bluuebunny/crossref_metadata_2025 huggingface dataset:
Stress Granule Assembly Is Mediated by Prion-like Aggregation of TIA-1
Authors: Gilks Natalie , Kedersha Nancy , Ayodele Maranatha , Shen Lily , Stoecklin Georg , Dember Laura M. , Anderson Paul
Published: September 2004

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level–expressing cells but prevent SG assembly in high-level–expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2α in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2α phosphorylation in response to stress.
Hydrogen sulfide‐induced apoptosis of human aorta smooth muscle cells via the activation of mitogen‐activated protein kinases and caspase‐3
Authors: Yang Guangdong , Sun Xianfeng , Wang Rui
Published: September 2004

The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, position this gas well in the family of gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). In this study, we demonstrate that H2S at physiologically relevant concentrations induced apoptosis of human aorta smooth muscle cells (HASMCs). Exposure of HASMCs to H2S did not induce necrosis as verified with Trypan blue exclusion and LDH release analysis. After inhibiting endogenous H2S production, exogenous H2S induced much more significant apoptosis, which was not altered by the presence of albumin or glutathione. H2S treatment increased the activities of ERK and p38 mitogen‐activated protein kinase (MAPK), but not c‐Jun N‐terminal kinase activity. Suppression of extracellular signal‐regulated kinase (ERK) activity, but not of p38 activity, inhibited the H2S‐induced apoptosis of HASMCs. The activation of ERK by H2S in HASMCs was accompanied by increased caspase‐3 activity. Inhibition of caspase‐3 by AC‐DEVD‐CHO attenuated the H2S‐induced cell apoptosis. Inhibition of ERK by U0126 decreased caspase‐3 activity, whereas AC‐DEVD‐CHO did not alter ERK activity. In conclusion, exogenous H2S induces apoptosis of HASMCs, which is significantly affected by the endogenous H2S level. Of the three investigated MAPKs, only ERK played an active role in mediating H2S‐induced apoptosis of HASMCs by activating caspase‐3. These findings may help reveal novel mechanisms for many diseases linked to H2S‐related abnormal cellular proliferation and apoptosis.
Evidence for altered interleukin (IL)‐18 pathway in human heart failure
Authors: Mallat Ziad , Heymes Christophe , Corbaz Anne , Logeart Damien , Alouani Sami , Cohen‐Solal Alain , Seidler Tim , Hasenfuss Gerd , Chvatchko Yolande , Shah Ajay M. , Tedgui Alain
Published: September 2004

Interleukin (IL)‐18 is the interferon‐γ‐inducing factor and has potent proinflammatory activities. IL‐18 has been recently implicated in atherosclerotic plaque instability and myocardial ischemia‐reperfusion injury. However, it is unknown whether IL‐18 expression is increased in human myocardium or if it has any role in heart failure. We analyzed the expression of IL‐18, its receptor IL‐18Rα, and its endogenous inhibitor, IL‐18 binding protein (IL‐18BP) in myocardial tissue from patients with end‐stage heart failure (ischemic or dilated cardiomyopathy) and controls by use of quantitative real‐time reverse transcriptase polymerase chain reaction, Western blot or immunohistochemical techniques. Plasma levels of IL‐18 were also determined in 48 patients with heart failure. IL‐18 mRNA and protein levels were up‐regulated in the myocardium of patients with ischemic cardiomyopathy. Both ischemic and dilated myocardium showed increased IL‐18Rα levels, suggesting potential biological effects. In addition, mRNA levels of IL‐18 BP were down‐regulated in the failing myocardium. Finally, plasma IL‐18 levels were significantly elevated in patients with heart failure and were higher in those who died at follow‐up than in survivors. The results suggest a potential role for the immunoinflammatory IL‐18 signaling pathway in the pathophysiology of heart failure and identify novel therapeutic targets for future testing.
A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe
Authors: Sigova Alla , Rhind Nicholas , Zamore Phillip D.
Published: September 2004

The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways.
Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular stabilization
Authors: Paik Ji-Hye , Skoura Athanasia , Chae Sung-Suk , Cowan Ann E. , Han David K. , Proia Richard L. , Hla Timothy
Published: September 2004

Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. Here we describe the molecular basis of vascular stabilization regulated by sphingosine 1-phosphate (S1P), a platelet-derived lipid mediator. S1P1 receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S1P1/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S1P treatment modulated the phosphorylation of N-cadherin as well as p120-catenin and induced the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S1P1 receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S1P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases.
Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival
Authors: Hock Hanno , Meade Eliza , Medeiros Sarah , Schindler Jeffrey W. , Valk Peter J.M. , Fujiwara Yuko , Orkin Stuart H.
Published: September 2004

Hematopoietic stem cells (HSCs) sustain blood formation throughout life. Pathways regulating maintenance of adult HSCs are largely unknown. Here we report that the Ets-related transcription factor Tel/Etv6, the product of a locus frequently involved in translocations in leukemia, is a selective regulator of HSC survival. Following inactivation of Tel/Etv6, HSCs are lost in the adult bone marrow but their progeny are unaffected and transiently sustain blood formation. Accordingly, absence of Tel/Etv6 after lineage commitment is ostensibly without consequence except for unexpected impairment of maturation of megakaryocytes. Thus, we establish Tel/Etv6 as a selective and essential regulator of postembryonic HSCs.
Down-Regulation of DELLA Genes Is Not Essential for Germination of Tomato, Soybean, and Arabidopsis Seeds
Authors: Bassel George W. , Zielinska Elzbieta , Mullen Robert T. , Bewley J. Derek
Published: September 2004

The relationship between expression of a negative regulator of GA signal transduction (RGL2) belonging to the DELLA gene family and repression of Arabidopsis seed germination has been studied (Lee S, Cheng H, King KE, Wang W, He Y, Hussain A, Lo J, Harberd NP, Peng J [2002] Genes and Development 16: 646–658). There is one DELLA gene (LeGAI) present in tomato (Lycopersicon esculentumMill.), which is expressed in both vegetative and reproductive tissues. During germination of wild-type tomato seed, there was no decline in the expression of LeGAI in either the embryo or the endosperm. Rather, LeGAI transcripts increased in these tissues following imbibition and remained high during and following germination. A similar increase in LeGAI transcripts occurred in the endosperm and embryo of GA-treated gib-1 mutant seed during and following germination. Likewise in soybean (Glycine max) seed, there was no decline in the expression of two DELLA genes in the radicle before or after germination. Upon reexamination of RGL2 in Arabidopsis seeds, a decline in its expression was noted but only after radicle emergence, i.e. after germination had been completed. Taken together, these data are consistent with GA-induced down-regulation of DELLA genes not being a prerequisite for germination of tomato, soybean, and Arabidopsis seeds.
Enhanced Photosynthesis and Redox Energy Production Contribute to Salinity Tolerance in Dunaliella as Revealed by Homology-Based Proteomics
Authors: Liska Adam J. , Shevchenko Andrej , Pick Uri , Katz Adriana
Published: September 2004

Salinity is a major limiting factor for the proliferation of plants and inhibits central metabolic activities such as photosynthesis. The halotolerant green alga Dunaliella can adapt to hypersaline environments and is considered a model photosynthetic organism for salinity tolerance. To clarify the molecular basis for salinity tolerance, a proteomic approach has been applied for identification of salt-induced proteins in Dunaliella. Seventy-six salt-induced proteins were selected from two-dimensional gel separations of different subcellular fractions and analyzed by mass spectrometry (MS). Application of nanoelectrospray mass spectrometry, combined with sequence-similarity database-searching algorithms, MS BLAST and MultiTag, enabled identification of 80% of the salt-induced proteins. Salinity stress up-regulated key enzymes in the Calvin cycle, starch mobilization, and redox energy production; regulatory factors in protein biosynthesis and degradation; and a homolog of a bacterial Na+-redox transporters. The results indicate that Dunaliella responds to high salinity by enhancement of photosynthetic CO2 assimilation and by diversion of carbon and energy resources for synthesis of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to enhance photosynthetic activity at high salinity is remarkable because, in most plants and cyanobacteria, salt stress inhibits photosynthesis. The results demonstrated the power of MS BLAST searches for the identification of proteins in organisms whose genomes are not known and paved the way for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.
Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds
Authors: Chen Guoping , Hackett Rachel , Walker David , Taylor Andy , Lin Zhefeng , Grierson Donald
Published: September 2004

