bluuebunny/crossref_metadata_2025_split huggingface dataset:
The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas
Authors: Schroda Michael , Blöcker Dagmar , Beck Christoph F. 
Published: March 2003

SummaryThe Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, β2TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A–RBCS2 promoter fusion as compared to the RBCS2 promoter alone.
Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1
Authors: Jones Huw D. , Kurup Smita , Peters Nicholas C. B. , Holdsworth Michael J. 
Published: March 2003

SummaryThe Avena fatua (wild oat) homologue of VIVIPAROUS 1 (AfVP1) has been implicated in controlling the maintenance of embryo dormancy in mature imbibed seeds, but the detailed mechanisms by which this transcription factor family activates embryo maturation pathways and simultaneously represses germination are not known. A two‐hybrid screen in yeast identified three proteins that interacted specifically with AfVP1 (AfVP1 interacting proteins; AfVIPs). AfVIPs 2 and 3 interacted with the C‐terminus of AfVP1, which contains the B2 + B3 domains, previously shown to bind DNA, whereas AfVIP1 interacted with the isolated B3 domain. Using purified proteins in in vitro experiments, all three AfVIPs were shown also to interact with the Arabidopsis homologue ABSCISIC ACID INSENSITIVE 3 (ABI3). The three AfVIPs were expressed in both dormant and non‐dormant embryos, but the abundance of AfVIP1 and 3 transcripts was greater in germinated than dormant seeds, whereas transcripts of AfVIP2 (and AfVP1) were more highly expressed in dormant embryos. The AfVIP3 protein has homology to a human cell‐crisis gene with a predicted role in the cell cycle; AfVIP2 contains a ring‐type zinc finger motif. These homologies, together with analysis of expression studies, suggest that these proteins may play specific roles in AfVP1‐mediated regulation of the dormancy to germination transition in A. fatua seeds.
Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback‐insensitive dihydrodipicolinate synthase
Authors: Vauterin Marc , Frankard Valérie , Jacobs Michel 
Published: March 2003

SummaryDihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5≈ 10 μM), whereas the bacterial enzymes are less (50‐fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine‐sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS‐deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.
Changes in GA 20‐oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants
Authors: Carrera Esther , Bou Jordi , García‐Martínez José Luis , Prat Salomé 
Published: March 2003

SummaryGene StGA20ox1 encoding potato GA 20‐oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over‐expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20‐oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.
Probing expansin action using cellulose/hemicellulose composites
Authors: Whitney Sarah E. C. , Gidley Mike J. , McQueen‐Mason Simon J. 
Published: March 2003

SummaryCellulose‐based composite materials containing xyloglucans or mannan‐based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical α‐expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose‐only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose‐only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1‐4) β‐glucan to (1‐4) β‐glucan hydrogen bonded contacts can also serve as substrates.
Interactions within a network of phytochrome, cryptochrome and UV‐B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue
Authors: Wade Helena K. , Bibikova Tatiana N. , Valentine William J. , Jenkins Gareth I. 
Published: March 2003

SummaryThe Arabidopsis gene encoding the key flavonoid biosynthesis enzyme chalcone synthase (CHS) is regulated by several environmental and endogenous stimuli. Here we dissect the network of light signalling pathways that control CHS expression in mature leaves using cryptochrome (cry) and phytochrome (phy) deficient mutants. The UV‐A/blue light induction of CHS is mediated principally by cry1, but neither cry1 nor cry2 is involved in UV‐B induction or in the UV‐A and blue light signalling pathways that interact synergistically with the UV‐B pathway to enhance CHS expression. Moreover, these synergistic responses do not require phyA or phyB. Phytochrome is a positive regulator of the cry1 inductive pathway, mediating distinct potentiation and coaction effects. A red light pretreatment enhances subsequent cry1‐mediated CHS induction. This potentiation is unaltered in phyA and phyB mutants but much reduced in a phyA phyB double mutant, indicating that it requires principally phyA or phyB. In contrast, the cry1‐mediated induction of CHS, without pretreatment, is much reduced in phyB but not phyA mutants, indicating coaction between cry1 and phyB. Further experiments with phy‐deficient mutants demonstrate that phyB is a negative regulator of the UV‐B inductive pathway. We further show that phyB acts upstream of the points of interaction of the UV‐A and blue synergism pathways with the UV‐B pathway. We propose that phyB functions to balance flux through the cry1 and UV‐B signalling pathways.
Analysis of the SINE S1 Pol III promoter from Brassica; impact of methylation and influence of external sequences
Authors: Arnaud Philippe , Yukawa Yasushi , Lavie Laurence , Pélissier Thierry , Sugiura Masahiro , Deragon Jean‐Marc 
Published: March 2003

