PubMed Health⌕ Search

Biomedical subjects

J Giraudat

Publications and source records attributed to J Giraudat.

At least 19 recordsLinked to original sources
PubMed2006

AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis.

EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A two-hybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.

Amino Acid Sequence↗
PubMed2001

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway.

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.

Abscisic Acid↗
PubMed2000

Interactions between abscisic acid and ethylene signaling cascades.

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.

Abscisic Acid↗
PubMed1999

Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation.

Genetic and molecular studies have shown that the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a prominent role in the control of seed maturation. The ABI3 protein and its orthologues from various other plant species share four domains of high sequence identity, including three basic domains designated as B1, B2 and B3. The leaky abi3-1 mutation is a single amino acid substitution within the B3 domain. A new abi3 allele, abi3-7, was generated by mutagenizing abi3-1 seeds. The abi3-7 line contains, in addition to the abi3-1 mutation, a point mutation that converts residue Ala-458 into Thr within the B2 domain of the ABI3 protein. This Ala residue is absolutely conserved in all known ABI3 orthologues. Abi3-7 seeds display reductions in dormancy and in sensitivity to abscisic acid which are intermediate between those of the leaky abi3-1 and of the severe abi3-4 and abi3-5 mutants. Accumulation and distribution of At2S1 and At2S2 albumin mRNA as well as of AtEm1 and AtEm6 late embryogenesis-abundant proteins and mRNA have been analyzed. Both At2S1 and At2S2 mRNA are reduced in abi3-7, but distribution of At2S2 is spatially restricted. Accumulation of AtEm6 protein is more sensitive to abi3-7 mutation than AtEm1. However both mRNAs are considerably reduced in this mutant. Their distribution is also differentially affected. These results provide genetic evidence for the importance of the conserved B2 domain for ABI3 function in vivo.

2S Albumins, Plant↗
PubMed1999

Mapping of 25 drought-inducible genes, RD and ERD, in Arabidopsis thaliana.

We mapped 25 Arabidopsis thaliana drought-inducible genes. Responsive to Dehydration (RD) and Early Responsive to Dehydration (ERD), to the Arabidopsis genome and compared map positions with those of mutants that show environmental stress response. We hybridized CIC yeast artificial chromosome (YAC) library filters with the cDNAs and determined the map positions of 18 corresponding genes. We screened the P1 library with 7 other clones and analyzed segregation of their restriction fragment length polymorphisms (RFLP) in recombinant inbred (RI) lines. One cDNA could be mapped because it had been sequenced by the Arabidopsis genome sequencing program.

Adaptation, Biological↗
PubMed1999

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.

Abscisic Acid↗
PubMed1998

Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis.

In plants, a number of MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) homologues have been reported. However, there have been no reports of protein-protein interactions between these kinases or molecular analysis of MAPK cascades in higher plants. To analyze a possible MAPK cascade in Arabidopsis thaliana, we took two molecular approaches. One is the two-hybrid screening of ATMEKK1 (a MAPKKK)-interacting proteins; the other is an analysis of physical and functional interactions among isolated MAPK, MAPKK, and MAPKKK homologues from Arabidopsis. In two-hybrid screening using ATMEKK1 as bait, we isolated a novel MAPKK homologue, ATMKK2, a MAPK homologue, ATMPK4, and an unknown protein. ATMKK2 has high sequence similarity with MEK1 (a MAPKK) in Arabidopsis. Based on yeast two-hybrid analysis, we detected protein-protein interactions between ATMEKK1 and ATMKK2/MEK1 (MAPKKs), between ATMKK2/MEK1 and ATMPK4 (a MAPK), and between ATMPK4 and ATMEKK1. ATMPK4 and ATMKK2/MEK1 interacted with two distinct regions of ATMEKK1, the N-terminal regulatory domain and the C-terminal kinase domain, respectively. Coexpression of ATMEKK1 increased the ability of two closely related MAPKKs, ATMKK2 and MEK1, to complement a growth defect of the yeast pbs2 mutant. Coexpression of ATMPK4 and MEK1 complemented a growth defect of the yeast mpk1 and bck1 mutants. By contrast, other combinations of MAPKs and MAPKKs did not suppress these yeast mutations. These results suggest that ATMEKK1, ATMKK2/MEK1, and ATMPK4 may constitute a MAP kinase cascade.

Alanine↗
PubMed1998

Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants.