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.
Genetic Elucidation of Nitric Oxide Signaling in Incompatible Plant-Pathogen Interactions
Authors: Zeier Jürgen , Delledonne Massimo , Mishina Tatiana , Severi Emmanuele , Sonoda Masatoshi , Lamb Chris
Published: September 2004

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H2O2 levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H2O2 levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.
A Complementary Bioinformatics Approach to Identify Potential Plant Cell Wall Glycosyltransferase-Encoding Genes
Authors: Egelund Jack , Skjøt Michael , Geshi Naomi , Ulvskov Peter , Petersen Bent Larsen
Published: September 2004

Plant cell wall (CW) synthesizing enzymes can be divided into the glycan (i.e. cellulose and callose) synthases, which are multimembrane spanning proteins located at the plasma membrane, and the glycosyltransferases (GTs), which are Golgi localized single membrane spanning proteins, believed to participate in the synthesis of hemicellulose, pectin, mannans, and various glycoproteins. At the Carbohydrate-Active enZYmes (CAZy) database where e.g. glucoside hydrolases and GTs are classified into gene families primarily based on amino acid sequence similarities, 415 Arabidopsis GTs have been classified. Although much is known with regard to composition and fine structures of the plant CW, only a handful of CW biosynthetic GT genes—all classified in the CAZy system—have been characterized. In an effort to identify CW GTs that have not yet been classified in the CAZy database, a simple bioinformatics approach was adopted. First, the entire Arabidopsis proteome was run through the Transmembrane Hidden Markov Model 2.0 server and proteins containing one or, more rarely, two transmembrane domains within the N-terminal 150 amino acids were collected. Second, these sequences were submitted to the SUPERFAMILY prediction server, and sequences that were predicted to belong to the superfamilies NDP-sugartransferase, UDP-glycosyltransferase/glucogen-phosphorylase, carbohydrate-binding domain, Gal-binding domain, or Rossman fold were collected, yielding a total of 191 sequences. Fifty-two accessions already classified in CAZy were discarded. The resulting 139 sequences were then analyzed using the Three-Dimensional-Position-Specific Scoring Matrix and mGenTHREADER servers, and 27 sequences with similarity to either the GT-A or the GT-B fold were obtained. Proof of concept of the present approach has to some extent been provided by our recent demonstration that two members of this pool of 27 non-CAZy-classified putative GTs are xylosyltransferases involved in synthesis of pectin rhamnogalacturonan II (J. Egelund, B.L. Petersen, A. Faik, M.S. Motawia, C.E. Olsen, T. Ishii, H. Clausen, P. Ulvskov, and N. Geshi, unpublished data).
AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes
Authors: Reumann Sigrun , Ma Changle , Lemke Steffen , Babujee Lavanya
Published: September 2004

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783–800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in β-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid α-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.
Microbial Products Trigger Amino Acid Exudation from Plant Roots
Authors: Phillips Donald A. , Fox Tama C. , King Maria D. , Bhuvaneswari T.V. , Teuber Larry R.
Published: September 2004

Plants naturally cycle amino acids across root cell plasma membranes, and any net efflux is termed exudation. The dominant ecological view is that microorganisms and roots passively compete for amino acids in the soil solution, yet the innate capacity of roots to recover amino acids present in ecologically relevant concentrations is unknown. We find that, in the absence of culturable microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 μ  m) exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial products, which are produced by common soil microorganisms such as Pseudomonas bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e. exudation) of amino acids from roots of these four plant species. In alfalfa, treating roots with 200 μ  m phenazine, 2,4-diacetylphloroglucinol, or zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h. Data from 15N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under normal conditions is a phenomenon that probably reflects both active manipulation and passive uptake by microorganisms, as well as diffusion and adsorption to soil, all of which help overcome the innate capacity of plant roots to reabsorb amino acids. The importance of identifying potential enhancers of root exudation lies in understanding that such compounds may represent regulatory linkages between the larger soil food web and the internal carbon metabolism of the plant.
Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves
Authors: Smith Steven M. , Fulton Daniel C. , Chia Tansy , Thorneycroft David , Chapple Andrew , Dunstan Hannah , Hylton Christopher , Zeeman Samuel C. , Smith Alison M.
Published: September 2004

