Christiane Valon

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.

An update on abscisic acid signaling in plants and more...

The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a beta-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H(+)-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.

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.

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.

Constitutive activation of a plasma membrane H+‐ATPase prevents abscisic acid‐mediated stomatal closure

Light activates proton (H+)‐ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO2 to photosynthetic tissues. Light to darkness transition, high CO2 levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H+‐ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO2 and darkness. The OST2 gene encodes the major plasma membrane H+‐ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H+‐ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA‐directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.

The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action

Stomatal guard cells are functionally specialized epidermal cells usually arranged in pairs surrounding a pore. Changes in ion fluxes, and more specifically osmolytes, within the guard cells drive opening/closing of the pore, allowing gas exchange while limiting water loss through evapo-transpiration. Adjustments of the pore aperture to optimize these conflicting needs are thus centrally important for land plants to survive, especially with the rise in CO(2) associated with global warming and increasing water scarcity this century. The basic biophysical events in modulating membrane transport have been gradually delineated over two decades. Genetics and molecular approaches in recent years have complemented and extended these earlier studies to identify major regulatory nodes. In Arabidopsis, the reference for guard cell genetics, stomatal opening driven by K(+) entry is mainly through KAT1 and KAT2, two voltage-gated K(+) inward-rectifying channels that are activated on hyperpolarization of the plasma membrane principally by the OST2 H(+)-ATPase (proton pump coupled to ATP hydrolysis). By contrast, stomatal closing is caused by K(+) efflux mainly through GORK, the outward-rectifying channel activated by membrane depolarization. The depolarization is most likely initiated by SLAC1, an anion channel distantly related to the dicarboxylate/malic acid transport protein found in fungi and bacteria. Beyond this established framework, there is also burgeoning evidence for the involvement of additional transporters, such as homologues to the multi-drug resistance proteins (or ABC transporters) as intimated by several pharmacological and reverse genetics studies. General inhibitors of protein kinases and protein phosphatases have been shown to profoundly affect guard cell membrane transport properties. Indeed, the first regulatory enzymes underpinning these transport processes revealed genetically were several protein phosphatases of the 2C class and the OST1 kinase, a member of the SnRK2 family. Taken together, these results are providing the first glimpses of an emerging signalling complex critical for modulating the stomatal aperture in response to environmental stimuli.

A Brand New START: Abscisic Acid Perception and Transduction in the Guard Cell

Understanding how plants respond to the hormone abscisic acid provides targets for designing crops resilient to drought. The combined daily consumption of fresh water ranges from 200 to 700 liters per capita per day in most developed countries, with ~70% used for agricultural needs. With the looming prospect of global water crisis, the success in deciphering the early steps in the signal transduction of the stress hormone abscisic acid (ABA) has ignited hopes that crops can be engineered with the capacity to maintain productivity while requiring less water input. ABA can accumulate 10- to 30-fold in plants under drought stress relative to unstressed conditions. The hormone triggers diverse adaptive pathways, permitting plants to withstand temporary bouts of water shortage. One experimental model to unravel the details of ABA signaling mechanisms is based on the hormone’s ability to elicit stomatal closure—a rapid cellular response used by plants to limit water loss through transpiration. Each stoma, or pore, is contoured by two specialized kidney-shaped cells called guard cells. The stomatal pores are the primary portals for photosynthetic carbon dioxide uptake and, by triggering closure of these pores, drought negatively affects photosynthesis and, consequently, biomass. The stomatal aperture widens and narrows by expansion and contraction, respectively, of these flanking guard cells caused by changes in the intracellular concentrations of ions and organic solutes. These events require coordination of ion channels, which generate a specific pattern of electrical signals that relay the ABA stimulus. A soluble ABA receptor that turns on and off the activities of a kinase and phosphatase pair is at the heart of the signaling complex. Results distilled from the latest structural studies on these ABA receptors, characterized by the so-called START domain, are beginning to tender exciting promise for rational design of agonists and antagonists for modulating stress adaptive ability in plants. With 4 figures, 2 tables, and 127 citations, this Review charts the extraordinary progress on understanding how ABA controls membrane transport mechanisms that evoke the stomatal closing pathway. More background details are available with the teaching tools at www.plantcell.org.gate1.inist.fr/site/teachingtools/teaching.xhtml. The soluble receptors of abscisic acid (ABA) have been identified in Arabidopsis thaliana. The 14 proteins in this family, bearing the double name of PYRABACTIN RESISTANCE/PYRABACTIN-LIKE (PYR/PYL) or REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) (collectively referred to as PYR/PYL/RCAR), contain between 150 and 200 amino acids with homology to the steroidogenic acute regulatory-related lipid transfer (START) protein. Structural studies of these receptors have provided rich insights into the early mechanisms of ABA signaling. The binding of ABA to PYR/PYL/RCAR triggers the pathway by inducing structural changes in the receptors that allows them to sequester members of the clade A negative regulating protein phosphatase 2Cs (PP2Cs). This liberates the class III ABA-activated Snf1-related kinases (SnRK2s) to phosphorylate various targets. In guard cells, a specific SnRK2, OPEN STOMATA 1 (OST), stimulates H2O2 production by NADPH oxidase respiratory burst oxidase protein F and inhibits potassium ion influx by the inward-rectifying channel KAT1. OST1, the kinase CPK23, the calcium-dependent kinase CPK21, and the counteracting PP2Cs modulate the slow anion channel SLAC1, a pathway that contributes to stomatal responses to diverse stimuli, including ABA and carbon dioxide. A minimal ABA response pathway that leads to activation of the SLAC1 homolog, SLAH3, and presumably stomatal closure has been reconstituted in vitro. The identification of the soluble receptors and core components of the ABA signaling pathway provides promising targets for crop design with higher resilience to water deficit while maintaining biomass.

Abscisic Acid Signal off the STARTing Block

The year 2009 marked a real turnaround in our understanding of the mode of abscisic acid (ABA) action. Nearly 25 years had elapsed since the first biochemical detection of ABA-binding proteins in the plasmalemma of Vicia guard cells was reported. This recent--and laudable--achievement is owed largely to the discovery of the soluble ABA receptors whose major interacting proteins happen to be some of the most well-established components of earliest steps in ABA signaling. These soluble receptors, with the double name of PYRABACTIN RESISTANCE (PYR) or REGULATORY COMPONENT OF ABA RECEPTOR (RCAR), are a family of Arabidopsis proteins of about 150-200 amino acids that share a conserved START domain. The ABA signal transduction circuitry under non-stress conditions is muted by the clade A protein phosphatases 2C (PP2C) (notably HAB1, ABI1, and ABI2). During the initial steps of ABA signaling, the binding of the hormone to the receptor induces a conformational change in the latter that allows it to sequester the PP2Cs. This excludes them from the negative regulation of the downstream ABA-activated kinases (OST1/SnRK2.6/SRK2E, SnRK2.2, and SnRK2.3), thus unleashing the pathway by freeing them to phosphorylate downstream targets that now include several b-ZIP transcription factors, ion channels (SLAC1, KAT1), and a NADPH oxidase (AtrbohF). The discovery of this family of soluble receptors and the rich insight already gained from structural studies of their complexes with different isoforms of ABA, PP2C, and the synthetic agonist pyrabactin lay the foundation towards rational design of chemical switches that can bolster drought hardiness in plants.

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.

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.