A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on the basis of both yeast 2-hybrid analysis and complementation analysis of yeast mutants. Specific protein-protein interactions between ATMPK4 (a MAPK) and MEK1 (a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by using the 2-hybrid system. A growth defect of the yeast mpk1delta mutant was reversed by coexpression of ATMPK4 and MEK1. Coexpression of the N-terminal deletion form of ATMEKK1 increased the ability of MEK1 to suppress a growth defect of the yeast pbs2delta mutant. These results suggest that ATMPK4, MEK1, and ATMEKK1 may interact with each other and constitute a specific MAPK cascade in Arabidopsis. This is the first demonstration of a possible MAPK cascade in plants.

Arabidopsis↗
PubMed1998

The role of ABI1 in abscisic acid signal transduction: from gene to cell.

The semi-dominant abi1-1 mutation of Arabidopsis interferes with multiple aspects of abscisic acid signal transduction resulting in reduced seed dormancy and sensitivity of root growth in ABA. Furthermore, the mutant transpires excessively as a result of abnormal stomatal regulation leading to a wilty phenotype. The ABI1 gene has been cloned. The carboxyl-terminal domain of the predicted ABI1 protein is related to the 2C class of serine-threonine phosphatases while no overt homology was found in the extended amino terminus. A combination of in vitro assays and yeast mutant complementation studies confirmed that ABI1 is a functional protein phosphatase 2C. The abi1-1 mutation converts the amino acid glycine180 to aspartic acid, and in the above test systems, causes a partial loss of the phosphatase activity. In transgenic Nicotiana benthamiana guard cells, the abi1-1 gene causes a reduction in the background current of the outward-rectifying potassium channels, and also in the abscisic acid-sensitivity of both the outward- and the inward-rectifying potassium channels in the plasma membrane. However, normal sensitivity of both potassium channels to, and stomatal closure in, abscisic acid was recovered in the presence of H7 and staurosporine, both broad-range protein kinase antagonists. These results suggest the aberrant potassium channel behavior as a major consequence of abi1-1 action and implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell potassium channels to abscisic acid-evoked signal cascades.

Abscisic Acid↗
PubMed1997

Cloning and characterisation of MEK1, an Arabidopsis gene encoding a homologue of MAP kinase kinase.

We report the cloning of a cDNA for MEK1, an Arabidopsis thaliana gene encoding a homologue of MAP kinase kinase (MEK). The predicted protein sequence shows 41% identity over 270 amino acids to vertebrate MEK proteins, and contains conserved features characteristic of MEK. Analysis of transcript levels show that expression of the gene is regulated by developmental processes (etiolation/de-etiolation) and by wounding. However in contrast to the rapid wound induction of MAP kinase transcripts in other plant species, MEK1 transcripts first accumulated 6-12 h after wounding.

Amino Acid Sequence↗
PubMed1997

Interactions between the ABI1 and the ectopically expressed ABI3 genes in controlling abscisic acid responses in Arabidopsis vegetative tissues.

In Arabidopsis, the abscisic acid (ABA)-Insensitive ABI1 and ABI3 genes have been proposed to act in separate ABA signalling cascades. Recessive mutations in ABI3 alter various physiological processes during seed development, whereas the dominant abi1 mutation inhibits ABA responses largely in vegetative tissues. The seed-specific ABI3 gene was ectopically expressed in the vegetative tissues of transgenic Arabidopsis plants carrying a transcriptional fusion between the CaMV 35S promoter and the ABI3 cDNA. Genetic interactions between the ectopically expressed ABI3 and endogenous ABI1 genes were investigated by monitoring diverse ABA responses in vegetative tissues. Ectopic expression of ABI3 conferred to plantlets the ability to accumulate the seed-specific At2S33 and AtEm1 mRNAs in response to ABA, and the abi1 mutation inhibited this ABI3-dependent induction of AtEm1 by ABA. Furthermore, ectopic expression of ABI3 also influenced ABI1-dependent responses that occur in wild-type vegetative tissues. Expression of ABI3 increased ABA induction of the Rab18 mRNA and ABA inhibition of root growth, and both responses were sensitive to the abi1 mutation in the presence as in the absence of ABI3. Finally, although ABI3 is thought to be a transcription activator and stomatal regulation is not known to involve transcriptional events, the ectopically expressed ABI3 gene suppressed the effect of the abi1 mutation on stomatal regulation. The present data demonstrate that ABI1 and ABI3 genetically interact in controlling diverse ABA responses in transgenic vegetative tissues. The possibility that the endogenous ABI1 and ABI3 genes may similarly act in a common ABA signalling pathway in seed is discussed with previous phenotypic studies of the abi1 and abi3 mutants.

