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. 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 forty identified network components, and accords well with previous experimental results at both the pathway and whole cell physiological level. 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 of 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. Our method can be readily applied to other biological signaling networks to identify key regulatory components in systems where quantitative information is limited.Comment: 17 pages, 8 figure

Calcium signals are necessary to establish auxin transporter polarity in a plant stem cell niche

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca^(2+)) and that transient Ca^(2+) response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca^(2+) occur during development of the SAM and this Ca^(2+) response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca^(2+) is not necessary for the response of MTs to mechanical perturbations revealing that Ca^(2+) specifically acts downstream of mechanics to regulate PIN1 polarity response

Development of novel proteomic strategies to dissect plant phosphoproteomic signaling under environmental stresses

Protein phosphorylation is one of the important signaling mechanisms in plants which transduces environmental stimuli such as salinity, microbes, and hormones into intracellular signals and activates plant defense mechanisms. Thus, understanding the correlation between environmental stresses and alteration of plant phosphorylation requires system-wide phosphoproteomic analysis, which includes identification of kinase-substrate complexes and measurement of phosphorylation-mediated signaling changes. However, identification and quantification of plant phosphoproteome remains challenging due to the highly dynamic nature of plant proteome, interferences of cell wall, pigments, and secondary metabolites. Recently, mass spectrometry (MS) has been integrated with phosphopeptide enrichment approaches for identifying thousands of phosphorylation sites and for quantifying phosphoprotein stoichiometry. Although MS-based phosphoproteomics has revealed the global phosphorylation changes related to different physiological states of plants, many kinase-substrate networks involved in essential signaling pathways, such as the ABA-induced SNF-1-related protein kinase 2 (SnRK2) pathway and the mitogen-activated protein kinases (MAPKs) cascades, are still not completely understood. This dissertation discusses strategies for improving plant sample preparation and for identifying the direct substrates of the plant kinases. Chapter one highlights the low phosphopeptide identification rate by mass spectrometry. Chapter two details the development of a sample preparation protocol for the plant phosphoproteome analysis, and the application of the protocol for the study of tomato cold-induced phosphoproteomic changes. Chapter three shows the development of a novel approach for identification of the direct substrates of the plant kinases, whose activation regulates the signaling transductions of plant stress defense mechanisms

Bound by Fate : The Role of Reactive Oxygen Species in Receptor-Like Kinase Signaling

In plants, receptor-like kinases (RLKs) and extracellular reactive oxygen species (ROS) contribute to the communication between the environment and the interior of the cell. Apoplastic ROS production is a frequent result of RLK signaling in a multitude of cellular processes; thus, by their nature, these two signaling components are inherently linked. However, it is as yet unclear how ROS signaling downstream of receptor activation is executed. In this review, we provide a broad view of the intricate connections between RLKs and ROS signaling and describe the regulatory events that control and coordinate extracellular ROS production. We propose that concurrent initiation of ROS-dependent and -independent signaling linked to RLKs might be a critical element in establishing cellular responses. Furthermore, we discuss the possible ROS sensing mechanisms in the context of the biochemical environment in the apoplast. We suggest that RLK-dependent modulation of apoplastic and intracellular conditions facilitates ROS perception and signaling. Based on data from plant and animal models, we argue that specific RLKs could be components of the ROS sensing machinery or ROS sensors. The importance of the crosstalk between RLK and ROS signaling is discussed in the context of stomatal immunity. Finally, we highlight challenges in the understanding of these signaling processes and provide perspectives for future research.Peer reviewe

Training Memory: Exploring the Intersection of Plant Stress Signalling and DNA Methylation

