bluuebunny/crossref_metadata_2025 huggingface dataset:
The maize RNA‐binding protein, MA16, is a nucleolar protein located in the dense fibrillar component
Authors: Albà M. Mar , Culiáñez‐Macià Francisco A. , Goday Adela , Freire Miguel Angel , Nadal Belén , Pagès Montserrat
Published: March 2003

A developmentally and environmentally regulated gene in maize, MA16, encoding an RNA‐binding protein that binds preferentially to uridine and guanosine‐rich RNAs has previously been described. To gain some insight into the function of MA16 the distribution of MA16 mRNA and protein during maize development was investigated using in situ hybridization, RNA and protein gel blot analysis and immunocytochemistry. The results show that MA16 is expressed throughout development of the embryo and seedling in different tissues and at different levels. The level of MA16 mRNA is higher in developing and expanding structures such as the root elongation zone and young leaves. After stress treatment MA16 mRNA increases in total and polysomal RNA, but no significant change in the level of the protein was detected. MA16 is a non‐ribosomal nucleolar protein. Using immunoelectron microscopy the MA16 protein has been located in the dense fibrillar component and to a lesser extent in the granular component of the nucleolus. It was found that MA16 contains the conserved sequence motifs R(G)nY(G)nR and RR(E/D)(G)nY(G)n repeated in the C‐terminal of the molecule that conforms imperfectly to the GAR motif proposed for nucleolar proteins. In light of these results the stress regulation of MA16 and a likely role for this protein in pre‐rRNA processing and/or ribosome assembly is discussed.
Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T‐DNA borders
Authors: Bhattacharyya Madan K. , Stermer Bruce A. , Dixon Richard A.
Published: March 2003

A new binary vector for Agrobacterium‐mediated plant transformation was constructed, in which two selectable markers, for kanamycin and hygromycin resistance, were placed next to the right and left T‐DNA borders, respectively, and a CaMV 35S promoter‐driven β‐glucuronidase (GUS) gene was placed between these markers as a reporter gene (transgene). Using double antibiotic selection, all transgenic tobacco plants carrying at least one intact copy of the T‐DNA expressed the transgene, and this population exhibited reduced variability in transgene expression as compared with that obtained from the parent vector pBI121. Absence of the intact transgene was the major reason for transgenic plants with little or no transgene expression. Integration of truncated T‐DNAs was also observed among transgenic plants that expressed the transgene and carried multiple T‐DNA inserts. The copy number of fully integrated T‐DNAs was positively associated with transgene expression levels in R0 plants and R1 progeny populations. Variability due to position effect was determined among 17 plants carrying a single T‐DNA insert. The coefficient of variability among these plants was only 35.5%, indicating a minor role for position effects in causing transgene variability. The new binary vector reported here can therefore be used to obtain transgenic populations with reduced variability in transgene expression.
Preparation of megabase‐size DNA from plant nuclei
Authors: Zhang Hong‐Bin , Zhao Xinping , Ding Xiaoling , Paterson Andrew H. , Wing Rod A.
Published: March 2003

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low‐melting‐point agarose microbeads or plugs, and DNA purification in situ. This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed‐field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.
Tissue‐specific DNase I‐sensitive sites of the maize P gene and their changes upon epimutation
Authors: Lund Gertrud , Prem Das O. , Messing Joachim
Published: March 2003

A map has been developed of nuclease‐hypersensitive sites of P‐rr, the standard allele of the P‐locus of Zea mays L. Using a traditional DNase I assay, eight such sites have been found that are specific for the expressing tissue and span a region of more than 25 kb of the P‐locus, making it one of the largest plant genes yet described. The maps of the standard allele have also been compared with the recently described moderately stable P‐pr allele, which arose from epimutation. Six of the eight sites exhibit the same tissue‐specificity in P‐pr plants, while two stay repressed as in non‐expressing tissues of plants with the standard allele. Interestingly, the two repressed sites coincide with two hypermethylated restriction sites that have previously been correlated with the expression potential of the P‐pr allele. On the other hand, four of the DNase I sites, coinciding with CpG islands that were not hypermethylated by the epimutation, also showed no differences in their sensitivity to DNase I between the standard allele and the P‐pr allele. This suggests that the epimutation affects both site‐specific methylation changes and a specific local chromatin structure of the P gene involved in its regulation.
A novel Arabidopsis type 1 protein phosphatase is highly expressed in male and female tissues and functionally complements a conditional cell cycle mutant of Aspergillus
Authors: Arundhati A. , Feiler H. , Traas J. , Zhang H. , Lunness P.A. , Doonan J.H.
Published: March 2003

