bluuebunny/crossref_metadata_2025 huggingface dataset:
Poised for Contagion: Evolutionary Origins of the Infectious Abilities of Invertebrate Retroviruses
Authors: Malik Harmit S. , Henikoff Steve , Eickbush Thomas H.
Published: July 2002

Phylogenetic analyses suggest that long-terminal repeat (LTR) bearing retrotransposable elements can acquire additional open-reading frames that can enable them to mediate infection. Whereas this process is best documented in the origin of the vertebrate retroviruses and their acquisition of an envelope (env) gene, similar independent events may have occurred in insects, nematodes, and plants. The origins of env-like genes are unclear, and are often masked by the antiquity of the original acquisitions and by their rapid rate of evolution. In this report, we present evidence that in three other possible transitions of LTR retrotransposons to retroviruses, an envelope-like gene was acquired from a viral source. First, the gypsy and related LTR retrotransposable elements (the insect errantiviruses) have acquired their envelope-like gene from a class of insect baculoviruses (double-stranded DNA viruses with no RNA stage). Second, the Cer retroviruses in the Caenorhabditis elegans genome acquired their envelope gene from a Phleboviral (single ambisense-stranded RNA viruses) source. Third, the Tas retroviral envelope (Ascaris lumricoides) may have been obtained fromHerpesviridae (double-stranded DNA viruses, no RNA stage). These represent the only cases in which the env gene of a retrovirus has been traced back to its original source. This has implications for the evolutionary history of retroviruses as well as for the potential ability of all LTR-retrotransposable elements to become infectious agents.
Potential Gene Conversion and Source Genes for Recently Integrated Alu Elements
Authors: Roy Astrid M. , Carroll Marion L. , Nguyen Son V. , Salem Abdel-Halim , Oldridge Michael , Wilkie Andrew O. M. , Batzer Mark A. , Deininger Prescott L.
Published: July 2002

Alu elements comprise >10% of the human genome. We have used a computational biology approach to analyze the human genomic DNA sequence databases to determine the impact of gene conversion on the sequence diversity of recently integrated Alu elements and to identify Alu elements that were potentially retroposition competent. We analyzed 269 Alu Ya5 elements and identified 23 members of a new Alu subfamily termed Ya5a2 with an estimated copy number of 35 members, including the de novo Alu insertion in the NF1 gene. Our analysis of Alu elements containing one to four (Ya1–Ya4) of the Ya5 subfamily-specific mutations suggests that gene conversion contributed as much as 10%–20% of the variation between recently integrated Alu elements. In addition, analysis of the middle A-rich region of the different Alu Ya5 members indicates a tendency toward expansion of this region and subsequent generation of simple sequence repeats. Mining the databases for putative retroposition-competent elements that share 100% nucleotide identity to the previously reported de novo Alu insertions linked to human diseases resulted in the retrieval of 13 exact matches to the NF1 Alu repeat, three to the Alu element in BRCA2, and one to the Alu element in FGFR2 (Apert syndrome). Transient transfections of the potential source gene for the Apert's Alu with its endogenous flanking genomic sequences demonstrated the transcriptional and presumptive transpositional competency of the element.
Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle
Authors: Smith Timothy P.L. , Grosse William M. , Freking Brad A. , Roberts Andrew J. , Stone Roger T. , Casas Eduardo , Wray James E. , White Joseph , Cho Jennifer , Fahrenkrug Scott C. , Bennett Gary L. , Heaton Michael P. , Laegreid William W. , Rohrer Gary A. , Chitko-McKown Carol G. , Pertea Geo , Holt Ingeborg , Karamycheva Svetlana , Liang Feng , Quackenbush John , Keele John W.
Published: July 2002

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000–23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.
Analysis of Conserved Noncoding DNA inDrosophilaReveals Similar Constraints in Intergenic and Intronic Sequences
Authors: Bergman Casey M. , Kreitman Martin
Published: July 2002

Comparative genomic approaches to gene andcis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation inDrosophilafor 40 loci with known or suspectedcis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%–26% of noncoding sequences surveyed are conserved inDrosophila, with median block length ∼19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models ofcis-regulatory DNA sequence evolution.
Argus—A New Database System for Web-Based Analysis of Multiple Microarray Data Sets
Authors: Comander Jason , Weber Griffin M. , Gimbrone Michael A. , García-Cardeña Guillermo
Published: July 2002