Plants are sessile organisms living in a dynamic environment to which they must continually acclimatize in order to maximise their reproductive potential. This plasticity is achieved through many complex and intricate signalling pathways that allow for the continuous perception, response, and adjustments to new environmental stimuli. A growing body of evidence suggests that such pathways are not merely static but dynamic and can be primed following repeated activation, thus affecting enhanced responses to recurring stresses. Such examples of priming have led to a notion that plants have some capacity to form stress memories of past environmental perturbations. However, the full extent and nature of such memory, and the machinery involved to store and transmit these, remain enigmatic. One prospective mechanism is the involvement of heritable, yet rapid and reversible, chromatin marks that, theoretically, could be shaped by the environment to convey a regulatory effect on the expression of the underlying genotype, thus acting as an epigenetic layer of regulation. This thesis explores the potential intersection of stress signalling pathways and chromatin variation, specifically DNA methylation, to co-ordinate plant stress responses. First, mechanistic insights into the operation of a SAL1-PAP-XRN retrograde signalling pathway to fine-tune plant physiology under drought are presented. A key finding was that this pathway complements canonical ABA signalling to induce stomatal closure, thus minimising water-loss under water limited conditions. Furthermore, the SAL1-PAP-XRN pathway was found to effect chromatin patterns, specifically DNA methylation at short transposable elements. These observations implicate cross-talk with the RNA directed DNA methylation pathway, however, the exact mechanism for this interaction remains to be identified. Multiple investigations were performed to test for stress-induced changes in DNA methylation that could potentially regulate responses to recurring stress, thus conveying a memory. A transgenerational recurring drought stress experiment tested whether descendants of drought-exposed lineages displayed greater drought tolerance (transgenerational memory). For the majority of traits tested, including plant growth rate and drought survival, offspring from plant lineages exposed to successive generations of repeated drought stress performed comparably to those from control lineages. However, memory was demonstrated in the form of enhanced seed dormancy, in drought stressed lineages, that persisted at least one generation removed from stress. Whether this capacity for memory could be related to the type or severity of stress applied, or species examined, remains to be investigated further. The transgenerational drought experiment was paired with a recurring excess-light stress experiment to investigate memory within a generation. Not only did this treatment lead to priming of plant photosynthetic behaviour, indicative of a greater capacity to withstand abrupt increases in light intensity, but new leaves from stressed plants, developed in the absence of stress, also showed altered photosynthetic characteristics compared to unstressed counterparts. Such observations are consistent with the mitotic transmission of stress-induced traits. Given multiple demonstrations of memory, comparisons were made to unstressed controls to test for any correlating changes in DNA methylation that might explain the phenomena observed. However, in both experiments, observations of memory were found to be independent of large-scale conserved changes in DNA methylation discounting it as a conveyor of plant stress memories, under these conditions, raising questions regarding the mechanism(s) responsible for the examples of memory observed herein. Ultimately, this thesis systematically evaluates the notion that plants are able to form genuine memories, potentially underpinned by reversible chromatin marks, that may facilitate acclimation to local environments on a relatively rapid scale compared to the fixation of adaptive genetic polymorphisms. Any capacity for plant stress memories may provide avenues for further epigenomic based agronomic tools to improve crop stress tolerance. However, the nature of such memories observed here appear subtle and nuanced, and are forgotten beyond a generation. Further characterisation and mechanistic understanding of mitotic memory mechanisms, however, may still hold potential. It was also observed that stress signalling pathways can interact with those involved in chromatin modification, giving novel insight into their mechanistic functioning and the how the onset of stress may induce chromatin changes. Despite this potential, the DNA methylome was found to be relatively impervious to stress-induced changes and, thus, is an unlikely memory mechanism

Boolean Networks as Predictive Models of Emergent Biological Behaviors

Interacting biological systems at all organizational levels display emergent behavior. Modeling these systems is made challenging by the number and variety of biological components and interactions (from molecules in gene regulatory networks to species in ecological networks) and the often-incomplete state of system knowledge (e.g., the unknown values of kinetic parameters for biochemical reactions). Boolean networks have emerged as a powerful tool for modeling these systems. We provide a methodological overview of Boolean network models of biological systems. After a brief introduction, we describe the process of building, analyzing, and validating a Boolean model. We then present the use of the model to make predictions about the system's response to perturbations and about how to control (or at least influence) its behavior. We emphasize the interplay between structural and dynamical properties of Boolean networks and illustrate them in three case studies from disparate levels of biological organization.Comment: Review, to appear in the Cambridge Elements serie

ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant

A distributed approach to underwater acoustic communications

Submitted in partial fulfillment of the requirements for the degree of Master of Science at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2003A novel distributed underwater acoustic networking (UAN) protocol suitable for ad-hoc deployments of both stationary and mobile nodes dispersed across a relatively wide coverage area is presented. Nodes are dynamically clustered in a distributed manner based on the estimated position of one-hop neighbor nodes within a shallow water environment. The spatial dynamic cellular clustering scheme allows scalable communication resource allocation and channel reuse similar in design to land-based cellular architectures, except devoid of the need for a centralized controlling infrastructure. Simulation results demonstrate that relatively high degrees of interference immunity, network connectivity, and network stability can be achieved despite the severe limitations of the underwater acoustic channel