bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial patterns of expression of flavonoid/isoflavonoid pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme
Authors: Harrison Maria J. , Dixon Richard. A.
Published: March 2003

A combination of Northern blot analysis and in situ hybridization was used to measure and localize changes in the levels of transcripts encoding five enzymes of phenylpropanoid/flavonoid/isoflavonoid metabolism in Medicago truncatula roots following colonization with the mycorrhizal fungus Glomus versiforme. In colonized roots, elevated levels of phenylalanine ammonia‐lyase (PAL) and chalcone synthase (CHS) transcripts were detected specifically in the cortical cells containing arbuscules. This localization was discrete, and elevated levels of transcripts were not observed in adjacent, non‐colonized cells. In contrast, isoflavone reductase (IFR) transcripts, which were detected at relatively high levels in the cortical cells of non‐colonized roots, were almost undetectable in the colonized cortical cells containing arbuscules and were also lowered in adjacent cells. The expression of chalcone isomerase (CHI) and isoliquiritigenin 2′‐O‐methyltransferase (ChalOMT) was unaffected by colonization, and the tissue‐specific patterns of gene expression were the same as in non‐colonized roots. It is concluded that the establishment of the mycorrhizal interaction results in cell type‐specific differential expression of genes of phenylpropanoid/flavonoid/isoflavonoid biosynthesis, which may be causally related to arbuscule development.
The developmental expression of the asparagus intracellular PR protein (AoPR1) gene correlates with sites of phenylpropanoid biosynthesis
Authors: Warner Simon A.J. , Gill Aideen , Draper John
Published: March 2003

Previous reports have described the induction, by either wounding or attempted pathogen invasion, of an Asparagus officinalis intracellular pathogenesis‐related (AoPR1) promoter—GUS gene fusion in transgenic tobacco. Here we describe the unexpected developmental expression pattern of the AoPR1—GUS gene which correlates well, temporally and spatially, with the developmental expression observed for GUS fusions with promoters derived from genes coding for enzymes in the ‘core phenylpropanoid pathway’. Analysis of endogenous AoPR1 gene expression in asparagus and both AoPR1—GUS and AoPR1—luciferase gene fusions in transgenic tobacco suggests that the AoPR1 promoter directs similar cell‐specific transcription patterns in both asparagus and transgenic tobacco. The AoPR1 promoter contains sequence motifs similar to those implicated as important in the regulation of phenylpropanoid pathway genes and another ‘intracellular’ PR gene. Treatment with salicylic acid enhances AoPR1 promoter gene activity both in tobacco and in asparagus.
Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide‐H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae
Authors: Stolz Jürgen , Stadler Ruth , Opekarová Miroslava , Sauer Norbert
Published: March 2003

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine‐tagged STP1‐His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild‐type STP1 and the recombinant his‐tagged protein were located in the plasma membranes of transformed yeast cells. The C‐terminal modification caused no loss of transport activity compared with the wild‐type protein. Anti‐STP1‐antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome‐c oxidase. Addition of ascorbate/TMPD/cytochrome‐c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [3H]tetraphenylphosphonium cations) and a simultaneous, uncoupler‐sensitive influx of d‐glucose into the energized vesicles. STP1‐His6 protein is functionally active after solubilization with octyl‐β‐d‐glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one‐step purification of the STP1‐His6 protein on Ni2+‐NTA columns. The identity of the purified protein was checked by immunoblotting and N‐terminal sequencing.
Analysis of phytochrome‐ and ABA‐deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia
Authors: Kraepiel Yvan , Rousselin Philippe , Sotta Bruno , Kerhoas Lucien , Einhorn Jacques , Caboche Michel , Miginiac Emile
Published: March 2003

