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B Weisshaar

Publications and source records attributed to B Weisshaar.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearance, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thaliana tt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds.

Arabidopsis↗
PubMed1999

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Arabidopsis↗
PubMed1998

Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes.

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or downstream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.

Arabidopsis↗
PubMed1998

CPRF4a, a novel plant bZIP protein of the CPRF family: comparative analyses of light-dependent expression, post-transcriptional regulation, nuclear import and heterodimerisation.

Several DNA-binding proteins with conserved basic region/leucine zipper domains (bZIP) have been isolated from parsley. They all recognise defined ACGT-containing elements (ACEs), including ACE(PcCHSII) in the Light Regulatory Unit LRU1 of the CHS promoter which confers light responsiveness. A new member of this Common Plant Regulatory Factor (CPRF) family, designated CPRF4a, has been cloned, which displays sequence similarity to HBP-1a from wheat, as well as to other plant bZIP proteins. CPRF4a specifically binds as a homodimer to ACE(PcCHSII) and forms heterodimers with CPRF1 but not with CPRF2. In adult parsley plants, CPRF2 and CPRF4a mRNAs are found in all tissues and organs in which the chalcone synthase gene CHS is expressed. In protoplasts from suspension cultured cells, UV irradiation (290-350 nm) did not cause an increase in levels of CPRF1, CPRF2, or CPRF4a mRNA, whereas the corresponding CPRF proteins accumulated within 15 min of light treatment. Furthermore, the rapid light-mediated increase of CPRF proteins was insensitive to transcriptional inhibitors, suggesting that a post-transcriptional mechanism controls CPRF accumulation. CPRFs as well as Arabidopsis thaliana G-box binding factors (GBFs) are selectively transported from the cytosol into the nucleus, as shown in an in vitro nuclear transport system prepared from evacuolated parsley protoplasts, indicating that cytosolic compounds are involved in regulated nuclear targeting of plant bZIP factors.

Acyltransferases↗
PubMed1998

Phenylpropanoid biosynthesis and its regulation.

In the past year progress has been made in the manipulation of phenylpropanoid metabolism but several studies highlight gaps in our understanding of the biochemistry of these pathways. New components involved in transcriptional regulation of phenylpropanoid genes have been identified, including transcription factors and novel proteins that function upstream of DNA-binding proteins.

Gene Expression Regulation, Plant↗
PubMed1998

Identification of UV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homologous protoplast transient expression system.

To identify DNA sequences of the Arabidopsis thaliana chalcone synthase gene (CHS) concerned with induction by UV-B and UV-A/blue light, AtCHS promoter constructions were assayed by transient expression in protoplasts prepared from two different lines of cultured A. thaliana cells. The protoplasts responded similarly to A. thaliana leaf tissue in light-dependent CHS transcript accumulation. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light-responsive promoter activity. A 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion to 164 bp resulted in reduced promoter strength but retention of responsiveness to UV-B and UV-A/blue light. Further deletion abolished transcriptional activity. The 164 bp promoter contains sequences closely resembling LRUPcCHS, (light-responsive unit of the Petroselinum crispum CHS promoter). This A. thaliana CHS promoter region, designated LRUAtCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in LRUAtCHS corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of the 164 bp and 335 bp promoter deletions. However, the mutant 668 bp promoter retained residual UV-B and UV-A/blue light-induced expression, indicating the presence of additional functional sequences upstream of -335. Mutation of a single G-box-like sequence around -442 had no effect on light responsiveness, indicating that it does not function in light regulation of this promoter. Since no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant, we conclude that the two phototransduction pathways regulate transcription factors which interact with common promoter elements. The results from-our analysis of a A. thaliana light-responsive promoter will facilitate the study of light-dependent gene regulation by genetic means in Arabidopsis thaliana.

Acyltransferases↗
PubMed1998

Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana.

Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

Amino Acid Sequence↗
PubMed1997

PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit.