The ongoing revolution in microarray technology allows biologists studying gene expression to routinely collect >105 data points in a given experiment. Widely accessible and versatile database software is required to process this large amount of raw data into a format that facilitates the development of new biological insights. Here, we present a novel microarray database software system, namedArgus, designed to process, analyze, manage, and publish microarray data. Argus imports the intensities and images of externally quantified microarray spots, performs normalization, and calculates ratios of gene expression between conditions. The database can be queried locally or over the Web, providing a convenient format for Web-publishing entire microarray data sets. Searches for regulated genes can be conducted across multiple experiments, and the integrated results incorporate images of the actual hybridization spots for artifact screening. Query results are presented in a clone- or gene-oriented fashion to rapidly identify highly regulated genes, and scatterplots of expression ratios allow an individual ratio to be interpreted in the context of all data points in the experiment. Algorithms were developed to optimize response times for queries of regulated genes. Supporting databases are updated easily to maintain current gene identity information, and hyperlinks to the Web provide access to descriptions of gene function. Query results also can be exported for higher-order analyses of expression patterns. This combination of features currently is not available in similar software. Argus is available athttp://vessels.bwh.harvard.edu/software/Argus.
Large-Scale Clustering of cDNA-Fingerprinting Data
Authors: Herwig Ralf , Poustka Albert J. , Müller Christine , Bull Christof , Lehrach Hans , O'Brien John
Published: July 2002

Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order of hundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting—a method for simultaneous determination of expression level for every active gene of a specific tissue—although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points,x and y, we introduce mutual information that can be interpreted as the amount of information about x iny, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed.
A Comprehensive Approach to Clustering of Expressed Human Gene Sequence: The Sequence Tag Alignment and Consensus Knowledge Base
Authors: Miller Robert T. , Christoffels Alan G. , Gopalakrishnan Chella , Burke John , Ptitsyn Andrey A. , Broveak Tania R. , Hide Winston A.
Published: July 2002

The expressed human genome is being sequenced and analyzed by disparate groups producing disparate data. The majority of the identified coding portion is in the form of expressed sequence tags (ESTs). The need to discover exonic representation and expression forms of full-length cDNAs for each human gene is frustrated by the partial and variable quality nature of this data delivery. A highly redundant human EST data set has been processed into integrated and unified expressed transcript indices that consist of hierarchically organized human transcript consensi reflecting gene expression forms and genetic polymorphism within an index class. The expression index and its intermediate outputs include cleaned transcript sequence, expression, and alignment information and a higher fidelity subset, SANIGENE. The STACK_PACK clustering system has been applied to dbEST release 121598 (GenBank version 110). Sixty-four percent of 1,313,103 Homo sapiens ESTs are condensed into 143,885 tissue level multiple sequence clusters; linking through clone-ID annotations produces 68,701 total assemblies, such that 81% of the original input set is captured in a STACK multiple sequence or linked cluster. Indexing of alignments by substituent EST accession allows browsing of the data structure and its cross-links to UniGene. STACK metaclusters consolidate a greater number of ESTs by a factor of 1.86 with respect to the corresponding UniGene build. Fidelity comparison with genome reference sequenceAC004106 demonstrates consensus expression clusters that reflect significantly lower spurious repeat sequence content and capture alternate splicing within a whole body index cluster and three STACK v.2.3 tissue-level clusters. Statistics of a staggered release whole body index build of STACK v.2.0 are presented.
Prediction of Gene Function by Genome-Scale Expression Analysis: Prostate Cancer-Associated Genes
Authors: Walker Michael G. , Volkmuth Wayne , Sprinzak Einat , Hodgson David , Klingler Tod
Published: July 2002

We wish to identify genes associated with disease. To do so, we look for novel genes whose expression patterns mimic those of known disease-associated genes, using a method we call Guilt-by-Association (GBA), on the basis of a combinatoric measure of association. Using GBA, we have examined the expression of 40,000 human genes in 522 cDNA libraries, and have discovered several hundred previously unidentified genes associated with cancer, inflammation, steroid-synthesis, insulin-synthesis, neurotransmitter processing, matrix remodeling, and other disease processes. The majority of the genes thus discovered show no sequence similarity to known genes, and thus could not have been identified by homology searches. We present here an example of the discovery of eight genes associated with prostate cancer. Of the 40,000 most-abundant human genes, these 8 are the most closely linked to the known diagnostic genes, and thus are prime targets for pharmaceutical research.

