bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipid peroxidation and the oxidative burst associated with infection of Capsicum annuum by Botrytis cinerea
Authors: Deighton Nigel , Muckenschnabel Ingo , Goodman Bernard A. , Williamson Brian 
Published: March 2003

SummaryA combination of electron paramagnetic resonance (EPR) spectroscopy and analytical chemistry has been used to study the changes in free radical content, transition metal ion status and lipid peroxidation following inoculation of fruits of sweet pepper (Capsicum annuum) with Botrytis cinerea. EPR detected a high concentration of an unidentified free radical associated with the spreading lesion that extends into the surrounding, healthy tissues. In addition, the EPR‐detectable iron(III) was highest at the centre of the lesion, again displaying a gradient out into the surrounding tissues. Analyses for aldehydic products of lipid peroxidation were performed to assess the accumulation and potential of these compounds to contribute to the cell death associated with necrotrophic pathogens. In contrast to the spectrum of aldehydes typically observed within peroxidized biological samples, no accumulation of malondialdehyde nor n‐hexanal was observed. Instead, high levels of two hydroxyalkenals (4‐hydroxy‐2‐hexenal and 4‐hydroxy‐2‐nonenal) were detected at concentra‐ tions up to 4000 and 20 000 pmol g− 1, respectively, at the host–pathogen interface. These results are discussed in terms of the likely mechanisms of formation of these aldehydes.
Graft transmission of post‐transcriptional gene silencing: target specificity for RNA degradation is transmissible between silenced and non‐silenced plants, but not between silenced plants
Authors: Sonoda Shoji , Nishiguchi Masamichi 
Published: March 2003

SummaryUsing the grafting procedure, we examined the transmission of post‐transcriptional gene silencing (PTGS) in Nicotiana benthamiana which had been transformed with the coat protein gene, including the 3′ non‐translated region of the sweet potato feathery mottle potyvirus. Transmission of PTGS from silenced lines to non‐silenced ones was bidirectional, but occurred efficiently from root stocks to scions. The level of transgene methylation in non‐silenced scions grafted onto silenced root stocks was not increased. When grafted scions which had become silenced were removed from silenced root stocks and regrafted onto non‐silenced or vector‐transformed root stocks, PTGS was maintained. However, their progeny did not show PTGS. Previously we reported that our transgenic lines had different target specificities of PTGS for RNA degradation: one line recognized only the 3′ part of the transgene mRNA while others involved the whole transgene mRNA (
Sonoda et al. 1999
, Phytopathology, 89, 385–391). Using these lines, we showed that target specificity of PTGS induced in non‐silenced scions after grafting was determined by that in silenced root stocks. However, unexpectedly, target specificity of PTGS induced in silenced scions after grafting was not changed by grafting onto silenced root stocks showing different target specificity, indicating that the second PTGS from silenced root stocks was not superimposed to silenced scions.
Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella
Authors: Acarkan Adile , Roßberg Mathias , Koch Marcus , Schmidt Renate 
Published: March 2003

SummaryGenome colinearity has been studied for two closely related diploid species of the Brassicaceae family, Arabidopsis thaliana and Capsella rubella. Markers mapping to chromosome 4 of A. thaliana were found on two linkage groups in Capsella and colinear segments spanning more than 10 c
m were revealed. Detailed analysis of a 60 kbp region in A. thaliana and its counterpart in C. rubella showed virtually complete conservation of gene repertoire, order and orientation. The comparison of orthologous genes revealed very similar exon–intron structures and sequence identities of 90% or more were found for exon sequences. This extensive genome colinearity at the genetic and molecular level allows the efficient transfer of data from the well‐studied A. thaliana genome to other species in the Brassicaceae family, substantially facilitating genome analysis studies for species of this family.
Resistance to the plant PR‐5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition
Authors: Ibeas José Ignacio , Yun Dae‐Jin , Damsz Barbara , Narasimhan Meena L. , Uesono Yukifumi , Ribas Juan Carlos , Lee Hyeseung , Hasegawa Paul M. , Bressan Ray A. , Pardo José M. 
Published: March 2003

SummaryThe capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR‐5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1‐v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1‐d allele or a null ssd1Δ mutation displayed high sensitivity to osmotin. The SSD1‐v protein mediates osmotin resistance in a cell wall‐dependent manner. Deletion of SSD1‐v or SSD1‐d impeded sorting of the PIR proteins (osmotin‐resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post‐transcriptional regulation of gene expression. The sensitivity of ssd1Δ cells to osmotin was only partially suppressed by over‐accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall‐mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell‐wall morphology and lower levels of alkali‐insoluble cell‐wall glucans. Therefore SSD1 is an important regulator of fungal cell‐wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR‐5 proteins.
Integration of the FISH pachytene and genetic maps of Medicago truncatula
Authors: Kulikova Olga , Gualtieri Gustavo , Geurts René , Kim Dong‐Jin , Cook Douglas , Huguet Thierry , De Jong J. Hans , Fransz Paul F. , Bisseling Ton 
Published: March 2003