Light regulatory unit 1 (LRU1) is necessary for and sufficient to mediate light-dependent activation of the chalcone synthase (CHS) minimal promoter in Petroselinum crispum. This composite promoter unit consists of at least two distinct cis-acting elements, designated ACECHS and MRECHS, both of which are required for light induction. The ACGT-containing element ACECHS interacts with common plant regulatory factors (CPRFs) which belong to the basic region/leucine zipper (bZIP) class of transcription factors. Here, we demonstrate that MRECHS, originally identified as an in vivo DNA footprint, is a MYB recognition element. This element possesses a functional core that is essential for light responsiveness and is specifically recognized by two distantly related MYB-like proteins: MYB305 and the novel factor MYB1 from P. crispum. PcMYB1 was identified by both its specific binding to MRECHS in vitro and recognition of MRECHS in vivo. The deduced amino acid sequence revealed that PcMYB1 contains only one MYB-like repeat. This portion of the protein constitutes the DNA-binding domain. Mutational analysis of PcMYB1 in combination with sequence comparison suggests the presence of a helix-turn-helix structure containing a recognition helix that is sufficient for sequence-specific binding. The structure of this distinct MYB-like DNA-binding domain appears to be conserved in proteins from all three eukaryotic phyla.

Acyltransferases↗
PubMed1996

The transcriptional regulator CPRF1: expression analysis and gene structure.

Many eukaryotic DNA-binding proteins share a conserved amino acid sequence known as the basic region leucine zipper (bZIP) domain. bZIP proteins recognise DNA, upon dimerization, in a sequence-specific manner. The Common Plant Regulatory Factor 1 (CPRF1) is a bZIP transcription factor from parsley (Petroselinum crispum), which recognises defined elements containing ACGT cores. CPRF1 genomic DNA was cloned and the gene was sequenced. Analysis of the sequence data revealed the existence of 12 exons and 11 introns within a stretch of about 9 kb. A second RNA species hybridising to CPRF1 probes was identified as an alternatively spliced, additional CPRF1 transcript containing intron 8. This polyadenylated RNA species showed accumulation characteristics very similar to those of the CPRF1 mRNA. CPRF1 specifically binds an ACGT-containing element which is located within the composite regulatory unit that is necessary and sufficient for light activation of the parsley chalcone synthase (CHS) minimal promoter. Expression studies at the mRNA level demonstrated that CPRF1 mRNA is present in all organs of light-grown plants in which CHS mRNA expression is detectable, and light-dependent CHS mRNA accumulation was shown to be blocked by cycloheximide. Therefore, translation of a protein factor, possibly CPRF1, may be a prerequisite for CHS promoter activation.

Acyltransferases↗
PubMed1995

Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants.

The expression of chalcone synthase (CHS) genes, which encode the first enzyme of the flavonoid pathway, is under developmental control as well as affected by external stimuli such as light. Varying fragments of the 1 kb upstream region of the CHS1 gene from white mustard (Sinapis alba L.) were fused to the GUS-coding region, and the light-regulated expression of these constructs was analysed in transgenic Arabidopsis and tobacco plants. Studies performed with Arabidopsis seedlings indicate the presence of two elements within the CHS1 promoter mediating light responses via different photoreceptors. One element, located about 150 bp upstream of the transcription start site, is homologous to Unit 1 of the parsley CHS gene, the second, far more upstream element carries sequences similar to Unit 2 of the same gene. Detailed studies on Unit 1-driven expression indicate that this element transfers the expression characteristics of the original gene to both Arabidopsis and tobacco. Although the expression characteristics of Unit 1 are indistinguishable from those of the full-length promoter within the same species, we observed differences in mustard CHS promoter regulation between Arabidopsis and tobacco plants transgenic for the identical construct. The difference in photoreceptor usage by the same promoter element in different transgenic species (Unit 1 from mustard in Arabidopsis vs. tobacco) was also observed for different but homologous promoter elements in the same transgenic species (Unit 1 from mustard and parsley in tobacco). We therefore conclude that the same promoter and even the same promoter element (Unit 1) can mediate different spatial patterns of expression and modes of light regulation in different transgenic species.