The phytochrome chromophore‐deficient mutant, pew1, of Nicotiana plumbaginifolia exhibited decreased germination and slower dehydration of detached leaves during water stress as compared with the wild‐type. These physiological processes are controlled by abscisic acid (ABA) and we examined, therefore, whether phytochrome plays a specific role in the regulation of ABA metabolism using the pew1 mutant. The ABA contents of mature seeds and young leaves were analysed and in both cases mutant material was found to contain higher amounts of ABA as compared with the wild‐type. This indicates that the phytochrome activation can lead to a decrease of the ABA level in the wild‐type plant. The role of phytochromes was investigated in greater detail using the ABA‐deficient mutant aba1 of N. plumbaginifolia exhibiting an early and synchronous germination. This mutant accumulates at very high levels a metabolite derived from a precursor (ABA‐aldehyde) in the ABA biosynthetic pathway. The first biochemical characterization of this molecule, which corresponds to the glucose‐conjugated ABA‐alcohol (ABA‐AG) is described. A pew1‐aba1 double mutant exhibiting both an etiolated growth and early germination was also obtained. The comparable accumulation of ABA‐AG in the pew1‐aba1 double mutant as compared with the aba1 mutant allowed the proposition that, in a wild‐type plant, the phytochrome‐mediated light signal enhances ABA degradation rather than inhibits its biosynthesis.
Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)
Authors: Kang Fan , Rawsthorne Stephen
Published: March 2003

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP‐glucose‐6‐phosphate dehydrogenase, NADP‐6‐phosphogluconate dehydrogenase, fructose‐1,6‐bisphosphatase, NADP‐malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl‐CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with 14C‐labelled carbon precursors and the incorporation of 14C into starch and fatty acids was determined. 14C from glucose‐6‐phosphate (G‐6‐P), fructose, glucose, fructose‐6‐phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness‐ and ATP‐dependent manner. The rate of starch synthesis was highest from G‐6‐P, although fructose gave rates which were 70% of those from G‐6‐P. Glucose‐1‐phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G‐6‐P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G‐6‐P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.
The blue light‐responsive AthH2 gene of Arabidopsis thaliana is primarily expressed in expanding as well as in differentiating cells and encodes a putative channel protein of the plasmalemma
Authors: Kaldenhoff R. , Kölling A. , Meyers J. , Karmann U. , Ruppel G. , Richter G.
Published: March 2003

According to our previous studies the Arabidopsis gene AthH2 which is inducible by blue light and phytohormones codes for an intrinsic membrane protein. It bears a resemblance to several distinct channel proteins of plant and animal species classified as the MIP/NOD‐26/GlpF family. In the present study biochemical analyses and electron microscopic immunochemistry were used to elucidate the subcellular location of the AthH2 protein. The results clearly demonstrate that it is an exclusive constituent of the plasmalemma. Furthermore, the expression of the AthH2 gene in transgenic Arabidopsis plants containing the promoter region of AthH2 fused to the β‐glucuronidase (gus) reporter gene was studied. The in situ localization of gus activity revealed that the specific promoter is temporally activated by light in expanding and/or differentiating cells comprising newly formed tissues and organs: root elongation zone, guard cells of stomata, vascular bundle sheaths, filaments of stamen and young siliques. Several sites of gus expression coincide spatially with those of in situ hybridization and the immunocytochemical reaction, respectively, suggesting that the AthH2 promoter had correctly responded to light as an important exogenous factor with relevance to the complex pattern of differentiation. Studies with protoplasts from plants transformed with an antisense construct revealed a water transport capacity of the AthH2 protein.
Signalling potential of iron in plant—microbe interactions: the pathogenic switch of iron transport in Erwinia chrysanthemi
Authors: Masclaux Céline , Expert Dominique
Published: March 2003

