bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant
Authors: Vogel Guido , Aeschbacher Roger A. , Müller Joachim , Boller Thomas , Wiemken Andres 
Published: March 2003

SummaryIt is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought‐resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose‐6‐phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat‐sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C‐terminal part of the yeast TPS2 gene and other microbial trehalose‐6‐phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose‐6‐phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose‐6‐phosphate but not glucose‐6‐phosphate or sucrose‐6‐phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose‐6‐phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.
Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato
Authors: Wolters Anne‐marie A. , Trindade Luisa M. , Jacobsen Evert , Visser Richard G. F. 
Published: March 2003

SummaryIn this study the T‐DNA composition of four antisense potato transformants showing complete or very strong inhibition of granule‐bound starch synthase (GBSS) activity was analysed in detail. By Southern blot hybridizations, it was determined that all four transformants contained T‐DNAs on multiple linkage groups and that most linkage groups contained multiple T‐DNA copies, often in combination with non‐T‐DNA vector sequences. Subsequently, fluorescence in situ hybridization was performed on extended DNA fibres (‘fibre‐FISH’) of three progeny plants each containing a single linkage group with a complex T‐DNA organization. By using two differently labelled probes, one consisting of T‐DNA sequences and the other of vector DNA sequences, it was possible to visualize the composition of complex loci. DNA sequences of 5–6 kb were well distinguishable. With this technique it is possible to determine T‐DNA copy number, and arrangement of T‐DNA and vector DNA sequences in a locus, more accurately than by Southern blot analysis alone. Therefore, fibre‐FISH is a valuable supplementary tool to study T‐DNA integrations in detail.
Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms
Authors: Xu Huiling , Swoboda Ines , Bhalla Prem L. , Sijbers Anneke M. , Zhao Chongxin , Ong Eng‐kok , Hoeijmakers Jan H. J. , Singh Mohan B. 
Published: March 2003

SummaryNucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross‐linking agent mitomycin C in ERCC1‐deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.
Cytogenetics for the model system Arabidopsis thaliana
Authors: Fransz Paul , Armstrong Sue , Alonso‐blanco Carlos , Fischer Thilo C. , Torres‐ruiz Ramón A. , Jones Gareth 
Published: March 2003

SummaryA detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm‐ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.
Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene
Authors: Kumpatla Siva P. , Hall Timothy C. 
Published: March 2003

SummaryExtensive methylation of the Ubi1 promoter has been shown to be associated with transcriptional silencing and bialaphos herbicide sensitivity in several R1 progeny derived from a transgenic rice line, JKA 52, containing multiple copies of the introduced genes (Kumpatla et al. 1997;Plant Physiol. 115, 361–373). All of the progeny from silenced R1 lines were silenced, and a proportion (24%) of the R2 seedlings from JKA 52–6, a non‐silenced homozygous R1 line, were found to be silenced. Several non‐silenced (bialaphos‐resistant) R2 progeny derived from the selfed seed of JKA 52–6 were tested at various stages throughout the growth period and found to be resistant to bialaphos, indicating the absence of silencing. Five of these were investigated further to determine whether silencing could recur in subsequent generations. R3 seedlings were raised from selfed R2 seed and herbicide sensitivity was detected in 5–40% of the plants in three of the five lines. This silencing was accompanied by methylation of the Ubi1 promoter and the absence of steady‐state bar mRNA. This recurrent onset of silencing and instability of expression associated with a transgene locus containing several repeated sequences is undesirable. The data strongly suggest that multi‐generation molecular analysis should be undertaken for transgenic lines to be used for agricultural purposes and that lines containing methylated inserts should be carefully evaluated before being included in further breeding strategies.
Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells
Authors: Hussey Patrick J. , Yuan Ming , Calder Grant , Khan Safina , Lloyd Clive W. 
Published: March 2003

SummaryMaize actin‐depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein‐conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re‐modelling the actin after ADF1 injection may be provided by the microtubular system.
Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron
Authors: Lumbreras Victoria , Stevens David R. , Purton Saul 
Published: March 2003

SummaryHeterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation‐independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high‐level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.
Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes
Authors: Ritt Christoph , Grimm Rudi , Fernández Silvia , Alonso Juan C. , Grasser Klaus D. 
Published: March 2003

SummaryIn contrast to other eukaryotes which usually express two closely related HMG1‐like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA‐binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini‐circles and supercoiled DNA. They induce DNA‐bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self‐associate, whereas they are monomeric in solution. The maize HMG1‐like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin‐associated protein required for the site‐specific β‐mediated recombination. A variable function of the HMG1‐like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.
Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina
Authors: Blackman Leila M. , Overall Robyn L. 
Published: March 2003

SummaryThe macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata‐associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.
The GCN4 motif in a rice glutelin gene is essential for endosperm‐specific gene expression and is activated by Opaque‐2 in transgenic rice plants
Authors: Wu Chuan‐Yin , Suzuki Akihiro , Washida Haruhiko , Takaiwa Fumio 
Published: March 2003