Type 1 protein phosphatases are very highly conserved throughout eukaryotes where they regulate a number of key metabolic and morphogenetic processes. A cDNA, AtPP1bg, representing a new member of the type 1 protein phosphatase gene family in Arabidopsis has been isolated on the basis of hybridization with the Aspergillus bimG protein phosphatase gene. The AtPP1bg gene potentially encodes a 37 kDa protein very closely related to PP1 but with divergent N‐ and C‐termini. The predicted amino acid sequence shows 71% identity to the ORF of the bimG gene. When expressed in Aspergillus under the alcA promoter, this phosphatase complements the temperature‐sensitive bimG11 mutation allowing nearly normal vegetative growth at 37°C (but not at 42°C). Notably, the plant PP1 does not support morphogenesis (conidiation) at 37°C. This may indicate that conidophore formation has particular phosphatase requirement(s) which the plant PP1 cannot supply.

The pattern of expression of the AtPP1bg transcript has been studied during development of the plant. In situ hybridization of Arabidopsis with antisense probes shows that this phosphatase gene is expressed at a low level throughout the plant, but is strongly upregulated within developing flowers, especially in the tapetum, the developing and mature pollen and in the ovaries. This implies that the AtPP1bg either has a specialized role in the formation of these organs, or that there is an increased requirement for protein phosphatase 1 at these stages. It was found that the level of AtPP1bg transcript, as judged by the relative intensity of staining in different cells within the floral meristems, did not vary during the cell cycle.
Somatic and germinal activities of maize Activator (Ac) transposase mutants in transgenic tobacco
Authors: Kunze Reinhard , Kühn Sandra , Jones Jonathan D.G. , Scofield Steven R.
Published: March 2003

The properties have been investigated of two deletion derivatives of the transposase protein (TPase) of maize transposable element Ac in transgenic tobacco. The wild‐type and mutant TPases were expressed as fusions to the cauliflower mosaic virus 35S promoter. A deletion of 102 amino acids from the N‐terminus, TPase(103–807), induces Ds excisions from a SPT::Ds reporter locus with a higher frequency than the wild‐type TPase. The increased transpositional activity of TPase(103–807) is a dominant trait, as seedlings coexpressing truncated and wild‐type TPase show the characteristic TPase(103–807) variegation pheno‐type. A transpositionally inactive TPase deletion derivative lacking 188 amino acids from the N‐terminus inhibits the transpositional activity of the wild‐type TPase.
Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6‐bisphosphate levels
Authors: Scott Peter , Lange Alex J. , Pilkis Simon J. , Kruger Nicholas J.
Published: March 2003

The aim of this work was to investigate the role of fructose 2,6‐bisphosphate (Fru 2,6‐P2) during photosynthesis. The level of Fru 2,6‐P2 in tobacco plants was elevated by the introduction of a modified mammalian gene encoding 6‐phosphofructo‐2‐kinase (6‐PF‐2‐K). Estimates of the metabolite control coefficient (C) for Fru 2,6‐P2 levels in response to increased 6‐PF‐2‐K activity, suggest that small increases in 6‐PF‐2‐K activity have little effect upon steady‐state Fru 2,6‐P2 levels (C = +0.08 for a 0–58% increase in 6‐PF‐2‐K activity). However, larger changes resulted in dramatic rises in Fru 2,6‐P2 levels (C = +3.35 for 206–268% increase in 6‐PF‐2‐K activity). Transgenic plants contained Fru 2,6‐P2 levels in the dark that ranged from 104 to 230% of the level in wild‐type tobacco. Plants with altered levels of Fru 2,6‐P2 were used to determine the effects of this signal metabolite upon carbohydrate metabolism during the initial phase of the light period. Here we provide direct evidence that Fru 2,6‐P2 contributes to the regulation of carbon partitioning in tobacco leaves by inhibiting sucrose synthesis.
Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes
Authors: Bohlmann Jörg , DeLuca Vincenzo , Eilert Udo , Martin William
Published: March 2003

Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670‐ and 1700‐fold, respectively. Glutamine‐ and ammonia‐ dependent AS activities were strictly co‐purified in all steps. Through cDNA cloning and complementation of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional ASα subunits, ASα1 and ASα2. The data indicate that ASα from Ruta requires an ASβ subunit with a native molecular weight of 60–65 kDa for the glutamine‐dependent reaction. Protein synthesized invitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature ASα subunits are active in plastids in vivo. ASα1 and ASα2 are constitutively expressed in Ruta cell cultures, but ASα1 steady‐state mRNA levels are increased 100‐fold 6 h subsequent to elicitation whereas ASα2 expression remains constitutive. Increased ASα1 transcription corresponds to elicitor‐induced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.
An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato
Authors: Bonnema Guusje , Hontelez Jan , Verkerk Ruud , Zhang Yong Qing , van Daelen Raymond , van Kammen Ab , Zabel Pim
Published: March 2003

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular‐weight DNA from agarose‐embedded leaf protoplasts, (ii) controlled partial digestion in situ using EcoRI endonuclease in the presence of EcoRI methylase (M. EcoRI), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker‐Cf4 and VFNT Cherry resulted in libraries (total 21 060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three‐dimensional PCR‐based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine‐rich repeat sequences located around the Cf4/ Cf9 locus.
A promoter for strong and ubiquitous anaerobic gene expression in tobacco
Authors: Köhler Uwe , Mendel Ralf R. , Cerff Rüdiger , Hehl Reinhard
Published: March 2003

The maize GapC4 promoter confers strong and ubiquitous anaerobic expression of the β‐glucuronidase reporter gene in transgenic tobacco. Among 21 transgenic tobacco lines harboring 785 or 461 bp from the GapC4 promoter, 18 show anaerobic gene expression. In six lines anaerobic fluorescence values were more than 1000‐fold over non‐specific background and in five lines anaerobic gene expression exceeded aerobic expression of a reporter gene construct under the control of the strong cauliflower mosaic virus 35S promoter. Aerobic gene expression was detected in only three lines. Anaerobic induction is observed in all tissues including mature leaves and flowers and is transcriptionally induced within at least 12 h. Within 3 h of transfer of tobacco seedlings from anaerobic to aerobic conditions reporter gene transcription is turned off. Among other environmental factors only wounding and UV light result in a 10‐ to 40‐fold induction of reporter gene expression above background. The strong inducibility, mode of expression under non‐inducing conditions, the specificity, and the ubiquitous induction distinguishes the GapC4 promoter from all previously analyzed anaerobic promoter‐reporter gene constructs.
T‐DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium
Authors: Escudero Jesús , Neuhaus Gunther , Schläppi Michael , Hohn Barbara
Published: March 2003

Agrobacterium has been established as a tool for gene delivery to most dicotyledonous plant species. However, it is not generally efficient in monocotyledonous plant species, especially not in Graminae. In maize, Agrobacterium‐mediated DNA transfer has been detected but early developmental stages in the plant proved incompetent as recipients. This research tests whether the lack of competence in young immature embryos of maize could be overcome by providing Agrobacterium in the interior of the plant cell. A microinjection technique was used to target single meristematic cells and prove competence to Agrobacterium. This response is dependent on the maize plant genotype.
Structural instability of a transgene locus in tobacco is associated with aneuploidy
Authors: Papp I. , Iglesias V.A. , Moscone E.A. , Michalowski S. , Spiker S. , Park Y.‐D. , Matzke M.A. , Matzke A.J.M.
Published: March 2003

