bluuebunny/crossref_metadata_2025 huggingface dataset:
The Comprehensive Mouse Radiation Hybrid Map Densely Cross-Referenced to the Recombination Map: A Tool to Support the Sequence Assemblies
Authors: Rowe Lucy B. , Barter Mary E. , Kelmenson Jennifer A. , Eppig Janan T.
Published: January 2003

We have developed a unique comprehensive mouse radiation hybrid (RH) map of nearly 23,000 markers integrating data from three international genome centers and over 400 independent laboratories. We have cross-referenced this map to the 0.5-cM resolution recombination-based Jackson Laboratory (TJL) backcross panel map, building a complete set of RH framework chromosome maps based on a high density of known-ordered anchor markers. We have systematically typed markers to improve coverage and resolve discrepancies, and have reanalyzed data sets as needed. The cross-linking of the RH and recombination maps has resulted in a highly accurate genome-wide map with consistent marker order. We have compared these linked framework maps to the Ensembl mouse genome sequence assembly, and show that they are a useful medium resolution tool for both validating sequence assembly and elucidating chromosome biology.

[Supplemental material is available online at www.genome.org.]
Comparative Gene Prediction in Human and Mouse
Authors: Parra Genı́s , Agarwal Pankaj , Abril Josep F. , Wiehe Thomas , Fickett James W. , Guigó Roderic
Published: January 2003

The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level with the mouse genome can help eliminate some of those false positives. Here we describe SGP2, a gene prediction program that combines ab initio gene prediction with TBLASTX searches between two genome sequences to provide both sensitive and specific gene predictions. The accuracy of SGP2 when used to predict genes by comparing the human and mouse genomes is assessed on a number of data sets, including single-gene data sets, the highly curated human chromosome 22 predictions, and entire genome predictions from ENSEMBL. Results indicate that SGP2 outperforms purely ab initio gene prediction methods. Results also indicate that SGP2 works about as well with 3x shotgun data as it does with fully assembled genomes. SGP2 provides a high enough specificity that its predictions can be experimentally verified at a reasonable cost. SGP2 was used to generate a complete set of gene predictions on both the human and mouse by comparing the genomes of these two species. Our results suggest that another few thousand human and mouse genes currently not in ENSEMBL are worth verifying experimentally.
The Companion Cell-Specific Arabidopsis Disaccharide Carrier AtSUC2 Is Expressed in Nematode-Induced Syncytia
Authors: Juergensen Katja , Scholz-Starke Joachim , Sauer Norbert , Hess Paul , van Bel Aart J.E. , Grundler Florian M.W.
Published: January 2003

Cyst nematodes induce a metabolically highly active syncytial cell complex in host roots. The syncytia are symplastically isolated. Because they form a strong sink, assimilates must be imported via the apoplast, thus suggesting that specific membrane-bound sugar transport proteins are expressed and activated. To identify possible candidate genes, transgenic Arabidopsis plants expressing different reporter genes under the control of different promoters from Arabidopsis sugar transporter genes were infected with the beet cyst nematode (Heterodera schachtii). With polymerase chain reaction, 13 additional sugar transporters were tested for their presence in the syncytia through the use of a syncytium-specific cDNA library. Analysis of the infected roots showed that the promoter of the sucrose (Suc) transporter AtSUC2 gene that codes for a companion cell-specific Suc transporter in noninfected plants was found to be expressed in syncytia. Its expression patterns in β-glucuronidase and green fluorescent protein plants were monitored. Syncytium-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction. Results support the idea that AtSUC2 mediates the transmembrane transfer of Suc. AtSUC2 is the first disaccharide carrier described to be activated by pathogens.
RNA Target Sequences Promote Spreading of RNA Silencing
Authors: Van Houdt Helena , Bleys Annick , Depicker Anna
Published: January 2003

