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J Giraudat

Publications and source records attributed to J Giraudat.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Abscisic Acid↗
PubMed1995

Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana.

Four clones corresponding to Arabidopsis thaliana transcripts regulated by progressive drought stress were isolated. Abundance of the AtDi8, AtDi19 and AtDi21 mRNAs increased in both roots and leaves during progressive drought. The AtDr4 mRNA was expressed in a root-specific manner in regularly watered plants, and became undetectable under drought conditions. In all cases, the drought-induced modifications of mRNA abundance could be reversed by subsequent rehydration. The predicted AtDr4 protein displays extensive similarity to various members of the Künitz protein family, suggesting that AtDr4 might be a root-specific protease inhibitor. Of these four genes, only AtDi8 and AtDi21 responded to an exogenous supply of abscisic acid (ABA). Analysis of the ABA-deficient aba mutant demonstrated that endogenous ABA indeed participates in the drought regulation of these two transcripts. This ABA-dependent response was differentially affected in the various classes of ABA-insensitive Arabidopsis mutants. The AtDi19 and AtDr4 mRNAs both responded to drought in an ABA-independent manner, but at distinct thresholds of the progressive drought stress. Regulation of these four target genes by progressive drought stress thus appears to be mediated by at least three distinct signals, only one of which is ABA.

Abscisic Acid↗
PubMed1995

Abscisic acid signaling.

The plant hormone abscisic acid (ABA) regulates the development and germination of seeds, as well as the adaptation of vegetative tissues to conditions of environmental stress. During the past year, considerable insights have been gained into the molecular nature of the complex signaling network that mediates the actions of ABA. Biophysical studies indicate that at least some of the effects of ABA in stomatal guard cells involve intracellular receptors. Also, increasing evidence supports the view that guard cells contain redundant ABA transduction pathways, and that cytoplasmic Ca2+ acts as a second messenger in at least one of these pathways. Finally, mutational analysis in Arabidopsis indicates that the multiple effects of ABA at the whole plant level are mediated by overlapping branches of a highly ramified signaling network. Two Arabidopsis loci that determine ABA sensitivity have already been cloned and found to encode a protein phosphatase and a transcriptional activator.

Abscisic Acid↗
PubMed1994

RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes.

Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.

Amino Acid Sequence↗
PubMed1994

Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.

The Arabidopsis ABI1 locus is essential for a wide spectrum of abscisic acid (ABA) responses throughout plant development. Here, ABI1 was shown to regulate stomatal aperture in leaves and mitotic activity in root meristems. The ABI1 gene was cloned and predicted to encode a signaling protein. Although its carboxyl-terminal domain is related to serine-threonine phosphatase 2C, the ABI1 protein has a unique amino-terminal extension containing an EF hand calcium-binding site. These results suggest that the ABI1 protein is a Ca(2+)-modulated phosphatase and functions to integrate ABA and Ca2+ signals with phosphorylation-dependent response pathways.

Abscisic Acid↗
PubMed1994

Conservation of the human breast basic conserved 1 gene in the plant kingdom: characterization of a cDNA clone from Arabidopsis thaliana.

Based on low-stringency genomic Southern analysis, the recently described human breast basic conserved 1 (BBC1) gene has been proposed to be conserved throughout Eukaryota [Adams et al., Hum. Mol. Genet. 1 (1992) 91-96]. We have isolated and characterized a cDNA clone (AtBBC1) which corresponds to a BBC1 homologue from the model plant species, Arabidopsis thaliana. Furthermore, upon Northern analysis, the AtBBC1 probe detects related transcripts in various dicotyledenous and monocotyledonous plant species, indicating that actively transcribed BBC1-related genes are widespread in the plant kingdom. BBC1 has been shown to be expressed in all mammalian tissues examined [Adams et al., Hum. Mol. Genet. 1 (1992) 91-96]; similarly, here the AtBBC1 transcript has been detected in a variety of A. thaliana organs. In addition, the abundance of the AtBBC1 mRNA is developmentally regulated during the course of fruit maturation, and is positively correlated with active cell division.

Amino Acid Sequence↗
PubMed1994

Current advances in abscisic acid action and signalling.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Abscisic Acid↗
PubMed1994

Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid.