This paper describes molecular and cytogenetic evidence for the instability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT‐rich regions. One of these regions binds to nuclear scaffolds in vitro, suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcino‐genesis are discussed.
Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines
Authors: Renckens Suzy , De Greve Henri , Beltrán‐Herrera Javier , Toong Lin Thia , Deboeck Francine , De Rycke Riet , Van Montagu Marc , Hernalsteens Jean‐Pierre
Published: March 2003

The new unstable virescent seedling (vis*) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene (nia) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1‐like transposable element dTph1–3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4, is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1. Insertion of dTph1–3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1. In contrast to dTph1‐like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1–3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.
Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines
Authors: Liu Yao‐Guang , Mitsukawa Norihiro , Lister Clare , Dean Caroline , Whittier Robert F.
Published: March 2003

A new collection of 129 Arabidopsis thaliana RFLP markers has been established based upon DNA fragments cloned in the pUC119 plasmid vector and insert end sequences of P1 clones. Dominant/null alleles affecting low‐copy number sequences account for nine of the mapped polymorphisms, suggesting that deletions are not rare in A. thaliana. Recombinant inbred (RI) lines were used for mapping these marker loci. RI line‐based mapping allows integration of this set of markers with markers previously reported as well as with any markers mapped in the future using this replenishable mapping resource. These markers are useful for map‐based gene isolation and genome physical mapping in A. thaliana as well as studies of chromosome colinearity (synteny) with related species.
Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties
Authors: Eberhard Jenny , Ehrler Thomas T. , Epple Petra , Felix Georg , Raesecke Hanns‐R. , Amrhein Nikolaus , Schmid Jürg
Published: March 2003

The three aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized in the plastids of higher plants. There is, however, biochemical evidence that a cytosolic isoform exists of the enzyme catalysing the first step of that branch of the pathway which is specific for the synthesis of phenylalanine and tyrosine, i.e. chorismate mutase (CM). We now report on the isolation of a cDNA clone encoding a cytosolic CM isozyme from Arabidopsis thaliana that was identified by complementing a CM‐deficient Escherichia coli strain. The deduced amino acid sequence of this isozyme was 50% identical to that of a previously isolated plastidic CM, and 41% identical to that of yeast CM. The organ‐specific expression patterns of the two CM genes were rather similar, but only the gene encoding the plastidic isozyme was elicitor‐ and pathogen‐inducible. The plastidic CM expressed in E. coli was activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas the cytosolic isozyme was insensitive. The existence of a cytosolic CM isozyme implies that either a cytosolic pathway (partial or complete) for the biosynthesis of phenylalanine and tyrosine exists, or that prephenate, originating from chorismate in the cytosol, is utilized for the synthesis of metabolites other than these two aromatic amino acids.
Identification of a salicylic acid‐responsive element in the promoter of the tobacco pathogenesis‐related β‐1,3‐glucanase gene, PR‐2d
Authors: Shah Jyoti , Klessig Daniel F.
Published: March 2003

The tobacco pathogenesis‐related PR‐2d gene encodes an acidic β‐1,3‐glucanase. Expression of the PR‐2d:uidA(GUS) chimeric gene is induced in leaves undergoing the hyper‐sensitive resistance response to tobacco mosaic virus and after treatment with salicylic acid (SA), a chemical believed to play an important role(s) in disease resistance. We have constructed transgenic tobacco plants which carry various segments of the PR‐2d promoter fused to a heterologous core 35S promoter driving the uidA(GUS) reporter gene. Their analysis indicates that sequences from −364 to −288 upstream of the PR‐2d transcription start site confer a high level of activation by SA (20‐fold). Mutations within this sequence, located between −339 and −333, depressed SA activation. This region is also required for the SA‐inducibility of a truncated PR‐2d:GUS chimeric gene. Contained within this region is a 25 bp element located between −348 and −324 which was specifically recognized by nuclear factors from tobacco leaves. No conclusive differences were observed in the ability of proteins in nuclear extracts from water‐treated versus SA‐treated plants to bind to this cis element in vitro.
Influence of auxin on the establishment of bilateral symmetry in monocots
Authors: Fischer Christiane , Neuhaus Gunther
Published: March 2003