It is generally recognized that a silencing-inducing locus can efficiently reduce the expression of genes that give rise to transcripts partially homologous to those produced by the silencing-inducing locus (primary targets). Interestingly, the expression of genes that produce transcripts without homology to the silencing-inducing locus (secondary targets) can also be decreased dramatically via transitive RNA silencing. This phenomenon requires primary target RNAs that contain sequences homologous to secondary target RNAs. Sequences upstream from the region homologous to the silencing inducer in the primary target transcripts give rise to approximately 22-nucleotide small RNAs, coinciding with the region homologous to the secondary target. The presence of these small RNAs corresponds with reduced expression of the secondary target whose transcripts are not homologous to the silencing inducer. The data suggest that in transgenic plants, targets of RNA silencing are involved in the expansion of the pool of functional small interfering RNAs. Furthermore, methylation of target genes in sequences without homology to the initial silencing inducer indicates not only that RNA silencing can expand across target RNAs but also that methylation can spread along target genes.
Sugar-Regulated Expression of a Putative Hexose Transport Gene in Grape
Authors: Atanassova Rossitza , Leterrier Marina , Gaillard Cécile , Agasse Alice , Sagot Emeric , Coutos-Thévenot Pierre , Delrot Serge
Published: January 2003

Different lengths of the promoter of grape (Vitis vinifera) VvHT1 (Hexose Transporter 1) gene, which encodes a putative hexose transporter expressed during the ripening of grape, have been transcriptionally fused to the β-glucuronidase reporter gene. In transgenic tobacco (Nicotiana tabacum) transformed with these constructs,VvHT1 promoters were clearly responsible for the sink organ preferential expression. The potential sugar effectors ofVvHT1 promoter were studied in tobacco cv Bright-Yellow 2 cells transformed with chimeric constructs. Glucose (56 mm), sucrose (Suc; 58 mm), and the non-transported Suc isomer palatinose doubled the β-glucuronidase activity conferred by the VvHT1 promoter, whereas fructose did not affect it. These effects were the strongest with the 2.4-kb promoter, which contains all putative sugar-responsive elements (activating and repressing), but they were also significant with the 0.3-kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both Suc and palatinose was confirmed in the homologous grape berry cell culture. The data provide the first example of a putative sugar transporter, which is induced by both glucose and Suc in higher plants. Although induction ofVvHT1 expression by Suc does not require transport, the presence of glucosyl moiety is necessary for Suc sensing. These results provide new insights into sugar sensing and signaling in plants.
Divergent Light-, Ascorbate-, and Oxidative Stress-Dependent Regulation of Expression of the Peroxiredoxin Gene Family in Arabidopsis
Authors: Horling Frank , Lamkemeyer Petra , König Janine , Finkemeier Iris , Kandlbinder Andrea , Baier Margarete , Dietz Karl-Josef
Published: January 2003

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q andprx II E, decreased upon application of ascorbate.prx Q also responded to peroxides and diamide treatment.prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (Em′) of Prx II B, Prx II C, and Prx II E were around −290 mV and, thus, less negative than Em′ of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (−307 to −322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.
trans-Resveratrol and Grape Disease Resistance. A Dynamical Study by High-Resolution Laser-Based Techniques
Authors: Montero C. , Cristescu S.M. , Jiménez J.B. , Orea J.M. , te Lintel Hekkert S. , Harren F.J.M. , González Ureña A.
Published: January 2003

Two modern laser-based techniques were synchronously applied to study the dynamics of the trans-resveratrol activity in Botrytis cinerea-infected grapes. Direct analysis of trans-resveratrol in both infected and noninfected grapes (Vitis vinifera, Aledo variety) was performed by using an analytical technique incorporating laser desorption coupled with laser resonant ionization and time-of-flight mass spectrometry. On the other hand, one of the most sensitive on-line methods for trace gas detection, laser photoacoustic spectroscopy, was used to investigate the involvement of the plant hormone ethylene (C2H4) in theB. cinerea grapes interaction and its temporal relationship with the trans-resveratrol content upon infection. The trans-resveratrol content and the ethylene released by noninfected grapes showed an opposite behavior. In this case, a high trans-resveratrol content corresponds to a low ethylene emission. For the B. cinerea-infected grapes, ethylene emission rises up after 48 h when the analogous content of trans-resveratrol started to decrease irreversibly. Moreover, the activity of trans-resveratrol as natural pesticide has been investigated by exogenous application on grapes. A short submerge (5 s) of the grapes in 1.6 × 10−4  m solution of trans-resveratrol delays the increase of C2H4 emission with about 48 h and produces a decrease of the C2H4 concentration and its emission rate. The treatment has positive effects on fruit conservation during storage; it doubled the normal shelf-life of grapes at room temperature, maintaining their post-harvest quality within 10 d.
Regulation of Early Tomato Fruit Development by theDiageotropicaGene
Authors: Balbi Virginia , Lomax Terri L.
Published: January 2003