To gain insight into the synthesis and functions of enzymes of starch metabolism in leaves of Arabidopsis L. Heynth, Affymetrix microarrays were used to analyze the transcriptome throughout the diurnal cycle. Under the conditions employed, transitory leaf starch is degraded progressively during a 12-h dark period, and then accumulates during the following 12-h light period. Transcripts encoding enzymes of starch synthesis changed relatively little in amount over 24 h except for two starch synthases, granule bound starch synthase and starch synthase II, which increased appreciably during the transition from dark to light. The increase in RNA encoding granule-bound starch synthase may reflect the extensive destruction of starch granules in the dark. Transcripts encoding several enzymes putatively involved in starch breakdown showed a coordinated decline in the dark followed by rapid accumulation in the light. Despite marked changes in their transcript levels, the amounts of some enzymes of starch metabolism do not change appreciably through the diurnal cycle. Posttranscriptional regulation is essential in the maintenance of amounts of enzymes and the control of their activities in vivo. Even though the relationships between transcript levels, enzyme activity, and diurnal metabolism of starch metabolism are complex, the presence of some distinctive diurnal patterns of transcripts for enzymes known to be involved in starch metabolism facilitates the identification of other proteins that may participate in this process.
The Fast and Transient Transcriptional Network of Gravity and Mechanical Stimulation in the Arabidopsis Root Apex
Authors: Kimbrough Jeffery M. , Salinas-Mondragon Raul , Boss Wendy F. , Brown Christopher S. , Sederoff Heike Winter
Published: September 2004

Plant root growth is affected by both gravity and mechanical stimulation (Massa GD, Gilroy S [2003] Plant J 33: 435–445). A coordinated response to both stimuli requires specific and common elements. To delineate the transcriptional response mechanisms, we carried out whole-genome microarray analysis of Arabidopsis root apices after gravity stimulation (reorientation) and mechanical stimulation and monitored transcript levels of 22,744 genes in a time course during the first hour after either stimulus. Rapid, transient changes in the relative abundance of specific transcripts occurred in response to gravity or mechanical stimulation, and these transcript level changes reveal clusters of coordinated events. Transcriptional regulation occurs in the root apices within less than 2 min after either stimulus. We identified genes responding specifically to each stimulus as well as transcripts regulated in both signal transduction pathways. Several unknown genes were specifically induced only during gravitropic stimulation (gravity induced genes). We also analyzed the network of transcriptional regulation during the early stages of gravitropism and mechanical stimulation.
Genomic Analysis of the Nitrate Response Using a Nitrate Reductase-Null Mutant of Arabidopsis
Authors: Wang Rongchen , Tischner Rudolf , Gutiérrez Rodrigo A. , Hoffman Maren , Xing Xiujuan , Chen Mingsheng , Coruzzi Gloria , Crawford Nigel M.
Published: September 2004

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.
Induction and Functional Analysis of Two Reduced Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutathione Peroxidase-Like Proteins in Synechocystis PCC 6803 during the Progression of Oxidative Stress
Authors: Gaber Ahmed , Yoshimura Kazuya , Tamoi Masahiro , Takeda Toru , Nakano Yoshihisa , Shigeoka Shigeru
Published: September 2004

Synechocystis PCC 6803 contains two types of glutathione peroxidase-like proteins (GPX-1 and GPX-2) that utilize NADPH but not reduced glutathione and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides. The steady-state transcript level of gpx-1 gradually increased under oxidative stress conditions imposed by high light intensity, high salinity, or application of methylviologen or t-butyl hydroperoxide in the wild-type and GPX-2 knock-out mutant (gpx-2Δ) cells. To examine the ability of GPX-1, GPX-2, and thioredoxin peroxidase to scavenge lipid hydroperoxide in vivo, we measured the photosynthetic evolution of O2 and the level of lipid peroxidation in the wild-type and each type of mutant cell after the application of t-butyl hydroperoxide or H2O2. The data reported here indicate that GPX-1 and GPX-2 are essential for the removal of lipid hydroperoxides under normal and stress conditions, leading to the protection of membrane integrity.
Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance
Authors: Kumar Shashi , Dhingra Amit , Daniell Henry
Published: September 2004

