Biomedical subjects
E Grill
Publications and source records attributed to E Grill.
Signalling of abscisic acid to regulate plant growth.
Abscisic acid (ABA) mediated growth control is a fundamental response of plants to adverse environmental cues. The linkage between ABA perception and growth control is currently being unravelled by using different experimental approaches such as mutant analysis and microinjection experiments. So far, two protein phosphatases, ABI1 and ABI2, cADPR, pH, and Ca2+ have been identified as main components of the ABA signalling pathway. Here, the ABA signal transduction pathway is compared to signalling cascades from yeast and mammalian cells. A model for a bifurcated ABA signal transduction pathway exerting a positive and negative control mechanism is proposed.
ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration.
The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, maintenance of seed dormancy, and inhibition of plant growth. All three responses are regulated by the ABI1 gene product. The ABI1 protein (ABI1p) has been characterized as a protein serine/threonine phosphatase of type 2C that is highly affected in its activity by changes in the proton and magnesium ion concentrations. In the ABA-insensitive mutant abi1 of Arabidopsis thaliana a single amino acid exchange in the primary structure results in both a dominant insensitive phenotype and a strongly reduced protein phosphatase activity in vitro by possibly impairing metal ion coordination.
ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis.
The abi2-1 (abscisic acid insensitive) mutant of Arabidopsis thaliana shows abscisic acid (ABA) insensitivity with respect to seed germination and vegetative ABA responses. We identified the ABI2 gene by a combination of positional mapping and homology to ABI1. The ABI2 protein shows 80% amino acid sequence identity to ABI1, a protein phosphatase 2C (PP2C) involved in ABA signaling. The mutation that confers the abi2-1 phenotype is equivalent to the mutation previously identified in abi1-1 and the resulting Gly168Asp abi2 protein shows a reduced PP2C activity. Thus, a pair of highly homologous PP2Cs regulate ABA signaling.
ABA signal transduction.
Recent advances in the study of abscisic acid signal transduction include the identification of cyclic ADP-ribose as a central mediator of abscisic acid responses. The characterisation of type 2C protein phosphatases, ABI1 and ABI2, implicates negative control and redundant action on the signal pathway of this hormone. In addition, abscisic acid-mediated inhibition of gibberellin-stimulated responses seems to depend on the activation of a phospholipase D during induction of alpha-amylase in barley aleurone cells as well as on a putative acetyltransferase involved in elongation growth.
Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2.
We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.
A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana.
The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.
Capillary zone electrophoresis and micellar electrokinetic chromatography of 4-aminobenzonitrile carbohydrate derivatives.
Aldoses, ketoses and uronic acids were derivatized successfully within 15 min at a temperature of 90 degrees C by reductive amination with 4-aminobenzonitrile. Subsequently, the derivatives were separated as their borate complexes by capillary zone electrophoresis, using 175 mM borate buffer, pH 10.5, as carrier. The electrophoretic mobilities were determined by the complex stability, which was found to depend on the number of hydroxyl groups on any given carbohydrate derivative, the presence of substituents, and most strongly on the configuration of the vicinal hydroxyl groups at C-3 and C-4 in aldoses and uronic acids, and with regard to ketoses on those at C-4 and C-5. Time of analysis could be reduced considerably by the use of micellar electrokinetic chromatography, which separated 4-aminobenzonitrile sugar derivatives on the basis of their differential partitioning into an electroendosmotically driven aqueous phase and into sodium dodecyl sulfate micelles. Optimum resolution was achieved with a Tris-phosphate buffer, pH 7.5, containing 100 mM of sodium dodecyl sulfate. The method made it possible to resolve several carbohydrates which had not been resolved successfully by means of capillary zone electrophoresis, such as glucose and fructose. Moreover, separation selectivity could be adjusted by varying the capillary temperature. Finally, on-column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 1 fmol and a concentration sensitivity of 0.3 microM.
Capillary zone electrophoresis of p-aminobenzoic acid derivatives of aldoses, ketoses and uronic acids.
Aldoses, ketoses and uronic acids were derivatized with p-aminobenzoic acid and separated as their borate complexes by capillary zone electrophoresis, using a capillary tube of fused silica containing 150 mM borate buffer, pH 10.0, as carrier. The electrophoretic mobilities of 22 carbohydrates were determined and found to increase with increasing stability of the borate complexes formed. Besides the number of hydroxyl groups and the presence of substituents, complex stability depended most strongly on the configuration of the three vicinal hydroxyl groups at C2, C3 and C4. On-column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 15 fmol and a concentration sensitivity of 4 microM. Reproducible quantification of carbohydrates was achieved at least in the concentration range of 0.1-10 mM in reaction solutions by the relative peak area method, using cinnamic acid as internal standard. The method was applied successfully to the determination of the monosaccharide composition of polysaccharides extracted from Radix althaeae.