SummaryA molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.
Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin‐treated oats
Authors: Yao Nan , Tada Yasuomi , Park Pyoyun , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki 
Published: March 2003

SummaryHistological and cytological evidence of where and when apoptotic cells occur in Pc‐2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM‐TUNEL was positive in the non‐collapsed cells but not in the collapsed cells in the treated leaves. However, the EM‐TUNEL assay was positive in the nuclei of the non‐collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin‐treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin‐treated non‐collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM‐TUNEL assay indicated that EGTA co‐incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.
‘Caged cytoskeletons’: a rapid method for the isolation of microtubule‐associated proteins from synchronized plant suspension cells
Authors: McCutcheon S. , Hemsley R. J. , Jopson M. F. , Lloyd C. W. 
Published: March 2003

SummaryIn the cytoskeleton method for isolating microtubule‐associated proteins MAP65, DcKRP120‐1 and DcKRP120‐2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti‐microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY‐2 cells for only several minutes allows extraction of detergent‐soluble proteins, leaving synchronized ‘caged cytoskeletons’ for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti‐microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin‐related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.
Transactivation of BARNASE under the AtLTP1 promoter affects the basal pole of the embryo and shoot development of the adult plant in Arabidopsis
Authors: Baroux Célia , Blanvillain Robert , Moore Ian R. , Gallois Patrick 
Published: March 2003

SummaryGenetically controlled expression of a toxin provides a tool to remove a specific structure and consequently study its role during a developmental process. The availability of many tissue‐specific promoters is a good argument for the development of such a strategy in plants. We have developed a conditional system for targeted toxin expression and demonstrated its use for generating embryo phenotypes that can bring valuable information about signalling during embryogenesis. The BARNASE gene was expressed in the Arabidopsis embryo under the control of two promoters, one from the cyclin AtCYCB1 gene and one from the AtLTP1 gene (LipidTransferProtein 1). One‐hundred percent seed abortion was obtained with the cyclin promoter. Surprisingly however, the embryos displayed a range of lethal phenotypes instead of a single arrested stage as expected from this promoter. We also show that BARNASE expression under the control of the AtLTP1 promoter affects the basal pole of the globular embryo. Together with reporter expression studies, this result suggests a role of the epidermis in controlling the development of the lower tier of the embryo. This defect was not embryo‐lethal and we show that the seedlings displayed a severe shoot phenotype correlated to epidermal defects. Therefore, the epidermis does not play an active role during organogenesis in seedlings but is important for the postgermination development of a viable plant.
Analysis of gene expression in Arabidopsis thaliana by array hybridization with genomic DNA fragments aligned along chromosomal regions
Authors: Hanano Shigeru , Amagai Masayuki , Kaneko Takakazu , Kuwata Chikara , Tsugane Taneaki , Sakurai Nozomu , Nakamura Yasukazu , Shibata Daisuke , Tabata Satoshi 
Published: March 2003

SummaryThe availability of the entire genomic sequence of the higher plant Arabidopsis thaliana prompted an analysis of chromosomal regions for gene expression with the use of high‐density DNA array filters spotted with genomic DNA fragments (genomic DNA array analysis). One TAC and nine P1 clones, each of which contains a genomic DNA insert of approximately 80 kb and was used for sequencing of chromosome 5, were arbitrarily selected for analysis. The total size of the genomic regions corresponding to these clones is 819 kb. A total of 339 DNA fragments (average size, 2.9 kb) that cover contiguously the 10 chromosomal regions was selected and spotted onto nylon filters. The filters were then subjected to hybridization with 33P‐labelled cDNA molecules that had been synthesized from polyadenylated RNA isolated from 3‐week‐old plants. Quantitative and reproducible measurement of hybridization signals allowed analysis of the transcription of all genes in the targeted regions that were expressed at a level above the limit of detection. The data revealed that the analysed chromosomal regions are rich in active genes, and that they also provided a basis for the identification of novel transcripts whose sequences are not represented in the expressed sequence tag (EST) database.
Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation
Authors: Merlot Sylvain , Mustilli Anna‐Chiara , Genty Bernard , North Helen , Lefebvre Valérie , Sotta Bruno , Vavasseur Alain , Giraudat Jérôme 
Published: March 2003

SummaryIn response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2–1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1–1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.
Intrinsic direct repeats generate consistent post‐transcriptional gene silencing in tobacco
Authors: Ma Chonglie , Mitra Amitava 
Published: March 2003