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93–101 μmol g−1 dry weight of β-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.
GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox
Authors: Zimmermann Philip , Hirsch-Hoffmann Matthias , Hennig Lars , Gruissem Wilhelm
Published: September 2004

High-throughput gene expression analysis has become a frequent and powerful research tool in biology. At present, however, few software applications have been developed for biologists to query large microarray gene expression databases using a Web-browser interface. We present GENEVESTIGATOR, a database and Web-browser data mining interface for Affymetrix GeneChip data. Users can query the database to retrieve the expression patterns of individual genes throughout chosen environmental conditions, growth stages, or organs. Reversely, mining tools allow users to identify genes specifically expressed during selected stresses, growth stages, or in particular organs. Using GENEVESTIGATOR, the gene expression profiles of more than 22,000 Arabidopsis genes can be obtained, including those of 10,600 currently uncharacterized genes. The objective of this software application is to direct gene functional discovery and design of new experiments by providing plant biologists with contextual information on the expression of genes. The database and analysis toolbox is available as a community resource at https://www.genevestigator.ethz.ch.
TheElm1(ZmHy2) Gene of Maize Encodes a Phytochromobilin Synthase
Authors: Sawers Ruairidh J.H. , Linley Philip J. , Gutierrez-Marcos Jose F. , Delli-Bovi Teegan , Farmer Phyllis R. , Kohchi Takayuki , Terry Matthew J. , Brutnell Thomas P.
Published: September 2004

The light insensitive maize (Zea mays) mutant elongated mesocotyl1 (elm1) has previously been shown to be deficient in the synthesis of the phytochrome chromophore 3E-phytochromobilin (PΦB). To identify the Elm1 gene, a maize homolog of the Arabidopsis PΦB synthase gene AtHY2 was isolated and designated ZmHy2. ZmHy2 encodes a 297-amino acid protein of 34 kD that is 50% identical to AtHY2. ZmHY2 was predicted to be plastid localized and was targeted to chloroplasts following transient expression in tobacco (Nicotiana plumbaginifolia) leaves. Molecular mapping indicated that ZmHy2 is a single copy gene in maize that is genetically linked to the Elm1 locus. Sequence analysis revealed that the ZmHy2 gene of elm1 mutants contains a single G to A transition at the 3′ splice junction of intron III resulting in missplicing and premature translational termination. However, flexibility in the splicing machinery allowed a small pool of in-frame ZmHy2 transcripts to accumulate in elm1 plants. In addition, multiple ZmHy2 transcript forms accumulated in both wild-type and elm1 mutant plants. ZmHy2 splice variants were expressed in Escherichia coli and products examined for activity using a coupled apophytochrome assembly assay. Only full-length ZmHY2 (as defined by homology to AtHY2) was found to exhibit PΦB synthase activity. Thus, the elm1 mutant of maize is deficient in phytochrome response due to a lesion in a gene encoding phytochromobilin synthase that severely compromises the PΦB pool.
Light Regulation of the Arabidopsis Respiratory Chain. Multiple Discrete Photoreceptor Responses Contribute to Induction of Type II NAD(P)H Dehydrogenase Genes
Authors: Escobar Matthew A. , Franklin Keara A. , Svensson Å. Staffan , Salter Michael G. , Whitelam Garry C. , Rasmusson Allan G.
Published: September 2004

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism.
Seed-Specific Heterologous Expression of a Nasturtium FAE Gene in Arabidopsis Results in a Dramatic Increase in the Proportion of Erucic Acid
Authors: Mietkiewska Elzbieta , Giblin E. Michael , Wang Song , Barton Dennis L. , Dirpaul Joan , Brost Jennifer M. , Katavic Vesna , Taylor David C.
Published: September 2004

