bluuebunny/crossref_metadata_2025_split huggingface dataset:
A tomato homeobox gene (HD‐Zip) is involved in limiting the spread of programmed cell death
Authors: Mayda Esther , Tornero Pablo , Conejero Vicente , Vera Pablo 
Published: March 2003

SummaryAntisense suppression in transgenic tomato plants ofH52, a gene encoding a new homeodomain protein of the HD‐Zip class, produces a conditional lethal phenotype. The transgenic lines that survive exhibit spontaneous misregulation of cell death control in leaves, which, once initiated, propagates and engulfs the entire leaf. Activation of defence genes, over‐accumulation of ethylene and conjugated salicylic acid, and growth reduction of virulent pathogens also occurs in these plants. In wild‐type plants,H52is up‐regulated upon infection, mirroring the generation of the oxidative burst which normally precedes the hypersensitive response (HR). Thus, H52 appears to be a transcription factor involved in cellular protection by limiting spread of programmed cell death in plants.
Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine‐rich nuclear export signals in Arabidopsis thaliana
Authors: Haasen Dorothea , Köhler Claudia , Neuhaus Gunther , Merkle Thomas 
Published: March 2003

SummaryTransport across the nuclear envelope is mediated by transport receptors from the Importin β family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine‐rich nuclear export signals (NESs). AtXPO1 shares 42–50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two‐hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine‐rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY‐2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.
Immunological analysis of the phosphorylation state of maize C4‐form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide
Authors: Ueno Yoshihisa , Imanari Eiji , Emura Junji , Yoshizawa‐Kumagaye Kumiko , Nakajima Kiichiro , Inami Kaoru , Shiba Tetsuo , Sakakibara Hitoshi , Sugiyama Tatsuo , Izui Katsura 
Published: March 2003

SummaryThe phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme’s sensitivity to its feedback inhibitor, L‐malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state‐specific antibodies to the C4‐form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15‐mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity‐chromatography. Specificity of the resulting antibodies to the C4‐form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root‐form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi‐quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13‐week‐old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid‐morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4‐form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen‐limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.
Post‐transcriptional defects in tobacco chloroplast mutants lacking the cytochrome b6/f complex
Authors: Monde Rita‐Ann , Zito Francesca , Olive Jacqueline , Wollman Francis‐André , Stern David B. 
Published: March 2003

SummaryA variety of post‐transcriptional mechanisms govern the synthesis and assembly of photosynthetic protein complexes in chloroplasts. To test whether such mechanisms are conserved between photosynthetic algae and vascular plants, we have interrupted the chloroplast petA, petB and petD genes of tobacco, which encode three subunits of the cytochrome b6/f complex, and compared our results to those previously obtained with Chlamydomonas reinhardtii. As expected, the mutants exhibited high chlorophyll fluorescence, consistent with the loss of a functional cytochrome b6/f complex. Unlike the corresponding mutants of Chlamydomonas, however, cytochrome f was barely detectable in the ΔpetB or ΔpetD mutants. The amounts of petB‐ and petD‐containing mRNAs were reduced in the mutants compared to wild‐type plants, but the remaining mRNA was normally associated with polysomes. In contrast, there was a decrease in polysome association of the polycistronic petA mRNA in the ΔpetB and ΔpetD mutants, suggesting that the synthesis of cytochrome f may be decreased in the absence of cytochrome b6 or SUIV. These results are discussed in light of the translational autoregulation model that has been proposed for cytochrome b6/f complex assembly in Chlamydomonas.
Lesions in the sterol Δ7 reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis
Authors: Choe Sunghwa , Tanaka Atsushi , Noguchi Takahiro , Fujioka Shozo , Takatsuto Suguru , Ross Amanda S. , Tax Frans E. , Yoshida Shigeo , Feldmann Kenneth A. 
Published: March 2003

SummaryThe brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Δ7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark‐green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C‐labeled precursors in dwf5 verified a deficiency in a sterol Δ7 reductase activity. All six independent alleles contain loss‐of‐function mutations in the sterol Δ7 reductase gene. These include a putative mRNA instability mutation in dwf5‐1, 3′ and 5′ splice‐site mutations in dwf5‐2 and dwf5‐6, respectively, premature stop codons in dwf5‐3 (R400Z) and dwf5‐5 (R409Z), and a mis‐sense mutation in dwf5‐4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild‐type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith–Lemli–Opitz syndrome are caused by loss‐of‐function mutations in a sterol Δ7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.
Heat‐shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T‐DNA insertions
Authors: Matsuhara Shio , Jingu Fumito , Takahashi Taku , Komeda Yoshibumi 
Published: March 2003