To study the influence of auxin on the shift from radial to bilateral symmetry during monocot embryogenesis, the fate of young wheat (Triticum aestivum L.) zygotic embryos has been manipulated in vitro by adding auxins, an auxin transport inhibitor and an auxin antagonist to the culture medium. The two synthetic auxins used, 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), induced identical phenotypes. In the most severe cases, the shift from radial to bilateral symmetry was blocked resulting in continuous uniform radial growth. The natural auxin indole‐3‐acetic acid (IAA) induced the same phenotype. The effect of 2,4,5‐T and 2,4D depended on their concentrations and on the developmental stage of the isolated embryos. In the presence of 2,3,5‐triiodobenzoic acid (TIBA), an auxin transport inhibitor, the overall embryo symmetry was abnormal. The relative position of the shoot apical meristem in comparison with the scutellum was anomalous. The quality of shoot apical meristem and the scutellum differentiation was altered compared with normal developed embryos. No root meristem was differentiated. The effect of TIBA depends on its concentration and on the developmental stage of the isolated embryos. By contrast, 2‐(pchlorophenoxy)‐2‐methylpropionic acid (PCIB) which is described as an auxin antagonist, has no visible direct effect on the embryonic symmetry. These observations indicate that auxin influences the change from radial symmetry to embryonic polarity during monocot embryogenesis. A model of auxin action during early wheat embryo development is proposed.
BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat
Authors: Albert Sylvie , Delseny Michel , Devic Martine
Published: March 2003

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T‐DNA‐transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls (ban). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT (Transparent Testa) genes to two groups according to their epistatic relationship to ban. The flavonoid content of germinated ban and wild‐type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild‐type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis.
A carbonic anhydrase gene is induced in the nodule primordium and its cell‐specific expression is controlled by the presence of Rhizobium during development
Authors: De La Peña Teodoro Coba , Frugier Florian , McKhann Heather I. , Bauer Petra , Brown Spencer , Kondorosi Adam , Crespi Martin
Published: March 2003

Under nitrogen starvation, Rhizobium meliloti is able to induce nitrogen‐fixing nodules on alfalfa roots. Certain alfalfa cultivars spontaneously develop pseudonodules in the absence of bacteria. A transcript, Msca1, expressed in spontaneous and R. meliloti‐induced nodules, that codes for a carbonic anhydrase (CA), an enzyme catalyzing the hydration of CO2 has been identified. This is the first CA gene cloned from a non‐photosynthetic tissue in plants. Msca1 was activated initially in all cells of the bacterium‐induced nodule primordium and was also induced by cytokinin treatment of alfalfa roots. The presence of CA enzymatic activity in different nodule types was demonstrated. Thus, Msca1 is a new early nodulin gene with a function possibly related to the increased amyloplast deposition of the dividing cortical cells. Msca1 transcripts were subsequently found mainly in a peripheral envelope of cells in developing and mature nodules. This novel pattern of gene expression is controlled by the presence of the bacterium inside the nodule. Sucrose synthase and phosphoenol pyruvate carboxylase (PEPC), other genes of the carbon fixation metabolism, were expressed in the same peripheral cells and even more strongly in the nitrogen‐fixing region. Analysis of expression patterns of these genes indicated that early CA function may not be related to carbon fixation through PEPC. CA might be acting in pH regulation and/or CO2/HCO3− transport during nodule initiation. Thus, carbonic anhydrase may play different roles at several stages of nodule development and function.
A defect in synapsis causes male sterility in a T‐DNA‐tagged Arabidopsis thaliana mutant
Authors: Peirson Brenda N. , Bowling Sarah E. , Makaroff Christopher A.
Published: March 2003

