bluuebunny/crossref_metadata_2025_split huggingface dataset:
Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation
Authors: Ye Guang‐Ning , Stone Deborah , Pang Sheng‐Zhi , Creely Wendi , Gonzalez Kathleen , Hinchee Maud 
Published: March 2003

SummaryThe visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post‐infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3–5 days post‐infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5–11 days post‐infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.
Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell‐type specific expression in plants
Authors: Kaltenbach Melanie , Schröder Gudrun , Schmelzer Elmon , Lutz Verena , Schröder Joachim 
Published: March 2003

SummaryWe investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68–78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3′,5′‐hydroxylase, but 3′‐hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3′‐ as well as 3′5′‐hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3′5′H was present in dark‐grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4‐hydroxylase and flavanone 3β‐hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3′5′H substrates. Immunohistochemical localization of F3′5′H had not been performed before. A comparison of CHS and F3′5′H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3′5′H was only detected in the phloem. The cell‐type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.
Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis
Authors: Hartmann Ulrike , Höhmann Susanne , Nettesheim Klaus , Wisman Ellen , Saedler Heinz , Huijser Peter 
Published: March 2003

SummaryFlowering time mutants represent genetic functions in control of the floral transition, an important developmental phase switch in the life cycle of higher plants. Many such mutants have been identified and characterized, particular in Arabidopsis. Here we describe the identification and initial characterization of a new early flowering mutant of Arabidopsis. The corresponding gene, SVP (SHORT VEGETATIVE PHASE), was cloned through transposon tagging and represents a new member of the MADS‐box gene family of transcription factors. Analysis of its transcriptional activity revealed the presence of differently sized transcripts that were confined to vegetative tissues and floral primordia and absent from developed flowers and siliques. The function of SVP as a repressor of the floral transition is discussed.
The Arabidopsis Cdc2a‐interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin‐dependent kinase activity in vitro
Authors: Lui Helen , Wang Hong , DeLong Catherine , Fowke Larry C. , Crosby William L. , Fobert Pierre R. 
Published: March 2003

SummaryCyclin‐dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein–protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two‐hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy‐terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK‐binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1‐associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.
Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1
Authors: Liu Zongrang , Hossain Gazi Showkat , Islas‐Osuna Maria A. , Mitchell David L. , Mount David W. 
Published: March 2003

SummaryTo analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV‐induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium‐mediated transformation of the uvh1‐1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1‐1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1‐1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3′ splice site and introducing an in‐frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.
The two largest chloroplast genome‐encoded open reading frames of higher plants are essential genes
Authors: Drescher Anja , Ruf Stephanie , Calsa Tercilio , Carrer Helaine , Bock Ralph 
Published: March 2003

SummaryThe chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild‐type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild‐type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild‐type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.
Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate
Authors: Munnik Teun , Meijer Harold J. G. , Ter Riet Bas , Hirt Heribert , Frank Wolfgang , Bartels Dorothea , Musgrave Alan 
Published: March 2003

SummaryIn mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1‐butanol, and part is due to diacylglycerol kinase activity as monitored via 32P‐PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell‐suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.
Developmental and light‐regulated post‐translational control of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase levels in potato
Authors: Korth Kenneth L. , Jaggard Daniel A. W. , Dixon Richard A. 
Published: March 2003

SummaryIn plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR). We have added an eight amino‐acid‐residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR–FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR–FLAG protein were found only in apical meristematic tissue, suggesting post‐translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR–FLAG transgenic and the non‐transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post‐translational level. After 2.5 h in darkness, levels of HMGR–FLAG are approximately one‐half of those in plants in the light; protein levels recover rapidly when dark‐treated plants are returned to the light. In non‐transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR–FLAG remain associated with polyribosomes in dark‐treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR–FLAG. The epitope‐tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light‐regulated control of HMGR occurs post‐translationally in vivo.
The alternative oxidase in roots of Poa annua after transfer from high‐light to low‐light conditions
Authors: Millenaar Frank F. , Roelofs Roeland , Gonzàlez‐Meler Miquel A. , Siedow James N. , Wagner Anneke M. , Lambers Hans 
Published: March 2003