Although competition for iron has long been recognized in bacterial virulence and animal host defence, its role in plant pathogenesis has only been clarified recently, with the soft rot pathogen Erwinia chrysanthemi. A functional genetic system encoding the production and utilization of the siderophore chrysobactin is required for symptom expansion on African violets and chrysobactin has been detected in 24‐h old diseased tissues. It was also established that the transcriptional activity of the fct cbs operon representative of this system, encoding transport and biosynthetic functions, is negatively regulated by iron. In this study, Escherichia coli lacZ was used as a reporter gene to analyse the expression of this operon at the onset of infection. A series of fct::lac chromosomal fusions regulated by iron were constructed and the behaviour of one engineered strain was analysed in planta; induction of LacZ activity appeared to occur after 10 h following leaf inoculation and to be subject to variations over time typical of the regulatory response to iron. In contrast to parental cells, engineered cells thus disrupted in the production and utilization of chrysobactin gave rise to a necrotic front associated with a downshift in viable counts. These data, for the first time, provide direct evidence of in vivo regulation of an iron‐controlled function associated with pathogenicity and demonstrate (i) that expression of the chrysobactin‐mediated iron uptake system contributes greatly to invasive growth of the pathogen, and (ii) that plant extracellular fluids can be detected by pathogens as iron‐limited environments.
Germin‐like oxalate oxidase, a H2O2‐producing enzyme, accumulates in barley attacked by the powdery mildew fungus
Authors: Zhang Ziguo , Collinge David B. , Thordal‐Christensen Hans
Published: March 2003

Germin‐like oxalate oxidase is an oligomeric enzyme which generates H2O2. This paper reports increased activity of this enzyme in association with the response of barley to the powdery mildew fungus, Erysiphe graminis f.sp. hordei. The increase is detected in a colorimetric assay as well as on activity blots using extracts of both resistant and susceptible leaves. The increase is generally apparent from 24 h after inoculation. From 48 h after inoculation, there is an approximately 10‐fold higher oxalate oxidase activity in the samples from inoculated plants compared with the controls. The oxalate oxidase activity increase appears 1–3 days earlier than PR‐1 protein accumulation. SDS—PAGE analysis suggests that this pathogen‐response oxalate oxidase is different from a commercially available barley oxalate oxidase, and from a constitutive barley root oxalate oxidase.
Organ‐specific differences in the ratio of alternatively spliced chorismate synthase (LeCS2) transcripts in tomato
Authors: Görlach Jörn , Raesecke Hanns‐R. , Abel Gernot , Wehrli Rolf , Amrhein Nikolaus , Schmid Jürg
Published: March 2003

The primary transcript of one of the two chorismate synthase genes (LeCS2) of tomato is differentially processed due to an alternative splicing of the third intron. A novel observation was made when the abundances of the two LeCS2‐specific transcripts in different organs were analysed. The ratio of these two transcripts differs in RNA populations from different organs. Possible explanations for this finding and its potential physiological impact for plant metabolism are discussed.
The axr4 auxin‐resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation
Authors: Hobbie Lawrence , Estelle Mark
Published: March 2003

To understand the molecular mechanism of auxin action, mutants of Arabidopsis thaliana with altered responses to auxin have been identified and characterized. Here the isolation of two auxin‐resistant mutants that define a new locus involved in auxin response, named AXR4, is reported. The axr4 mutations are recessive and map near the ch1 mutation on chromosome 1. Mutant plants are specifically resistant to auxin and defective in root gravitropism. Double mutants between axr4 and the recessive auxin‐resistant mutants axr1‐3 and aux1‐7 were characterized to ascertain possible genetic interactions between the mutations. The roots of the axr4 axr1‐3 double mutant plants are less sensitive to auxin, respond more slowly to gravity, and form fewer lateral roots than either parental single mutant. These results suggest that the two mutations have additive or even synergistic effects. The AXR1 and AXR4 gene products may therefore act in separate pathways of auxin response or perhaps perform partially redundant functions in a single pathway. The axr4 aux1‐7 double mutant has the same sensitivity to auxin as the aux1‐7 mutant but forms far fewer lateral roots than either parental single mutant. The aux1‐7 mutation thus appears to be epistatic to axr4 with respect to auxin‐resistant root elongation, whereas in lateral root formation, the effects of the two mutations are additive. The complexity of the genetic interactions indicated by these results may reflect differences in the mechanism of auxin action during root elongation and the formation of lateral roots. The AXR4 gene product, along with those of the AXR1 and AUX1 genes, is important for normal auxin sensitivity, gravitropic response in roots and lateral root formation.
A constituent of the chloroplast import complex represents a new type of GTP‐binding protein
Authors: Seedorf Matthias , Waegemann Karin , Soll Jürgen
Published: March 2003

