bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of a 70 kDa heat‐shock protein of bean mitochondria
Authors: Vidal Vincent , Ranty Benoit , Dillenschneider Marietta , Charpenteau Martine , Ranjeva Raoul 
Published: March 2003

SummaryA bean cDNA clone that specifies a 70 kDa heat‐shock protein (hsp70) has been isolated and sequenced. The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family. The predicted protein was found to contain an amino‐terminal extension typical of transit sequences. The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame. This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product. The imported protein is identical in size to a mitochondrial protein that cross‐reacts with hsp70‐specific antibodies. The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family. Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa. Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium‐dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only. The data are discussed with respect to the chaperone function of hsp70 in mitochondria.
Fruit‐specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense‐related gene expression in leaves
Authors: Martineau Belinda , Houck Catherine M. , Sheehy Raymond E. , Hiatt William R. 
Published: March 2003

SummaryThis paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit‐specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100‐fold higher than in control fruit. In the leaves of a fruit‐bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR‐1 and chitinase mRNAs, which encode defense‐related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.
A cis‐acting element and a trans‐acting factor involved in the wound‐induced expression of a horseradish peroxidase gene
Authors: Kawaoka Akiyoshi , Kawamoto Tomohiro , Sekine Masami , Yoshida Kazuya , Takano Mitsuo , Shinmyo Atsuhiko 
Published: March 2003

SummaryThe mechanisms that control the wound‐induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′‐deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β‐glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP‐1, that bound specifically to the Box 1 sequence was identified. The putative TFHP‐1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP‐1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP‐1 suppressed the expression of prxC2 (−529)/GUS.
Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol‐14‐demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol‐14‐demethylase herbicide inhibitor
Authors: Grausem Bernard , Chaubet Nicole , Gigot Claude , Loper John C. , Benveniste Pierre 
Published: March 2003

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T‐DNA in which the gene CYP51A1 encoding lanosterol‐14‐demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol‐14‐demethylase (OBT14DM) and LAN14DM) but were resistant to γ‐ketotriazole (γ‐kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ‐kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective.Further analysis of the transgenic calli grown in the presence of γ‐kt showed that their Δ5‐sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ‐kt, displayed a sterol content strongly enriched in 14α‐methyl sterols and depressed in physiological Δ5‐sterols. When the transgenic calli were cultured in mixtures of γ‐kt and LAN14DM inhibitors sterol compositions enriched in 14α‐methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM.Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14‐demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.
A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre—lox
Authors: Osborne Brian I. , Wirtz Uwe , Baker Barbara 
Published: March 2003

SummaryA system for insertional mutagenesis and chromosomal rearrangement in Arabidopsis has been developed. The T‐DNA vectors are based on the maize transposon Ds, lox sites from the Cre—lox site‐specific recombination system, and transcriptional fusions expressing Ac transposase or Cre recombinase. The engineered transposon is termed Dslox. Transposed Dslox insertions were created by crossing plants bearing Dslox with plants expressing Ac transposase, then simultaneously selecting for excision and re‐insertion in F2 seedlings using the herbicides chlorsulfuron and phosphonothricin, respectively. F2 plants bearing stable Dslox insertions were identified by scoring for the absence of the Ac transposase T‐DNA, using a novel, visual marker in that T‐DNA. Two independent Dslox insertions were characterized and placed 5.6 and 16.5 cM from their T‐DNAlox, which mapped close to m506 on chromosome 4. Plants bearing either of the two different transposed Dsloxs and T‐DNAlox were crossed to plants expressing Cre recombinase, which catalyzed recombination between the lox site in transposed Dslox and the lox site in T‐DNAlox. Lox—lox recombinants were identified selectively amongst progeny of these crosses. Molecular and genetic analysis of the lox—lox rearrangements indicated that both were inversions. The smaller inversion was germinally transmitted from generation to generation as a simple trait, whereas the larger inversion was not transmitted to progeny of plants bearing the rearrangement.
Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta
Authors: Faiss Martin , Strnad Miroslav , Redig Pascale , Doležal Karel , Hanuš Jan , Van Onckelen Harry , Schmülling Thomas 
Published: March 2003

