bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential expression and regulation of K+ channels in the maize coleoptile: molecular and biophysical analysis of cells isolated from cortex and vasculature
Authors: Bauer Claudia S. , Hoth Stefan , Haga Ken , Philippar Katrin , Aoki Naohiro , Hedrich Rainer 
Published: March 2003

SummaryRecently, two K+ channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K+ channel activated by external acidification, while ZMK2 mediates voltage‐independent and proton‐inhibited K+ currents. In search of the related gene products in planta, we applied the patch–clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6–8 pS K+ channel gave rise to inwardly rectifying K+ currents. Like ZMK1, this channel was activated by apoplastic acidification. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltage‐independent K+ currents with a single‐channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca2+, Cs+ and Ba2+ is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K+ channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2‐like channels represent the first in planta analysis of a K+ channel that shares properties with the AKT3 K+ channel family.Keywords: K+ channel, voltage‐independent, proton block, maize coleoptile.
FRL1 is required for petal and sepal development in Arabidopsis
Authors: Hase Yoshihiro , Tanaka Atsushi , Baba Tomohiro , Watanabe Hiroshi 
Published: March 2003

SummaryA novel flower mutant, frl1 (frill 1) was isolated in Arabidopsis thaliana. The frl1 mutant has serrated petals and sepals but the other floral and vegetative organs appear to be normal. To analyse the role of the FRL1 gene, morphological, cytological and double mutant analyses were carried out. The frl1 flower had broader petals and sepals as compared with the wild‐type. The distal region of frl1 petals contained fewer epidermal cells but their size was variable and generally larger than that in the wild‐type. However, no significant difference was found in the basal region. Observations of the early petal development revealed that the morphology of the developing frl1 petal was normal until the middle of stage 9, but the frl1 phenotype became apparent in stages later than 10
. Furthermore, larger nuclei with varied sizes were observed in the distal region of frl1 petals, but not in this region in wild‐type petals. This strongly suggests that abnormal endo‐reduplication had occurred. These observations indicate that the frl1 mutation affects the number of cell divisions and the subsequent cell expansion during the late stage of petal lamina formation, and that FRL1 might be maintaining the mitotic state or suppressing the transition to the endo‐reduplication cycle. Double mutants with the homeotic mutants apetala3‐1 and agamous showed additive phenotypes. Ectopic petals in the third whorl of fr11 ag flowers were serrated, indicating that the FRL1 gene acts in petal and sepal development in an organ‐specific manner.
The hormonal regulation of axillary bud growth in Arabidopsis
Authors: Chatfield Steven P. , Stirnberg Petra , Forde Brian G. , Leyser Ottoline 
Published: March 2003

SummaryApically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild‐type and hormone‐signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin‐mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin‐signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.
Size constraints for targeting post‐transcriptional gene silencing and for RNA‐directed methylation in Nicotiana benthamiana using a potato virus X vector
Authors: Thomas Carole L. , Jones Louise , Baulcombe David C. , Maule Andrew J. 
Published: March 2003

SummaryUsing a recombinant potato virus X (PVX) vector, we investigated the relationship between the length of RNA sequence identity with a transgene and the ability to promote post‐transcriptional gene silencing (PTGS) and transgene methylation. The lower size limit required for targeting reporter transgene mRNA de novo using PTGS was 23 nucleotides (nt) of complete identity, a size corresponding to that of small RNAs associated with PTGS in plants and RNA interference (RNAi) in animals. The size and sequence specificity were also explored for PTGS‐associated transgene methylation and for the targeting of the vector RNA. The PTGS‐competent short sequences resulted in similar patterns of methylation. In all cases, including specific sequences of 33 nt with or without symmetrical cytosine residues, the methylation was distributed throughout the transcribed region of the transgene. In contrast, short sequences lacking symmetrical cytosines were less efficient at promoting PTGS of the transgene mRNA. Short gfp sequences in the PVX vector provided as effective a target for the degradation of viral RNA as was found for PVX carrying the complete gfp cDNA. Short sequences were able to initiate PTGS of an endogenous gene, phyotene desaturase, although this occurred in the absence of DNA methylation. This experimental approach provides important insights into the relationship between short RNA sequences and PTGS.
Quantitative in vivo measurement of glutathione in Arabidopsis cells
Authors: Meyer Andreas J. , May Mike J. , Fricker Mark 
Published: March 2003