The vegetative phenotype of the auxin-resistantdiageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) includes reduced gravitropic response, shortened internodes, lack of lateral roots, and retarded vascular development. Here, we report that early fruit development is also dramatically altered by the single-genedgt lesion. Fruit weight, fruit set, and numbers of locules and seeds are reduced in dgt. In addition, time to flowering and time from anthesis to the onset of fruit ripening are increased by the dgt lesion, whereas ripening is normal. The dgt mutation appears to affect only the early stages of fruit development, irrespective of allele or genetic background. Expression of members of the LeACS(1-aminocyclopropane-1-carboxylic acid synthase, a key regulatory enzyme of ethylene biosynthesis) and LeIAA(Aux/IAA, auxin-responsive) gene families were quantified via real-time reverse transcriptase-polymerase chain reaction in both dgt and wild-type fruits, providing the first analysis of Aux/IAA gene expression in fruit. Thedgt lesion affects the expression of only certain members of both the LeACS and LeIAAmultigene families. Different subsets of LeIAA gene family members are affected by the dgt mutation in fruits and hypocotyls, indicating that the DGT gene product functions in a developmentally specific manner. The differential expression of subsets of LeIAA and LeACS gene family members as well as the alterations in dgt fruit morphology and growth suggest that the early stages of fruit development in tomato are regulated, at least in part, by auxin- and ethylene-mediated gene expression.
Ethylene Biosynthesis in Detached Young Persimmon Fruit Is Initiated in Calyx and Modulated by Water Loss from the Fruit
Authors: Nakano Ryohei , Ogura Emi , Kubo Yasutaka , Inaba Akitsugu
Published: January 2003

Persimmon (Diospyros kaki Thunb.) fruit are usually classified as climacteric fruit; however, unlike typical climacteric fruits, persimmon fruit exhibit a unique characteristic in that the younger the stage of fruit detached, the greater the level of ethylene produced. To investigate ethylene induction mechanisms in detached young persimmon fruit, we cloned three cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DK-ACS1, 2, and -3) and two encoding ACC oxidase (DK-ACO1 and -2) genes involved in ethylene biosynthesis, and we analyzed their expression in various fruit tissues. Ethylene production was induced within a few days of detachment in all fruit tissues tested, accompanied by temporally and spatially coordinated expression of all theDK-ACS andDK-ACO genes. In all tissues except the calyx, treatment with 1-methylcyclopropene, an inhibitor of ethylene action, suppressed ethylene production and ethylene biosynthesis-related gene expression. In the calyx, one ACC synthase gene (DK-ACS2) exhibited increased mRNA accumulation accompanied by a large quantity of ethylene production, and treatment of the fruit with 1-methylcyclopropene did not prevent either the accumulation of DK-ACS2transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2in the calyx. These results indicate that ethylene biosynthesis in detached young persimmon fruit is initially induced in calyx and is modulated by water loss through transcriptional activation ofDK-ACS2. The ethylene produced in the calyx subsequently diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis in these tissues, leading to a burst of ethylene production.
Metabolism of Sugars in the Endosperm of Developing Seeds of Oilseed Rape
Authors: Hill Lionel M. , Morley-Smith Edward R. , Rawsthorne Stephen
Published: January 2003

