Sarah M. Assmann

PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid

Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants.

Two Novel GPCR-Type G Proteins Are Abscisic Acid Receptors in Arabidopsis

In plants, G proteins modulate signaling by the stress hormone, abscisic acid (ABA). We identify and characterize two novel Arabidopsis proteins that show homology to an orphan vertebrate GPCR (GPR89) and interact with the sole Arabidopsis G protein alpha subunit, GPA1, but also have intrinsic GTP-binding and GTPase activity. We have named these proteins GPCR-type G proteins (GTG1 and GTG2). Arabidopsis mutants lacking both GTG1 and GTG2 exhibit ABA hyposensitivity. GTG1 and GTG2 bind ABA specifically. The GDP-bound form of the GTGs exhibits greater ABA binding than the GTP-bound form, the GTPase activity of the GTGs is inhibited by GPA1, and gpa1 null mutants exhibit ABA-hypersensitive phenotypes. These results predict that, unusually, it is the GDP-bound, not the GTP-bound, form of the GTGs that actively relays the signal. We propose that GTG proteins function both as a new type of G protein and as a class of membrane-localized ABA receptors.

Predicting essential components of signal transduction networks: a dynamic model of guard cell abscisic acid signaling.

Plants both lose water and take in carbon dioxide through microscopic stomatal pores, each of which is regulated by a surrounding pair of guard cells. During drought, the plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure, thereby promoting water conservation. Dozens of cellular components have been identified to function in ABA regulation of guard cell volume and thus of stomatal aperture, but a dynamic description is still not available for this complex process. Here we synthesize experimental results into a consistent guard cell signal transduction network for ABA-induced stomatal closure, and develop a dynamic model of this process. Our model captures the regulation of more than 40 identified network components, and accords well with previous experimental results at both the pathway and whole-cell physiological level. By simulating gene disruptions and pharmacological interventions we find that the network is robust against a significant fraction of possible perturbations. Our analysis reveals the novel predictions that the disruption of membrane depolarizability, anion efflux, actin cytoskeleton reorganization, cytosolic pH increase, the phosphatidic acid pathway, or K+ efflux through slowly activating K+ channels at the plasma membrane lead to the strongest reduction in ABA responsiveness. Initial experimental analysis assessing ABA-induced stomatal closure in the presence of cytosolic pH clamp imposed by the weak acid butyrate is consistent with model prediction. Simulations of stomatal response as derived from our model provide an efficient tool for the identification of candidate manipulations that have the best chance of conferring increased drought stress tolerance and for the prioritization of future wet bench analyses. Our method can be readily applied to other biological signaling networks to identify key regulatory components in systems where quantitative information is limited.

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features

RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5′ splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

Hormone interactions in stomatal function

Research in recent years on the biology of guard cells has shown that these specialized cells integrate both extra- and intra-cellular signals in the control of stomatal apertures. Among the phytohormones, abscisic acid (ABA) is one of the key players regulating stomatal function. In addition, auxin, cytokinin, ethylene, brassinosteroids, jasmonates, and salicylic acid also contribute to stomatal aperture regulation. The interaction of multiple hormones can serve to determine the size of stomatal apertures in a condition-specific manner. Here, we discuss the roles of different phytohormones and the effects of their interactions on guard cell physiology and function.

The Arabidopsis Putative G Protein-Coupled Receptor GCR1 Interacts with the G Protein a Subunit GPA1 and Regulates Abscisic Acid Signaling

Heterotrimeric G proteins composed of α, β, and γ subunits link ligand perception by G protein–coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Gα (GPA1) and Gβ (AGB1) subunits, and two probable Gγ subunits (AGG1 and AGG2). One Arabidopsis gene, GCR1, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR1 actually interacts with GPA1 was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR1 and GPA1 are indeed physically coupled. GCR1–GPA1 interaction depends on intracellular domains of GCR1. gcr1 T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr1 guard cells are also hypersensitive to the lipid metabolite, sphingosine-1-phosphate (S1P), which is a transducer of the ABA signal upstream of GPA1. Because gpa1 mutants exhibit insensitivity in aspects of guard cell ABA and S1P responses, whereas gcr1 mutants exhibit hypersensitivity, GCR1 may act as a negative regulator of GPA1-mediated ABA responses in guard cells.

Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins

In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K+ channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein α-subunit gene, GPA1 (refs 5, 6, 7–8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.

Plants: the latest model system for G‐protein research

In humans, heterotrimeric G proteins couple stimulus perception by G‐protein‐coupled receptors (GPCRs) with numerous downstream effectors. By contrast, despite great complexity in their signal‐transduction attributes, plants have a simpler repertoire of G‐signalling components. Nonetheless, recent studies on Arabidopsis thaliana have shown the importance of plant G‐protein signalling in such fundamental processes as cell proliferation, hormone perception and ion‐channel regulation.

G-Protein Complex Mutants Are Hypersensitive to Abscisic Acid Regulation of Germination and Postgermination Development

Abscisic acid (ABA) plays regulatory roles in a host of physiological processes throughout plant growth and development. Seed germination, early seedling development, stomatal guard cell functions, and acclimation to adverse environmental conditions are key processes regulated by ABA. Recent evidence suggests that signaling processes in both seeds and guard cells involve heterotrimeric G proteins. To assess new roles for the Arabidopsis (Arabidopsis thaliana) Gα subunit (GPA1), the Gβ subunit (AGB1), and the candidate G-protein-coupled receptor (GCR1) in ABA signaling during germination and early seedling development, we utilized knockout mutants lacking one or more of these components. Our data show that GPA1, AGB1, and GCR1 each negatively regulates ABA signaling in seed germination and early seedling development. Plants lacking AGB1 have greater ABA hypersensitivity than plants lacking GPA1, suggesting that AGB1 is the predominant regulator of ABA signaling and that GPA1 affects the efficacy of AGB1 execution. GCR1 acts upstream of GPA1 and AGB1 for ABA signaling pathways during germination and early seedling development: gcr1 gpa1 double mutants exhibit a gpa1 phenotype and agb1 gcr1 and agb1 gcr1 gpa1 mutants exhibit an agb1 phenotype. Contrary to the scenario in guard cells, where GCR1 and GPA1 have opposite effects on ABA signaling during stomatal opening, GCR1 acts in concert with GPA1 and AGB1 in ABA signaling during germination and early seedling development. Thus, cell- and tissue-specific functional interaction in response to a given signal such as ABA may determine the distinct pathways regulated by the individual members of the G-protein complex.

Functional Proteomics of Arabidopsis thaliana Guard Cells Uncovers New Stomatal Signaling Pathways

We isolated a total of 3 × 108 guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range two-dimensional (2D) gels, and 2D liquid chromatography–matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K+ channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.