SummaryA new, non‐destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH–bimane (GSB) conjugate. At low (10–100 µm) concentrations of MCB, labelling was mediated by a glutathione S‐transferase, which confers specificity for GSH. HPLC analysis of MCB‐labelled low molecular‐weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis–Menten kinetics with an apparent KM of 40 µm and Vmax of 470 µmol lcyt −1 min−1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of γ‐glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830–942 nmol g−1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane‐labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two‐photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 ± 3% cytoplasm was determined by manual segmentation, and a value of 37 ± 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 ± 0.3 and 3.2 ± 0.3 mm for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 ± 0.5 and 3.5 ± 0.7 mm, respectively.
A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence‐ and defence‐related processes
Authors: Robatzek Silke , Somssich Imre E. 
Published: March 2003

SummaryWRKY proteins constitute a large family of plant‐specific transcription factors whose precise functions have yet to be elucidated. Here we show that expression of one representative in Arabidopsis, AtWRKY6, is influenced by several external and internal signals often involved in triggering senescence processes and plant defence responses. Progressive 5′ deletions of the AtWRKY6 promoter allowed separation of defined regions responsible for the expression in distinct organs or upon pathogen challenge. Nuclear localization of AtWRKY6 was demonstrated; protein truncations and gain‐of‐function studies enabled delineation of a region harbouring a novel type of functional nuclear localization signal (NLS).
In vivo target promoter‐binding activities of a xenobiotic stress‐activated TGA factor
Authors: Johnson Christopher , Boden Erin , Desai Mihir , Pascuzzi Pete , Arias Jonathan 
Published: March 2003

SummaryXenobiotic chemicals induce the expression of nuclear detoxification genes. A full understanding of this protective response will require characterization of its transcriptional regulatory machinery. We describe here the use of a recently developed plant chromatin immunoprecipitation (ChIP) assay to define nuclear promoter targets of TGA1a, a tobacco basic/leucine zipper transcription factor whose activity is potentiated by herbicide‐induced xenobiotic stress. TGA1a selectively binds as‐1‐type cis‐elements, which regulate transcription of putative detoxification and defense genes. With ChIP, we show that endogenous TGA1a binds as‐1‐containing promoter sequences of two tobacco glutathione S‐transferase genes, GNT1 and GNT35. This binding activity is strongly enhanced by xenobiotic stress, as is expression of these genes. In contrast, TGA1a apparently does not bind in vivo to functional as‐1 elements in promoters of PR‐1a and PG13, genes whose expression is insensitive to this stimulus. The findings here thus discriminate between a number of possible functional promoter binding sites for a trans‐regulatory factor, within the context of a signal response pathway.
A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible
Authors: Belbahri Lassaad , Boucher Christian , Candresse Thierry , Nicole Michel , Ricci Pierre , Keller Harald 
Published: March 2003

SummaryPlants activate disease resistance responses when they recognize pathogen‐derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro‐organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen‐inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1–PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.
Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds
Authors: Tierens Koenraad F. M.‐J. , Thomma Bart P. H. J. , Bari Rajendra P. , Garmier Marie , Eggermont Kristel , Brouwer Margreet , Penninckx Iris A. M. A. , Broekaert Willem F. , Cammue Bruno P. A. 
Published: March 2003

SummaryAn Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild‐type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild‐type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.
High free‐methionine and decreased lignin content result from a mutation in the Arabidopsis S‐adenosyl‐L‐methionine synthetase 3 gene
Authors: Shen Bo , Li Changjiang , Tarczynski Mitchell C. 
Published: March 2003

SummaryAs an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over‐accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met – more than 200‐fold that observed for wild type – yet showed little or no difference in the concentrations of other protein amino‐acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S‐adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino‐acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S‐adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.
AtMYB21, a gene encoding a flower‐specific transcription factor, is regulated by COP1
Authors: Shin Byongchul , Choi Goh , Yi Hankuil , Yang Seungchan , Cho Insook , Kim Jonghyun , Lee Seunghee , Paek Nam‐Chon , Kim Jae‐Hong , Song Pill‐Soon , Choi Giltsu 
Published: March 2003