The sugars in the endosperm of a developing seed have many potential roles, including the supply of carbon to the developing embryo and controlling gene expression in it. Our understanding of their metabolism is, however, fragmentary and is confined to a very few species (especially Vicia spp.). To develop a quantitative understanding of the regulation of sugars in seeds of oilseed rape (Brassica napus), we measured relevant enzyme activities, the sizes of the pools of sugars in the liquid endosperm, and the flux of sugars from the endosperm into the embryo. The concentrations of hexose sugars in the liquid endosperm decreased, and sucrose (Suc) increased through development. The overall osmotic potential also fell. The timing of the changes was not precise enough to determine whether they signaled the onset of rapid accumulation of storage products. Changes in endosperm invertase activity were complex and quantitatively do not explain the changes in sugars. The embryo can metabolize hexose sugars in addition to Suc, and possibly at higher rates. Therefore, in addition to invertase, the growing embryo itself has a potential to influence the balance of sugars in the endosperm. The activity of Suc synthase in the embryo was greater than that of invertase during development. This observation and a higher activity of fructokinase than glucokinase in the embryo are both consistent with the embryo using Suc as a carbon source.
Modulation of CYP79 Genes and Glucosinolate Profiles in Arabidopsis by Defense Signaling Pathways
Authors: Mikkelsen Michael Dalgaard , Petersen Bent Larsen , Glawischnig Erich , Jensen Anders Bøgh , Andreasson Erik , Halkier Barbara Ann
Published: January 2003

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 andCYP79B3 were both highly induced. Specifically, the indole glucosinolateN-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutantcoi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducingmpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.
Functional Mitochondrial Complex I Is Required by Tobacco Leaves for Optimal Photosynthetic Performance in Photorespiratory Conditions and during Transients
Authors: Dutilleul Christelle , Driscoll Simon , Cornic Gabriel , De Paepe Rosine , Foyer Christine H. , Noctor Graham
Published: January 2003

The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO2showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO2 levels. The inhibition of photosynthesis was alleviated by high CO2 or low O2. The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO2 evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously.
Spatial and Temporal Effects of Free-Air CO2Enrichment (POPFACE) on Leaf Growth, Cell Expansion, and Cell Production in a Closed Canopy of Poplar
Authors: Taylor Gail , Tricker Penny J. , Zhang Fang Z. , Alston Victoria J. , Miglietta Franco , Kuzminsky Elena
Published: January 2003

Leaf expansion in the fast-growing tree,Populus × euramericana was stimulated by elevated [CO2] in a closed-canopy forest plantation, exposed using a free air CO2 enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO2] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0–3) and late (LPI, 6–8) stages in development. Early and late effects of elevated [CO2] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO2] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO2]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO2] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO2] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO2].
Alanine Aminotransferase Homologs Catalyze the Glutamate:Glyoxylate Aminotransferase Reaction in Peroxisomes of Arabidopsis
Authors: Liepman Aaron H. , Olsen Laura J.
Published: January 2003

