bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intron‐tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium‐transformed plant cells
Authors: Ferrando Alejandro , Farràs Rosa , Jásik Ján , Schell Jeff , Koncz Csaba 
Published: March 2003

SummaryEpitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T‐DNA‐mediated transformation, the detection of epitope‐tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope‐tagged proteins in Agrobacterium‐infected plant cells. We have developed a method for labelling plant coding sequences with intron‐tagged epitope‐coding domains that are not processed in Agrobacterium. Here we show that the expression of HA‐epitope‐tagged constructs encoding β‐glucuronidase and S‐phase kinase‐associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope‐tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium‐transformed plant cells.
Carotenoid biosynthesis during tomato fruit development: regulatory role of 1‐deoxy‐D‐xylulose 5‐phosphate synthase
Authors: Lois Luisa María , Rodríguez‐Concepción Manuel , Gallego Francesca , Campos Narciso , Boronat Albert 
Published: March 2003

SummaryPlant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate‐independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ‐specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1‐Deoxy‐D‐xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit‐specific isoform of phytoene synthase) in wild‐type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.
Induction of dwarfism in transgenic Solanum dulcamara by over‐expression of a gibberellin 20‐oxidase cDNA from pumpkin
Authors: Curtis Ian S. , Ward Dennis A. , Thomas Stephen G. , Phillips Andrew L. , Davey Michael R. , Power J. Brian , Lowe Kenneth C. , Croker Stephen J. , Lewis Mervyn J. , Magness Simon L. , Hedden Peter 
Published: March 2003

SummaryThe gibberellin (GA) 20‐oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi‐dwarfs with smaller, deep‐green leaves and highly pigmented stems compared to the wild‐type. Such transformants flowered earlier than the wild‐type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA1 was reduced in leaves of the transformants to approximately 20% or less of that in the wild‐type and to about 40% or less in stems. The concentrations of other 13‐hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA17, which accumulated in the transformants due to 13‐hydroxylation of GA25. By contrast, the concentrations of non‐13‐hydroxylated GAs, GA4 and GA34, were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20‐oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild‐type, reflecting the feedback control of 20‐oxidase gene expression that serves as a homeostatic mechanism for GAs.
Cell‐type‐specific calcium responses to drought, salt and cold in the Arabidopsis root
Authors: Kiegle Edward , Moore Catherine A. , Haseloff Jim , Tester Mark A. , Knight Marc R. 
Published: March 2003

SummaryLittle is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer‐trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.
Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia
Authors: Metzlaff Michael , O'dell Michael , Hellens Roger , Flavell Richard B. 
Published: March 2003

SummaryThe introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post‐transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template‐specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post‐transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA.
Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype
Authors: Wopereis Judith , Pajuelo Eloisa , Dazzo Frank B. , Jiang Qunyi , Gresshoff Peter M. , De Bruijn Frans J. , Stougaard Jens , Szczyglowski Krzysztof 
Published: March 2003

SummaryLegume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1‐1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1‐1 mutant root. No significant differences between har1‐1 mutant and wild‐type plants were detected with respect to root growth responses to 1‐aminocyclopropane1‐carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1‐1 mutant but not the wild‐type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non‐symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.
An Ry‐mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y
Authors: Mestre Pere , Brigneti Gianinna , Baulcombe David C. 
Published: March 2003

Summary Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry‐mediated resistance against PVY in potato, we expressed each of the PVY‐encoded proteins in leaves of PVY‐resistant (Ry) and ‐susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry‐mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry‐mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host‐encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry‐mediated resistance response.
Cre‐lox site‐specific recombination between Arabidopsis and tobacco chromosomes
Authors: Koshinsky Heather A. , Lee Elsa , Ow David W. 
Published: March 2003

SummaryTo create hybrid chromosomes, we tested the Cre‐lox system to mediate recombination between Arabidopsis thaliana and Nicotiana tabacum chromosomes. Protoplasts of the two plants were fused to allow site‐specific recombination to join a promoter from tobacco to a hygromycin resistance coding‐region from Arabidopsis. The expected recombination junction was detected in hygromycin‐resistant calli. Analysis of one hybrid suspension cell line revealed the presence of markers corresponding to the north arm of Arabidopsis chromosome III, but not markers from other chromosome arms. However, these markers were not detected in regenerated plants. With a second hybrid cell line we obtained a single hygromycin‐resistant progeny from approximately 18 000 self‐fertilized seeds of one regenerated plant. Molecular analysis of this hybrid indicated that a small portion of the north arm of Arabidopsis chromosome V is present in the tobacco genome. However, neither the recombination junction nor Arabidopsis DNA was detected in tissue from the plant grown without selection or in the subsequent generation. Thus interspecies transfer of a chromosome arm between plant cells is possible, but maintenance of the hybrid chromosome in a plant is unlikely. The feasibility of site‐specific recombination between genomes of different species offers new possibilities for engineering hybrid chromosomes that may be maintained in cell culture.
Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity
Authors: Örvar Björn Lárus , Sangwan Veena , Omann Franz , Dhindsa Rajinder S. 
Published: March 2003

SummaryMany plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non‐freezing temperatures at the onset of winter. CA is associated with gene expression that requires transient calcium influx into the cytosol. Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold‐acclimatize. Conversely, chemical agents causing increased calcium influx induce cold acclimatization‐specific (cas) gene expression in alfalfa at 25°C. How low temperature triggers calcium influx is, however, unknown. We report here that induction of a CA‐specific gene (cas30), calcium influx and freezing tolerance at 4°C are all prevented by cell membrane fluidization, but, conversely, are induced at 25°C by membrane rigidification. cas30 expression and calcium influx at 4°C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25°C by the actin microfilament destabilizer cytochalasin D (CD). JK blocked the membrane rigidifier‐induced, but not the calcium channel agonist‐induced cas30 expression at 25°C. These findings indicate that cytoskeleton re‐organization is an integral component in low‐temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidification and calcium influx in CA.
Antisense‐inhibition of ADP‐glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation
Authors: Weber Hans , Rolletschek Hardy , Heim Ute , Golombek Sabine , Gubatz Sabine , Wobus Ulrich 
Published: March 2003

