bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents
Authors: Véry Anne‐Aliénor , Gaymard Frédéric , Bosseux Corinne , Sentenac Hervé , Thibaud Jean‐Baptiste 
Published: March 2003

SummaryThe open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3′ and 5′ untranslated regions of a β‐globin cDNA from Xenopus oocyte. The polyadenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly‐ or outwardly‐directed, depending on K+ external concentration. Predictions of the voltage‐gated channel theory were shown to fit the data well. The equivalent gating charge and the half‐activation potential ranged around 2 and −145 mV, respectively. KAT1 gating characteristics did not depend on K+ external concentration nor on external pH in the 5.0–7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K+ lay in the range 15–30 mM, at external pH 7.4. As for many K+ channels of animal cells, external caesium caused a voltage‐dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage‐independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward‐rectifying voltage‐gated channels described in both plant cells (namely guard cells) and animal cells.
Generation of a high‐quality P1 library of Arabidopsis suitable for chromosome walking
Authors: Liu Yao‐Guang , Mitsukawa Norihiro , Vazquez‐Tello Alejandro , Whittier Robert F. 
Published: March 2003

SummaryUsing improved techniques, a representative P1 library of Arabidopsis was constructed and characterized. Megabase genomic DNA was prepared from nuclei and partially digested with Sau3AI. DNA fragments of 75–100 kb were selected by size fractionation in low melting agarose, concentrated by a spot‐evaporation/dialysis method, and cloned in the pAd10sacBII P1 vector. The library contains 10 080 clones individually stored in microtiter plates. With an average insert size of about 80 kb, the library represents about eight haploid genomic equivalents of this plant. This library can be screened rapidly by dot hybridization of plate and well position pools. Characterization of the library by restriction analysis, screening with RFLP probes, RFLP mapping of insert end sequences, and chromosome walking shows that the library is of high quality with respect to insert site, completeness, and absence of chimeric artifacts. With this library a contig of about 600 kb has been constructed in the cer9 locus region. This P1 library is expected to be useful for genome mapping and gene cloning in Arabidopsis research programs.
Three families of thiol peptides are induced by cadmium in maize
Authors: Meuwly Philippe , Thibault Pierre , Schwan Adrian L. , Rauser Wilfried E. 
Published: March 2003

SummaryPhytochelatins ((γGluCys)nGly) are synthesized from glutathione by plants exposed to metals like Cd2+, Cu2+ and Zn2+. An intracellular complex formed by phytochelatins with Cd2+ and sulfide is thought to detoxify the metal possibly by sequestration in the vacuole. It was found that maize seedlings exposed to Cd2+ produced phytochelatins and two additional families of cysteina‐containing peptides, (γGluCys)n and (γGluCys)nGlu. All thiol peptides with n = 2 and 3 were purified and their structure characterized by tandem mass spectrometry. For maize plants exposed to Cd2+ for 7 days, phytochelatins were synthesized preferentially in the first 24 h whereas the amounts of (γGluCys)n and (γGluCys)nGlu were the highest thereafter. This was probably due to an initial large pool of glutathione available in control plants compared with a dearth of γGluCys and no detectable γGluCysGlu. The (γGluCys)nGlu peptides were induced exclusively by Cd2+ as they were below the detection limit in control seedlings that contained low amounts of phytochelatins and (γGluCys)n. Since the Cys moiety of the peptides is essential for binding Cd2+, a role for accumulated (γGluCys)n and (γGluCys)nGlu in detoxifying Cd2+ in plants must be considered.
Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels
Authors: Kelly Walter B. , Esser John E. , Schroeder Julian I. 
Published: March 2003

SummaryThe cellular mechanisms that regulate potassium (K+) channels in guard cells have been the subject of recent research, as K+ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca2+ concentrations, Ca2+ buffers and GTP‐binding protein modulators on inward‐rectifying K+ channels. Ca2+ inhibition of inward‐rectifying K+ currents depended strongly on the concentration and effectiveness of the Ca2+ buffer used, indicating a large Ca2+ buffering capacity and pH increases in guard calls. When the cytosolic Ca2+ concentration was buffered to micromolar levels using BAPTA, inward‐rectifying K+ channels were strongly inhibited. However, when EGTA was used as the Ca2+ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca2+. Under the imposed conditions, GTPγS did not significantly inhibit inward‐rectifying K+ channel currents when cytosolic Ca2+ was buffered to low levels or when using EGTA as the Ca2+ buffer. Furthermore, GDPβS reduced inward K+ currents at low cytosolic Ca2+, indicating a novel mode of inward K+ channel regulation by G‐protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca2+ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward‐rectifying K+ channel currents in a population of cells, indicating possible Ca2+‐dependent action of GTP‐binding protein modulators in K+ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K+ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K+ channel down‐regulation alone may not be rate‐limiting, and it is proposed that the concerted action of proton‐pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G‐protein modulators regulate inward K+ channels in a more complex and limited, possibly Ca2+‐dependent, manner than previously proposed.
Mechanisms of chilling‐induced oxidative stress injury and tolerance in developing maize seedlings: changes in antioxidant system, oxidation of proteins and lipids, and protease activities
Authors: Prasad Tottempudi K. 
Published: March 2003