[The sequence data described in this paper have been submitted to the GenBank data library under accession nos.AF109298–AF109303.]
Cellular and Molecular Mechanisms of Estrogen Regulation of Memory Function and Neuroprotection Against Alzheimer's Disease: Recent Insights and Remaining Challenges
Authors: Brinton Roberta Diaz
Published: July 2002

This review focuses on recent advances in our knowledge of estrogen action in the brain. The greatest amount of attention was devoted to those studies that impact our understanding of estrogen regulation of memory function and prevention of degenerative diseases associated with memory systems, such as Alzheimer's disease. A review of recent advances in our understanding of estrogen receptors, both nuclear and membrane, is also presented. Finally, these data are considered in regard to their relevancy to the use of estrogen replacement therapy for cognitive health throughout menopause and the development of an estrogen replacement therapy designed for the unique requirements of the brain.
Plant Expansins Are a Complex Multigene Family with an Ancient Evolutionary Origin
Authors: Li Yi , Darley Catherine P. , Ongaro Verónica , Fleming Andrew , Schipper Ori , Baldauf Sandra L. , McQueen-Mason Simon J.
Published: July 2002

Expansins are a group of extracellular proteins that directly modify the mechanical properties of plant cell walls, leading to turgor-driven cell extension. Within the completely sequenced Arabidopsis genome, we identified 38 expansin sequences that fall into three discrete subfamilies. Based on phylogenetic analysis and shared intron patterns, we propose a new, systematic nomenclature of Arabidopsis expansins. Further phylogenetic analysis, including expansin sequences found here in monocots, pine (Pinus radiata, Pinus taeda), fern (Regnellidium diphyllum, Marsilea quadrifolia), and moss (Physcomitrella patens) indicate that the three plant expansin subfamilies arose and began diversifying very early in, if not before, colonization of land by plants. Closely related “expansin-like” sequences were also identified in the social amoeba,Dictyostelium discoidium, suggesting that these wall-modifying proteins have a very deep evolutionary origin.
Accumulation of Ricinoleic, Lesquerolic, and Densipolic Acids in Seeds of Transgenic Arabidopsis Plants That Express a Fatty Acyl Hydroxylase cDNA from Castor Bean
Authors: Broun P. , Somerville C.
Published: July 2002

A cDNA encoding the oleate 12-hydroxylase from castor bean (Ricinus communis L.) has previously been shown to direct the synthesis of small amounts of ricinoleic acid (12-hydroxyoctadeccis-9-enoic acid) in seeds of transgenic tobacco plants. Expression of the cDNA under control of the Brassica napus napin promoter in transgenic Arabidopsis thaliana plants resulted in the accumulation of up to 17% of seed fatty acids as ricinoleate and two novel fatty acids that have been identified by gas chromatography-mass spectrometry as lesquerolic (14-hydroxyeicos-cis-11-enoic acid) and densipolic (12-hydroxyoctadec-cis-9,15-dienoic acid) acids. Traces of auricolic acid were also observed. These results suggest that either the castor hydroxylase can utilize oleic acid and eicosenoic acid as substrates for ricinoleic and lesquerolic acid biosynthesis, respectively, or Arabidopsis contains an elongase that accepts ricinoleic acid as a substrate. These observations are also consistent with indirect biochemical evidence that an n-3 desaturase is capable of converting ricinoleic acid to densipolic acid. Expression of the castor hydroxylase also caused enhanced accumulation of oleic acid and a corresponding decrease in the levels of polyunsaturated fatty acids. Since the steady-state level of mRNA for the oleate-12 desaturase was not affected, it appears that the presence of the hydroxylase, directly or indirectly, causes posttranscriptional inhibition of desaturation.
A cDNA Clone Highly Expressed in Ripe Banana Fruit Shows Homology to Pectate Lyases
Authors: Dominguez-Puigjaner E. , Llop I. , Vendrell M. , Prat S.
Published: July 2002

A cDNA clone (Banl7), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Banl7 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Banl7 transcript can be induced in green banana fruit by exogenous application of ethylene. This demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Banl7 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Banl7 might play a role in the loss of mesocarp firmness during fruit ripening.
Expression and Native Structure of Cytosolic Class II Small Heat-Shock Proteins
Authors: Helm K. W. , Lee G. J. , Vierling E.
Published: July 2002