Plant peroxisomal glyoxylate aminotransferases play central roles within the photorespiratory pathway. Genes encoding glyoxylate aminotransferases have been isolated from several animals and microbes, but only recently have plant homologs been identified. Three Arabidopsis homologs of alanine (Ala):glyoxylate aminotransferase 2 (AGT2) contain a putative type 1 peroxisomal targeting signal (PTS1), but the metabolic significance of these AGT2 homologs is unknown. GGT1 and GGT2 are Ala aminotransferase (AlaAT) homologs from Arabidopsis that represent another type of glyoxylate aminotransferase. These proteins are class I aminotransferases, each containing a putative PTS1. GGT1 and GGT2 are members of a small family of AlaATs in Arabidopsis. When expressed as recombinant proteins inEscherichia coli, GGT1 and GGT2 displayed biochemical characteristics very similar to one another, and to the Arabidopsis protein purified from leaves. Four aminotransferase activities were specifically associated with GGT1 and GGT2, using the substrate pairs glutamate (Glu):glyoxylate, Ala:glyoxylate, Glu:pyruvate, and Ala:2-oxoglutarate. GGT1 and GGT2 may have partially redundant functions; transcripts of both genes were detected in many of the same tissues. Although Glu:glyoxylate aminotransferase (GGT) activity has been observed in several locations in different plants and algae, including the cytoplasm and mitochondria, our subcellular fractionation data indicate that GGT activity was exclusively peroxisomal in Arabidopsis. Thus, glyoxylate aminotransferase reactions in plant peroxisomes appear to be catalyzed by at least two distinct types of aminotransferases: an AGT1 homolog with serine:glyoxylate aminotransferase activity (A.H. Liepman, L.J. Olsen [2001] Plant J 25: 487–498), and a pair of closely related, potentially redundant AlaAT homologs with GGT activity.
The Arabidopsis AtIPT8/PGA22 Gene Encodes an Isopentenyl Transferase That Is Involved in De Novo Cytokinin Biosynthesis
Authors: Sun Jiaqiang , Niu Qi-Wen , Tarkowski Petr , Zheng Binglian , Tarkowska Danuse , Sandberg Göran , Chua Nam-Hai , Zuo Jianru
Published: January 2003

Cytokinin plays a critical role in plant growth and development by stimulating cell division and cell differentiation. Despite many years' research efforts, our current understanding of this hormone is still limited regarding both its biosynthesis and signaling. To genetically dissect the cytokinin pathway, we have used a functional screen to identify Arabidopsis gain-of-function mutations that enable shoot formation in the absence of exogenous cytokinins. By using a chemical-inducible activation tagging system, we have identified over 40 putative mutants, designated as pga(plant growth activators), which presumably were affected in key components of cytokinin biosynthesis and signaling pathway. Here, we report a detailed characterization ofpga22, a representative mutant from this collection. A gain-of-function mutation in the PGA22 locus resulted in typical cytokinin responses. Molecular and genetic analyses indicated that PGA22 encodes an isopentenyl transferase (IPT) previously identified as AtIPT8. Plants of the pga22mutant accumulated at remarkably higher levels of isopentenyladenosine-5′-monophosphate and isopentenyladenosine when analyzed by mass spectrometry, suggesting that AtIPT8/PGA22 is a functional IPT that may direct the biosynthesis of cytokinins in planta via an isopentenyladenosine-5′-monophosphate-dependent pathway.
ARAMEMNON, a Novel Database for Arabidopsis Integral Membrane Proteins
Authors: Schwacke Rainer , Schneider Anja , van der Graaff Eric , Fischer Karsten , Catoni Elisabetta , Desimone Marcelo , Frommer Wolf B. , Flügge Ulf-Ingo , Kunze Reinhard
Published: January 2003

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.
Elicitor Activity of a Fungal Endopolygalacturonase in Tobacco Requires a Functional Catalytic Site and Cell Wall Localization
Authors: Boudart Georges , Charpentier Myriam , Lafitte Claude , Martinez Yves , Jauneau Alain , Gaulin Elodie , Esquerré-Tugayé Marie-Thérèse , Dumas Bernard
Published: January 2003

CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared:CLPG1, with or without its signal peptide (SP; PG1, PG1ΔSP); CLPG1 with the tobaccoexpansin1 SP instead of its own SP (Exp::PG1ΔSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and β-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco.
The Sucrose TransporterStSUT1Localizes to Sieve Elements in Potato Tuber Phloem and Influences Tuber Physiology and Development,
Authors: Kühn Christina , Hajirezaei Mohammad-Reza , Fernie Alisdair R. , Roessner-Tunali Ute , Czechowski Tomasz , Hirner Brigitte , Frommer Wolf B.
Published: January 2003

The sucrose (Suc) H+-cotransporterStSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. ReducedSUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc- and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm.
Regulatory Networks in Seeds Integrating Developmental, Abscisic Acid, Sugar, and Light Signaling
Authors: Brocard-Gifford Inès M. , Lynch Tim J. , Finkelstein Ruth R.
Published: January 2003