SummaryThe small subunit of a Vicia faba ADP‐glucose pyrophosphorylase (AGP) cDNA was expressed in antisense orientation in Vicia narbonensis under the control of the seed‐specific legumin B4 promoter. From several independent transgenic lines both ADP‐glucose pyrophosphorylase AGP‐mRNA and AGP enzyme activity were reduced by up to 95% in the cotyledons during the mid‐ to late‐maturation phase. Starch was moderately decreased and sucrose was increased. In two of three lines, transcripts encoding the large subunit of AGP and the storage protein vicilin were increased, whereas legumin B‐mRNA was decreased. Transcripts of other storage‐associated genes were not altered. The cotyledons contained more protein and total nitrogen. Despite the reduction in starch, total carbon was not decreased and dry weight was unchanged. Compared to the wild type, transgenic seeds contained more water and accumulated dry weight during a longer period, and therefore had a prolonged seed‐filling period. Transgenic cotyledon cells of comparable age to the wild type were more highly vacuolated and contained smaller starch grains, indicating a delay in cellular differentiation. We conclude that a specific alteration in carbon metabolism can have pleiotropic effects on water and nitrogen content and induces temporal changes in seed development.
cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol
Authors: Hertzberg Magnus , Sievertzon Maria , Aspeborg Henrik , Nilsson Peter , Sandberg Göran , Lundeberg Joakim 
Published: March 2003

SummaryMicroarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50–200 µg of total RNA and 1–2 µg of mRNA is required for each hybridisation, which is equivalent to 50–100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub‐mg amounts of plant tissue. Using 0.1 µg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2–4 cell layers with a fresh weight of ∼0.5 mg.
Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress
Authors: Hong Suk‐Whan , Vierling Elizabeth 
Published: March 2003

SummaryHsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report thatArabidopsis thalianaHsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid‐maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated fromArabidopsisT‐DNA mutant populations. One allele,hot1‐3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assayshot1‐3shows a similar, though somewhat more severe, phenotype to the previously described missense allelehot1‐1, revealing that thehot1‐1mutation is also close to null for protein activity. The second new mutant allele,hot1‐2,has an insertion in the promoter 101 bp 5′ to the putative TATA element. During heat stress thehot1‐2mutant produces normal levels of protein in hypocotyls and 10‐day‐old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. Thehot1‐2mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.
Silencing of a meristematic gene using geminivirus‐derived vectors
Authors: Peele Charles , Jordan Chad V. , Muangsan Nooduan , Turnage Michael , Egelkrout Erin , Eagle Patricia , Hanley‐Bowdoin Linda , Robertson Dominique 
Published: March 2003

SummaryGeminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome‐mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down‐regulated throughout meristematic tissues. Our results demonstrate that geminivirus‐derived vectors can be used to study genes involved in meristem function in intact plants.
Establishment of gene‐trap and enhancer‐trap systems in the moss Physcomitrella patens
Authors: Hiwatashi Yuji , Nishiyama Tomoaki , Fujita Tomomichi , Hasebe Mitsuyasu 
Published: March 2003

SummaryBecause of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene‐trap and enhancer‐trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene‐trap and enhancer‐trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene‐trap and 1073 enhancer‐trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene‐trap line YH209, which shows rhizoid‐specific expression, was obtained by 5′ and 3′ RACE. This gene was named PpGLU, and forms a clade with plant acidic α‐glucosidase genes. Thus, these gene‐trap and enhancer‐trap systems should prove useful to identify tissue‐ and cell‐specific genes in Physcomitrella.
Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley
Authors: Zhang Yuhua , Shewry Peter R. , Jones Huw , Barcelo Pilar , Lazzeri Paul A. , Halford Nigel G. 
Published: March 2003

SummaryA chimaeric gene was constructed comprising a wheat high molecular weight glutenin subunit gene promoter, a 304‐bp sucrose non‐fermenting‐1‐related (SnRK1) protein kinase sequence in the antisense orientation, and the cauliflower mosaic virus 35S RNA gene terminator. Transgenic barley plants containing the antisense SnRK1 chimaeric gene were produced by particle bombardment of barley immature embryos with the aim of obtaining plants expressing the antisense SnRK1 sequence in the seeds. Despite the fact that the promoter was expected to be active only in seeds, two independent transgenic lines were found to fail to transmit the transgene to the T1 generation. These T0 plants had matured and died before this was discovered, but subsequently four other independent transgenic lines were found to be affected in the same way. Cytological analysis of the pollen grains in these lines showed that about 50% were normal but the rest had arrested at the binucleate stage of development, were small, pear‐shaped, contained little or no starch and were non‐functional. The presence of antisense SnRK1 transcripts was detected in the anthers of the four lines analyzed and a ubiquitin promoter/UidA (Gus) gene, one of the marker genes codelivered with the antisense gene, was found to be expressed only in the abnormal pollen. Expression analyses confirmed that SnRK1 is expressed in barley anthers and that expression of one class of SnRK1 transcripts (SnRK1b) was reduced in the abnormal lines. All of the abnormal lines showed approximately 50% seed set, and none of the transgenes were detected in the T1 generation.