Higher plants synthesize small heat-shock proteins (smHSPs) from five related gene families. The class I and II families encode cytosolic smHSPs. We characterized the class II smHSPs of pea (Pisum sativum) and compared them with class I smHSPs. Antibodies against recombinant HSP17.7, a class II smHSP, recognized four heat-inducible 17- to 18-kD polypeptides and did not cross-react with class I smHSPs. On sucrose gradients the class II smHSPs sedimented primarily at 8 Svedberg units, indicating that they are components of large complexes similar in size to class I smHSP complexes. However, the class I and II complexes were readily distinguishable by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Nondenaturing immune precipitations using anti-HSP17.7 or anti-HSP18.1 (a class I smHSP) antiserum provide further evidence that the class I and II smHSPs exist in different complexes, composed primarily of smHSPs. Recombinant HSP17.7 and HSP18.1 formed complexes of sizes similar to those formed in vivo. When these two smHSPs were mixed, denatured with urea, and then dialyzed, the distinct class I and II complexes again formed, each containing only HSP18.1 or HSP17.7. Thus, cytosolic smHSPs from two related gene families expressed simultaneously form distinct complexes in vivo, suggesting that they have subtly different functions.
LHT1, A Lysine- and Histidine-Specific Amino Acid Transporter in Arabidopsis
Authors: Chen L. , Bush D. R.
Published: July 2002

We have identified a new amino acid transporter from the Arabidopsis thaliana expressed sequence tag cDNA collection by functional complementation of a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 [mu]M, respectively. This transporter (LHT1, lysine histidine transporter) has little affinity for arginine when measured directly in uptake experiments or indirectly with substrate competition. The cDNA is 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHT1 is an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT1 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter is present in all tissues, with the strongest expression in young leaves, flowers, and siliques. Wholemount, in situ hybridization revealed that expression is further localized on the surface of roots in young seedlings and in pollen. Overall, LHT1 belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given its substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains.
Regulation of Leaf Senescence by Cytokinin, Sugars, and Light
Authors: Wingler Astrid , von Schaewen Antje , Leegood Richard C. , Lea Peter J. , Paul Quick W.
Published: July 2002

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.
Herbicide Safener-Binding Protein of Maize1
Authors: Scott-Craig John S. , Casida John E. , Poduje Lisa , Walton Jonathan D.
Published: July 2002

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicidesS-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolicO-methyltransferase, but SafBP does notO-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.
Epicuticular Wax Accumulation and Fatty Acid Elongation Activities Are Induced during Leaf Development of Leeks1
Authors: Rhee Yoon , Hlousek-Radojcic Alenka , Ponsamuel Jayakumar , Liu Dehua , Post-Beittenmiller Dusty
Published: July 2002

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.
Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat1
Authors: Piñeros Miguel A. , Shaff Jon E. , Kochian Leon V.
Published: July 2002

A Cd2+-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species ofThlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO3)2solutions, the electrode exhibited a Nernstian response in solutions with Cd2+ activities as low as 50 nm. Addition of Ca2+ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd2+. Addition of high concentrations of K+ and Mg2+ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd2+ than for any of those potentially interfering ions tested. Flux measurements along the roots of the twoThlaspi species showed no differences in the pattern or the magnitude of Cd2+ uptake within the time frame considered. The Cd2+-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.
Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1
Authors: Zhou Fasong , Zhang Ziguo , Gregersen Per L. , Mikkelsen Jørn D. , de Neergaard Eigil , Collinge David B. , Thordal-Christensen Hans
Published: July 2002

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn.Erysiphe] graminis f.sp.hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encodingHvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.
A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1
Authors: Hung Chen-Yi , Lin Yan , Zhang Meng , Pollock Susan , David Marks M. , Schiefelbein John
Published: July 2002

A position-dependent pattern of epidermal cell types is produced during root development inArabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUSfusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximalGL2 activity but is not required for the pattern ofGL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation andGL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotylGL2 activity was shown to be influenced byttg and 35S::R but not bygl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG andGL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.
Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores ofPhytophthora parasitica varnicotianae1
Authors: Able Amanda J. , Guest David I. , Sutherland Mark W.
Published: July 2002

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2−) production based on reduction of the tetrazolium dye sodium,3′-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2·/O2−) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2·/O2− production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2·/O2− was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10−15 mol min−1 cell−1 were observed. The HO2·/O2− scavengers O2− dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2−production is a necessary precursor to the HR.
Expression of the YeastFREGenes in Transgenic Tobacco
Authors: Samuelsen Andrew I. , Martin Ruth C. , Mok David W.S. , Mok Machteld C.
Published: July 2002