SummaryIt is well documented that transgenes with inverted repeats can efficiently trigger post‐transcriptional gene silencing (PTGS), presumably via a double stranded RNA induced by complementary sequences in their transcripts. We show here that transgenes with intrinsic direct repeats can also induce PTGS at a very high frequency (80–100%). A transgene with three or four repeats induced PTGS in almost 100% of the primary transformants, regardless of whether a strong (enhanced 35S promoter) or a relatively weak (chlorophyll a/b binding protein promoter) promoter was used. The PTGS induced by three or four repeats is consistently inherited in subsequent generations, and can inactivate homologous genes in trans. Based on the high frequency and consistent heritability, we propose that the intrinsic direct repeat within a transgene may act as a primary determinant of PTGS referred to as direct repeat‐induced PTGS (driPTGS). Silencing occurred in all five genes, in this and two previous reports, suggesting that driPTGS might be a universal gene silencing mechanism both in dicotyledonous tobacco plants and monocotyledonous rice cells. In addition, driPTGS may help dissect the gene silencing mechanism and generate silenced phenotypes useful for research and plant biotechnology products.
Searching limiting steps in the expression of chloroplast‐encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii
Authors: Eberhard Stephan , Drapier Dominique , Wollman Francis‐André 
Published: March 2003

SummaryWe performed a systematic investigation of the quantitative relationship between genome copy number, transcription, transcript abundance and synthesis of photosynthetic proteins in the chloroplast of the green algae Chlamydomonas reinhardtii grown either in mixotrophic or phototrophic conditions. The chloroplast gene copy number is lower in the latter condition and the half‐life and accumulation levels of most chloroplast transcripts are significantly reduced, although the relative rates of protein synthesis remain similar. Our study shows that, in most instances, chloroplast protein synthesis is poorly sensitive to changes in gene copy number or transcript abundance in the chloroplast. Treatment with 5‐fluoro‐2′‐deoxyuridine, that inhibits chloroplast DNA replication and decreases extensively the number of copies of the chloroplast genome, had limited effects on the abundance of most chloroplast transcripts and little if any effect on the rates of protein synthesis. When using rifampicin, that selectively inhibits chloroplast transcription, we found no direct correlation between the level of transcripts remaining in the chloroplast and the rates of chloroplast protein synthesis. For two chloroplast genes, a 90% decrease in the amount of transcript did not cause a drop in the rate of synthesis of the corresponding protein product. Overall, our results demonstrate that there is no gene dosage effect in the chloroplast and that transcript abundance is not limiting in the expression of chloroplast‐encoded protein.
Knock‐out of a putative transporter results in altered blue‐light signalling in Chlamydomonas
Authors: Dame Gregory , Gloeckner Gernot , Beck Christoph F. 
Published: March 2003

SummaryNitrogen starvation and blue light are the two environmental cues that control sexual differentiation in Chlamydomonas reinhardtii. Insertional mutagenesis was applied to generate mutants that still require nitrogen starvation as the initiating signal for gametogenesis but were no longer dependent on irradiation. In one mutant analysed, sequences adjacent to the site of insertion were cloned and used for the isolation of a genomic clone that, upon transformation, could complement the mutant phenotype. The gene identified (LRG6) encodes two mRNAs that appear to be the products of differential splicing. The two putative gene products derived from these mRNAs differ in their C‐terminal ends. Both predicted gene products exhibit multiple hydrophobic domains with α‐helical secondary structure typical for integral membrane proteins. These proteins may form pores, and may function as transporters of as‐yet unknown substrates. Since rendering the LRG6 gene non‐functional resulted in light‐independence of gamete formation, it is suggested that this transporter may inhibit signal flux from the photoreceptor to target genes – either directly by its activity or indirectly by serving as a scaffold for signalling proteins. Shutting off this transporter may be required for the activation of signal flux in this pathway. This concept is supported by the observed reduction in LRG6 mRNA levels during the first phase of gametic differentiation.
Arabidopsis thaliana root non‐selective cation channels mediate calcium uptake and are involved in growth
Authors: Demidchik Vadim , Bowen Helen C. , Maathuis Frans J.M. , Shabala Sergey N. , Tester Mark A. , White Philip J. , Davies Julia M. 
Published: March 2003

SummaryCalcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage‐independent rapidly activating Ca2+‐permeable non‐selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium‐permeable NSCCs co‐existed with hyperpolarisation‐activated Ca2+ channels (HACCs), which activated 40–60 min after forming the whole‐cell configuration. HACCs activated at voltages <−130 to −150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin‐expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+‐selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co‐existence is proposed. In mature epidermal cells, NSCC‐mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC‐mediated Ca2+ influx predominates.