SummaryThe rolC gene of Agrobacterium rhizogenes T‐DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC β‐glucosidase is due to an alteration of the cytokinin balance in planta. HPLC radiocounting assays of [3H]‐labeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coli cells or cell‐free extracts showed that cytokinin N3‐ and O‐glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7‐ and N9‐glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC‐expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline‐inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin‐synthezising ipt gene and the rolC gene showed added phenotypes indicating that the rolC phenotype is mediated on a signalling pathway different from those of cytokinins. RolC/ipt hybrids also accumulated high levels of cytokinin O‐glucosides. It is concluded that the phenotypic alterations caused by the rolC gene product are not due to a release of free cytokinins from inactive conjugates, most likely because of subcellular compartmentation of the putative substrate.
Salt regulation of transcript levels for the c subunit of a leaf vacuolar H+‐ATPase in the halophyte Mesembryanthemum crystallinum
Authors: Tsiantis Miltos S. , Bartholomew Dolores M. , Smith J. Andrew C. 
Published: March 2003

SummaryThe halophyte Mesembryanthemum crystallinum is an inducible crassulacean acid metabolism (CAM) plant native to seasonally arid coastal environments that has been widely used to study plant responses to environmental stress. On exposure of plants to salt, the activities of both the tonoplast (vacuolar) H+‐ATPase (V‐ATPase) and Na+/ H+ antiporter increase in leaf cells, thereby energizing vacuolar salt accumulation. To investigate the molecular basis of this response, a cDNA (Vmac1) encoding the H+‐conducting c subunit (16.6 kDa) of an M. crystallinum V‐ATPase has been cloned. Northern analysis of RNA from leaves of plants treated with NaCl or with isoosmotic mannitol solutions demonstrated (i) that NaCl increased steady‐state transcript levels for the V‐ATPase c subunit, and (ii) that this effect was caused by the ionic rather than the osmotic component of salt stress. Southern analysis of genomic DNA suggested the probable existence of more than one gene for this subunit of the V‐ATPase in M. crystallinum. Expression studies using the 3′‐untranslated region of the Vmac1 cDNA as a probe showed that the corresponding salt‐inducible transcript was preferentially expressed in leaves. Induction by salt was also observed in juvenile plants in addition to adult ones. These findings, as well as the inability of mannitol to upregulate mRNA levels for this gene, clearly differentiate between the induction of transcript for the V‐ATPase c subunit and for genes involved in the CAM pathway in M. crystallinum. Further, the plant growth regulator abscisic acid (ABA) was able to mimic the effect of salt on transcript levels for the V‐ATPase c subunit, suggesting the possible involvement of ABA in a distinct signal‐transduction pathway linked to vacuolar salt accumulation in this highly salt‐tolerant species.
Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus
Authors: Wang Tzann‐Wei , Balsamo Ronald A. , Ratnayake Chandra , Platt Kathryn A. , Ting Julie T.L. , Huang Anthony H.C. 
Published: March 2003

SummarymRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15‐residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS‐PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of ∼ 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N‐terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of low‐density organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule‐filled plastids and lipid‐containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low‐density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil‐body oleosins share common epitopes at the generally non‐conserved C‐terminal domain with the sporophytic tapetum oleosins.
Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers
Authors: Serna Laura , Fenoll Carmen 
Published: March 2003

SummaryThe origin and process by which the mosaic of the different cell types is established during the development of the leaf epidermis in Arabidopsis are largely unknown, although the recent characterization of two mutants which develop stomatal clusters (four lips (flp) and too many mouths (tmm)) has opened up the possibility for genetic dissection of the stomata spacing. By using growth conditions which limit gas exchange with the open atmosphere, stomatal clusters that look like phenocopies of flp and tmm have been induced, suggesting that stomata spacing is under environmental as well as genetic control in Arabidopsis. The origin of these clusters has been addressed by following promoter activity for genes that are markers for competence for cell division (cdc2aAt), mitotic activity (cyc1aAt), and guard mother cell and developing guard cell identity (rha1). Their different expression patterns in the various cell types during epidermal differentiation and the asynchrony in the development of the various stomata that constitute each cluster suggest that these stomatal clusters derive from a single protodermal cell through a process that involves changes in cell fate in a subset of subsidiary cells. It was also found that guard cells express cdc2aAt and cyc1aAt, supporting the idea that they may remain competent for cell division.
Chromosome micro‐dissection and region‐specific libraries from pachytene chromosomes of maize (Zea mays L.)
Authors: Stein  , Ponelies  , Musket  , McMullen  , Weber  
Published: March 2003