Progression through embryogenesis and the transition to germination is subject to regulation by many transcription factors, including those encoded by the Arabidopsis LEC1 (LEAFY COTYLEDON1), FUS3 (FUSCA3), and abscisic acid-insensitive (ABI) ABI3,ABI4, and ABI5 loci. To determine whether the ABI4, ABI5, LEC1, andFUS3 loci interact or act independently, we analyzedabi fus3 and abi lec1 double mutants. Our results show that both ABI4 and ABI5interact genetically with both LEC1 andFUS3 in controlling pigment accumulation, suppression of vivipary, germination sensitivity to abscisic acid, gene expression during mid- and late embryogenesis, sugar metabolism, sensitivity to sugar, and etiolated growth. However, the relative strengths of the observed interactions vary among responses and may even be antagonistic. Furthermore, the interactions reveal cryptic effects of individual loci that are not detectable by analyses of single mutants. Despite these strong genetic interactions, but consistent with the disparities in peak expression of these loci, none of the ABI transcription factors appear to interact directly with either FUS3 or LEC1 in a yeast (Saccharomyces cerevisiae) two-hybrid assay system.
Molecular Cloning and Biological Activity of α-, β-, and γ-Megaspermin, Three Elicitins Secreted by Phytophthora megasperma H20
Authors: Baillieul Fabienne , de Ruffray Patrice , Kauffmann Serge
Published: January 2003

We report on the molecular cloning of thePhytophthora megasperma H20 (PmH20) glycoprotein shown previously as an inducer of the hypersensitive response, of localized acquired resistance and of systemic acquired resistance in tobacco (Nicotiana tabacum), and of the PmH20 α- and β-megaspermin, two elicitins of class I-A and I-B, respectively. The structure of the glycoprotein shows a signal peptide of 20 amino acids followed by the typical elicitin 98-amino acid-long domain and a 77-amino acid-long C-terminal domain carrying anO-glycosylated moiety. The molecular mass deduced from the translated cDNA sequence is 14,920 and 18,676 D as determined by mass spectrometry. This structure together with multiple sequence alignments and phylogenetic analyses indicate that the glycoprotein belongs to class III elicitins. It is the first class III elicitin protein characterized, which we named γ-megaspermin. We compared the biological activity of the three PmH20 elicitins when applied to tobacco cv Samsun NN plants. Although α- and γ-megaspermin were similarly active, β-megaspermin was the most active in inducing the hypersensitive response and localized acquired resistance, which was assessed by measuring the levels of acidic and basic pathogenesis-related proteins and of the antioxidant phytoalexin scopoletin. The three elicitins induced similar levels of systemic acquired resistance measured as the expression of acidic PR proteins and is increased resistance to challenge tobacco mosaic virus infection.
AtSTP6, a New Pollen-Specific H+-Monosaccharide Symporter from Arabidopsis
Authors: Scholz-Starke Joachim , Büttner Michael , Sauer Norbert
Published: January 2003

This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H+/monosaccharide transporter family. The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices. Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K  m = 20 μm), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes. Analyses ofAtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6expression only during the late stages of pollen development. A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6mutation on pollen viability, pollen germination, fertilization, and seed production. However, differences between wild-type and mutant plants could not be observed.
Do Phytotropins Inhibit Auxin Efflux by Impairing Vesicle Traffic?
Authors: Petrášek Jan , Černá Adriana , Schwarzerová Kateřina , Elčkner Miroslav , Morris David A. , Zažı́malová Eva
Published: January 2003

Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.
Identification and Characterization of the ARIADNEGene Family in Arabidopsis. A Group of Putative E3 Ligases
Authors: Mladek Christina , Guger Klaus , Hauser Marie-Theres
Published: January 2003