SummaryLight is an important environmental signal that governs plant growth and development. One important light‐signalling component involved in plant light responses is COP1. The pleiotropic phenotypes of the cop1 mutant suggest that COP1 regulates not only photomorphogenesis, but also other developmental processes. We investigated the role of COP1 by identifying genes that are regulated by COP1. We report that AtMYB21, a gene encoding a flower‐specific transcription factor, is ectopically expressed in the cop1 mutant. Analysis shows that dark‐grown transgenic seedlings expressing AtMYB21‐GR fusion protein display some features of the cop1 mutant, including decreased hypocotyl cell expansion, open cotyledons in the dark, and seedling lethality in the presence of dexamethasone. Light‐grown adult transgenic plants expressing AtMYB21 have shorter stems, smaller and narrower leaves, narrower petals, and malformed carpels. In addition, we show that AtMYB21 directly regulates two genes that are also expressed more abundantly in the cop1 mutant. The results indicate that COP1 is required to repress the AtMYB21 gene in seedlings, and the pleiotropic phenotypes shown in the cop1 mutant are due to the combination of misregulation of genuine light‐signalling components and other tissue‐specific factors.
Barley stripe mosaic virus‐induced gene silencing in a monocot plant
Authors: Holzberg Steve , Brosio Paul , Gross Cynthia , Pogue Gregory P. 
Published: March 2003

SummaryRNA silencing of endogenous plant genes can be achieved by virus‐mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus‐induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive‐sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the γb gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo‐bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo‐bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild‐type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo‐bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology‐dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA‐silencing mechanisms in monocot plant species.
Revival of a resurrection plant correlates with its antioxidant status
Authors: Kranner Ilse , Beckett Richard P. , Wornik Sabine , Zorn Margret , Pfeifhofer Hartwig W. 
Published: March 2003

Summary Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, β‐carotene, ascorbate, α‐tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, γ‐tocopherol, lutein, neoxanthin and glucose‐6‐phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re‐watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of α‐tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, β‐carotene and α‐tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.
A nucleus‐encoded maize protein with sigma factor activity accumulates in mitochondria and chloroplasts
Authors: Beardslee Thomas A. , Roy‐Chowdhury Sanchita , Jaiswal Pankaj , Buhot Laurence , Lerbs‐Mache Silva , Stern David B. , Allison Lori A. 
Published: March 2003

SummaryPlants contain nuclear gene families that encode proteins related to the principal sigma factors of eubacteria. As sigma factors function in transcription, the plant proteins have been presumed or demonstrated to associate with the eubacteria‐like RNA polymerase of chloroplasts. In maize, five sig cDNA sequences have been reported, and four of the products are present in plastids as predicted. However, in vitro chloroplast import assays and computer algorithms gave ambiguous results with the fifth protein, ZmSig2B. Unlike the other maize sigma factors, ZmSig2B is expressed throughout developing seedling leaves, as well as in roots and etiolated tissues. To determine the subcellular location of ZmSig2B, we have now used immunoblot assays to show that it co‐purifies with both mitochondria and plastids. Its NH2‐terminal 153 amino acids, translationally fused to green fluorescent protein (GFP), targeted GFP to chloroplasts and mitochondria in bombarded maize leaves. A putative role for ZmSig2B in mitochondrial transcription is supported by its presence in a maize mitochondrial transcription extract. ZmSig2B also exhibits the expected properties of a chloroplast sigma factor: recombinant ZmSig2B binds to a chloroplast promoter and initiates transcription in vitro when combined with Escherichia coli core RNA polymerase. Therefore ZmSig2B is an unusual nucleus‐encoded sigma factor that appears to function in both chloroplasts and mitochondria.
A novel method for the construction of genome wide transcriptome maps
Authors: Brugmans Bart , Del Carmen Asun Fernandez , Bachem Christian W. B. , Van Os Hans , Van Eck Herman J. , Visser Richard G. F. 
Published: March 2003

SummaryExpression profiling by cDNA‐AFLP is commonly used to display the transcriptome of a specific tissue, treatment or developmental stage. In this paper, cDNA‐AFLP has been used to study transcripts expressed in segregating populations from Arabidopsis thaliana and potato (Solanum tuberosum). The genetic differences between the offspring genotypes are thus visualized as polymorphisms in the transcriptome. We show that polymorphic transcripts can be used as genetic markers and allow the construction of a linkage map. The resulting map shows that, in contrast to genomic markers, the transcriptome‐derived markers did not cluster in particular areas of the chromosome, and that cDNA‐AFLP markers are targeted specifically to transcriptionally active regions. The cDNA‐AFLP markers used in mapping are derived from DNA polymorphisms in transcripts, rather than differences in expression regulation. The high potential of transcriptome markers as opposed to (anonymous) genomic markers for applications in genetic analyses, marker‐assisted breeding and map‐based cloning is discussed.