Jérôme Giraudat

Arabidopsis OST1 Protein Kinase Mediates the Regulation of Stomatal Aperture by Abscisic Acid and Acts Upstream of Reactive Oxygen Species Production

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO2, suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H2O2 or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.

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.

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.

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.

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.

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.

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.

A Network of Local and Redundant Gene Regulation Governs Arabidopsis Seed Maturation

In Arabidopsis thaliana, four major regulators (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], LEAFY COTYLEDON1 [LEC1], and LEC2) control most aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance, and dormancy. The molecular basis for complex genetic interactions among these regulators is poorly understood. By analyzing ABI3 and FUS3 expression in various single, double, and triple maturation mutants, we have identified multiple regulatory links among all four genes. We found that one of the major roles of LEC2 was to upregulate FUS3 and ABI3. The lec2 mutation is responsible for a dramatic decrease in ABI3 and FUS3 expression, and most lec2 phenotypes can be rescued by ABI3 or FUS3 constitutive expression. In addition, ABI3 and FUS3 positively regulate themselves and each other, thereby forming feedback loops essential for their sustained and uniform expression in the embryo. Finally, LEC1 also positively regulates ABI3 and FUS3 in the cotyledons. Most of the genetic controls discovered were found to be local and redundant, explaining why they had previously been overlooked. This works establishes a genetic framework for seed maturation, organizing the key regulators of this process into a hierarchical network. In addition, it offers a molecular explanation for the puzzling variable features of lec2 mutant embryos.

The Homologous ABI5 and EEL Transcription Factors Function Antagonistically to Fine-Tune Gene Expression during Late Embryogenesis

In Arabidopsis, the basic leucine zipper transcription factor ABI5 activates several late embryogenesis–abundant genes, including AtEm1 and AtEm6. However, the expression of many other seed maturation genes is independent of ABI5. We investigated the possibility that ABI5 homologs also participate in the regulation of gene expression during seed maturation. We identified 13 ABI5-related genes in the Arabidopsis genomic sequence. RNA gel blot analysis showed that seven of these genes are active during seed maturation and that they display distinct expression kinetics. We isolated and characterized two mutant alleles of one of these genes, AtbZIP12/EEL. Unlike abi5, the eel mutations did not inhibit the expression of any of the maturation marker genes that we monitored. On the contrary, the accumulation of the AtEm1 and AtEm6 mRNAs was enhanced in eel mutant seeds compared with wild-type seeds. Gel mobility shift assays, combined with analysis of the genetic interactions among the eel and abi5 mutations, indicated that ABI5 and EEL compete for the same binding sites within the AtEm1 promoter. This study illustrates how two homologous transcription factors can play antagonistic roles to fine-tune gene expression.

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 AtDiB, 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 Kunitz 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.