SummaryThis paper describes expression profiles of the Arabidopsis HSP18.2 heat‐shock gene promoter by using three different reporter genes, and the application of this promoter to a method we have developed to drive heat‐shock‐dependent transcription of plant genome DNA flanked by T‐DNA insertions. We show that, irrespective of the location of the T‐DNA insertion, an HSP18.2 promoter towards the left border of the T‐DNA effectively induces transcription of flanking genome sequences in response to heat shock. If polyadenylated, tagged transcripts can be easily identified by RT–PCR.
Heterotrimeric G‐proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR‐5 protein
Authors: Coca María A. , Damsz Barbara , Yun Dae‐Jin , Hasegawa Paul M. , Bressan Ray A. , Narasimhan Meena L. 
Published: March 2003

SummaryMembrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR‐5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G‐protein mediated signaling pathway. The guanidine nucleotide GDPβS, that locks G‐proteins in a GDP‐bound inactive form, inhibits osmotin‐induced conidial lysis. A dominant interfering mutation in FadA, the α‐subunit of a heterotrimeric G‐protein, confers resistance to osmotin. A deletion mutation in SfaD, the β‐subunit of a heterotrimeric G‐protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.
Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum
Authors: Bharti Kapil , Schmidt Enrico , Lyck Ruth , Heerklotz Dirk , Bublak Daniela , Scharf Klaus‐Dieter 
Published: March 2003

SummaryStress‐induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs). Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family. HsfA3 was cloned from a tomato heat stress cDNA library by yeast two‐hybrid screening, using HsfA1 as a bait. HsfA3 is a single‐copy gene with all the conserved sequence elements characteristic of a heat stress transcription factor. The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions. Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family. It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf‐dependent reporter constructs both in tobacco protoplasts and yeast. Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C‐terminal domain of HsfA3.
T‐DNA insertional mutagenesis for functional genomics in rice
Authors: Jeon Jong‐Seong , Lee Sichul , Jung Ki‐Hong , Jun Sung‐Hoon , Jeong Dong‐Hoon , Lee Jinwon , Kim Chanhong , Jang Seonghoe , Lee Shinyoung , Yang Kiyoung , Nam Jongmin , An Kyungsook , Han Min‐Jung , Sung Ryo‐Jin , Choi Hyun‐Sook , Yu Jung‐Hwa , Choi Jung‐Hwan , Cho Se‐Yu , Cha Sang‐Su , Kim Shi‐In , An Gynheung 
Published: March 2003

SummaryWe have produced 22 090 primary transgenic rice plants that carry a T‐DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel‐blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T‐DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T‐DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless β‐glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T‐DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6–2.1% of tested organs were GUS‐positive in the tested organs, and that their GUS expression patterns were organ‐ or tissue‐specific or ubiquitous in all parts of the plant. The large population of T‐DNA‐tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.
Simultaneous analysis of metabolites in potato tuber by gas chromatography–mass spectrometry
Authors: Roessner Ute , Wagner Cornelia , Kopka Joachim , Trethewey Richard N. , Willmitzer Lothar 
Published: March 2003

SummaryA new method is presented in which gas chromatography coupled to mass spectrometry (GC–MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC–MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70–140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil‐ and in vitro‐grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.
Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana
Authors: Cheng Ning‐Hui , Su Chun‐Lin , Carter Shelly A. , Nelson Richard S. 
Published: March 2003

SummaryPlant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non‐vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non‐vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem‐associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem‐associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular‐associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.
Knockout of the regulatory site of 3‐ketoacyl‐ACP synthase III enhances short‐ and medium‐chain acyl‐ACP synthesis
Authors: Abbadi Amine , Brummel Monika , Spener Friedrich 
Published: March 2003

Summary3‐ketoacyl‐acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl‐acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium‐chain acyl‐ACP end products of the FAS reaction, i.e. inhibition by lauroyl‐ACP was uncompetitive towards acetyl CoA and non‐competitive with regard to malonyl‐ACP. This indicated a distinct attachment site for regulatory acyl‐ACPs. Based on alignment of primary structures of various KAS IIIs and 3‐ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl‐ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl‐ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium‐chain fatty acid‐producing Cuphea lanceolata seeds and long‐chain fatty acid‐producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short‐chain acyl‐ACPs in the case of C. lanceolata, and of medium‐chain acyl‐ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non‐transgenic plants for production of seed oils with new qualities.
Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose‐1,6‐bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana
Authors: Strand Åsa , Zrenner Rita , Trevanion Stephen , Stitt Mark , Gustafsson Petter , Gardeström Per 
Published: March 2003

SummaryPhotosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose‐1,6‐bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi‐limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose‐1,5‐bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose‐P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end‐product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP‐glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.
Arabidopsis DNA ligase IV is induced by γ‐irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4
Authors: West Christopher E. , Waterworth Wanda M. , Jiang Qing , Bray Clifford M. 
Published: March 2003

SummaryRejoining of single‐ and double‐strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double‐strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by γ‐irradiation, represent one of the most serious threats to genomic integrity. Non‐homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP‐dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino‐acid sequence to the human DNA ligase IV. Yeast two‐hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV‐binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C‐terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by γ‐irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.