SummaryTranscription is an important control point in the transposable element mobilization process. To better understand the regulation of the plant SINE (Short Interspersed Elements) S1, its promoter sequence was studied using an in vitro pol III transcription system derived from tobacco cells. We show that the internal S1 promoter can be functional although upstream external sequences were found to enhance this basal level of transcription. For one putative ‘master’ locus (na7), three CAA triplets (in positions –12, –7 and –2) and two overlapping TATA motifs (in positions –54 to –43) were important to stimulate transcription. For this locus, two transcription initiation regions were characterized, one centered on position + 1 (first nucleotide of the S1 element) and one centered on position − 19 independently of the internal motifs. The CAA triplets only influence transcription in + 1 and work in association with the internal motifs. We show that methylation can inhibit transcription at the na7 locus. We also observe that S1 RNA is cleaved in a smaller Poly (A) minus product by a process analogous to the maturation of mammalian SINEs.
Generation and properties of ascorbic acid‐deficient transgenic tobacco cells expressing antisense RNA for L‐galactono‐1,4‐lactone dehydrogenase
Authors: Tabata Kazufumi , Ôba Kazuko , Suzuki Kanichi , Esaka Muneharu 
Published: March 2003

SummaryIn higher plants, the terminal step of l‐ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L‐galactono‐1,4‐lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA‐deficient transgenic tobacco BY‐2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY‐2 cells using PCR. Two transgenic cell‐lines, AS1–1 and AS2–2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild‐type BY‐2 cells. In synchronous cultures, division of AS1–1 and AS2–2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild‐type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild‐type cells. The calli of AS1–1 and AS2–2 appeared to be sticky and soft. Back extrusion method also showed that AsA‐deficient BY‐2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1–1 and AS2–2 cells were abnormally slender, suggesting a potential for a significant and a uni‐axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.
Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor
Authors: Karsies Aletta , Hohn Thomas , Leclerc Denis 
Published: March 2003

SummaryTargeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N‐terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N‐terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.
Analysis of heat‐shock transcription factor–DNA binding in Arabidopsis suspension cultures by UV laser crosslinking
Authors: Zhang Lemin , Eggers‐Schumacher Gabriele , Schöffl Fritz , Prändl Ralf 
Published: March 2003

SummaryCrosslinking techniques are important in examining protein–DNA interactions in living cells. Formaldehyde and UV light emitted by conventional lamps are the most commonly used crosslinking agents. The crosslinking step is followed by immunoprecipitation of specific protein–DNA adducts, and by analysis and quantification of the co‐immunoprecipitated DNA. There are a few reported cases of fruitful in vivo protein–DNA crosslinking experiments, but not in plants. In this report, we analyse the binding of heat‐shock transcription factor (HSF) to heat‐shock promoters in intact Arabidopsis cells. Efficient protein–DNA crosslinking by irradiation of Arabidopsis suspension culture tissue was achieved using UV laser pulses. In addition, methods for immunoprecipitation and detection of the co‐immunoprecipitated DNA are reported. Our results suggest that Arabidopsis HSFs immunoreactive for HSF1 antibodies bind constitutively to the HSP18.2 gene.
BZI‐1 specifically heterodimerises with the tobacco bZIP transcription factors BZI‐2, BZI‐3/TBZF and BZI‐4, and is functionally involved in flower development
Authors: Strathmann Anne , Kuhlmann Markus , Heinekamp Thorsten , Dröge‐Laser Wolfgang 
Published: March 2003