Fluorescence microscopy was used to study meiosis in microsporocytes from wild‐type Arabidopsis thaliana and a T‐DNA‐tagged meiotic mutant. Techniques for visualizing chromosomes and β‐tubulin in other plant species were evaluated and modified in order to develop a method for analyzing meiosis in A. thaliana anthers. Like most dicots, A. thaliana microsporocytes undergo simultaneous cytokinesis in which both meiotic divisions are completed prior to cytokinesis. However, two unique events were observed in wild‐type A. thaliana that have not been reported in other angiosperms: (1) polarization of the microsporocyte cytoskeleton during prophase I prior to nuclear envelope breakdown, and (2) extensive depolymerization of microtubules just prior to metaphase II. The first observation could have implications regarding a previously uncharacterized mechanism for determining the axis of the metaphase I spindle during microsporogenesis. The second observation is peculiar since microtubules are known to be involved in chromosome alignment in other species; possible explanations will be discussed. A T‐DNA‐tagged meiotic mutant of A. thaliana (syn1), which had previously been shown to produce abnormal microspores with variable DNA content, was also cytologically characterized. The first observable defect occurs in microsporocytes at telophase I, where some chromosomes are scattered throughout the cytoplasm, usually attached to stray microtubules. Subsequent developmental stages are affected, leading to complete male sterility. Based on similarities to synaptic mutants that have been described in other species, it is suggested that this mutant is defective in synaptonemal complex formation and/or cohesion between sister chromatids.
Seal‐promoting solutions and pipette perfusion for patch clamping plant cells
Authors: Maathuis Frans J.M. , Taylor Alison R. , Assmann Sarah M. , Sanders Dale
Published: March 2003

Patch‐clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (GΩ) seal between the membrane and the patch‐clamp pipette. This can prove problematic in many plant‐cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl− concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl− had no effect. To reap the benefit of seal‐promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell‐attached patch, excised‐patch and whole‐cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.
Characterization of a DNA‐binding protein that interacts with 5′ flanking regions of two fruit‐ripening genes
Authors: Coupe Simon A. , Deikman Jill
Published: March 2003

The E4/E8 binding protein (E4/E8BP) interacts with sequences in the 5′ flanking regions of two genes, E4 and E8, that are coordinately regulated by ethylene during tomato fruit ripening. The DNA‐binding activity of this protein increases during fruit ripening, and it may play a role in regulation of these genes. To begin to understand the function of this protein, a cDNA has been isolated that encodes a protein, E4/E8BP‐1, with DNA‐binding specificity similar to that of E4/E8BP. This DNA‐binding protein is closely related to a DNA binding protein from tobacco, 3AF1, that interacts with the promoter of the pea rbcS‐3A gene. A repeated domain was identified within the predicted 3AF1 amino acid sequence, which includes a series of histidines and cysteines, suggestive of zinc binding, and this repeat is conserved in E4/E8BP‐1. Interaction of both E4/E8BP‐1 and nuclear extracts from ripening fruit with the E8 recognition sequence is sensitive to 1,10‐phenanthroline, indicating that a metal is required for binding of both the native and recombinant proteins. The mRNA for E4/E8BP‐1 is moderately abundant in fruit, and increases slightly during fruit ripening, consistent with a role in fruit ripening. A truncated version of E4/E8BP‐1 was able to transactivate the E4 promoter in transient assay, demonstrating that this DNA‐binding protein can interact with the E4 promoter in vivo to enhance gene transcription.
Retrofitting YACs for direct DNA transfer into plant cells
Authors: Adam Gerhard , Mullen Jeffrey A. , Kindle Karen L.
Published: March 2003

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant‐selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6‐ to 12‐fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.
Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism
Authors: Duwenig Elke , Steup Martin , Willmitzer Lothar , Kossmann Jens
Published: March 2003

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild‐type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild‐type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild‐type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.
A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice
Authors: Kusumi Kensuke , Mizutani Akiko , Nishimura Mitsuo , Iba Koh
Published: March 2003