ARIADNE (ARI) proteins were recently identified in fruitfly (Drosophila melanogaster), mouse, and man because of their specific interaction with the ubiquitin-conjugating (E2) enzymes UbcD10, UbcM4, UbcH7, and UbcH8. They are characterized by specific motifs and protein structures that they share with PARKIN, and there is increasing evidence that ARI/PARKIN proteins function as E2-dependent ubiquitin-protein ligases. On the basis of homology and motif searches, 16 AtARI genes were identified in Arabidopsis. Analysis of the position of exons/introns and their chromosomal localization indicates that the AtARI gene family expanded via larger and smaller genome duplications. We present evidence that retroposition of processed mRNA may have also contributed to enlarging this gene family. Phylogenetic analyses divides the AtARI proteins into three subgroups. Two groups are absent in yeast, invertebrates, and vertebrates and may therefore represent new plant-specific subfamilies. Examination of the predicted protein sequences revealed that the ARI proteins share an additional leucine-rich region at the N terminus that is highly conserved in all phyla analyzed. Furthermore, conserved consensus signals for casein kinase II-dependent phosphorylation and for nuclear localization were identified. The in silico-based analyses were complemented with experimental data to quantify expression levels. Using real-time polymerase chain reaction, we show that theARI genes are differentially transcribed.AtARI1 is highly expressed in all organs, whereas no transcripts could be detected for AtARI11,AtARI13, and AtARI14.AtARI12 and AtARI16 are expressed in an organ-specific manner in the roots and siliques, respectively.
Proteomics of Light-Harvesting Proteins in Different Plant Species. Analysis and Comparison by Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Photosystem II
Authors: Zolla Lello , Timperio Anna-Maria , Walcher Wolfgang , Huber Christian G.
Published: January 2003

An overview of the intact molecular masses and the hydrophobic properties of the photosystem II (PSII) light-harvesting proteins in 14 different plant species is presented. The protein separation and identification was achieved by means of reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry. The good correspondence of the molecular masses measured by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry with those deduced from the DNA sequence (0.008%–0.016% relative deviation in Arabidopsis) enabled the identification of the different protein types. Utilizing this correlation, it was possible in several cases to spot a gene product for the previously cloned genes. In PSII, all antenna proteins show hydrophobic properties considerably different within the same as well as among various species, in contrast to observations made previously with PSI. These differences might reflect a tuning of protein-protein interactions that play a role in inducing different supramolecular organizations of PSII: within the same species as a consequence of short-term adaptations, and among species for seasonal species adaptation. The relative antenna stoichiometry was readily established on the basis of relative peak areas of the separated proteins in the ultraviolet chromatograms. The correspondence found between the high copy number of genes with the gene products reveals that the genes are not silent in their protein expression. Moreover, the high copy number of gene products as well as protein heterogeneity observed in PSII suggest a possible plant strategy to realize the high degree of organization and interconnection of the light-harvesting systems under any environmental conditions.
The Pea Gene NA Encodesent-Kaurenoic Acid Oxidase
Authors: Davidson Sandra E. , Elliott Robert C. , Helliwell Chris A. , Poole Andrew T. , Reid James B.
Published: January 2003

The gibberellin (GA)-deficient dwarf namutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 hadent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway froment-kaurenoic acid to GA12 when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7α-hydroxykaurenoic acid and GA12-aldehyde, some additional products of the pea KAO activity were detected, includingent-6α,7α-dihydroxykaurenoic acid and 7β-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 andna-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with thena-1 allele cosegregated with the dwarfna phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants.PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds ofna plants.
Organ-Specific Expression of Brassinosteroid-Biosynthetic Genes and Distribution of Endogenous Brassinosteroids in Arabidopsis
Authors: Shimada Yukihisa , Goda Hideki , Nakamura Ayako , Takatsuto Suguru , Fujioka Shozo , Yoshida Shigeo
Published: January 2003