The fatty acid elongase [often designated FAE or β-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium β-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.
Embryo-Specific Reduction of ADP-Glc Pyrophosphorylase Leads to an Inhibition of Starch Synthesis and a Delay in Oil Accumulation in Developing Seeds of Oilseed Rape
Authors: Vigeolas Helene , Möhlmann Torsten , Martini Norbert , Neuhaus H. Ekkehard , Geigenberger Peter
Published: September 2004

In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting 14C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of 14C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation.
Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen
Authors: Scheible Wolf-Rüdiger , Morcuende Rosa , Czechowski Tomasz , Fritz Christina , Osuna Daniel , Palacios-Rojas Natalia , Schindelasch Dana , Thimm Oliver , Udvardi Michael K. , Stitt Mark
Published: September 2004

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.
The Capacity of Green Oilseeds to Utilize Photosynthesis to Drive Biosynthetic Processes
Authors: Ruuska Sari A. , Schwender Jörg , Ohlrogge John B.
Published: September 2004

Seeds of many plant species are green during embryogenesis. To directly assess the influence of light on the physiological status of green oilseeds in planta, Brassica napus and soybean (Glycine max) seeds were rapidly dissected from plants growing in the light or dark. The activation state of malate dehydrogenase, which reflects reduced thioredoxin and NADP/NADPH ratios, was found to be as high in seeds exposed to light as in leaves and to decrease in the dark. Rubisco was highly activated (carbamylated) in both light and dark, most likely reflecting high seed CO2 concentrations. Activities of Rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO2 produced during B. napus oil biosynthesis. To determine the influence of light on oil synthesis in planta, siliques on intact plants in full sunlight or detached siliques fed 3H2O were partly covered with aluminum foil. Seeds from light and dark sections were analyzed, and fatty acid accumulation was found to be higher in seeds exposed to light than seeds from dark sections. The spectrum of light filtering through silique walls and the pigment composition of developing B. napus embryos were determined. In addition to a low chlorophyll a/b ratio, the carotenoid pigments of seeds can provide additional capture of the green light that filters through siliques. Together, these results demonstrate that even the low level of light reaching seeds plays a substantial role in activating light-regulated enzymes, increasing fatty acid synthesis, and potentially powering refixation of CO2.
Protection of Plasma Membrane K+ Transport by the Salt Overly Sensitive1 Na+-H+ Antiporter during Salinity Stress
Authors: Qi Zhi , Spalding Edgar P.
Published: September 2004

Physicochemical similarities between K+ and Na+ result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K+ nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K+ transport mutants akt1 (Arabidopsis K+ transporter) and skor (shaker-like K+ outward-rectifying channel). The K+-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na+-free media displayed wild-type K+ dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K+ permeability and growth rate in K+-limiting conditions of sos1 but not wild-type plants. Increasing K+ availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na+, the SOS1 Na+-H+ antiporter is necessary for protecting the K+ permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na+ levels predicted to result from loss of SOS1 function impaired the K+ permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K+ channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K+ uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na+ on K+ transport is probably more important at the uptake stage than at the xylem loading stage.
A macroscale analysis of coastal steepening around the coast of England and Wales
Authors: Taylor J A , Murdock A P , Pontee N I
Published: September 2004

Coastal steepening potentially presents an array of management issues in the form of financial implications of sea defence degradation, increased risk posed to the hinterland as wave attenuation is reduced, ‘coastal squeeze’ and statutory requirements in the light of the Habitats Directive. The extent to which coastal steepening has occurred throughout England and Wales has been investigated through use of a GIS and dataset based on historical Ordnance Survey map information. Data were collected along 1084 selected profile lines, positioned so as to be geomorphologically representative of the coast. Features recorded from each map year included the positions of mean high water (MHW) and mean low water (MLW), the relative movements of which infer changing intertidal gradients. The results presented in this paper are on a subject and scale not previously published. It is revealed that 61% of the coastline studied has experienced a tendency towards steepening. Of the remainder, 33% has flattened, and 6% has experienced no rotational movement. This tendency towards steepening has been the dominant movement on each of the west, south, and east coasts.