bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose‐6‐phosphate
Authors: Tauberger Eva , Fernie Alisdair R. , Emmermann Michael , Renz Andreas , Kossmann Jens , Willmitzer Lothar , Trethewey Richard N. 
Published: March 2003

SummaryThe aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole‐plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose‐6‐phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.
Ectopic expression of a tomato 9‐cis‐epoxycarotenoid dioxygenase gene causes over‐production of abscisic acid
Authors: Thompson Andrew J. , Jackson Alison C. , Symonds Rachael C. , Mulholland Barry J. , Dadswell A. Ruth , Blake Patrick S. , Burbidge Alan , Taylor Ian B. 
Published: March 2003

SummaryThe tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild‐type allele of notabilis, LeNCED1, encodes a putative 9‐cis‐epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline‐inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10‐fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric ‘Super‐Promoter’. Many of these plants were wilty, suggesting co‐suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.
Development of an efficient maintenance and screening system for large‐insert genomic DNA libraries of hexaploid wheat in a transformation‐competent artificial chromosome (TAC) vector
Authors: Liu Yao‐Guang , Nagaki Kiyotaka , Fujita Masako , Kawaura Kanako , Uozumi Masahiko , Ogihara Yasunari 
Published: March 2003

SummaryThree large‐insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium‐mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96‐well format, each well containing approximately 300–600 colonies (12 plates for small library, four for medium‐size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.
Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene
Authors: Hanin Moez , Mengiste Tesfaye , Bogucki Augustyn , Paszkowski Jerzy 
Published: March 2003

SummaryThe Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA‐damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA‐damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.
An estrogen receptor‐based transactivator XVE mediates highly inducible gene expression in transgenic plants
Authors: Zuo Jianru , Niu Qi‐Wen , Chua Nam‐Hai 
Published: March 2003

SummaryWe have developed an estrogen receptor‐based chemical‐inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA‐binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10‐90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol‐activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the −46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical‐inducible system for regulating transgene expression in plants.
The S‐methylmethionine cycle in angiosperms: ubiquity, antiquity and activity
Authors: Ranocha Philippe , McNeil Scott D. , Ziemak Michael J. , Li Changjiang , Tarczynski Mitchell C. , Hanson Andrew D. 
Published: March 2003

SummaryAngiosperms synthesize S‐methylmethionine (SMM) from methionine (Met) and S‐adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S‐methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S‐methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co‐expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short‐term control of AdoMet level.
Form I Rubiscos from non‐green algae are expressed abundantly but not assembled in tobacco chloroplasts
Authors: Whitney Spencer M. , Baldet Pierre , Hudson Graham S. , Andrews T. John 
Published: March 2003

SummaryNon‐green algae have Rubiscos that are phylogenetically distinct from their counterparts in green algae and higher plants. Some non‐green‐algal Rubiscos are more specific for CO2, relative to O2, than higher‐plant Rubiscos, sometimes coupled with lower Michaelis constants for CO2. If these Rubiscos could be substituted for the higher‐plant enzyme, and if they functioned successfully in the higher‐plant chloroplast and were regulated appropriately, they would improve the CO2 use and quantum efficiency of higher‐plant photosynthesis. To assess the feasibility of expressing non‐green algal Rubiscos in higher‐plant chloroplasts, we inserted the rbcLS operons from the rhodophyte Galdieria sulphuraria and the diatom Phaeodactylum tricornutum into the inverted repeats of the plastid genome of tobacco, leaving the tobacco rbcL gene unaltered. Homoplasmic transformants were selected. The transgenes directed the synthesis of abundant amounts of transcripts and both subunits of the foreign Rubiscos. In some circumstances, leaves of the transformants with the P. tricornutum Rubisco contained as much foreign Rubisco protein as endogenous tobacco Rubisco (>30% of the soluble leaf protein). However, the subunits of the foreign Rubiscos were not properly folded and/or assembled. All the foreign large subunits and most of the foreign small subunits were recovered in the insoluble fractions of leaf extracts. Edman sequencing yielded the expected N‐terminal sequences for the foreign small subunits but the N‐termini of the foreign large subunits were blocked. Accumulation of large amounts of denatured foreign Rubisco in the leaves, particularly of the P. tricornutum transformants, caused a reduction in the amount of tobacco Rubisco present, with concomitant reductions in leaf CO2 assimilation and plant growth.
Efficient elimination of selectable marker genes from the plastid genome by the CRE‐lox site‐specific recombination system
Authors: Corneille Sylvie , Lutz Kerry , Svab Zora , Maliga Pal 
Published: March 2003

SummaryIncorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE‐lox site‐specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear‐encoded plastid‐targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA‐ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build‐up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE‐lox system described here will be a highly efficient tool to obtain marker‐free transplastomic plants.
Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli
Authors: González‐Melendi Pablo , Wells Brian , Beven Alison F. , Shaw Peter J. 
Published: March 2003

SummaryThe rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50–100 RNA polymerases together with the nascent pre‐rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as ‘Christmas trees’ 2–3 µm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver‐enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post‐embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread‐like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.
Assessment of the food safety issues related to genetically modified foods
Authors: Kuiper Harry A. , Kleter Gijs A. , Noteborn Hub P. J. M. , Kok Esther J. 
Published: March 2003

SummaryInternational consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post‐marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.
Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants
Authors: Papenbrock Jutta , Mishra Sanjay , Mock Hans‐Peter , Kruse Elisabeth , Schmidt Eva‐Kathrin , Petersmann Astrid , Braun Hans‐Peter , Grimm Bernhard 
Published: March 2003

SummaryProtoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid‐located ferrochelatase (FeChII). A 1700‐bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity‐dependent formation of necrotic leaf lesions. In comparison with wild‐type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.
Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol‐secreting fungal pathogen Alternaria alternata
Authors: Jennings Dianne B. , Daub Margaret E. , Pharr D. Mason , Williamson John D. 
Published: March 2003

SummaryOur previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen‐mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non‐mannitol‐containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol‐ and non‐mannitol‐secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non‐mannitol‐secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol‐secreting fungal plant pathogens.
Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis
Authors: Linkohr Birgit I. , Williamson Lisa C. , Fitter Alastair H. , Leyser H.M. Ottoline 
Published: March 2003

SummaryPlant root systems can respond to nutrient availability and distribution by changing the three‐dimensional deployment of their roots: their root system architecture (RSA). We have compared RSA in homogeneous and heterogeneous nitrate and phosphate supply in Arabidopsis. Changes in nitrate and phosphate availability were found to have contrasting effects on primary root length and lateral root density, but similar effects on lateral root length. Relative to shoot dry weight (DW), primary root length decreased with increasing nitrate availability, while it increased with increasing phosphate supply. Lateral root density remained constant across a range of nitrate supplies, but decreased with increasing phosphate supply. In contrast, lateral root elongation was suppressed both by high nitrate and high phosphate supplies. Local supplies of high nitrate or phosphate in a patch also had different effects. Primary root growth was not affected by a high nitrate patch, but growth through a high phosphate patch reduced primary root growth after the root left the patch. A high nitrate patch induced an increase in lateral root density in the patch, whereas lateral root density was unaffected by a high phosphate patch. However, both phosphate‐ and nitrate‐rich patches induced lateral root elongation in the patch and suppressed it outside the patch. This co‐ordinated response of lateral roots also occurs in soil‐grown plants exposed to a nutrient‐rich patch. The auxin‐resistant mutants axr1, axr4 and aux1 all showed the wild‐type lateral root elongation responses to a nitrate‐rich patch, suggesting that auxin is not required for this response.
Single‐step transformation for generating marker‐free transgenic rice using the ipt‐type MAT vector system
Authors: Endo Saori , Sugita Koichi , Sakai Miho , Tanaka Hiroshi , Ebinuma Hiroyasu 
Published: March 2003

AbstractThe ipt‐type MAT vector uses the ipt gene for regeneration of marker‐free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin‐dependent embryogenesis. We report a single‐step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre‐cultured rice seeds, marker‐free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt‐intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker‐free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker‐free transgenic plants in economically important crops.