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non‐reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co‐immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full‐length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP‐binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C‐terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N‐terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C‐terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease‐sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo‐affinity labelling in the presence of [α‐32P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.
Altered lignin composition in transgenic tobacco expressing O‐methyltransferase sequences in sense and antisense orientation
Authors: Atanassova Rossitza , Favet Noëlle , Martz Françoise , Chabbert Brigitte , Tollier Marie‐Thérèse , Monties Bernard , Fritig Bernard , Legrand Michel
Published: March 2003

The monomeric composition of tobacco lignin has been modified by genetic engineering. Sense or antisense expression of sequences encoding O‐methyltransferase (OMT), a lignin biosynthetic enzyme, was shown to modulate enzyme activity. Ten constructs harboring the entire or a partial OMT cDNA were used. Populations of 20 trensgenic plants per construct were analyzed for OMT activity and compared with untransformed controls. As expected, expression of only the full‐length sense construct led to an increase in OMT activity. An important reduction of activity was found in a variable number of plantlets from all other transgenic populations but the inhibition was sustained through the adult stage only in plants transformed with the complete cDNA. T‐DNA genes were shown to be stably integrated into the tobacco genome and to be transmitted to the progeny. By using gene‐specific probes, OMT inhibition in stems was correlated to a parallel disappearance of OMT transcripts originating from both the resident gene and the transgene. In contrast, transgene transcripts were detected in leaf tissues where the resident gene is poorly expressed, thus indicating that relative expression of the two OMT genes controls transcript turnover. In stems of inhibited plants, a marked decrease of syringyl units and the appearance of 5‐hydroxy guaiacyl units were demonstrated. These two structural features are also characteristic of natural mutants of maize with an improved digestibility compared with wild lines. These data demonstrate the feasibility and the potential benefits of lignin manipulation.
The spinach AtpC and AtpD genes contain elements for light‐regulated, plastid‐dependent and organ‐specific expression in the vicinity of the transcription start sites
Authors: Bolle Cordelia , Kusnetsov Victor V. , Herrmann Reinhold G. , Oelmüller Ralf
Published: March 2003

Run‐on assays with isolated nuclei demonstrate that the transcription rates of AtpC and AtpD (gene products: the CF1 subunits γ and δ of the chloroplast ATP synthase) are comparable in spinach seedlings. However, chimeric GUS gene fusions with 5′‐flanking regions of the AtpC gene direct an approximately 10‐fold lower GUS level in transgenic tobacco compared with equivalent fragments from the AtpD gene. Both promoters contain sequences in the vicinity of the respective TATA boxes, which are sufficient to direct light‐regulated, plastid‐dependent and organ‐specific expression of the GUS gene. In contrast, the upstream regions of both promoters differ: the higher GUS level directed by the AtpD promoter is caused by enhancer‐like elements located upstream of the region involved in the regulated expression, while nucleotides upstream of −73 in the AtpC promoter contribute relatively little to the promoter activity. 5′‐Deletion analyses and site‐directed mutagenesis studies indicated that the −73/ −48 bp AtpC region contains cis‐elements crucial for this regulated expression. If five nucleotides within this region (−59/−55) are exchanged, the GUS gene is constitutively expressed and the activity in etiolated seedlings, in seedlings with photobleached plastids and in roots increases to the level detectable in green cotyledons. It is concluded that signal transduction pathways from different regulators converge prior to gene regulation and that these five nucleotides are part of a cis‐element which functions as a repressor in darkness, in tissues with impaired plastids and in roots.
The amino‐terminus of phytochrome A contains two distinct functional domains
Authors: Jordan Emily T. , Cherry Joel R. , Walker Joseph M. , Vierstra Richard D.
Published: March 2003