SummaryThe activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high‐light to low‐light conditions, and at the same time from long‐day to short‐day conditions for four days. As a result of the low‐light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O‐isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low‐light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.
Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa
Authors: Horie Tomoaki , Yoshida Kazuya , Nakayama Hideki , Yamada Katsuyuki , Oiki Shigetoshi , Shinmyo Atsuhiko 
Published: March 2003

SummaryIt is thought that Na+ and K+ homeostasis is crucial for salt‐tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+‐Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni‐OsHKT1). We also isolated two cDNAs homologous to Ni‐OsHKT1 from salt‐tolerant indica rice, cv Pokkali (Po‐OsHKT1, Po‐OsHKT2). The predicted amino acid sequence of Ni‐OsHKT1 shares 100% identity with Po‐OsHKT1 and 91% identity with Po‐OsHKT2, and they are 66–67% identical to wheat HKT1. Low‐K+ conditions (less than 3 mm) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mm Na+. We further characterized the ion‐transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+‐uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+‐limiting conditions. When OsHKTs were expressed in Na+‐sensitive yeast, OsHKT1 rendered the cells more Na+‐sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+‐ and K+‐coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.
Requirement for 3‐ketoacyl‐CoA thiolase‐2 in peroxisome development, fatty acid β‐oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings
Authors: Germain Véronique , Rylott Elizabeth L. , Larson Tony R. , Sherson Sarah M. , Bechtold Nicole , Carde Jean‐Pierre , Bryce James H. , Graham Ian A. , Smith Steven M. 
Published: March 2003

Summary3‐ketoacyl‐CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid β‐oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T‐DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long‐chain (C16 to C20) fatty acyl‐CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long‐chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto‐acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal β‐oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.
Reversible redox control of plant vacuolar H+–ATPase activity is related to disulfide bridge formation in subunit E as well as subunit A
Authors: Tavakoli Nastaran , Kluge Christoph , Golldack Dortje , Mimura Tetsuro , Dietz Karl‐Josef 
Published: March 2003

SummaryThe plant vacuolar proton pump can be subjected to reversible redox regulation in vitro. The redox‐dependent activity change involves disulfide bridge formation not only in Vatp A, as reported for bovine V–ATPase, but also in the stalk subunit Vatp E. Microsomal membranes isolated from barley leaves were analysed for their activity of bafilomycin‐sensitive ATP hydrolysis and proton pumping using quinacrine fluorescence quenching in vesicle preparations. ATP hydrolysis and proton pumping activity were inhibited by H2O2. H2O2‐deactivated ATPase was reactivated by cysteine and glutathione. The glutathione concentration needed for half maximal reactivation was 1 mmol l−1. The activity loss was accompanied by shifts in electrophoretic mobility of Vatp A and E which were reversed upon reductive reactivation. The redox‐dependent shift was also seen with recombinant Vatp E, and was absent following site‐directed mutagenesis of either of the two cys residues conserved throughout all plant Vatp E sequences. V–ATPase was also inhibited by oxidized thioredoxin. These results support the hypothesis that tuning of vacuolar ATPase activity can be mediated by redox control depending on the metabolic requirements.
The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes
Authors: Ballvora Agim , Ercolano Maria Raffaella , Weiß Julia , Meksem Khalid , Bormann Christina Angelika , Oberhagemann Petra , Salamini Francesco , Gebhardt Christiane 
Published: March 2003

SummaryLate blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single‐gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single‐gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine‐rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.
A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket
Authors: Carlsson Anders S. , LaBrie Samuel T. , Kinney Anthony J. , Von Wettstein‐Knowles Penny , Browse John 
Published: March 2003

SummaryThe fab1 mutant of Arabidopsis is partially deficient in activity of β‐ketoacyl‐[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low‐temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild‐type, the Leu207Phe protein showed a 10‐fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size‐exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.