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Plant voltage-gated K⁺ channels have been referred to as “plant Shakers” in reference to animal Shaker channels, the first K⁺ channels identified. Recent advances in our knowledge of K⁺ channel evolution and structure have significantly deepened the divide between these plant and animal K⁺ channels, suggesting that it is time to completely retire the “plant Shaker” designation. Evolutionary genomics reveals that plant voltage-gated K⁺ channels and metazoan Shakers derive from distinct prokaryotic ancestors. The plant channels belong to a lineage that includes cyclic nucleotide-gated channels and metazoan ether-à-go-go and hyperpolarization-activated, cyclic nucleotide-gated channels. We refer to this lineage as the CNBD channel superfamily, because all these channels share a cytoplasmic gating domain homologous to cyclic nucleotide binding domains. The first structures of CNBD superfamily channels reveal marked differences in coupling between the voltage sensor and ion-conducting pore relative to metazoan Shaker channels. Viewing plant voltage-gated K⁺ channel function through the lens of CNBD superfamily structures should lead to insights into how these channels are regulated.

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K+ and Ca2+ currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K+ channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca2+-permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K+ channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network's domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.

Stomata are pores on plant aerial surfaces, each bordered by a pair of guard cells. They control gas exchange vital for plant survival. Understanding how guard cells respond to environmental signals such as atmospheric carbon dioxide (CO 2 ) levels is not only insightful to fundamental biology but also relevant to real-world issues of crop productivity under global climate change. In the past decade, multiple important signaling elements for stomatal closure induced by elevated CO 2 have been identified. Yet, there is no comprehensive understanding of high CO 2 -induced stomatal closure. In this work, we assemble a cellular signaling network underlying high CO 2 -induced stomatal closure by integrating evidence from a comprehensive literature analysis. We further construct a Boolean dynamic model of the network, which allows in silico simulation of the stomatal closure response to high CO 2 in wild-type Arabidopsis thaliana plants and in cases of pharmacological or genetic manipulation of network nodes. Our model has a 91% accuracy in capturing known experimental observations. We perform network-based logical analysis and reveal a feedback core of the network, which dictates cellular decisions in closure response to high CO 2 . Based on these analyses, we predict and experimentally confirm that applying nitric oxide (NO) induces stomatal closure in ambient CO 2 and causes hypersensitivity to elevated CO 2 . Moreover, we predict a negative regulatory relationship between NO and the protein phosphatase ABI2 and find experimentally that NO inhibits ABI2 phosphatase activity. The experimental validation of these model predictions demonstrates the effectiveness of network-based modeling and highlights the decision-making role of the feedback core of the network in signal transduction. We further explore the model’s potential in predicting targets of signaling elements not yet connected to the CO 2 network. Our combination of network science, in silico model simulation, and experimental assays demonstrates an effective interdisciplinary approach to understanding system-level biology.

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγsubunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gαsubunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγdimers (AGB1 plus a Gγsubunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγbinding in yeast. We observed interaction of the XLGs with all three Gβγdimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed ingpa1mutants but are recapitulated inxlgmutants. Thus, XLG-Gβγheterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.

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.

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.

Stomatal pores, vital for CO 2 uptake and water loss regulation in plants, are formed by two specialized guard cells. Despite their importance, there is limited understanding of how guard cells sense and respond to changes in vapor pressure difference (VPD). This study leverages a selection of CO 2 hyposensitive and abscisic acid (ABA) signaling mutants in Arabidopsis, including heterotrimeric G protein mutants and RLK (receptor-like kinase) mutants, along with a variety of canola cultivars to delve into the intracellular signaling mechanisms prompting stomatal closure in response to high VPD. Stomatal conductance response to step changes in VPD was measured using the LI-6800F gas exchange system. Our findings highlight that stomatal responses to VPD utilize intracellular signaling components. VPD hyposensitivity was particularly evident in mutants of the ht1 ( HIGH LEAF TEMPERATURE1 ) gene, which encodes a protein kinase expressed mainly in guard cells, and in gpa1-3 , a null mutant of the sole canonical heterotrimeric Gα subunit, previously implicated in stomatal signaling. Consequently, this research identifies a nexus in the intricate relationships between guard cell signal perception, stomatal conductance, environmental humidity, and CO 2 levels.

The plant hormone abscisic acid (ABA) promotes stomatal closure via multifarious cellular signaling cascades. Our previous comprehensive reconstruction of the stomatal closure network resulted in an 81-node network with 153 edges. Discrete dynamic modeling utilizing this network reproduced over 75% of experimental observations but a few experimentally supported results were not recapitulated. Here we identify predictions that improve the agreement between model and experiment. We performed dynamics-preserving network reduction, resulting in a condensed 49 node and 113 edge stomatal closure network that preserved all dynamics-determining network motifs and reproduced the predictions of the original model. We then utilized the reduced network to explore cases in which experimental activation of internal nodes in the absence of ABA elicited stomatal closure in wet bench experiments, but not in our in silico model. Our simulations revealed that addition of a single edge, which allows indirect inhibition of any one of three PP2C protein phosphatases (ABI2, PP2CA, HAB1) by cytosolic Ca2+ elevation, resolves the majority of the discrepancies. Consistent with this hypothesis, we experimentally show that Ca2+ application to cellular lysates at physiological concentrations inhibits PP2C activity. The model augmented with this new edge provides new insights into the role of cytosolic Ca2+ oscillations in stomatal closure, revealing a mutual reinforcement between repeated increases in cytosolic Ca2+ concentration and a self-sustaining feedback circuit inside the signaling network. These results illustrate how iteration between model and experiment can improve predictions of highly complex cellular dynamics.

Background In the presence of drought and other desiccating stresses, plants synthesize and redistribute the phytohormone abscisic acid (ABA). ABA promotes plant water conservation by acting on specialized cells in the leaf epidermis, guard cells, which border and regulate the apertures of stomatal pores through which transpirational water loss occurs. Following ABA exposure, solute uptake into guard cells is rapidly inhibited and solute loss is promoted, resulting in inhibition of stomatal opening and promotion of stomatal closure, with consequent plant water conservation. There is a wealth of information on the guard cell signaling mechanisms underlying these rapid ABA responses. To investigate ABA regulation of gene expression in guard cells in a systematic genome-wide manner, we analyzed data from global transcriptomes of guard cells generated with Affymetrix ATH1 microarrays, and compared these results to ABA regulation of gene expression in leaves and other tissues. Results The 1173 ABA-regulated genes of guard cells identified by our study share significant overlap with ABA-regulated genes of other tissues, and are associated with well-defined ABA-related promoter motifs such as ABREs and DREs. However, we also computationally identified a unique cis -acting motif, GTCGG, associated with ABA-induction of gene expression specifically in guard cells. In addition, approximately 300 genes showing ABA-regulation unique to this cell type were newly uncovered by our study. Within the ABA-regulated gene set of guard cells, we found that many of the genes known to encode ion transporters associated with stomatal opening are down-regulated by ABA, providing one mechanism for long-term maintenance of stomatal closure during drought. We also found examples of both negative and positive feedback in the transcriptional regulation by ABA of known ABA-signaling genes, particularly with regard to the PYR/PYL/RCAR class of soluble ABA receptors and their downstream targets, the type 2C protein phosphatases. Our data also provide evidence for cross-talk at the transcriptional level between ABA and another hormonal inhibitor of stomatal opening, methyl jasmonate. Conclusions Our results engender new insights into the basic cell biology of guard cells, reveal common and unique elements of ABA-regulation of gene expression in guard cells, and set the stage for targeted biotechnological manipulations to improve plant water use efficiency.