SummaryThe mechanisms involved in chilling injury or tolerance in developing maize seedlings subjected to low temperature stress have been investigated. Although acclimation‐induced chilling tolerance was developmentally regulated, no tolerance was observed in non‐acclimated developing seedlings subjected to 7 days of 4°C stress. Consistent with previous results, chilling induced oxidative stress in the seedlings. Whereas acclimated seedlings had elevated levels (35–120%) of antioxidant enzymes, such as catalase, glutathione reductase and guaiacol peroxidase, non‐acclimated seedlings had lower levels of these enzyme activities during chilling conditions. The increase in the activity of mitochondrial catalase3 isozyme, a major anti‐oxidant enzyme in maize, was due to an increase in cat3 transcripts and CAT3 protein, as indicated by Northern and Western blot analyses. Besides, non‐acclimated seedlings had increased levels of oxidized proteins (two to threefold) and lipids (35–65%) during chilling and recovery compared with control or acclimated seedlings. Low‐temperature stress also inhibited two proteases, endopeptidase and aminopeptidase, by 35% in non‐acclimated seedlings. Since seedlings treated with prooxidants also promoted the oxidation of proteins and lipids and the inhibition of proteases, it was concluded that in vivo generated oxidative stress was likely responsible for affecting these processes. These results provide a correlative evidence to suggest that: (i) in non‐acclimated seedlings, chilling injury was partly due to the build up of reactive oxygen species (ROS) that promoted the oxidation of proteins and lipids and inhibited protease activity; (ii) in acclimated seedlings, chilling tolerance was partly due to an enhanced anti‐oxidant system that prevented the accumulation of ROS and therefore, prevented the oxidation of proteins and lipids and the inhibition of protease activities during 4°C stress and recovery.
The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time
Authors: Devlin Paul F. , Halliday Karen J. , Harberd Nicholas P. , Whitelam Garry C. 
Published: March 2003

SummaryA major function of phytochromes in light‐grown plants involves the perception of changes in the relative amounts of red and far‐red light (R:FR ratio) and the initiation of the shade‐avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end‐of‐day far‐red light (EOD FR) treatments. Phytochrome B‐deficient (phyB) mutants, which have a constitutive elongated‐petiole and early‐flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild‐type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild‐type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.
Detection of single‐copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization
Authors: Fransz Paul F. , Stam Maike , Montijn Bob , Hoopen Rogier Ten , Wiegant Joop , Kooter Jan M. , Oud Oof , Nanninga Nanne 
Published: March 2003

SummaryDNA sequences homologous to single‐copy genes were labelled with biotinylated dUTP or digoxygenin‐labelled dUTP and hybridized to chromosome spreads. The hybridization signals were visualized with fluorescent avidin‐ or antibody‐conjugates. This method allowed the detection of DNA targets on metaphase chromosomes as small as 1.4 kb. The hybridization signals were identified as fluorescent spots on both sister chromatids. Using an 18S rDNA probe as marker to identify chromosomes II and III it was possible to assign single‐copy genes to these chromosomes. In the line V30 the endogenous chalcone synthase gene (chsA) was mapped at the distal end of the short arm of chromosome 5. The cDNA probe for this single‐copy gene was 1.4 kb. In contrast, in the lines Mitchell and V26 chsA was localized at the distal end of the long arm of chromosome 3, suggesting that a chromosomal rearrangement had taken place. In a transformed Petunia uidA, transgenes were detected using a 2.7 kb probe. One transgene was mapped on one of the homologues of chromosome II proximal to the ribosomal genes. This homologue could be distinguished from the other by having the ribosomal genes at the distal end of the long arm. Using multicolour fluorescence in situ hybridization it was shown that it is possible to detect the endogenous chsA genes and both transgenes simultaneously.
Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5
Authors: Schmidt Renate , Love Karina , West Joanne , Lenehan Zoë , Dean Caroline 
Published: March 2003

SummaryIn order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome‐walking experiments, provide substrates for large‐scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.
Intron‐mediated enhancement of transgene expression in maize is a nuclear, gene‐dependent process
Authors: Rethmeier Nikki , Seurinck Jef , Van Montagu Marc , Cornelissen Marc 
Published: March 2003

SummaryIn monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5′ untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5′ UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron‐mediated enhancement of expression (IME) is gene‐dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability, it may mean that IME rescues rather than stimulates gene expression.
Transposon Display identifies individual transposable elements in high copy number lines
Authors: Va Dirk , De N , Broeck N , Maes Tamara , Sauer Matt , Zethof Jan , D Peter , Keukeleire E , D’hauw Mariëlla , Va Marc , Montagu N , Gerats Tom 
Published: March 2003