Reactivation of metal-requiring apoenzymes by phytochelatin-metal complexes.
The enzymatically inactive, metal-requiring apoforms of diamino oxidase and of carbonic anhydrase were reactivated by copper-and zinc-phytochelatin complexes, respectively. The level and the rate of reactivation effected by metal complexes consisting of poly(gamma-glutamylcysteinyl)glycine as well as by the respective free metal ion were compared. An efficient transfer of zinc and copper from phytochelatin-complexes to apoenzymes was observed in vitro.
Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking.
A yeast artificial chromosome (YAC) genomic library of Arabidopsis thaliana was constructed in a derivative of the vector pYAC4 which was modified to facilitate the production of end-specific probes for chromosome walking. Experiments in which a subset of 2300 clones from the library were probed with 30 restriction fragment length polymorphism (RFLP) markers indicated that, on the average, the entire genome is represented once in each 800 YAC clones. Thus, the complete library of more than 20,000 YACs is expected to contain most or all of the Arabidopsis genome with a high probability. The YAC clones examined in the sample had an average insert size of approximately 150 kb +/- 10 and represented more than 5% of the Arabidopsis genome. Based on the properties of the library and the currently available RFLP maps for Arabidopsis, only one or two steps from flanking RFLPs should be sufficient to isolate an average gene in Arabidopsis by chromosome walking with the YACs. In order to facilitate chromosome walking, a method for the production of hybridization probes from the ends of the inserts was employed that is based on a combination of pre-amplification of the vector/insert junction sequences via the polymerase chain reaction and specific transcription from T3 or T7 RNA polymerase promoters flanking the cloning site in the YAC vector.
Development of a system for efficient chromosome walking in Arabidopsis.
The small genome size of Arabidopsis and the low level of repetitive DNA sequences make this crucifer an attractive system for chromosome walking to isolate genes. Mapping of a mutant locus relative to restriction fragment length polymorphism (RFLP) markers provides the first step towards isolating the corresponding gene. The RFLP marker closest to the target gene serves as a starting point. The distance between gene and marker is generally in the range of 50-200 kb. In order to facilitate chromosome walking of this magnitude, we constructed a yeast artificial chromosome (YAC) library of Arabidopsis. Large fragments of Arabidopsis DNA were cloned into a YAC vector and transformed into yeast. The library contains more than 10 equivalents of the Arabidopsis genome. YACs containing sequences of RFLP markers of Arabidopsis revealed an average insert size of 150 kb. Thus, 1-3 (contiguous) YACs should be sufficient to clone genes from Arabidopsis by chromosome walking. In order to use the system to isolate genes involved in the signal transduction of abscisic acid, we fine-mapped the mutant loci abi-1 and abi-2, which confer abscisic acid insensitivity, relative to RFLPs and isolated the corresponding YACs.
Phytochelatins, the heavy metal binding peptides of plants: characterization and sequence determination.
The isolation of a homologous series of five novel heavy metal complexing peptides from plant cell suspension cultures is reported. Their structures were established as (gamma-Glu-Cys)n Gly, n = 3-7. These peptides appear upon induction with heavy metals and represent the principal metal binding activity in induced cells. The name phytochelatin (PC) is proposed for this new class of natural products in higher plants. The hitherto postulated metallothioneins do not exist in plants but their function of chelating heavy metals via metal-thiolate coordination is fulfilled by the simpler phytochelatins. They are not primary gene products because of their repetition of gamma-glutamyl linkages.
Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus.
The thermostability of the staphylococcal plasmids pC194 and pUB110 and their antibiotic-resistance determinants was examined upon transfer to Bacillus stearothermophilus CU21. Plasmid pGS13, a pUB110 derivative carrying the chloramphenicol acetyltransferase (CAT) gene of pC194, could be maintained up to the maximum growth temperature (68 degrees C) by selection for chloramphenicol resistance. In the absence of selective pressure, pGS13 was lost at temperatures above 60 degrees C. Segregational instability of pGS13 was accompanied by a progressive loss of negative superhelicity at elevated temperatures. Thermostable mutants of pGS13 were isolated by screening for expression of the antibiotic-resistance determinants after growth under non-selective conditions. These mutants were found to contain an insertion of a 1.7 kb DNA sequence derived from the cryptic B. stearothermophilus plasmid pBS02. Increased thermostability correlated with preservation of plasmid superhelicity at elevated temperatures.
Receptors and signalling components of plant hormones.
Recent advances in understanding plant hormonal signalling has resulted in the identification of a variety of signalling components including receptor kinases with homology to the bacterial two component system as well as serine/threonine kinases and protein phosphatases. In addition, the existence of MAP kinase pathways in plants indicates a similar role of these signalling cascades in the relay of exogenous signals into the nucleus as has been disclosed in animal cells. The emerging signalling pathways of the plant hormone abscisic acid and ethylene are presented.