SummaryThe tobacco (Nicotiana tabacum) protein BZI‐1 is closely related to the plant bZIP transcription factors CPRF2, G/HBF‐1 and OHP1. Using the C‐terminal part of BZI‐1, which includes the bZIP domain, as a bait in a yeast two‐hybrid screen, three BZI‐1 interacting bZIP transcription factors, referred to as BZI‐2, BZI‐3/TBZF and BZI‐4, were isolated. The observed interactions are due to the leucine zipper dimerisation domain and have been found to be specific, in so far as other bZIP transcription factors do not interact with BZI‐1. The formation of heterodimers is favoured to homodimerisation. Furthermore, physical protein–protein interaction was confirmed by in vitro binding studies. Expression analysis reveals that BZI‐2 mRNA is predominantly located in the stems and parts of the flower. BZI‐3/TBZF expression has been observed predominantly in flowers and, to a lesser extent, in the vegetative parts of the plant. In particular, BZI‐4 is transcribed specifically in the stamen, the petals and the pistils of the tobacco flower. Since BZI‐1 is expressed ubiquitously in tobacco plants, co‐localisation with BZI‐2, BZI‐3/TBZF or BZI‐4 might influence BZI‐1 heterodimerisation and consequently target gene selection. Analysis of transgenic plants displaying changes in BZI‐1 protein level revealed that BZI‐1 regulates BZI‐4 expression. Moreover, a reduction in functional BZI‐1 protein resulted in flowers having a reduced size; in particular, the stamen and the petals are affected. Consequently, BZI‐1 homo‐ or heterodimers are involved in the control of the size of the organs of flowers. Based on these data, we discuss a model postulating BZI transcription factor heterodimerisation as a mechanism determining target gene selection and regulating processes involved in plant development.
Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes fromChlamydomonas reinhardtii
Authors: Hippler Michael , Klein Jens , Fink Andreas , Allinger Tanja , Hoerth Patric 
Published: March 2003

SummaryFunctional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild‐type and mutant strains ofChlamydomonas reinhardtii.Thylakoid membrane proteins were separated by high‐resolution two‐dimensional gel electrophoresis (2‐DE) and analysed by immuno‐blotting and mass spectrometry for the presence of membrane‐spanning proteins. Our data show that light‐harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2‐DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2‐DE revealed the presence of 18 LHCI protein spots. The use of two peptide‐specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post‐translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2‐DE. Comparison between 2‐DE maps from thylakoid membrane proteins isolated from a PSI‐deficient (Δycf4)andacrd1mutant, which is conditionally reduced in PSI and LHCI under copper‐deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2‐DE, and (ii) that high‐resolution 2‐DE facilitates the comparative analysis of membrane protein complexes in wild‐type and mutants cells.
Hydroxyl radical‐induced cell‐wall loosening in vitro and in vivo: implications for the control of elongation growth
Authors: Schopfer Peter 
Published: March 2003

SummaryHydroxyl radicals (OH) are capable of unspecifically cleaving cell‐wall polysaccharides in a site‐specific reaction. I investigated the hypothesis that cell‐wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load‐bearing cell‐wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre‐loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin‐induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2–), an OH precursor, in the growth‐controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall‐loosening factor acting in auxin‐mediated elongation growth of plant species with widely differing cell‐wall polysaccharide compositions.
Ribozyme termination of RNA transcripts down‐regulate seed fatty acid genes in transgenic soybean
Authors: Buhr Tony , Sato Shirley , Ebrahim Farida , Xing Aiqiu , Zhou You , Mathiesen Michelle , Schweiger Bruce , Kinney Anthony , Staswick Paul ,   Tom Clemente
Published: March 2003

SummaryWe investigated whether termination of transcripts with a self‐cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease specific plant gene expression. Nuclear retention was first monitored in tobacco using the β‐glucuronidase gene terminated with either the 35S CaMV 3′ untranslated sequence (UTR) or a cis‐acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme‐terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme‐terminated transcripts were subsequently tested as a gene down‐regulation strategy in soybean. The embryo‐specific Δ‐12 fatty acid desaturase FAD2‐1 gene was targeted because its down‐regulation elevates oleic acid content of seed storage lipids. Both ribozyme‐terminated antisense and standard antisense constructs were capable of gene down‐regulation, producing over 57% oleic acid compared with less than 18% in wild‐type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene down‐regulation and the simultaneous down‐regulation of two embryo‐specific genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2‐1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2‐1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2‐1 and the FatB gene encoding a palmitoyl‐thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to specifically down‐regulate endogenous gene expression in soybean.