Jeffrey Leung

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.

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.

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

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.

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase.

MINT‐7260208: OST1 (uniprotkb:Q940H6) and ATRBOHF (uniprotkb:O48538) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)

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.

The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover

Background Genetic evidence in Arabidopsis thaliana indicates that members of the Snf1-Related Kinases 2 family (SnRK2) are essential in mediating various stress-adaptive responses. Recent reports have indeed shown that one particular member, OPEN STOMATA (OST)1, whose kinase activity is stimulated by the stress hormone abscisic acid (ABA), is a direct target of negative regulation by the core ABA co-receptor complex composed of PYR/PYL/RCAR and clade A Protein Phosphatase 2C (PP2C) proteins. Methodology/Principal Findings Here, the substrate preference of OST1 was interrogated at a genome-wide scale. We phosphorylated in vitro a bank of semi-degenerate peptides designed to assess the relative phosphorylation efficiency on a positionally fixed serine or threonine caused by systematic changes in the flanking amino acid sequence. Our results designate the ABA-responsive-element Binding Factor 3 (ABF3), which controls part of the ABA-regulated transcriptome, as a genuine OST1 substrate. Bimolecular Fluorescence Complementation experiments indicate that ABF3 interacts directly with OST1 in the nuclei of living plant cells. In vitro, OST1 phosphorylates ABF3 on multiple LXRXXpS/T preferred motifs including T451 located in the midst of a conserved 14-3-3 binding site. Using an antibody sensitive to the phosphorylated state of the preferred motif, we further show that ABF3 is phosphorylated on at least one such motif in response to ABA in vivo and that phospho-T451 is important for stabilization of ABF3. Conclusions/Significance All together, our results suggest that OST1 phosphorylates ABF3 in vivo on T451 to create a 14-3-3 binding motif. In a wider physiological context, we propose that the long term responses to ABA that require sustained gene expression is, in part, mediated by the stabilization of ABFs driven by ABA-activated SnRK2s.

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.

A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates

Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.