The N‐terminus of phytochrome A is important for the structural integrity and biological activity of the photoreceptor. Mutational analysis of the N‐terminus by two different strategies created two distinct photoreceptors, one inactive and the other hyperactive when expressed in transgenic tobacco, suggesting that this region has multiple functional domains. To identify critical residues within this N‐terminal region, a series of smaller deletions of oat phytochrome A were created, designated NB (Δ49–62), NC (Δ6–47), ND (Δ7–21), NE (Δ2–5), and NF (Δ6–12), and compared with a previously characterized deletion mutant NA (Δ7–69) and full‐length oat phytochrome A. Using photochemical properties as a measure of chromoprotein structure, it was found that the region between residues 13 and 62 was important for the spectral integrity of the photoreceptor. These deletion mutants were also biologically inactive when expressed in both mature tobacco plants and seedlings grown under continuous far‐red or red light. In contrast, deletion of the serine‐rich region between residues 6 and 12 did not alter the photochemical properties but did produce a hyperactive photoreceptor, indicating this region may be involved in down‐regulating phytochrome A activity. The data show that the N‐terminus of phytochrome A contains two functional domains, one necessary for conformational stability and biological activity (residues 13–62), and the other involved in attenuating phytochrome responses (residues 6–12).
In vitro binding of the tomato bZIP transcriptional activator VSF‐1 to a regulatory element that controls xylem‐specific gene expression
Authors: Torres‐Schumann Sonia , Ringli Christoph , Heierli Daniel , Amrhein Nikolaus , Keller Beat
Published: March 2003

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs‐1) specifically activated both the −82 CaMV 35S and the −76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs‐1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs‐1 in vitro. To study the molecular basis of xylem‐specific expression mediated by the vs‐1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs‐1 in vitro. This protein, designated VSF‐1, contains a bZIP motif close to the C‐terminus. Mutated vs‐1 elements were no longer bound by VSF‐1 and also failed to activate the minimal −82 CaMV 35S promoter in vivo. Transient expression of VSF‐1 in protoplasts stimulated vs‐1 dependent activation of the −76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF‐1 provided further evidence that vs‐1 is a potential in vivo target site, as VSF‐1 was a part of the observed complex formed between vs‐1 and nuclear protein extract. vs‐1 does not contain the 5′‐ACGT‐3′ core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF‐1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.
Isolation and characterization of an Arabidopsis thaliana cDNA encoding a Δ7‐sterol‐C‐5‐desaturase by functional complementation of a defective yeast mutant
Authors: Gachotte Daniel , Husselstein Tania , Bard Martin , Lacroute François , Benveniste Pierre
Published: March 2003

A yeast null mutant (erg 3) defective in ERG 3, the gene encoding the C‐5 sterol desaturase required for ergosterol synthesis was transformed with an Arabidopsis thaliana cDNA library inserted in a yeast vector. Transformants (4×105) were screened for cycloheximide (CH) resistance and 400 possible clones were analyzed to determine their sterol profile. Low levels of ergosterol in addition to Δ7‐ and Δ8‐sterols normally present in erg3 were isolated in three yeast transformants. Characterization of one transformant indicated a cDNA of 1141 bp. Transformation of an erg 3 strain with this plasmid led to CH resistance, nystatin sensitivity and an ergosterol profile. After subcloning in a pBluescript vector and subsequent sequencing, an ORF of 843 bp encoding a possible 281 amino acid polypeptide was deduced. Three histidine‐rich motifs (HX3H, HX2HH and HX2HH) were found in the A. thaliana ORF which are also present in the yeast ERG 3 gene. These histidine‐rich motifs are also characteristic of many membrane‐bound fatty acid desaturases from higher plants. These data strongly suggest that the A. thaliana cDNA encodes Δ7‐sterol‐C‐5‐desaturase.
High‐resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres
Authors: Fransz Paul F. , Alonso‐Blanco Carlos , Liharska Tsvetana B. , Peeters Anton J.M. , Zabel Pim , de Jong J. Hans
Published: March 2003

A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 µm, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multi‐colour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb µm−1, which is close to the Watson‐Crick DNA length estimate of 2.9 kb µm−1. Other experiments on small size molecular probes with both lambda clones (13.5–17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high‐resolution physical mapping of DNA sequences by FISH on plant DNA.
A protocol for obtaining embryogenic cell lines from Arabidopsis
Authors: Pillon Elisa , Terzi Mario , Baldan Barbara , Mariani Paola , Schiavo Fiorella Lo
Published: March 2003

