Electron Transport Components Research Articles

Plant signaling systems. Plastid-generated signals and their role in nuclear gene expression

Current concepts are outlined regarding the chloroplast effects on expression of the nuclear genes encoding plastid proteins. The major types of plastid-generated signals are considered. The signal molecules are shown to include the reactive oxygen species, the redox state of the components of photosynthetic electron transport, in particular plastoquinones, the redox-active molecules of plastid stroma, such as thioredoxin and glutathione, and also the intermediates of tetrapyrrole biosynthesis (Mg-protoporphyrin IX and its monomethyl ester). The sophisticated regulatory network is emphasized as a channel matching up the expression of nuclear and plastid genes. The plastid-generated signals help plants adapt to the changing and frequently adverse environmental conditions.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 A and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 158.7, c = 81.4 A. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 A resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 A.

Effects of electron donors and acceptors on anaerobic reduction of azo dyes by Shewanella decolorationis S12

Shewanella decolorationis S12 was able to reduce various azo dyes in a defined medium with formate, lactate, and pyruvate or H(2) as electron donors under anaerobic conditions. Purified membranous, periplasmic, and cytoplasmic fractions from strain S12 analyzed, respectively, only membranous fraction was capable of reducing azo dye in the presence of electron donor, indicating that the enzyme system for anaerobic azoreduction was located on cellular membrane. Respiratory inhibitor Cu(2+), dicumarol, stigmatellin, and metyrapone inhibited anaerobic azoreduction by purified membrane fraction, suggesting that the bacterial anaerobic azoreduction by strain S12 was a biochemical process that oxidizes the electron donors and transfers the electrons to the acceptors through a multicompound system related to electron transport chain. Dehydrogenases, cytochromes, and menaquinones were essential electron transport components for the azoreduction. The electron transport process for azoreduction was almost fully inhibited by O(2), 6 mM of NO3-, and 0.9 mM of NO2-, but not by 10 mM of Fe(3+). The inhibition may be a result from the competition for electrons from electron donors. These findings impact on the understanding of the mechanism of bacterial anaerobic azoreduction and have implication for improving treatment methods of wastewater contaminated by azo dyes.

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina acetivorans Grown with Acetate versus Methanol

Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs methanol-grown cells metabolically labeled with 14N vs 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were > or =3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were > or =2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate.

Gravity-induced absorption changes in Phycomyces blakesleeanus during parabolic flights: first spectral approach in the visible

Gravity-induced absorption changes as experienced during a series of parabolas on the Airbus 300 Zero-G have been measured previously pointwise on the basis of dual-wavelength spectroscopy. Only the two wavelengths of 460 and 665 nm as generated by light-emitting diodes have been utilised during our first two parabolic-flight campaigns. In order to gain complete spectral information throughout the wavelength range from 400 to 900 nm, a miniaturized rapid scan spectrophotometer was designed. The difference of spectra taken at 0 g and 1.8 g presents the first gravity-induced absorption change spectrum measured on wild-type Phycomyces blakesleeanus sporangiophores, exhibiting a broad positive hump in the visible range and negative values in the near infrared with an isosbestic point near 735 nm. The control experiment performed with the stiff mutant A909 of Phycomyces blakesleeanus does not show this structure. These results are in agreement with those obtained with an array spectrophotometer. In analogy to the more thoroughly understood so-called light-induced absorption changes, we assume that gravity-induced absorption changes reflect redox changes of electron transport components such as flavins and cytochromes localised within the plasma membrane.

The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA

The periplasmic nitrate reductase of Escherichia coli is important during anaerobic growth in low-nitrate environments. The nap operon encoding this nitrate reductase comprises seven genes including a gene, napF, that encodes a putative cytoplasmic iron-sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the E. coli cytoplasm. A bacterial two-hybrid protein-protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His(6)-tagged protein, exhibited spectral properties characteristic of an iron-sulphur protein. This protein was able to pull down NapA from soluble extracts of E. coli. A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm.

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12.

BackgroundIn Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated.ResultsThe pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC).ConclusionpH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm.

Photosynthesis, growth and structural characteristics of holm oak resprouts originated from plants grown under elevated CO2

The physiological characteristics of holm oak (Quercus ilex L.) resprouts originated from plants grown under current CO2 concentration (350 μl l−1) (A‐resprouts) were compared with those of resprouts originated from plants grown under elevated CO2 (750 μl l−1) (E‐resprouts). At their respective CO2 growth concentration, no differences were observed in photosynthesis and chlorophyll fluorescence parameters between the two kinds of resprout. E‐resprouts appeared earlier and showed lower stomatal conductance, higher water‐use efficiency and increased growth (higher leaf, stem and root biomass and increased height). Analyses of leaf chemical composition showed the effect of elevated [CO2] on structural polysaccharide (higher cellulose content), but no accumulation of total non‐structural carbohydrate on area or dry weight basis was seen. Four months after appearance, downregulation of photosynthesis and electron transport components was observed in E‐resprouts: lower photosynthetic capacity, photosystem II quantum efficiency, photochemical quenching of fluorescence and relative electron transport rate. Reduction in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCo) activity, deduced from the maximum carboxylation velocity of RuBisCo, accounts for the observed acclimation. Increased susceptibility of photosynthetic apparatus to increasing irradiance was detected in E‐resprouts.