SummaryThe dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation‐mediated PCR. The resulting fragments are locus‐specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1‐tagged genes. Fragments that co‐segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.
Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism
Authors: Vo Klaus , Schwartzenberg  , Kruse Sven , Reski Ralf , Moffatt Barbara , Laloue Michel 
Published: March 2003

SummaryAdenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.
Carrot DNA‐methyltransferase is encoded by two classes of genes with differing patterns of expression
Authors: Bernacchia Giovanni , Primo Antonella , Giorgetti Lucia , Pitto Letizia , Cella Rino 
Published: March 2003

SummaryIn the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine‐5)‐methyltransferase (DNA‐METase) are reported. The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size. Met1–5 and Met2–21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N‐terminus. The larger size of the Met2–21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171 bp sequence present only once in the Met1–5 cDNA. Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordia), but differ quantitatively and spatially in their mode of expression. Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1–5). In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size. These results provide the first direct demonstration that DNA‐METases of a higher eukaryote are encoded by a gene family.
Gypsy‐like retrotransposons are widespread in the plant kingdom
Authors: Suoniemi Annu , Tanskanen Jaakko , Schulman Alan H. 
Published: March 2003

SummaryRetrotransposons propagate via an RNA intermediate which is then reverse‐transcribed and packaged into virus‐like particles. They are either copia‐ or gypsy‐like in coding domain order and sequence similarity, the gypsy‐like elements sharing their organization with the retroviruses but lacking retroviral envelope domains. Copia‐like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants, but gypsy‐like elements have been reported only for scattered species. The authors have exploited the difference in domain order between these groups to amplify and clone segments bridging the reverse transcriptase – integrase region of specifically gypsy‐like retrotransposons. Species representative of the diversity of higher plants yielded products whose sequences establish that gypsy‐like transposons are dispersed throughout the plant genomes. This class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the fungi, plants and animals support their existence as ancient distinct lineages and subsequent, vertical radiation.
Molecular cloning and functional heterologous expression of two alleles encoding (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1), a new methyl jasmonate‐inducible cytochrome P‐450‐dependent mono‐oxygenase of benzylisoquinoline alkaloid biosynthesis
Authors: Pauli Hubert H. , Kutchan Toni M. 
Published: March 2003

SummaryAlkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (S)‐N‐methylcoclaurine represent a vast and varied structural array of physiologically active molecules. These compounds range from the dimeric bisbenzylisoquinolines, such as the muscle relaxant (+)‐tubocurarine, to the powerful anaesthetic opiate morphine, the anti‐microbial berberine and the anti‐microbial benzo[c]phenanthridine sanguinarine. The 3′‐hydroxylation of (S)‐N‐methylcoclaurine is a branch point that is the penultimate step in the biosynthesis of the central alkaloidal intermediate (S)‐reticuline. This study identified this enzyme as a cytochrome P‐450‐dependent mono‐oxygenase that has until now eluded attempts at identification using in vitro enzyme assays. Two alleles encoding this new enzyme (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1) were isolated from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate‐induced cell‐suspension cultures of the California poppy Eschscholzia californica. Partial clones generated by RT–PCR with cytochrome P‐450‐specific primers were used as hybridization probes. RNA gel‐blot hybridization indicated that the transcripts for CYP80B1 accumulate in response to the addition of methyl jasmonate to the cell culture medium. Both alleles were functionally expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda Sf9 cells in the presence and absence of the E. californica cytochrome P‐450 reductase. The enzyme was found to hydroxylate exclusively (S)‐N‐methylcoclaurine with a pH optimum of 7.5, temperature optimum of 35°C and a Km of 15 μm. In addition to the CYP80B1 alleles, another cytochrome P‐450 with an inducible transcript (CYP82B1) was isolated and expressed in the same manner, but was not found to be involved in alkaloid biosynthesis in this plant.
Identification of cat sequences required for intron‐dependent gene expression in maize cells
Authors: Rethmeier Nikki , Kramer Evelien , Va Marc , Montagu N , Cornelissen Marc 
Published: March 2003

SummaryTransgene expression in maize cells changed from intron‐independent to intron‐dependent by an exact exchange of the bar coding region for that of cat. By deletion mapping an approximately 100 nucleotide sequence element at the 5′ end of the cat coding region was identified that, when inserted at the translation start site of the bar gene, impaired expression. Successive inclusion of the salT intron in the 5′ untranslated region (UTR) restored expression near to wild‐type bar expression levels. A chimeric gfp gene, but not nptII gene, behaved similarly. These observations are in agreement with the view that intron‐mediated enhancement of transgene expression does not enhance, but rather restores expression of an impaired gene.