Embryogenic cell lines with lasting embryogenic potential can be obtained from somatic embryos induced directly from zygotic embryos of Arabidopsis thaliana, ecotype Columbia. The response to a critical concentration of auxin, which seems to be the all‐important factor in the generation of embryogenic cell lines, is exhibited by somatic embryos but not by zygotic ones. The basis for this differential response remains obscure and will be discussed in relation to other systems.
Low threshold levels of ultraviolet‐B in a background of photosynthetically active radiation trigger rapid degradation of the D2 protein of photosystem‐II
Authors: Jansen Marcel A.K. , Gaba Victor , Greenberg Bruce M. , Mattoo Autar K. , Edelman Marvin
Published: March 2003

The photosystem II reaction centre has at its core a heterodimer made up of two proteins, D1 and D2. The D1 protein is known to be rapidly degraded by photosyn‐thetically active radiation while the D2 protein is relatively stable. This paper reports that when the aquatic higher plant, Spirodela was exposed to ultraviolet‐B radiation, D2 degradation accelerated markedly and half life times approached those of the D1 protein. Moreover, in the presence of an environmentally relevant background of photosynthetically active radiation, low fluxes of ultraviolet‐B (but not ultraviolet‐A) radiation synergistically stimulated degradation of the D2 protein within functional reaction centres. Thus, above a critical threshold, ultraviolet‐B specifically targets the D1–D2 heterodimer for accelerated degradation.
Regulation of extent of vegetative development of the maize shoot meristem
Authors: Irish Erin , Jegla Dorothy
Published: March 2003

In maize plants (Zea mays L.), the extent of vegetative development in the shoot is precisely regulated such that the apical meristem produces a predictable number of leaves before converting to tassel development. In previous experiments using shoot apex culture, we showed that the developmental program that limits vegetative development in maize is not intrinsic to the shoot apical meristem. Rather, the meristem receives information from elsewhere in the plant and responds by either continuing leaf initiation or becoming determined for determinate growth and forming an inflorescence, the tassel. Here we examine leaf primordia as potential sources for that information using shoot apex culture. Our results show that the presence of the four to six youngest leaf primordia on the shoot apex is sufficient to provide such information. The ability to reset shoot development by meristem culture also allows us to examine the basis for expression of a specific phenotype at a particular developmental stage. We found that the mutation hcf106, which is typically expressed only during seedling stages, is not re‐expressed when the shoot morphogically has regained a juvenile phase.
Tissue‐specific expression of ATCYS‐3A, a gene encoding the cytosolic isoform of O‐acetylserine(thiol)lyase in Arabidopsis
Authors: Gotor Cecilia , Cejudo Francisco J. , Barroso Consuelo , Vega José M.
Published: March 2003

Atcys‐3A from Arabidopsis encodes the cytosolic isoform of O‐acetylserine(thiol)lyase that catalyzes the last step of cysteine biosynthesis. The Atcys‐3A transcript is present in different organs of mature plants, being more abundant in roots and declining to 40–50% in rosette leaves and flowers. In situ hybridization studies have shown a high Atcys‐3A signal in root tissues, mainly localized to the cortex and xylem parenchyma. In a flower before anthesis, the transcript is detected exclusively in anthers and sepals and evenly distributed throughout the receptacle of the flower. An unexpected observation from these studies is the highest expression of Atcys‐3A mRNA found in trichomes of either leaf or stem. The presence of high levels of the transcript is observed very early in trichome cell development. This is the first report describing the cellular localization of any plant O‐acetylserine(thiol)lyase mRNA. The high level of Atcys‐3A expression in trichomes raises new aspects to the biological function of trichomes, related to sulfate metabolism.
Localization of expression of KNAT3, a class 2 knotted1‐like gene
Authors: Serikawa Kyle A. , Martinez‐Laborda Antonio , Kim Han‐Suk , Zambryski Patricia C.
Published: March 2003

