in components relevant for retrograde signalling, namely stn7, ex1, tpt1, and a mutant expressing E .coli catalase in the chloroplast showed unaltered transcriptional responses of water-water cycle enzymes. These findings, together with the response of marker transcripts, indicate that abscisic acid is not involved and that the plastoquinone redox state and reactive oxygen species do not play a major role in regulating the transcriptional response at t=6 h, while other marker transcripts suggest a major role for reductive power, metabolites, and lipids as signals for the response of the water-water cycle.

New insights into the function of the iron deficiency-induced protein C from Synechococcus elongatus PCC 7942.

Iron limitation has a strong impact on electron transport reactions of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942 (thereafter referred to as S. elongatus). Among the various adaptational processes on different cellular levels, iron limitation induces a strongly enhanced expression of IdiC (iron-deficiency-induced protein C). In this article, we show that IdiC is loosely attached to the thylakoid and to the cytoplasmic membranes and that its expression is enhanced during conditions of iron starvation and during the late growth phase. The intracellular IdiC level was even more increased when additional iron was replenished in the late growth phase. On the basis of its amino acid sequence and of its absorbance spectrum, IdiC can be classified as a member of the family of thioredoxin (TRX)-like (2Fe-2S) ferredoxins. The presence of an iron cofactor in IdiC was detected by inductive coupled plasma optical emission spectrometry (ICP-OES). Comparative measurements of electron transport activities of S. elongatus wild type (WT) and an IdiC-merodiploid mutant called MuD, which contained a strongly reduced IdiC content under iron-sufficient as well as iron-deficient growth conditions, were performed. The results revealed that MuD had a strongly increased light sensitivity, especially under iron limitation. The measurements of photosystem II (PS II)-mediated electron transport rates in WT and MuD strain showed that PS II activity was significantly lower in MuD than in the WT strain. Moreover, P(700) (+) re-reduction rates provided evidence that the respiratory activities, which were very low in the MuD strain in the presence of iron, significantly increased in iron-starved cells. Thus, an increase in respiration may compensate for the drastic decrease of photosynthetic electron transport activity in MuD grown under iron starvation. Based on the similarity of the S. elongatus IdiC to the NuoE subunit of the NDH-1 complex in Escherichia coli, it is likely that IdiC has a function in the electron transport processes from NAD(P)H to the plastoquinone pool. This is in agreement with the up-regulation of IdiC in the late growth phase as well as under stress conditions when PS II is damaged. As absence or high reduction of the IdiC level would prevent or reduce the formation of functional NDH-1 complexes, under such conditions electron transport routes via alternative substrate dehydrogenases, donating electrons to the plastoquinone pool, can be assumed to be up-regulated.

Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56.

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.

Tuber-specific cphA expression to enhance cyanophycin production in potatoes.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

The vesicle-inducing protein 1 from Synechocystis sp. PCC 6803 organizes into diverse higher-ordered ring structures.

The vesicle-inducing protein in plastids 1 (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. In contrast to chloroplasts, it has been suggested that in cyanobacteria the protein is only tightly associated with the cytoplasmic membrane. In the present study we analyze and describe the subcellular localization and the oligomeric organization of Vipp1 from the cyanobacterium Synechocystis PCC 6803. Vipp1 forms stable dimers and higher-ordered oligomers in the cytoplasm as well as at both the cytoplasmic and thylakoid membrane. Vipp1 oligomers are organized in ring structures with a variable diameter of 25-33 nm and corresponding calculated molecular masses of approximately 1.6-2.2 MDa. Six different types of rings were found with an unusual 12-17-fold symmetrical conformation. The simultaneous existence of multiple types of rings is very unusual and suggests a special function of Vipp1. Involvement of diverse ring structures in vesicle formation is suggested.

Detection of an L-amino acid dehydrogenase activity in Synechocystis sp. PCC 6803.

The protein Slr0782 from Synechocystis sp. PCC 6803, which has similarity to L-amino acid oxidase from Synechococcus elongatus PCC 6301 and PCC 7942, has been characterized in part. Immunoblot blot analysis showed that Slr0782 is mainly thylakoid membrane-associated. Moreover, expression of slr0782 mRNA and Slr0782 protein were analyzed and an activity assay was developed. Utilizing toluene-permeabilized cells, an L-arginine-stimulated O(2) uptake became detectable in Synechocystis sp. PCC 6803. Besides oxidizing the basic L-amino acids L-arginine, L-lysine, L-ornithine, and L-histidine, a number of other L-amino acids were also substrates, while D-amino acids were not. The best substrate was L-cysteine, and the second best was L-arginine. The L-arginine-stimulated O(2) uptake was inhibited by cations. The inhibition by o-phenanthroline and salicylhydroxamic acid suggested the presence of a transition metal besides FAD in the enzyme. Moreover, it is shown that inhibitors of the respiratory electron transport chain, such as KCN and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, also inhibited the L-arginine-stimulated O(2) uptake, suggesting that Slr0782 functions as an L-arginine dehydrogenase, mediating electron transfer from L-arginine into the respiratory electron transport chain utilizing O(2) as electron acceptor via cytochrome oxidase. The results imply that Slr0782 is an additional substrate dehydrogenase being able to interact with the electron transport chain of the thylakoid membrane.

Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum.

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphA(Te)) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphA(Te), pCP24-cphA(Te), pFNR-cphA(Te) and pPsbY-cphA(Te). These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphA(Te) led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphA(Te) and pFNR-cphA(Te) constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphA(Te) produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T(0)) transformants. Specific T(2) plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

L-amino acid oxidases with specificity for basic L-amino acids in cyanobacteria.

The two closely related fresh water cyanobacteria Synechococcus elongatus PCC 6301 and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containing L-amino acid oxidase with high specificity for basic L-amino acids (L-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluation of the nucleotide/amino acid sequence of the L-amino acid oxidase of Synechococcus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site and a translocation pathway signal sequence will be given. Moreover, the genome sequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an L-amino acid oxidase-similar protein will be found.

The redox imbalanced mutants of Arabidopsis differentiate signaling pathways for redox regulation of chloroplast antioxidant enzymes.

A network of enzymatic and nonenzymatic antioxidants protects chloroplasts from photooxidative damage. With all enzymatic components being nuclear encoded, the control of the antioxidant capacity depends on chloroplast-to-nucleus redox signaling. Using an Arabidopsis (Arabidopsis thaliana) reporter gene line expressing luciferase under control of the redox-sensitive 2-cysteine peroxiredoxin A (2CPA) promoter, six mutants with low 2CPA promoter activity were isolated, of which five mutants show limitations in redox-box regulation of the 2CPA promoter. In addition to 2CPA, the transcript levels for other chloroplast antioxidant enzymes were decreased, although a higher oxidation status of the ascorbate pool, a higher reduction state of the plastoquinone pool, and an increased oxidation status of the 2-Cys peroxiredoxin pool demonstrated photooxidative stress conditions. Greening of the mutants, chloroplast ultrastructure, steady-state photosynthesis, and the responses to the stress hormone abscisic acid were wild type like. In the rosette state, the mutants were more sensitive to low CO2 and to hydrogen peroxide. Comparison of gene expression patterns and stress sensitivity characterizes the mutants as redox imbalanced in the regulation of nuclear-encoded chloroplast antioxidant enzymes and differentiates redox signaling cascades.

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare.

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.

Regulation of peroxiredoxin expression versus expression of Halliwell-Asada-Cycle enzymes during early seedling development of Arabidopsis thaliana.

During early seedling development of oil seed plants, the transition from lipid based heterotrophic to photoautotrophic carbohydrate metabolism is accompanied with a biphasic control of the chloroplast antioxidant system. In continuous light, organellar peroxiredoxins (Prx) and thylakoid-bound ascorbate peroxidase (tAPx) are activated early in seedling development, while stromal ascorbate peroxidase (sAPx), Cu/Zn-superoxide dismutase-2 (Csd2) and monodehydroascorbate reductase (MDHAR) and the cytosolic peroxiredoxins PrxIIB, PrxIIC and PrxIID are fully activated between 2.5 and 3 days after radicle emergence (DARE). Discontinuous light synchronized the expression of chloroplast antioxidant enzymes, but defined diurnally specific typeII-Prx-patterns in the cytosol and initiated chloroplast senescence around 2.5 DARE. Carbohydrate feeding uncoupled sAPx expression from the light pattern. In contrast, sucrose-feeding did not significantly impact on Prx transcript amounts. It is concluded that upon post-germination growth Prxs are activated endogenously to provide early antioxidant protection, which is supported by the Halliwell-Asada-Cycle, whose expressional activation depends on metabolic signals provided only later in development or in day-night-cycles.

NAB1 is an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii.

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their light-harvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA-protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

Localization and function of the IdiA homologue Slr1295 in the cyanobacterium Synechocystis sp. strain PCC 6803.

Slr1295 (and Slr0513) in the cyanobacterium Synechocystis sp. PCC 6803 has amino acid similarity to the bacterial FbpA protein family and also to IdiA of Synechococcus PCC 6301/PCC 7942. To determine whether Slr1295 is the periplasm-located component of an iron transporter, or has a function in protecting photosystem (PS) II, subcellular localization and Deltaslr1295 mutant characterization studies were performed. Localization of Slr1295 provided evidence that it has an intracellular function, since virtually no Slr1295 was detected in the soluble protein fraction of the periplasm or in the cytoplasmic membrane. Characterization of a Deltaslr1295 Synechocystis PCC 6803 mutant indicated that PS II is more susceptible to inactivation in the mutant than in the wild-type (WT). Under mild iron limitation, modification of PS I to the PS I-IsiA complex is more advanced in the Deltaslr1295 mutant, indicating that iron deficiency leads more rapidly to changes in the photosynthetic apparatus in the mutant than in the WT. Biochemical fractionation procedures provide evidence that Slr1295 co-purifies with PS II. These results suggest a function of Slr1295 that is comparable to the function of IdiA in Synechococcus PCC 6301/PCC 7942 being a protein that protects PS II under iron limitation in an as yet unknown way.