SummaryThe GCN4 motif is conserved in a number of seed storage protein genes, and promoter fragments containing this motif have been shown to be involved in controlling seed‐specific expression of the genes studied. All genes encoding the rice seed storage protein glutelin contain the GCN4 motif at similar sites in their 5′ flanking regions. Using a stable homologous transgenic system, we have analysed the promoter of the rice glutelin gene GluB‐1 and demonstrated that the GCN4 motif functions as an essential cis‐element for endosperm‐specific gene expression. Moreover, a 21 bp GluB‐1 promoter fragment spanning the GCN4 motif, as a multimer, directed GUS gene expression in endosperm of transgenic rice plants, when fused directly to the core promoter (–46) of CaMV 35S. In transiently transfected rice protoplasts, over a hundred‐fold transactivation was observed from the 21 bp sequence by the bZIP type transcriptional activator Opaque‐2 (O2) co‐expressed under a CaMV 35S promoter. The transactivation was also evident in transgenic plants containing both O2 and the 21 bp sequence/GUS fusion. The O2‐mediated activation requires binding of O2 to an intact GCN4 motif. Our results suggest that a bZIP protein functionally similar to O2 may exist in rice and participate in controlling the endosperm‐specific expression of GluB‐1 through the GCN4 motif.
A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone
Authors: Crombie Hazel J. , Chengappa Sumant , Hellyer Amanda , Reid J. S. Grant 
Published: March 2003

SummaryA ‐D‐glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (Mr 76 000; a glycoprotein; pI > 9.5; apparent pH optimum 4.5; temperature optimum 30°C) catalysed the hydrolysis of p‐nitrophenyl‐‐D‐glucopyranoside, cello‐oligosaccharides, ‐linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1ν3) > (1ν4) > (1ν2) > (1ν6)] with significant transglycosylation occurring in the early stages of the reaction. Cello‐oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1ν6)‐. Xyloglucan oligosaccharides xylose‐substituted at the non‐reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose‐substituted glucose residue, and L is a galactosylxylose‐substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non‐reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non‐reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium ‐D‐galactosidase (
Edwards et al. (1988
) J. Biol. Chem. 263, 4333–4337), followed by alternations of nasturtium xyloglucan‐specific α‐xylosidase (
Fanutti et al. (1991
) Planta 184, 137–147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT–PCR and by screening cDNA libraries. A composite, full‐length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N‐terminal and internal sequences obtained from the purified ‐glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with ‐glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of ‘Family 3’, according to the classification of Henrissat (
Henrissat (1991) Biochem. J. 280, 309–316). There was strong sequence homology with a ‐glucan exo‐hydrolase from barley (
Hrmova et al. (1996
) J. Biol. Chem. 271, 5277–5286). The nasturtium ‐glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the α‐xylosidase and the ‐galactosidase is modelled.
Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin‐dependent heme oxygenase from Synechocystis sp. PCC 6803
Authors: Cornejo Juan , Willows Robert D. , Beale Samuel I. 
Published: March 2003

SummaryThe phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXα, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IXα. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin‐dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IXα by spectrophotometry and reverse‐phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100 000 g. Expression of HO1 increased several‐fold during incubation of the cells for 72 h in iron‐deficient medium.
The ArabidopsisERECTAgene is expressed in the shoot apical meristem and organ primordia
Authors: Yokoyama Ryusuke , Takahashi Taku , Kato Atsushi , Torii Keiko U. , Komeda Yoshibumi 
Published: March 2003

Summary In

Arabidopsis thaliana

(L.) Heynh, the mutation in

ERECTA

is known to confer a compact inflorescence by a reduction in the lengths of internodes and pedicels. We analyzed the expression pattern of this gene during plant development.

In situ

hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with the

ERECTA

promoter fused to the β‐glucuronidase (GUS) gene, showed that

ERECTA

was predominantly expressed in the shoot apical meristems and organ primordia.

ERECTA

expression in the shoot apical meristem was weak early in plant development but increased with the transition from the vegetative to the reproductive growth phase.

ERECTA

was also strongly expressed in organ primordia and immature organs but weakly in mature organs. Thus,

ERECTA

was expressed in a cell‐specific and developmentally regulated manner. In order to identify the regulatory mechanism responsible for the expression pattern of

ERECTA

, the

cis

‐acting regions in the

ERECTA

promoter were defined by study of the expression of the chimeric genes that consist of the 5′‐ or internal deleted promoter and a GUS reporter gene in transgenic plants. The results showed that the essential

cis

‐regulatory elements governing the spatially and temporally specific expression of

ERECTA

are located between positions –462 and – 228 bp and between positions –228 and –153 bp with respect to the transcriptional initiation site.
Over‐expression of a tobacco homeobox gene,NTH15, decreases the expression of a gibberellin biosynthetic gene encoding GA 20‐oxidase
Authors: Tanaka‐Ueguchi Miyako , Itoh Hironori , Oyama Naomi , Koshioka Masaji , Matsuoka Makoto 
Published: March 2003

Summary Ectopic expression of the homeobox gene,

NTH15

(

Nicotiana tabacum

homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA1. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA19 to GA20 was blocked in transgenic tobacco plants overexpressing

NTH15

. To investigate the relationship between the expression of

NTH15

and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20‐oxidase and a 3β‐hydroxylase. RNA gel blot analysis revealed that the expression of one gene (

Ntc12

, encoding GA 20‐oxidase), which in wild‐type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of

Ntc12

decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA20 or GA1 but not by GA19. These data strongly suggest that overexpression of

NTH15

inhibits the expression of

Ntc12

, resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants.

In situ

hybridization in wild‐type tobacco revealed that expression of

Ntc12

occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which

NTH15

was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.