Energy balance, organellar redox status, and acclimation to environmental stress

In plants and algal cells, changes in light intensity can induce intrachloroplastic and retrograde regulation of gene expression in response to changes in the plastoquinone redox status. We review the evidence in support of the thesis that the chloroplast acts as a general sensor of cellular energy imbalance sensed through the plastoquinone pool. Alteration in cellular energy balance caused by chloroplast or mitochondrial metabolism can induce intracellular signalling to affect chloroplastic and nuclear gene expression in response, not only to light intensity, but to a myriad of abiotic stresses. In addition, this chloroplastic redox sensing also appears to have a broader impact, affecting long-distance systemic signalling related to plant growth and development. The organization of the respiratory electron transport chains of mitochondria and heterotrophic prokaryotes is comparable to that of chloroplast thylakoid membranes, and the redox state of the respiratory ubiquinone pool is a well-documented cellular energy sensor. Thus, modulation of electron transport component redox status by abiotic stress regulates organellar as well as nuclear gene expression. From the evidence presented, we suggest that the photosynthetic and respiratory machinery in prokaryotic and eukaryotic organisms have a dual function: primary cellular energy transformation, and global environmental sensing.

PLASTID-TO-NUCLEUS RETROGRADE SIGNALING

Plant cells store genetic information in the genomes of three organelles: the nucleus, plastid, and mitochondrion. The nucleus controls most aspects of organelle gene expression, development, and function. In return, organelles send signals to the nucleus to control nuclear gene expression, a process called retrograde signaling. This review summarizes our current understanding of plastid-to-nucleus retrograde signaling, which involves multiple, partially redundant signaling pathways. The best studied is a pathway that is triggered by buildup of Mg-ProtoporphyrinIX, the first intermediate in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In addition, there is evidence for a plastid gene expression-dependent pathway, as well as a third pathway that is dependent on the redox state of photosynthetic electron transport components. Although genetic studies have identified several players involved in signal generation, very little is known of the signaling components or transcription factors that regulate the expression of hundreds of nuclear genes.

Blue polymer electrophosphorescent devices with different electron-transporting oxadiazoles

We report that the performances of blue polymer electrophosphorescent devices are crucially depending on the choice of the electron transporting material incorporated into the emissive layer. Devices with 1,3-bis[(4-tert-butylphenyl)-1,3,4-oxidiazolyl]phenylene (OXD-7) doped at ∼40wt% into a poly(vinylcarbazole) matrix exhibited significantly higher efficiencies than those with 2-(4-biphenylyl)-5-(4-tert-butylphenyl)-1,3,4-oxadiazole (PBD), yielding maximum luminous and power efficiency values of 18.2 Cd∕A and 8.8 lm∕W, respectively. Time resolved photoluminescence measurements revealed a long lifetime phosphorescence component in layers with PBD, which we assign to significant triplet harvesting by this electron-transporting component.

Spectral analysis of the bc1 complex components in situ: Beyond the traditional difference approach

The cytochrome (cyt) bc 1 complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc 1 complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc 1 complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc 1 turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b L. From LS analysis of the chromophoric components (RC, c tot, b H and b L), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc 1 complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.

Characterization of Transformed Arabidopsis with Altered Alternative Oxidase Levels and Analysis of Effects on Reactive Oxygen Species in Tissue

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinone pool to oxygen without energy conservation. AOX can use reductant in excess of cytochrome pathway capacity, preventing reactive oxygen species (ROS) formation from an over-reduced ubiquinone pool, and thus may be involved in acclimation to oxidative stresses. The AOX connection with mitochondrial ROS has been investigated only in isolated mitochondria and suspension culture cells. To study ROS and AOX in whole plants, transformed lines of Arabidopsis (Arabidopsis thaliana) were generated: AtAOX1a overexpressors, AtAOX1a anti-sense plants, and overexpressors of a mutated, constitutively active AtAOX1a. In the presence of KCN, leaf tissue of either mutant or wild-type AOX overexpressors showed no increase in oxidative damage, whereas anti-sense lines had levels of damage greater than those observed for untransformed leaves. Similarly, ROS production increased markedly in anti-sense and untransformed, but not overexpressor, roots with KCN treatment. Thus, AOX functions in leaves and roots, as in suspension cells, to ameliorate ROS production when the cytochrome pathway is chemically inhibited. However, in contrast with suspension culture cells, no changes in leaf transcript levels of selected electron transport components or oxidative stress-related enzymes were detected under nonlimiting growth conditions, regardless of transformation type. Further, a microarray study using an anti-sense line showed AOX influences outside mitochondria, particularly in chloroplasts and on several carbon metabolism pathways. These results illustrate the value of expanding AOX transformant studies to whole tissues.

