bluuebunny/crossref_metadata_2025_split huggingface dataset:
Derepression of the activity of genetically engineered heat shock factor causes constitutive synthesis of heat shock proteins and increased thermotolerance in transgenic Arabidopsis
Authors: Lee Jeong Hee , Hübel Anja , Schöffl Fritz 
Published: March 2003

SummaryATHSF1 is a heat shock transcription factor (HSF) of Arabidopsis that is constitutively expressed but its activity for DNA binding, trimer formation and transcriptional activation of heat shock (hs) genes is repressed at normal temperatures. In this study the functional properties of chimeric HSF‐glucuronidase (GUS) fusion proteins were tested. Ectopic expression of HSF—GUS or GUS—HSF in transgenic Arabidopsis plants resulted in a derepression of HSF functions as shown by trimer formation, specific DNA binding, and the constitutive expression of heat shock proteins (HSPs) at normal temperature. A novel GUS activity‐staining protocol was used to show the specific binding of trimeric HSF fusion proteins to DNA and following hs, an interaction between chimeric HSF—GUS and authentic HSF proteins. The chimeric HSFs were insensitive to the negative regulation that counteracts activation of the authentic HSF at normal temperature. Heterotrimer complexes were reconstituted in vitro from recombinant ATHSF1 and HSF—GUS proteins expressed in Escherichia coli and using this protocol, the temperature‐dependent activation of wt HSF was monitored in vivo and in vitro. Transgenic plants expressing constitutively active HSF—GUS fusion proteins are also constitutive for HSPs. Approximately 20% of the maximum heat‐inducible levels of HSP18 were already present at normal temperature. The level of basic thermotolerance was significantly enhanced in these plants. The results indicate that genetic engineering using protein fusion is a very effective means to derepress the activity of an important regulatory protein in plants, that consequently activates a constitutive hs response in the absence of heat stress and eventually alters the thermotolerance phenotype.
Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin
Authors: Kortstee Anne J. , Vermeesch Angela M.S. , De Vries Beja J. , Jacobsen Evert , Visser Richard G.F. 
Published: March 2003

SummaryIn order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coli was expressed in the amylose‐free potato mutant. The E. coli glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence. The E. coli glgB was cloned behind the two N‐terminal amino acids of the GBSS mature protein, creating a chimeric protein. Transgenic plants were obtained which expressed the E. coli branching enzyme as was shown by the presence of mRNA and protein in the tubers. Correctly processed protein was found both in the soluble and starch granule bound protein fraction. Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints. The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose‐residues or less in the amylopectin. Changes in other characteristics of the starch, such as average chain length (CL) and λmax, indicated a more branched structure for starch of transformed plants as well.
Evidence that actin filaments are involved in controlling the permeability of plasmodesmata in tobacco mesophyll
Authors: Ding Biao , Kwon Myoung‐Ok , Warnberg Leif 
Published: March 2003

SummaryThe role of actin filaments in regulating plasmodesmal transport has been studied by microinjection experiments in mesophyll cells of tobacco (Nicotiana tabacum L. cv. Samsun). When fluorescent dextrans of various molecular sizes were each co‐injected with specific actin filament perturbants cytochalasin D (CD) or profilin into these cells, dextrans up to 20 kilodalton (kDa) moved from the injected cell into surrounding cells within 3–5 min. In contrast, when such dextrans were injected alone or co‐injected with phalloidin into the mesophyll cells, they remained in the injected cells. Phalloidin co‐injection slowed down or even inhibited CD‐ or profilin‐elicited dextran cell‐to‐cell movement. Dextrans of 40 kDa or larger were unable to move out of the injected cell in the presence of CD or profilin. These data suggest that actin filaments may participate in the regulation of plasmodesmal transport by controlling the permeability of plasmodesmata.
fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression
Authors: Barnes Simon A. , Quaggio Ronaldo B. , Whitelam Garry C. , Chua Nam‐Hai 
Published: March 2003

SummaryPhysiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome‐regulated genes in fhy1. In far‐red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A‐deficient mutant, confirming that gene expression in far‐red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light‐induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A‐mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue‐light signaling pathway for CHS expression.
Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G‐fructosyltransferase
Authors: Vijn I. , Van Dijken A. , Sprenger N. , Van Dun K. , Weisbeek P. , Wiemken A. , Smeekens S. 
Published: March 2003

SummaryFructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G‐fructosyltransferase (6G‐FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G‐FFT from onion by screening a cDNA library using barley sucrose:fructan 6‐fructosyltransferase (6‐SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6‐SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1‐kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G‐FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.
Introns act post‐transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1
Authors: Rose Alan B. , Last Robert L. 
Published: March 2003

SummaryThe expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1‐GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1‐GUS fusions, and they act post‐transcriptionally to increase the steady‐state level of mRNA.
Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants
Authors: Masgrau Carles , Altabella Teresa , Farrás Rosa , Flores Dante , Thompson Andrew J. , Besford Robert T. , Tiburcio Antonio F. 
Published: March 2003

SummaryTo test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline‐inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline‐induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.
Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco
Authors: Fischer Regina , Budde Ina , Hain Rüdiger 
Published: March 2003

SummaryStilbene synthase genes (STS) have previously been used to enhance disease resistance in plants. In order to study the effects of modified STS expression patterns in plants, heterologous promoters were fused to an STS gene and the chimeric genes were transferred to tobacco. Very high constitutive expression of STS mediated by a duplicated upstream region of the 35S RNA promoter from CaMV affected secondary biosynthetic pathways. STS over‐expression caused altered flower pigmentation and male sterility, probably due to competition between the introduced STS and the endogenous chalcone synthase for the substrates 4‐coumaroyl CoA and malonyl CoA. Furthermore, tobacco plants with tapetum‐specific STS expression were male‐sterile. Thus, STS genes are promising tools in strategies for engineering altered flower colours and the development of a novel hybrid seed system.
Light modulates the spatial patterns of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana
Authors: Learned R. Marc , Connolly Erin L. 
Published: March 2003

SummaryAlthough the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for β‐glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light‐mediated regulation of HMG1 expression is shown to be an organ‐autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark‐induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.
A glucocorticoid‐mediated transcriptional induction system in transgenic plants
Authors: Aoyama Takashi , Chua Nam‐Hai 
Published: March 2003

SummaryA novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors. A chimeric transcription factor, designated GVG was constructed, consisting of the DNA‐binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence. Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid. Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 µM and the maximum expression level was over 100 times that of the basal level. Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h. The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used. The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants. The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system. Advantages and potential uses of this system are also discussed.
Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases?
Authors: Bonas Ulla , Van den Ackerveken Guido 
Published: March 2003

SummaryOne of the recent exciting developments in the research area of plant‐microbe interactions is a breakthrough in understanding part of the initial signalling between avirulent Gram‐negative bacteria and resistant plants. For resistance to occur, both interacting organisms need to express matching genes, the plant resistance gene and the bacterial avirulence gene. The biochemical function of bacterial avirulence genes and the nature of the signal molecules recognized by the plant have been a mystery for a long time. Recently, several laboratories have shown that bacterial avirulence proteins function as elicitors that are perceived within the plant cell.
Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae
Authors: Hiraiwa Nagako , Nishimura Mikio , Hara‐Nishimura Ikuko 
Published: March 2003

SummaryVacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N‐linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self‐catalytic proteolysis within the vacuoles.
Efficient in vitro transcription of plant nuclear tRNASer genes in a nuclear extract from tobacco cultured cells
Authors: Yukawa Yasushi , Sugita Mamoru , Sugiura Masahiro 
Published: March 2003

SummaryA nuclear extract derived from tobacco cultured BY‐2 cells supports RNA polymerase III‐dependent transcription of Arabidopsis tRNASer genes. Primer extension analysis indicated that the transcription starts at 6 bp upstream from the 5′ end of tRNA coding region. Procedures for nuclear extraction and in vitro reaction conditions have been optimized for tRNA transcription, which allows direct detection of de novo synthesized tRNA by gel electrophoresis. This improved in vitro system yields a mature‐sized tRNA of 85 nucleotides from the Arabidopsis tRNASer gene, indicating that efficient processing of the pre‐tRNA also occurs in the tobacco nuclear extract.
Light‐induced transcriptional repression of the pea AS1 gene: identification of cis‐elements and transfactors
Authors: Ngai Nora , Tsai Fong‐Ying , Coruzzi Gloria 
Published: March 2003