SummaryFor the first time, pachytene chromosomes have been used for micro‐dissection, amplification and cloning using the short arm of maize chromosome 6. For this purpose, methods for the preparation of unstained pachytene chromosomes were developed. In two separate experiments, 10 and 12 segments of the satellite region were isolated utilizing glass needles and DNA was amplified by linker‐adaptor‐PCR. After amplification, maize‐specific DNA was verified by genomic Southern hybridizations. Chromosomal in situ suppression hybridization confirmed that the adaptor PCR products originated from the micro‐dissected 6S region. The adaptor PCR products were cloned into a plasmid vector and the chromosome region‐specific libraries were characterized.
Transposase binding site methylation in the epigenetically inactivated Ac derivative Ds‐cy
Authors: Wang Lihua , Kunze Reinhard 
Published: March 2003

SummaryThe authors have determined the C‐methylation pattern of the non‐autonomous transposable element Ds‐cy, which is an epigenetically inactivated, transcriptionally silent derivative of the maize Activator (Ac) element. Like Ac, Ds‐cy is hypermethylated at the 3′‐end. However, in Ds‐cy the 5′‐end is also hypermethylated, including all subterminal binding sites for the Ac‐encoded transposase protein. As Ds‐cy transposes in the presence of an active Ac in the genome, the authors conclude that methylation of the 5′‐end TPase binding sites does not interfere with transposition, but correlates with inactivity of the Ac promoter. The authors discuss the implications of these results for the chromatid selectivity of Ac/Ds transposition and the mechanism of Ac‐induced chromosome breakage events.
Gene silencing from plant DNA carried by a Geminivirus
Authors: Kjemtrup Susanne , Sampson Kim S. , Peele Charles G. , Nguyen Long V. , Conkling Mark A. , Thompson William F. , Robertson Dominique 
Published: March 2003

SummaryThe geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open‐reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3‐5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti‐sense orientation, and a 403 bp 3' fragment. TGMV::su‐induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti‐sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.
Glucocorticoid‐inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death
Authors: McNellis Timothy W. , Mudgett Mary Beth , Li Karen , Aoyama Takashi , Horvath Diana , Chua Nam‐Hai , Staskawicz Brian J. 
Published: March 2003

SummaryPathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid‐inducible promoter. Dexamethasone‐induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae‐induced hypersensitive response and also induced the expression of a pathogenesis‐related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid‐inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene‐specified cell death response in both resistant and susceptible plants.
Transcription pattern of a FIM homologue in Impatiens during floral development and reversion
Authors: Pouteau Sylvie , Nicholls David , Tooke Fiona , Coen Enrico , Battey Nicholas 
Published: March 2003

SummaryFlowering and reversion in Impatiens are characterised by gradual transitions of organ identity and constitute a unique system for the molecular and physiological study of floral organogenesis. The authors have isolated an Impatiens homologue of the FIM gene of Antirrhinum (UFO in Arabidopsis), Imp‐FIM, and analysed its expression in three states of the terminal meristem: vegetative, floral, and reverted. In floral meristems, Imp‐FIM transcription is associated with petal identity, as in Antirrhinum and Arabidopsis, but this is achieved through a novel transcription pattern, characterised by a high level of transcript within petal primordia. This novel transcription pattern could contribute to the more diffuse boundaries between organ types in Impatiens. In vegetative meristems, Imp‐FIM is expressed in the axils of leaf primordia which are arranged in a spiral. A similar pattern is observed in reverted meristems in which leaf primordia are initiated in a whorled arrangement. This result indicates that the maintenance of floral phyllotaxis is not associated with a specific pattern of Imp‐FIM transcription. Transcription of Imp‐FIM in a non‐reverting line is no different from that in the reverting line. Therefore, the lack of floral commitment in the reverting line does not seem to be responsible for Imp‐FIM transcription within petals. The novel transcription pattern in petals, together with features of Impatiens that are reminiscent of fim and ufo mutant phenotypes suggest an evolutionary divergence for Imp‐FIM regulation in this species.