bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and characterization of irregular xylem4 (irx4): a severely lignin‐deficient mutant of Arabidopsis
Authors: Jones Louise , Ennos A. Roland , Turner Simon R. 
Published: March 2003

SummaryA severe lignin mutant, irx4, has been identified in Arabidopsis thaliana as a result of its collapsed xylem phenotype. In contrast to previously described irx mutants, irx4 plants have 50% less lignin than wild‐type plants, whilst the cellulose and hemicellulose content remained unchanged. These alterations in the composition of irx4 secondary cell walls had a dramatic effect on the morphology and architecture of the walls, which expand to fill most of the cell, and also on the physical properties of irx4 stems. Further analysis indicated that the irx4 mutation occurred in a cinnamoyl‐CoA reductase (CCR) gene within a highly conserved intron splice site sequence of intron 2. As a result, CCR mRNA transcripts were incorrectly spliced. Transgenic plants expressing an IRX3 promoter–CCR cDNA construct were used to generate a series of plants with varying degrees of lignin content in order to assess the role of lignin content in determining the physical properties of Arabidopsis stems.
Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine‐insensitive (coi1) mutation occurs through two distinct mechanisms
Authors: Kloek Andrew P. , Verbsky Michelle L. , Sharma Shashi B. , Schoelz James E. , Vogel John , Klessig Daniel F. , Kunkel Barbara N. 
Published: March 2003

SummaryA new allele of the coronatine‐insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1‐20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1‐20 plants is correlated with hyperactivation of PR‐1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA‐mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1‐20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA‐mediated defenses are required for restriction of PstDC3000 growth in coi1‐20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1‐20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1‐20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA‐dependent defense pathway; and (ii) an SA‐independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.
Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat
Authors: Engdahl Sheila , Aronsson Henrik , Sundqvist Christer , Timko Michael P. , Dahlin Clas 
Published: March 2003

SummaryMembrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro‐expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide‐binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co‐factor is essential for proper membrane association. POR protein carrying a mutation in the substrate‐binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein–membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.
Construct design for efficient, effective and high‐throughput gene silencing in plants
Authors: Wesley S. Varsha , Helliwell Christopher A. , Smith Neil A. , Wang MingBo , Rouse Dean T. , Liu Qing , Gooding Paul S. , Singh Surinder P. , Abbott David , Stoutjesdijk Peter A. , Robinson Simon P. , Gleave Andrew P. , Green Allan G. , Waterhouse Peter M. 
Published: March 2003

SummaryPost‐transcriptional silencing of plant genes using anti‐sense or co‐suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self‐complementary ‘hairpin’ RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti‐sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron‐containing constructs (ihpRNA) generally gave 90–100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co‐suppression or anti‐sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high‐throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large‐scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
Identification of 4‐coumarate:coenzyme A ligase (4CL) substrate recognition domains
Authors: Ehlting Jürgen , Shin Jane J. K. , Douglas Carl J. 
Published: March 2003

Summary4‐coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4‐coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate‐forming enzymes such as firefly luciferases, non‐ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4‐coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate‐forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.
Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell‐specific gene expression
Authors: Plesch Gunnar , Ehrhardt Thomas , Mueller‐Roeber Bernd 
Published: March 2003

SummaryDue to their unique structure and function, guard cells have attracted much attention at the physiological level. Very little, however, is known about the molecular events involved in the determination and maintenance of guard cell specificity. The KST1 gene encodes a K+ influx channel of guard cells in potato, and was therefore chosen as a model to study regulation of guard cell‐specific gene expression. Transgenic potato plants carrying a fusion between the KST1 promoter and the E. coli uidA (β‐glucuronidase) reporter gene revealed promoter activity in guard cells and in flowers. A detailed dissection of the KST1 promoter led to the discovery of two independent small TATA box‐proximal regulatory units, each of which was sufficient to direct guard cell‐specific gene transcription. Both fragments contain the sequence motif, 5′‐TAAAG‐3′, which is related to known target sites for a novel class of zinc finger transcription factors, called Dof proteins. Block mutagenesis of these Dof target sites in the context of different promoter constructs dramatically reduced guard cell promoter activity. A Dof gene, StDof1, was cloned and shown to be expressed in epidermal fragments highly enriched for guard cells. In gel retardation experiments, the StDof1 protein interacted in a sequence‐specific manner with a KST1 promoter fragment containing the TAAAG motif. These results provide evidence that TAAAG elements are target sites for trans‐acting Dof proteins controlling guard cell‐specific gene expression. Our data will add to the design of tailor‐made guard cell promoters as a further tool in molecular engineering of guard cell function and, hence, control of stomatal carbon dioxide (CO2) uptake and water loss in crop plants.
Evidence for expression level‐dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants
Authors: Hofius Daniel , Herbers Karin , Melzer Michael , Omid Ayelet , Tacke Eckhard , Wolf Shmuel , Sonnewald Uwe 
Published: March 2003