KNAT3 is a class 2 kn1‐like gene in Arabidopsis thaliana. The RNA expression patterns of KNAT3 were characterized through the use of promoter‐GUS fusion analysis and in situ hybridization. KNAT3 is expressed in several tissues and at several times during development. There are three main expression patterns: (1) during early organ development in young leaves, buds and pedicels; (2) at and near the junction between two organs at specific times during development, including the hypocotyl‐root boundary in young seedlings, the anther‐filament junction in mature flowers, and the ovule‐funiculus and peduncle‐silique boundaries in elongating siliques; and (3) in maturing tissues such as the style of elongating siliques, the petioles of maturing leaves, and most of the root. The varied expression patterns may indicate that KNAT3 plays several different roles in plants, depending on when and where it is expressed. Previous work on KNAT3 (Serikawa et al., 1996) indicated that expression of its RNA is regulated by light. Promoter‐GUS seedlings were grown under different light conditions (continuous white, red and far‐red light) to examine more closely the light regulation of the KNAT3 promoter. Continuous white light resulted in stronger overall GUS staining in the same patterns seen in seedlings grown under long‐day conditions (cotyledons, upper hypocotyl and roots). Continuous red light resulted in reduced GUS expression in those same tissues. Continuous far‐red light led to seedlings showing stronger staining in the hypocotyl and cotyledons than red light‐grown plants but no staining in the roots. Thus, the KNAT3 promoter responds differently to red and far‐red light.
Integration of T‐DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration
Authors: Kononov Maria E. , Bassuner Burgund , Gelvin Stanton B.
Published: March 2003

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor‐inducing (Ti) plasmid, the T‐DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T‐DNA borders, define the left and the right ends of the T‐DNA. The T‐DNA can be physically separated from the remainder of the Ti‐plasmid, creating a ‘binary vector’ system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T‐DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non‐T‐DNA binary vector ‘backbone’ sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T‐DNA binary vectors containing a nos‐nptll gene within the T‐DNA and a mas2′‐gusA (β‐glucuronidase) gene outside the T‐DNA borders. We regenerated kanamycin‐resistant transgenic tobacco plants and analyzed these plants for the expression of the vector‐localized gusA gene and for the presence of binary vector backbone sequences. Approximately one‐fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T‐DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T‐DNA. Although we could readily detect T‐strands containing the T‐DNA within the bacterium, we could not detect T‐strands containing only the vector backbone sequences or these vector sequences linked to the T‐DNA.
Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant
Authors: Barbacar Nicolae , Hinnisdaels Stefan , Farbos Isabelle , Monéger Françoise , Lardon André , Delichère Catherine , Mouras Armand , Negrutiu Ioan
Published: March 2003

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre‐meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.
Cell‐to‐cell movement of potato spindle tuber viroid
Authors: Ding Biao , Kwon Myoung‐Ok , Hammond Rosemarie , Owens Robert
Published: March 2003

Viroids are non‐translatable, autonomously replicating circular RNAs that infect only plants. An important component of the viroid infection process is cell‐to‐cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. In this study, potato spindle tuber viroid (PSTVd) has been used as a model system to investigate the mechanism of viroid cell‐to‐cell transport. Infectious RNA transcripts were produced from PSTVd cDNA clones in vitro, labeled with the nucleotide‐specific fluorescent dye TOTO‐1 iodide, and used for micro‐injection. When injected into symplasmically isolated guard cells of mature tomato and tobacco leaves, PSTVd remained in the injected cells; in contrast, PSTVd injected into symplasmically connected mesophyll cells moved rapidly from cell to cell. A 1400 nt RNA containing only vector sequences was unable to move out of the injected mesophyll cells, but when PSTVd was fused to this transcript, the fusion RNA moved from cell to cell. At the DNA level, PSTVd cDNA also appears able to mediate cell‐to‐cell movement of plasmid DNA. These data indicate that (i) PSTVd moves from cell to cell via plasmodesmata, and (ii) this movement may be mediated by a specific sequence or structural motif.
Functional complementation of an oxa1− yeast mutation identifies an Arabidopsis thaliana cDNA involved in the assembly of respiratory complexes
Authors: Hamel Patrice , Sakamoto Wataru , Wintz Henri , Dujardin Geneviève
Published: March 2003