Novel Regulatory Function for the CCAAT-Binding Factor in Candida albicans

Candida albicans is an opportunistic human pathogen that can sense environmental changes and respond by altering its cell morphology and physiology. A number of environmental factors have been shown to influence this dimorphic transition, including pH, starvation, serum, and amino acids. In this report, we investigate the function of the C. albicans CCAAT-binding factor. In Saccharomyces cerevisiae, this heterooligomeric transcriptional activator stimulates the expression of genes that encode proteins involved in respiration. To examine the function of this transcription factor in C. albicans, we cloned CaHAP5 and generated a hap5delta/hap5delta mutant of C. albicans. Using mobility shift studies, we identified four separate complexes from C. albicans cell extracts whose DNA-binding activities were abolished in the hap5delta/hap5delta mutant, suggesting that they represented sequence-specific CCAAT-binding complexes. We found that the C. albicans hap5delta homozygote was defective in hyphal development under a variety of conditions, and the mutant displayed a carbon source-dependent "hyperfilamentation" phenotype under certain growth conditions. In addition, the mRNA levels for two enzymes involved in respiration, encoded by COX5 and CYC1, were overexpressed in the hap5delta/hap5delta mutant when grown in medium containing amino acids as the sole carbon and nitrogen source. Thus, the C. albicans CCAAT-binding factor appeared to function as a repressor of genes encoding mitochondrial electron transport components, in contrast to its activator function in S. cerevisiae. These data provide the first evidence that the CCAAT-binding factor can act as a transcriptional repressor and raise new and interesting questions about how carbon metabolism is regulated in this opportunistic human pathogen.

Maximum photosynthetic efficiency of size-fractionated phytoplankton assessed by 14 C uptake and fast repetition rate fluorometry

Under high nutrient concentrations and sufficient light conditions, large phytoplankton may display higher photosynthetic efficiency than smaller cells. This is unexpected since smaller phytoplankton, because of their higher surface to volume ratio, possess a greater ability to take up nutrients and absorb light. In order to investigate the causes of the increased photosynthetic efficiency in larger phytoplankton, we assessed the maximum photosynthetic efficiency of coastal assemblages in three size classes (<5, 5–20, and >20 µm) by concurrently conducting 14C-based photosynthesis-irradiance experiments and fast repetition rate fluorescence measurements. The light-saturated, chlorophyll-specific photosynthesis (Pbmax) and the maximum photosystem II (PSII) photochemical efficiency (Fv/Fm) of each size class were determined during winter mixing (March 2003) and summer stratification (June 2003). During winter mixing, size-fractionated Pbmax and Fv/Fm were similar in all size classes. In contrast, during summer stratification, size-fractionated Pbmax and Fv/Fm were significantly higher in the >20-µm size class. In the entire data set, size-fractionated Pbmax and Fv/Fm were not significantly correlated. However, a significant relationship was found between size-fractionated Pbmaxand Fv/Fm for phytoplankton assemblages acclimated to low light conditions. Under high light, an excess PSII capacity may be responsible for the discrepancy between size-fractionated Pbmax and Fv/Fm measurements, whereas under low light conditions, photosynthetic electron transport chain and components downstream of PSII become more balanced, which results in a tight covariation between both variables. Higher maximum photosynthetic efficiencies of large-sized phytoplankton might be associated with a higher PSII photochemical efficiency characteristic of certain taxonomic groups such as diatoms.

A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe (III) and Mn (IV) oxides in Geobacter sulfurreducens

BackgroundMetal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration.ResultsWhen outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III) and insoluble Mn (IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction.ConclusionOmpJ is a putative porin found in the outer membrane of the model metal reducer G. sulfurreducens that is required for respiration of extracellular electron acceptors such as soluble and insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components. The exclusive presence of ompJ in members of the Geobacteraceae family as well as its role in metal reduction suggest that the ompJ sequence may be useful in tracking the growth or activity of Geobacteraceae in sedimentary environments.

Structure of the Terminal Oxygenase Component of Angular Dioxygenase, Carbazole 1,9a-Dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

Redox Regulation and Modification of Proteins Controlling Chloroplast Gene Expression

Chloroplasts are typical organelles of plant cells and represent the site of photosynthesis. As one very remarkable feature, they possess their own genome and a complete machinery to express the genetic information in it. The plastid gene expression machinery is a unique assembly of prokaryotic-, eukaryotic-, and phage-like components because chloroplasts acquired a great number of regulatory proteins during evolution. Such proteins can be found at all levels of gene expression. They significantly expand the functional and especially the regulatory properties of the "old" gene expression system that chloroplasts inherited from their prokaryotic ancestors. Recent results show that photosynthesis has a strong regulatory effect on plastid gene expression. The redox states of electron transport components, redox-active molecules coupled to photosynthesis, and pools of reactive oxygen species act as redox signals. They provide a functional feedback control, which couples the expression of chloroplast genes to the actual function of photosynthesis and, by this means, helps to acclimate the photosynthetic process to environmental cues. The redox signals are mediated by various specific signaling pathways that involve many of the "new" regulatory proteins. Chloroplasts therefore are an ideal model to study redox-regulated mechanisms in gene expression control. Because of the multiple origins of the expression machinery, these observations are of great relevance for many other biological systems.

Stress-induced co-expression of alternative respiratory chain components in Arabidopsis thaliana

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.