Acyltransferases↗
PubMed1994

Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter.

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.

Arabidopsis Proteins↗
PubMed1994

Functional analysis of a light-responsive plant bZIP transcriptional regulator.

Common plant regulatory factor 1 (CPRF1) is a parsley basic region/leucine zipper (bZIP) transcription factor that recognizes specific nucleotide sequences containing ACGT cores. Such a sequence is contained within LRU1, the composite light regulatory unit that is necessary and sufficient for light-dependent activity of the parsley chalcone synthase (CHS) promoter. After light treatment of both etiolated and green seedlings, CPRF1 mRNA levels increased prior to CHS mRNA accumulation. The change in CPRF1 mRNA leads to a light-responsive increase in CPRF1 protein. Transient expression analysis in parsley protoplasts using the CPRF1 promoter fused to the beta-glucuronidase (GUS) open reading frame indicated that light-dependent CPRF1 mRNA accumulation was under transcriptional control. The 5' untranslated region of the CPRF1 gene includes a cis-acting nucleotide sequence that contains two ACGT elements at a distance of 12 bp between their palindromic centers. This feature is reminiscent of as-1 and octopine synthase (ocs) elements identified in promoters from plant pathogens. This double ACGT Element element, designated dACECPRF1, stimulated transcription when placed 5' to a heterologous core promoter. CPRF1 bound to dACECPRF1 DNA as well as to the ACGT element from the CHS promoter in vitro. Cotransfection experiments demonstrated that CPRF1 interacts with these elements in vivo and that overexpression of CPRF1 actually reduced light-dependent transcription from the CHS promoter. CPRF1 thus appears to contribute to the regulation of the CPRF1 gene and to interfere with the activities of light-regulated promoters.

Acyltransferases↗
PubMed1993

Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum.

Ubiquitin is an omnipresent protein found in all eukaryotes so far analysed. It is involved in several important processes, including protein turnover, chromosome structure and stress response. Parsley (Petroselinum crispum) contains at least two active polyubiquitin (ubi4) genes encoding hexameric precursor proteins. The deduced amino acid sequences of the ubiquitin monomers are identical to one another and to ubiquitin sequences from several other plant species. Analysis of the promoter region of one ubi4 gene revealed putative regulatory elements. In parsley plants, the ubi4 mRNAs were the predominant ubiquitin mRNAs and were present at comparable levels in all plant organs tested. In cultured parsley cells, high levels of ubiquitin gene expression remained unaffected by heat shock, elicitor or light treatment.

Amino Acid Sequence↗
PubMed1993

Microtubule-associated protein 2 and the organization of cellular microtubules.

Microtubule-associated proteins (MAPs) are prominent components of the neuronal cytoskeleton that can promote microtubule formation and whose expression is under strong developmental regulation. They are thought to be involved in organizing the structure of microtubule fascicles in axons and dendrites, although whether they form active cross-links between microtubules or serve as strut-like spacer elements has yet to be resolved. In the experiments reported here we explored their influence on microtubules by expressing them in non-neuronal cells using DNA transfection techniques. We confirm earlier reports that microtubule-associated proteins of the MAP2/tau class can induce bundling of microtubules. In addition we find that MAP2 causes the rearrangement of microtubules in the cytoplasm in a manner that is dependent on the length of the microtubule bundles. Short bundles are straight and run across the cytoplasm whereas long bundles form a marginal band-like array at the periphery. We suggest that the latter arrangement is produced when microtubule bundles that are too long to fit inside the diameter of the cell bend under the restraining influence of the cortical cytoskeleton. In confirmation of this, we show that when the cortical actin network is depolymerized by cytochalasin B the MAP2-containing microtubule bundles push out cylindrical extensions from the cell surface. These results suggest that the induction of stiff microtubules bundles by MAP2, coupled with a breach in the cortical actin network, can confer two of the properties characteristic of neuronal processes; their cylindrical form and the presence of fasciculated microtubules.