Abscisic Acid↗
PubMed1997

Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid.

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase. Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl) to eliminate K+ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tall current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics. Pronounced activation of ICl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau approximately equal to 0.3 sec at -150 mV). Challenge with 20 microM ABA increased the steady-state current conductance, gCl, near 0 mV by 1.2- to 2.6-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations. Neither the steady-state and kinetic characteristics of ICl nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three- to four-fold. Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly. These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.

Abscisic Acid↗
PubMed1997

The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction.

Abscisic acid (ABA) mediates seed maturation and adaptive responses to environmental stress. In Arabidopsis, the ABA-INSENSITIVE1 (ABI1) protein phosphatase 2C is required for proper ABA responsiveness both in seeds and in vegetative tissues. To determine whether the lack of recessive alleles at the corresponding locus could be explained by the existence of redundant genes, we initiated a search for ABI1 homologs. One such homolog turned out to be the ABI2 locus, whose abi2-1 mutation was previously known to decrease ABA sensitivity. Whereas abi1-1 is (semi)dominant, abi2-1 has been described as recessive and maternally controlled at the germination stage. Unexpectedly, the sequence of the abi2-1 mutation showed that it converts Gly-168 to Asp, which is precisely the same amino acid substitution found in abi1-1 and at the coincidental position within the ABI1 phosphatase domain (Gly-180 to Asp). In vitro assays and functional complementation studies in yeast confirmed that the ABI2 protein is an active protein phosphatase 2C and that the abi2-1 mutation reduced phosphatase activity as well as affinity to Mg2+. Although a number of differences between the two mutants in adaptive responses to stress have been reported, quantitative comparisons of other major phenotypes showed that the effects of both abi1-1 and abi2-1 on these processes are nearly indistinguishable. Thus, the homologous ABI1 and ABI2 phosphatases appear to assume partially redundant functions in ABA signaling, which may provide a mechanism to maintain informational homeostasis.

Abscisic Acid↗
PubMed1997

The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development.

Previous studies have shown that recessive mutations at the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON1 (LEC1) loci lead to various abnormalities during mid-embryogenesis and late embryogenesis. In this study, we investigated whether these loci act in independent regulatory pathways or interact in controlling certain facets of seed development. Several developmental responses were quantified in abi3, fus3, and lec1 single mutants as well as in double mutants combining either the weak abi3-1 or the severe abi3-4 mutations with either fus3 or lec1 mutations. Our data indicate that ABI3 interacts genetically with both FUS3 and LEC1 in controlling each of the elementary processes analyzed, namely, accumulation of chlorophyll and anthocyanins, sensitivity to abscisic acid, and expression of individual members of the 12S storage protein gene family. In addition, both FUS3 and LEC1 regulate positively the abundance of the ABI3 protein in the seed. These results suggest that in contrast to previous models, the ABI3, FUS3, and LEC1 genes act synergistically to control multiple elementary processes during seed development.

Abscisic Acid↗
PubMed1996

Protein phosphatase activity of abscisic acid insensitive 1 (ABI1) protein from Arabidopsis thaliana.

Mutations at the ABI1 (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid. The sequence of ABI1 predicts a protein composed of an N-terminal domain that contains motifs for an EF-hand Ca(2+)-binding site, and a C-terminal domain with similarities to protein serine/threonine phosphatases 2C. We report here two sets of experimental evidence that indicate that ABI1 has typical protein phosphatase 2C activity. First, expression of the ABI1 C-terminal domain partially complemented the temperature-sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant. Second, recombinant proteins that contained the ABI1 C-terminal domain displayed in vitro phosphatase activity towards 32P-labelled casein, and this activity displayed Mg2+ or Mn2+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C. Characterisation of recombinant proteins that contained various portions of ABI1 indicated that the putative EF-hand motif is unlikely to mediate Ca2+ regulation of the ABI1 phosphatase activity at physiological Ca2+ concentrations, and may represent in EF-hand analogue rather than an EF-hand homologue. The abil-l mutation appeared to cause significant reduction in the phosphatase activity of ABI1. These results are discussed in relation to the dominant phenotype of abil-l over the wild-type allele in plants, and to the possible role of ABI1 in abscisic acid signalling.

Abscisic Acid↗
PubMed1996

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

Amino Acid Sequence↗