SummaryHigh‐level constitutive expression of the cell‐to‐cell movement protein from the phloem‐restricted potato leafroll virus (PLRV‐MP17) in transgenic tobacco plants leads to growth retardation and severe phenotypic changes of source leaves paralleled by a drastic accumulation of soluble sugars and starch (Herbers et al., 1997). To investigate whether the MP17‐induced alteration in carbon metabolism is related to the targeting and modification of specific plasmodesmata (Pd) or is rather due to pleiotropic effects caused by high MP17 protein amounts, non‐phenotypic tobacco plants expressing a MP17:GFP fusion protein were obtained and compared with previously described MP17 transgenic lines. Confocal laser scanning microscopy and immunogold labelling studies revealed an overall affinity of MP17 to Pd in vascular and non‐vascular tissue of source leaves, whereas in sink leaves GFP fluorescence was restricted to Pd of trichomes. In source leaves, plasmodesmal size exclusion limits of mesophyll cells were likewise increased by MP17 and MP17:GFP independent from steady‐state levels of the protein amount and phenotypic alteration. Conversely, carbohydrate contents in source leaves strictly correlated with quantified MP17 protein levels. Low expression of MP17 and MP17:GFP decreased soluble sugars and starch contents in leaves possibly due to changes in plasmodesmal permeability while increasing MP17 protein levels led to carbohydrate accumulation and a stunted growth. Infection of transgenic lines with the unrelated potato virus Y (PVY)N revealed an expression level‐dependent mode of MP17‐mediated resistance. Phenotypic changes and carbohydrate‐mediated defence responses as indicated by elevated levels of PR‐protein transcripts were crucial for increased viral resistance, whereas plasmodesmal targeting and modification by MP17 per se had either no effect or even increased susceptibility to PVY. Thus, our results implicate that the absolute level of expression needs to be critically considered when elucidating the effect of MPs on carbon metabolism, biomass allocation and virus resistance.
Control of flowering time by FLC orthologues in Brassica napus
Authors: Tadege Million , Sheldon Candice C. , Helliwell Chris A. , Stoutjesdijk Peter , Dennis Elizabeth S. , Peacock W. James 
Published: March 2003

Summary FLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. We isolated five FLC‐related sequences from Brassica napus (BnFLC1–5). Expression of each of the five sequences in Arabidopsis delayed flowering significantly, with the delay in flowering time ranging from 3 weeks to more than 7 months, relative to the flowering time of 3 weeks in untransformed Ler. In the reciprocal experiment, expression of Arabidopsis FLC (AtFLC) in an early flowering B. napus cultivar delayed flowering by 2–6 weeks, confirming the requirement of this gene for floral repression. In B. napus, we show that late flowering and responsiveness to vernalization correlate with the level of BnFLC mRNA expression. The different BnFLC genes show differential expression in leaves, stems and shoot tips, but expression is not detectable in roots. Vernalization dramatically reduces the level of BnFLC transcript and restores early flowering in the winter cultivar Colombus. We conclude that BnFLC genes confer winter requirement in B. napus and account for the major vernalization‐responsive flowering time differences in the different cultivars of B. napus in a manner analogous to that of AtFLC in Arabidopsis ecotypes.
Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease‐1 (FEN‐1), and their distribution in mitotic and meiotic cell cycles
Authors: Kimura Seisuke , Suzuki Tadzunu , Yanagawa Yuki , Yamamoto Taichi , Nakagawa Hiroki , Tanaka Ichiro , Hashimoto Junji , Sakaguchi Kengo 
Published: March 2003

SummaryThe biochemical and cell cycle‐dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease‐1 (OsFEN‐1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN‐1 and increased the flap‐endonuclease activity of OsFEN‐1 by 2.5‐fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN‐1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN‐1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN‐1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10–20 intense spots at leptotene. The number of spots decreased to 1–5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN‐1 in mitotic and meiotic cell cycles are discussed.
Infra‐red thermography revealed a role for mitochondria in pre‐symptomatic cooling during harpin‐induced hypersensitive response
Authors: Boccara Martine , Boué Christine , Garmier Marie , De Paepe Rosine , Boccara Albert‐Claude 
Published: March 2003

SummaryThe establishment of Erwinia amylovora harpin‐induced hypersensitive response (HR) in Nicotiana sylvestris was followed by infra‐red thermography (IRT). Three to four hours after elicitation, the temperature decreased in the harpin‐infiltrated zone associated to stomatal opening. The marked drop in temperature which reached 2°C and preceded necrosis symptoms for several hours, is thus likely caused by higher transpiration. Neither of these effects was observed in a respiratory mutant, affected in complex I structure and function and over‐expressing alternative oxidase, indicating that they are directly or indirectly mediated by mitochondrial function. However, as the HR establishment was similar in both wild type and mutant, cell death was either uncorrelated with the observed epidermal changes or occurred by a different signalling pathway in the two genotypes. IRT revealed a novel aspect of plant–pathogen interactions and could be applied to screen for mutants affected in elicitor signalling and/or for respiratory mutants.
Chromosome painting in Arabidopsis thaliana
Authors: Lysak Martin A , Fransz Paul F , Ali Hoda B. M , Schubert Ingo 
Published: March 2003

SummaryChromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome‐specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C−1). Pools of contiguous 113–139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.
Cloning and characterization of rice (Oryza sativa L) telomerase reverse transcriptase, which reveals complex splicing patterns
Authors: Heller‐Uszynska Katarzyna , Schnippenkoetter Wendelin , Kilian Andrzej 
Published: March 2003

SummaryPlant chromosomes terminate in telomeres as in other eukaryotes. Telomeres are vital to genome stability and their malfunctioning is lethal. One of the core components of the telomere complex is telomerase. The enzyme activity depends on RNA (TER) and reverse transcriptase (TERT) subunits. We describe here the isolation, sequencing and characterization of the telomerase reverse transcriptase catalytic subunit from the monocot plant Oryza sativa L. (OsTERT). A single copy of this gene is present in the rice genome. The protein predicted from the OsTERT sequence has all the signature motifs of the TERT family members. Our data indicate that rice telomerase activity is developmentally regulated and is high in in vitro tissue and cell culture. However, steady‐state transcript levels of the TERT gene do not seem to correlate with enzyme activity. Northern and RT–PCR analyses of the OsTERT gene transcript profile show multiple differentially spliced transcripts in both telomerase‐positive and telomerase‐negative tissues. Based on quantitative analysis of these transcripts, we speculate that the overall balance between the quantities of particular alternatively spliced transcripts may determine whether the TERT protein(s) is active or not. The diversity of splicing variants detected suggests that, as recently discovered for mammalian TERT proteins, rice TERT protein variants may perform functions other than telomere maintenance.
Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1‐dependent in cpr5
Authors: Clarke Joseph D. , Aarts Nicole , Feys Bart J. , Dong Xinnian , Parker Jane E. 
Published: March 2003

SummaryThe systemic acquired resistance (SAR) response in Arabidopsis is characterized by the accumulation of salicylic acid (SA), expression of the pathogenesis‐related (PR) genes, and enhanced resistance to virulent bacterial and oomycete pathogens. The cpr (constitutive expressor of PR genes) mutants express all three SAR phenotypes. In addition, cpr5 and cpr6 induce expression of PDF1.2, a defense‐related gene associated with activation of the jasmonate/ethylene‐mediated resistance pathways. cpr5 also forms spontaneous lesions. In contrast, the eds1 (enhanced disease susceptibility) mutation abolishes race‐specific resistance conferred by a major subclass of resistance (R) gene products in response to avirulent pathogens. eds1 plants also exhibit increased susceptibility to virulent pathogens. Epistasis experiments were designed to explore the relationship between the cpr‐ and EDS1‐mediated resistance pathways. We found that a null eds1 mutation suppresses the disease resistance phenotypes of both cpr1 and cpr6. In contrast, eds1 only partially suppresses resistance in cpr5, leading us to conclude that cpr5 expresses both EDS1‐dependent and EDS1‐independent components of plant disease resistance. Although eds1 does not prevent lesion formation on cpr5 leaves, it alters their appearance and reduces their spread. This phenotypic difference is associated with increased pathogen colonization of cpr5 eds1 plants compared to cpr5. The data allow us to place EDS1 as a necessary downstream component of cpr1‐ and cpr6‐mediated responses, but suggest a more complex relationship between EDS1 and cpr5 in plant defense.
Spatial analysis of plant metabolism: Sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns
Authors: Borisjuk Ljudmilla , Walenta Stefan , Rolletschek Hardy , Mueller‐Klieser Wolfgang , Wobus Ulrich , Weber Hans 
Published: March 2003

SummaryDuring legume embryogenesis the differentiation of the cotyledons proceeds gradually in a wave‐like manner. The process is metabolically and genetically controlled and regulated by sugars. In order to perform a spatial and temporal analysis of the sugar distribution pattern a new method was developed to specifically measure sucrose directly in tissues via bioluminescence and single photon counting. This enabled a quantitative sucrose imaging with a resolution close to the single cell level. The procedure was applied on sections of Vicia faba cotyledons covering the main stages of histodifferentiation. Young embryos before the storage phase contained moderate levels of sucrose, which were evenly distributed. At the onset of maturation high concentrations were present within a tissue layer covering the outwarded half of the coytledons. This layer was directly underneath the epidermis expressing a sucrose transporter gene indicating that epidermal transporters caused the high sucrose accumulation in the underlying tissue. At that stage the sucrose gradient was inversely oriented compared with cell size and the starch content. Cells within the interior were larger, contained starch but low sucrose. Thus, the sucrose pattern is controlled by uptake activity and permeability within the parenchyma. However, during the main storage phase actively elongating and starch accumulating cells contain highest sucrose concentrations indicating that differences in growth and starch accumulation also affect intracotyledonary sugar distribution. High sucrose concentrations were correlated with transcript levels of sucrose synthase and ADP‐Glc pyrophosphorylase indicating a signaling function for sucrose to induce starch biosynthesis on the gene expression level. Carbon flux through the sucrose synthase pathway towards starch increased when hexoses levels decreased.