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. There is only limited information on where BRs are synthesized and used. We studied the organ specificity of BR biosynthesis in Arabidopsis, using two different approaches: We analyzed the expression of BR-related genes using real-time quantitative reverse transcriptase-polymerase chain reaction, and analyzed endogenous BRs using gas chromatography-mass spectrometry. Before starting this study, we cloned the second BR-6-oxidase (BR6ox2) gene from Arabidopsis and found that the encoded enzyme has the same substrate specificity as the enzyme encoded by the previously isolated 6-oxidase gene (BR6ox1) of Arabidopsis. Endogenous BRs and the expression of BR-related genes were detected in all organs tested. The highest level of endogenous BRs and the highest expression of the BR6ox1,BR6ox2, and DWF4 genes were observed in apical shoots, which contain actively developing tissues. These genes are important in BR biosynthesis because they encode the rate-limiting or farthest downstream enzyme in the BR biosynthesis pathway. The second highest level of endogenous BRs and expression ofBR6ox1 and DWF4 were observed in siliques, which contains actively developing embryos and seeds. These findings indicate that BRs are synthesized in all organs tested, but are most actively synthesized in young, actively developing organs. In contrast, synthesis was limited in mature organs. Our observations are consistent with the idea that BRs function as the growth-promoting hormone in plants.
Polyamine Metabolism Is Altered in Unpollinated Parthenocarpicpat-2Tomato Ovaries
Authors: Fos Mariano , Proaño Karina , Alabadı́ David , Nuez Fernando , Carbonell Juan , Garcı́a-Martı́nez José L.
Published: January 2003

Facultative parthenocarpy induced by the recessive mutationpat-2 in tomato (Lycopersicon esculentum Mill.) depends on gibberellins (GAs) and is associated with changes in GA content in unpollinated ovaries. Polyamines (PAs) have also been proposed to play a role in early tomato fruit development. We therefore investigated whether PAs are able to induce parthenocarpy and whether thepat-2 mutation alters the content and metabolism of PAs in unpollinated ovaries. Application of putrescine, spermidine, and spermine to wild-type unpollinated tomato ovaries (cv Madrigal [MA/wt]) induced partial parthenocarpy. Parthenocarpic growth of MA/pat-2 (a parthenocarpic near-isogenic line to MA/wt) ovaries was negated by paclobutrazol (GA biosynthesis inhibitor), and this inhibition was counteracted by spermidine. Application of α-difluoromethyl-ornithine (-Orn) and/or α-difluoromethyl-arginine (-Arg), irreversible inhibitors of the putrescine biosynthesis enzymes Orn decarboxylase (ODC) and Arg decarboxylase, respectively, prevented growth of unpollinated MA/pat-2 ovaries. α-Difluoromethyl-Arg inhibition was counteracted by putrescine and GA3, whereas that of α-difluoromethyl-Orn was counteracted by GA3 but not by putrescine or spermidine. In unpollinated MA/pat-2 ovaries, the content of free spermine was significantly higher than in MA/wt ovaries. ODC activity was higher in pat-2 ovaries than in MA/wt. Transcript levels of genes encoding ODC and spermidine synthase were also higher in MA/pat-2. All together, these results strongly suggest that the parthenocarpic ability of pat-2 mutants depends on elevated PAs levels in unpollinated mutant ovaries, which correlate with an activation of the ODC pathway, probably as a consequence of elevated GA content in unpollinated pat-2tomato ovaries.
Metabolic Origin of Carbon Isotope Composition of Leaf Dark-Respired CO2 in French Bean
Authors: Tcherkez Guillaume , Nogués Salvador , Bleton Jean , Cornic Gabriel , Badeck Franz , Ghashghaie Jaleh
Published: January 2003

The carbon isotope composition (δ13C) of CO2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The δ13C of CO2 linearly decreased when temperature increased, from −19‰ at 10°C to −24‰ at 35°C. It also progressively decreased from −21‰ to −30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO2 was enriched in 13C compared with carbohydrates, the most 13C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO2 was depleted in 13C even when compared with the composition of total organic matter. In the two types of experiment, the variations of δ13C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of δ13C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13C-enriched CO2 and β-oxidation of fatty acids producing 13C-depleted CO2 when compared with total organic matter (−27.5‰). These results are consistent with the assumption that the δ13C of dark respired CO2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.