SummaryHere, we examine the cis‐elements and trans‐factors affecting the expression of asparagine synthetase (AS) genes whose transcription is negatively regulated by light. The promoters for the AS1 and AS2 genes of pea were isolated, sequenced, and functionally dissected for their ability to confer regulated expression to the GUS reporter gene in transgenic tobacco. Histochemical analysis of transgenic plants demonstrated that the AS1 and AS2 promoters show identical patterns of cell‐specific expression. The more highly active AS1 promoter was further demonstrated to confer negative light‐regulation to the GUS gene in transgenic tobacco. Deletion analysis and gain‐of‐function experiments showed that 124 bp of the AS1 promoter was sufficient to confer light‐activated repression to a heterologous promoter. Potential conserved transcription regulatory elements, Box B, Box C, and Box C′ within this region were shown to bind to nuclear proteins by gel shift analysis. A light‐specific DNA:protein interaction was detected with Box B. The nuclear factors that bind to Box C and C′ elements of AS1 are competed by a putative repressor element ‘RE1’ defined previously in the oat phytochrome gene whose transcription is also repressed by light. The Box B and C/ C′‐Box/REl‐binding factors were found in nuclear extracts of tobacco, pea, and Arabidopsis and may therefore be universal factors involved in light‐activated transcriptional repression.
Nicotiana plumbaginifolia hlg mutants have a mutation in a PHYB‐type phytochrome gene: they have elongated hypocotyls in red light, but are not elongated as adult plants
Authors: Hudson Matthew , Robson Paul R.H. , Kraepiel Yvan , Caboche Michel , Smith Harry 
Published: March 2003

SummaryTwo new allelic mutants of Nicotiana plumbaginifolia have been isolated which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light (W). This can be accounted for by the decreased response to red light (R) of the hypocotyl elongation rate in these mutants. Responses to other wavelengths are unaffected in the mutants. When grown in white light, mature hlg mutants are not elongated with respect to the wild‐type; they also bolt and flower later. The shade‐avoidance responses to red/far red ratio (R:FR) are intact in these mutants. Both mutants are deficient in a phyB‐like polypeptide that is immunodetectable in the wild‐type; both have wild‐type levels of a phyA‐like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single‐base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome B‐type photoreceptor. One allele, hlg‐1, has an introduced amino acid substitution; this may define a residue essential for phytochrome protein stability. The other allele, hlg‐2, has a stop codon introduced C‐terminal to the chromophore binding domain. As these phyB mutants are unaffected in shade‐avoidance responses, but deficient in perception of R, it is concluded that the phyB absent in these mutants is responsible for R perception in the N. plumbaginifolia seedling, but is not a R:FR sensor in light‐grown plants.
Seven zinc‐finger transcription factors are expressed sequentially during the development of anthers in petunia
Authors: Kobayashi Akira , Sakamoto Ayako , Kubo Kenichi , Rybka Zbigniew , Kanno Yoshiaki , Takatsuji Hiroshi 
Published: March 2003

SummaryThe development of anthers and pollen involves several stages. In an attempt to identify the transcription factors that are involved in the regulation of gene expression associated with the development of anthers, seven genes for new zinc‐finger proteins that are expressed in anthers were detected using RT‐PCR. Subsequent cloning and sequence analysis of the full‐length cDNA clones revealed that they all encoded zinc‐finger proteins of the EPF type. However, the encoded proteins were very different from each other in terms of size, number of zinc fingers (two, three and four) and the spacings between the fingers. Northern blot analysis revealed that the genes were expressed preferentially in anthers. Moreover, it was found that the seven genes were expressed transiently for short periods that corresponded to different developmental stages. It appeared that the genes were activated sequentially during the development of the anther. Considering these observations, a possible mechanism that could account for the autonomous progression of the development of the anther via a regulatory cascade of these transcription factors is discussed.