Two yeast genes, FRE1 and FRE2 (encodingFe(III) reductases) were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into tobacco (Nicotiana tabacum L.) viaAgrobacterium tumefaciens-mediated transformation. Homozygous lines containing FRE1, FRE2, or FRE1 plus FRE2 were generated. Northern-blot analyses revealed mRNA of two different sizes in FRE1 lines, whereas all FRE2 lines had mRNA only of the expected length. Fe(III) reduction, chlorophyll contents, and Fe levels were determined in transgenic and control plants under Fe-sufficient and Fe-deficient conditions. In a normal growth environment, the highest root Fe(III) reduction, 4-fold higher than in controls, occurred in the double transformant (FRE1 + FRE2). Elevated Fe(III) reduction was also observed in all FRE2 and some FRE1 lines. The increased Fe(III) reduction occurred along the entire length of the roots and on shoot sections. FRE2 and double transformants were more tolerant to Fe deficiency in hydroponic culture, as shown by higher chlorophyll and Fe concentrations in younger leaves, whereas FRE1 transformants did not differ from the controls. Overall, the beneficial effects ofFRE2 were consistent, suggesting thatFRE2 may be used to improve Fe efficiency in crop plants.
Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds1
Authors: Yim Kyu-Ock , Bradford Kent J.
Published: July 2002

Semipermeable cell walls or apoplastic “membranes” have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (β-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by β-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.
Arabidopsis Rho-Related GTPases: Differential Gene Expression in Pollen and Polar Localization in Fission Yeast
Authors: Li Hai , Wu Guang , Ware Doreen , Davis Keith R. , Yang Zhenbiao
Published: July 2002

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases fromplants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps,Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation forRop1At is much higher than that forRop3At and Rop5At. Furthermore,Rop1At is specifically expressed in anthers, whereasRop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing theRop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.
A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding
Authors: Olsson Anne , Svennelid Fredrik , Ek Bo , Sommarin Marianne , Larsson Christer
Published: July 2002

We have isolated the plasma membrane H+−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H+-ATPase, thus activating H+ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H+-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H+-ATPase.
Differential Expression and Internal Feedback Regulation of 1-Aminocyclopropane-1-Carboxylate Synthase, 1-Aminocyclopropane-1-Carboxylate Oxidase, and Ethylene Receptor Genes in Tomato Fruit during Development and Ripening
Authors: Nakatsuka Akira , Murachi Shiho , Okunishi Hironori , Shiomi Shinjiro , Nakano Ryohei , Kubo Yasutaka , Inaba Akitsugu
Published: July 2002

We investigated the feedback regulation of ethylene biosynthesis in tomato (Lycopersicon esculentum) fruit with respect to the transition from system 1 to system 2 ethylene production. The abundance of LE-ACS2, LE-ACS4, andNR mRNAs increased in the ripening fruit concomitant with a burst in ethylene production. These increases in mRNAs with ripening were prevented to a large extent by treatment with 1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts for the LE-ACS6 gene, which accumulated in preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit with propylene prevented the accumulation of transcripts for this gene.LE-ACS1A, LE-ACS3, andTAE1 genes were expressed constitutively in the fruit throughout development and ripening irrespective of whether the fruit was treated with MCP or propylene. The transcripts forLE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also prevented by treatment with MCP. The results suggest that in tomato fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A andLE-ACS3 and negatively feedback-regulatedLE-ACS6 genes with preexisting LE-ACO1and LE-ACO4 mRNAs. At the onset of the climacteric stage, it shifts to system 2 ethylene, with a large accumulation ofLE-ACS2, LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs as a result of a positive feedback regulation. This transition from system 1 to system 2 ethylene production might be related to the accumulated level ofNR mRNA.
Inhibition of Auxin Movement from the Shoot into the Root Inhibits Lateral Root Development in Arabidopsis
Authors: Reed Robyn C. , Brady Shari R. , Muday Gloria K.
Published: July 2002

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [3H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.
Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants1
Authors: Curioni Paola M.G. , Hartwig Ueli A. , Nösberger Josef , Schuller Kathryn A.
Published: July 2002

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH4+ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O2-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O2-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.
Complementary DNA Cloning and Characterization of Ferredoxin Localized in Bundle-Sheath Cells of Maize Leaves1
Authors: Matsumura Tomohiro , Kimata-Ariga Yoko , Sakakibara Hitoshi , Sugiyama Tatsuo , Murata Hiroshi , Takao Toshifumi , Shimonishi Yasutsugu , Hase Toshiharu
Published: July 2002

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of −423 and −406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.
Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, CytochromecOxidase, and Adenine Nucleotide Translocase Activities1
Authors: De Santis Aurelio , Landi Pierangelo , Genchi Giuseppe
Published: July 2002

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome coxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.