Differentiation of proplastids into functionally active chloroplasts is one of the most significant changes in cellular organization associated with leaf development in higher plants. This process involves activation of a large number of nuclear and chloroplast genes. A central question, therefore, concerns the nature and origin of the signals that initiate and control this process. The rice nuclear mutant, virescent‐1 (v1), is temperature‐conditional and develops chlorotic leaves when grown at restrictive temperatures. We report here the effects of v1 mutation on the expressions of plastid and nuclear genes during leaf development. In the wild‐type rice seedlings, the transcripts of the plastid RNA polymerase gene (rpoB) and ribosomal protein genes (rps7, rps15) accumulated during a strictly limited period of early leaf development, prior to the accumulation of the transcripts of photosynthetic genes (rbcL, RbcS, psbA, Lhc). This period coincides very closely with the leaf developmental stage (late P4) at which the V1 gene gives the signal that determines the virescent phenotype. On the contrary, in the v1 seedlings grown at a restrictive temperature (20°C), this stage‐specific accumulation of the rpo and rps transcripts was missing. Instead, the accumulation of these transcripts occurred during a later stage of leaf maturation. In such mutant seedlings, the expression of other plastid genes (psbA, rbcL, 16S rDNA) was strongly suppressed and the normal chloroplast development was disturbed. These data indicate that the V1 gene controls the timing of expression of the key plastid genes for the transcription/ translation apparatus that are essential for the subsequent activation of other plastid genes.
Expression of a soybean nodule‐enhanced phosphoenolpyruvate carboxylase gene that shows striking similarity to another gene for a house‐keeping isoform
Authors: Hata Shingo , Izui Katsura , Kouchi Hiroshi
Published: March 2003

Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full‐length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez‐Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non‐coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non‐coding regions as isoform‐specific probes showed that GmPEPC7 is nodule‐enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule‐enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium‐infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule‐enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule‐enhanced PEPCs of alfalfa and pea suggested that the soybean nodule‐enhanced isoform evolved from the housekeeping PEPC gene after the ureid‐translocating and amide‐translocating legumes diverged from each other.
The maize Lc regulatory gene up‐regulates the flavonoid biosynthetic pathway of Petunia
Authors: Bradley J. Marie , Davies Kevin M. , Deroles Simon C. , Bloor Stephen J. , Lewis David H.
Published: March 2003

Petunia (cv. Mitchell) transformants were produced which expressed the maize Leaf colour (Lc) cDNA under the control of the CaMV 35S promoter and had enhanced pigmentation in both vegetative and floral tissues. In flowering transgenic lines, intense pigmentation of leaves, stems and sepals occurred, along with lower levels of pigmentation in the flower tube and limb. No anthocyanins were detected in leaves, sepals and flower limb of wild‐type Mitchell petunia. Of the 12 phenylpropanoid genes examined, transcript levels for nine flavonoid‐specific biosynthetic genes were up‐regulated in the transgenic Lc lines, including weak activation of CHS, CHI and F3H along with stronger activation of DFR, F3′H, F3′5′H, ANS, UFGT and 3RT. PAL, C4H and FLS were unaffected by Lc. Lc‐induced pigmentation in Mitchell petunia appears to be specific to the sub‐epidermal layer in all tissues examined, including the flower limb. The results show that the monocot Lc regulatory gene from maize can up‐regulate the expression of both early and late flavonoid biosynthetic genes and enhance anthocyanin production in the dicot species Petunia.
Two isoforms of plant RAD23 complement a UV‐sensitive rad23 mutant in yeast
Authors: Sturm Arnd , Lienhard Susanne
Published: March 2003

DNA repair by nucleotide excision (NER) has been demonstrated in plant cells at the biochemical level but until now none of the molecular components of the plant NER complex has been identified. In this paper, the cloning and characterization of two isoforms of RAD23 from carrot (Daucus carota L.) are reported. It has been suggested that RAD23 in yeast is an assembly factor of the NER complex required for transcription‐coupled repair as well as efficient overall genome repair. A functional assay demonstrated that both plant homologues complement the UV‐sensitive phenotype of a rad23 deletion mutant of yeast. This result suggests that homologous polypeptides may catalyse NER in plants and yeast and, possibly, by a similar mechanism.
5′‐ and 3′‐sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco
Authors: Meulewaeter Frank , Danthinne Xavier , Va Marc , Montagu  , Cornelissen Marc
Published: March 2003

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5′ cap and a 3′ poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5′‐terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5′ region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5′ 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV‐cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED‐dependent translation in tobacco was stimulated further by the STNV 5′ 38 nt.
More than 80R2R3‐MYB regulatory genes in the genome of Arabidopsis thaliana
Authors: Romero  , Fuertes  , Benito  , Malpica  , Leyva  , Paz‐Ares
Published: March 2003