The nuclear gene OXA1 is essential for respiratory growth in yeast. It codes for a chaperon‐like protein, and has pleiotropic effects on the assembly of cytochrome c oxidase and ATP synthase of the mitochondrial respiratory chain. To study respiratory complex formation in plants, we have cloned a homolog of the yeast oxa1− in Arabidopsis thaliana, OXA1At, by functional complementation of a yeast oxa1− mutant. OXA1At is a single copy gene and appears to be constitutively expressed in A. thaliana. Although OXA1At encodes a protein sharing only 30% amino acid identity with the yeast Oxa1 protein, hydrophobic domains likely corresponding to trans‐membrane domains are strictly conserved. Cytochrome spectra and measurements of respiratory activities show that replacement of the yeast Oxa1 protein with the A. thaliana homolog leads to correct assembly and activity of cytochrome c oxidase, but to partial restoration of ATPase activity. Our results suggest that the Oxa1At protein is essential for the respiratory complex assembly in A. thaliana, and that genes involved in mitochondrial multiprotein complex formation can be conserved between plants and other organisms.
Slowly activating vacuolar channels can not mediate Ca2+‐induced Ca2+ release
Authors: Pottosin Igor I. , Tikhonova Lioubov I. , Hedrich Rainer , Schönknecht Gerald
Published: March 2003

In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca2+‐induced Ca2+ release the voltage‐ and Ca2+‐dependence of these K+ and Ca2+‐ permeable channels were studied in a quantitative manner. The patch‐clamp technique was applied to barley (Hordeum vulgare L.) mesophyll vacuoles in the whole vacuole and vacuolar‐free patch configuration. Under symmetrical ionic conditions the current‐voltage relationship of the open SV channel was characterized by a pronounced inward rectification. The single channel current amplitude was not affected by changes in cytosolic Ca2+ whereas an increase in vacuolar Ca2+ decreased the unitary current in a voltage‐dependent manner. The SV channel open‐probability increased with positive potentials and elevated cytosolic Ca2+, but not with elevated cytosolic Mg2+. An increase of cytosolic Ca2+ shifted the half‐activation potential to more negative voltages, whereas an increase of vacuolar Ca2+ shifted the half‐activation potential to more positive voltages. At physiological vacuolar Ca2+ activities (50 μM to 2 mM) changes in cytosolic Ca2+ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open‐probability on the electrochemical potential gradient for Ca2+ (ΔμCa). At the Ca2+ equilibrium potential (ΔμCa = 0) the open‐probability was as low as 0.4%. Higher open‐probabilities required net Ca2+ motive forces which would drive Ca2+ influx into the vacuole. Under conditions favouring Ca2+ release from the vacuole, however, the open‐probability further decreased. Based on quantitative analysis, it was concluded that the SV channel is not suited for Ca2+‐induced Ca2+ release from the vacuole.
Localization of a kinesin‐like calmodulin‐binding protein in dividing cells of Arabidopsis and tobacco
Authors: Bowser Jonathan , Reddy A.S.N.
Published: March 2003

The cloning and characterization of a novel kinesin‐like protein (kinesin‐like calmodulin‐binding protein, KCBP) from Arabidopsis and other plants has recently been described. Unlike all other known kinesin‐like proteins, KCBP interacts with calmodulin in the presence of micromolar calcium. An antibody specific to KCBP was raised using a calmodulin‐binding synthetic peptide that is unique to KCBP. The KCBP antibody detected a single protein of about 140 kDa in Arabidopsis and tobacco, the size predicted from cDNA sequences. In synchronized cell cultures, the amount of KCBP was abundant during M‐phase and very low in interphase. To get some insight into the function of this novel motor protein, KCBP in Arabidopsis and tobacco cells was localized by indirect immunofluorescence microscopy using affinity‐purified anti‐KCBP antibody. The KCBP was localized to the pre‐prophase band, the mitotic spindle and the phragmoplast. The association of KCBP with microtubule arrays in dividing cells suggests that this minus‐end‐directed microtubule motor protein is likely to be involved in the formation of these microtubule arrays and/or functions associated with these structures.
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

It 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

In 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

Nucleotide 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.