Cytochalasin B↗
PubMed1993

The low molecular weight form of microtubule-associated protein 2 is transported into both axons and dendrites.

In the developing brain microtubule-associated protein MAP2 occurs as both a high molecular weight form, MAP2b, which is present only in dendrites, and a low molecular weight form, MAP2c, which is also present in axons. Because the MAP2c amino acid sequence is entirely contained within that of MAP2b it is not possible to raise a MAP2c-specific antibody, so that it has been impossible to determine whether MAP2c is present in dendrites along with MAP2b. To answer this question we have generated a MAP2c cDNA clone tagged with a 10 amino acid epitope from human c-myc. This additional sequence does not alter either the binding of MAP2c to microtubules or its effects on microtubules in non-neuronal cells. When expressed in cultured primary neurons by transfection, the myc tag allowed the distribution of MAP2c to be determined independently of endogenous MAP2 protein by immunostaining with an anti-myc antibody. This showed that MAP2c is present in all processes, indicating that it can enter all kinds of processes and is stable in their cytoplasm. The results further suggest that the selective association of high molecular weight MAP2 with dendrites depends on a mechanism that prevents either its entrance or survival in the axonal compartment.

Amino Acid Sequence↗
PubMed1993

Actin depolymerisation induces process formation on MAP2-transfected non-neuronal cells.

We have previously shown that microtubules in nonneuronal cells form long, stable bundles after transfection with the embryonic neuronal microtubule-associated protein MAP2c. In this study, we found that treating MAP2c-transfected cells with the actin depolymerising drug cytochalasin B led to the outgrowth of microtubule-containing processes from the cell surface. This effect was specific to MAP2c and did not occur in untransfected cells whose microtubules had been stabilised by treatment with taxol. The outgrowth and retraction of these processes during repeated cycles of cytochalasin addition and removal was followed by video time-lapse microscopy and was suggestive of a physical interaction between compressive forces exerted by the MAP2c-stabilised microtubule bundles and tensile forces originating in the cortical actin network. We suggest that MAP2c confers three properties on cellular microtubules that are essential for process outgrowth: stability, bundling and stiffness. The latter probably arises from the linking together of neighbouring tubulin subunits by three closely spaced tubulin-binding motifs in the MAP2 molecule that limits their motion relative to one another and thus reduces the flexibility of the polymer. Similar multimeric tubulin-binding domains in other proteins of the MAP2 class, including tau in axons and MAP4 in glial cells, may play the same role in the development and support of asymmetric cell morphology. Axial bundles of microtubules are found in growing neurites but not in growth cones, suggesting that the regulated expression of these MAP-induced properties makes an important contribution to the establishment of a stable process behind the advancing growth cone.

Actins↗
PubMed1992

Homodimeric and heterodimeric leucine zipper proteins and nuclear factors from parsley recognize diverse promoter elements with ACGT cores.

Four short nucleotide sequences (boxes I to IV) contribute to the light responsiveness of the parsley chalcone synthase promoter. The sequence-related boxes II and III resemble several plant, viral, and bacterial promoter elements that share ACGT core sequences and are associated with diversely regulated genes. We have analyzed the binding characteristics and protein-protein interactions of factors from nuclear extracts and of three putative leucine zipper (bZIP) transcription factors potentially involved in the regulation of this promoter. These common plant regulatory factors (CPRFs) bind specifically to boxes II and III as well as other ACGT-containing promoter elements (hex1, Em1a, and as-1), though with markedly different affinities. Intact bZIP domains are crucial for CPRF binding to DNA. Distinct ensembles of nuclear factors bind to boxes II and III, despite their sequence similarity. The parsley CPRFs bind to DNA as dimers, selectively form heterodimeric DNA binding complexes, and interact with nuclear proteins.

Acyltransferases↗