Transcription factors belonging to the R2R3‐MYB family contain the related helix‐turn‐helix repeats R2 and R3. The authors isolated partial cDNA and/or genomic clones of 78 R2R3‐MYB genes from Arabidopsis thaliana and found accessions corresponding to 31 Arabidopsis genes of this class in databanks, seven of which were not represented in the authors’ collection. Therefore, there are at least 85, and probably more than 100, R2R3‐MYB genes present in the Arabidopsis thaliana genome, representing the largest regulatory gene family currently known in plants. In contrast, no more than three R2R3‐MYB genes have been reported in any organism from other phyla. DNA‐binding studies showed that there are differences but also frequent overlaps in binding specificity among plant R2R3‐MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins. This large‐sized gene family may contribute to the regulatory flexibility underlying the developmental and metabolic plasticity displayed by plants.
Genetic evidence for an essential role for potyvirus CI protein in cell‐to‐cell movement
Authors: Carrington James C. , Jensen Patricia E. , Schaad Mary C.
Published: March 2003

The potyvirus cylindrical inclusion (CI) protein, an RNA helicase required for genome replication, was analyzed genetically using alanine‐scanning mutagenesis. Thirty‐one mutations were introduced into the CI protein coding region of modified tobacco etch virus (TEV) genomes expressing either β‐glucuronidase or green fluorescent protein reporters. Twelve of the mutants were replication‐defective in protoplast inoculation assays. Among the 19 replication‐competent mutants, several possessed cell‐to‐cell or long‐distance movement defects in tobacco plants. Two mutants, AS1 and AS8, were restricted to single cells in inoculated leaves despite genome amplification levels that were equivalent to that of parental virus. Other mutants, such as AS9 and AS14, were able to move cell to cell slowly but were debilitated in long‐distance movement. These data provide genetic evidence for a direct role of CI protein in potyvirus intercellular movement, and for distinct roles of the CI protein in genome replication and movement. In combination with high‐resolution ultrastructural analyzes and previous genetic data, these results support a model in which CI protein interacts directly with plasmodesmata and capsid protein‐containing ribonucleoprotein complexes to facilitate potyvirus cell‐to‐cell movement.
Oxidative stress tolerance and longevity in Arabidopsis: the late‐flowering mutant gigantea is tolerant to paraquat
Authors: Kurepa Jasmina , Smalle Jan , Va Marc , Montagu N , Inzé Dirk
Published: March 2003

Recent genetic analyses of longevity in animals have revealed that long‐lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late‐flowering mutants to the superoxide radical‐generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi‐3) with the longest delay in flowering time had a high tolerance level to paraquat‐induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild‐type, although the actual levels of tolerance differed. In addition, the gi‐3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.
The untranslated leader sequence of the barleylipoxygenase 1(Lox1) gene confers embryo‐specific expression
Authors: Rouster Jacques , Van Mechelen Jan , Cameron‐Mills Verena
Published: March 2003

The barley

lipoxygenase 1

(

Lox1

) gene encodes a protein expressed in embryos during grain development and germination and in leaves after methyl‐jasmonate (MeJA) treatment. Transient gene expression assays in germinating barley embryos were used to identify

cis

‐regulatory elements involved in the embryo‐specific expression of the

Lox1

gene. Analysis of transcriptional or translational fusions between

Lox1

5′ upstream sequences and the

gusA

reporter gene indicated that the 5′‐untranslated leader sequence was involved in embryo‐specific expression. Replacement of the leader sequence from the aleurone‐specific

Chi26

gene with the

Lox1

leader sequence resulted in a chimeric gene expressed at high levels in embryo as well as in aleurone cells. Insertion of the

Lox1

leader sequence between the 35S minimum promoter (A domain –90/+8) and the

gusA

reporter gene greatly enhanced promoter activity in a tissue‐specific manner. Deletion/replacement analysis of the

Lox1

leader sequence, combined with transient expression in germinating embryos and

in vitro

transcription/translation assays, suggests that the

Lox1

leader sequence contains

cis

‐elements regulating qualitative (tissue‐specific) and quantitative gene expression.