The accumulation kinetics of 18 mRNAs were characterized during Arabidopsis silique development. These marker mRNAs could be grouped in distinct classes according to their coordinate temporal expression in the wild type and provided a basis for further characterization of the corresponding regulatory pathways. The abscisic acid (ABA)-insensitive abi3-4 mutation modified the expression pattern of several but not all members of each of these wild-type temporal mRNA classes. This indicates that the ABI3 protein directly participates in the regulation of several developmental programs and that multiple regulatory pathways can lead to the simultaneous expression of distinct mRNA markers. The ABI3 gene is specifically expressed in seed, but ectopic expression of ABI3 conferred the ability to accumulate several seed-specific mRNA markers in response to ABA in transgenic plantlets. This suggested that expression of these marker mRNAs might be controlled by an ABI3-dependent and ABA-dependent pathway(s) in seed. However, characterization of the ABA-biosynthetic aba mutant revealed that the accumulation of these mRNAs is not correlated to the ABA content of seed. A possible means of regulating gene expression by developmental variations in ABA sensitivity is apparently not attributable to variations in ABI3 cellular abundance. The total content of ABI3 protein per seed markedly increased at certain developmental stages, but this augmentation appears to result primarily from the simultaneous multiplication of embryonic cells. Our current findings are discussed in relation to their general implications for the mechanisms controlling gene expression programs in seed.

Abscisic Acid↗
PubMed1993

Characterization of an Arabidopsis thaliana gene (TMKL1) encoding a putative transmembrane protein with an unusual kinase-like domain.

We have characterized a novel Arabidopsis gene, designated TMKL1 (for Transmembrane Kinase-Like). The encoded protein is predicted to contain an N-terminal signal peptide, an extracellular domain with seven imperfect leucine-rich repeats, a single hydrophobic transmembrane segment, and a kinase-like intracellular domain which lacks, however, several of the key conserved residues essential for catalytic activity. The TMKL1 protein thus represents a unique and functionally intriguing member of the family of plant proteins related to animal receptor kinases. Northern blot analysis indicates that the TMKL1 gene is transcriptionally active in a variety of organs, and is developmentally regulated during silique maturation. Its possible functions are discussed.

Amino Acid Sequence↗
PubMed1992

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Amino Acid Sequence↗
PubMed1992

A Brassica napus transcript encoding a protein related to the Künitz protease inhibitor family accumulates upon water stress in leaves, not in seeds.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.

Amino Acid Sequence↗
PubMed1991

Mapping the Arabidopsis genome.

We are engaged in a project to assemble a complete physical map of the Arabidopsis thaliana (L.) Heynh. genome. The first stage of this project involved the analysis of approximately 20,000 random cosmid clones representing an 8- to 10-fold sampling redundancy. Using computer matching programs, these clones have been assembled into some 750 contigs, encompassing 90-95% of the Arabidopsis genome. We are currently attempting to bridge the gaps by selecting the missing clones by hybridization. As a complement to this project we have constructed an RFLP map which currently contains 175 markers. The RFLP map provides contact points between the physical map and classical genetic map. Our main objective for undertaking this project is to simplify the cloning of genes where only the locus and not the product of the gene is known. In other words, the combined RFLP/physical map serves as a general cloning tool by simplifying the movement from the genetic locus to the cloned gene.

Abscisic Acid↗
PubMed1990

The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.

Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker [3H]chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The [3H]chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.

Allosteric Site↗
PubMed1989

The noncompetitive blocker [(3)H]chlorpromazine labels segment M2 but not segment M1 of the nicotinic acetylcholine receptor alpha-subunit.

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker ]3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity side for noncompetitive blockers. The alpha-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the alpha-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta-chain [1] and Ser-254 and Leu-257 in the beta-chain [2]. Extended sequence analysis of the hydrophobic segment M1 further showed that no labeling-occurred in this region.

Amino Acid Sequence↗
PubMed1988

Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site.

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.

Acetylcholine↗
PubMed1987

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

Amino Acid Sequence↗
PubMed1986

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII.

Affinity Labels↗