Guard cells are a well-suited model for understanding how signals are transduced by molecular components within a signal transduction network, and for dissecting the functions of molecular components in the network at the single cell level. In addition, many powerful techniques have been developed that allow combined molecular genetic, cell biological, electrophysiological and functional genomic analyses, enabling detailed genetic dissection of guard cell signal transduction. For example, the study of ABA signal transduction in guard cells has been a cornerstone in discovering molecular components that play an important role in stomatal movements (Figure 2).

Figure 1. Guard cells are located in the leaf epidermis and form stomatal pores. Arabidopsis guard cells stained with the fluorescent dye 2’,7’-dichlorofluorescin diacetate (Data from Kwak et al., 2003).   Movie 1. Arabidopsis guard cells expressing yellow cameleon show changes in cytosolic calcium concentration in reponse to external calcium (time compression, 120X; Cho et. al., 2009 Plant J.)

Genetic Dissection of Cytosolic Calcium-Controlled Cellular Respons
Cytosolic Ca2+ ([Ca2+]cyt) mediates diverse cellular responses in both animal and plant cells and has been shown to contribute to regulation of stomatal movements. In plant cells, an increase in [Ca2+]cyt can transduce cellular responses to various biotic and abiotic stimuli including light, gravity, oxidative stress, cold shock, drought, hormones, salt stress, and fungal elicitors. Cytosolic calcium ([Ca2+]cyt) concentration dramatically changes when an extracellular signal is received at the cell surface. Such cytosolic Ca2+ increases are achieved by Ca2+ influx from the extracellular space and Ca2+ release from internal stores, which generate subsequent cellular signal responses. The orchestrated regulation of Ca2+ increase and decrease mechanisms often occurs as repetitive Ca2+ transients or oscillations and play a central role in Ca2+ homeostasis which is necessary for diverse and normal cellular activities. In guard cells, ABA induces increases in the [Ca2+]cyt concentration which result in stomatal closure, and Ca2+ oscillations encode necessary information for stomatal movements (Movie 1). However, molecular mechanisms by which Ca2+ mediates specific cellular responses remain largely unknown in both animal and plant kingdoms. We try to understand how this universal signal controls the specific cellular responses to various stimuli, by combining the use of newly developed techniques such as FRET-based Ca2+ imaging in plant cells that are easily genetically modified in order to facilitate dissection of Ca2+ signaling.

Dissection of ROS-mediated Guard Cell ABA Signaling
Reactive oxygen species (ROS) function as secondary messengers that mediate cellular responses to various stimuli in both animal and plant cells. In guard cells, ROS have been suggested to function in ABA signaling (Figure 2 ). However molecular components working downstream or upstream of ABA-induced ROS signaling remain to be identified. Previously we have identified two NADPH oxidases that are responsible for ABA-induced ROS production required for ABA signaling in guard cells. However, molecular players functioning downstream of ABA-triggered ROS production remain to be identified. Our preliminary data show that two specific MAP kinases are positive regulators of ABA signaling working downstream of ROS in guard cells. We are currently characterizing these two MAPK kinases to provide further insights into ABA signaling networks in guard cells.

Figure 2 ABA signaling network in guard cells (Kwak et al., 2008, The Clickable Guard cell, Version II, The Arabidopsis Book)

Brassica Guard Cell Project
Drought causes severe damage to crops, resulting in major losses in yield. In addition, fresh water scarcity is one of the major global problems of the 21st century, affecting more than 1.1 billion people worldwide. Climate experts predict that as global temperatures rise, there will be more areas affected by drought globally and that there will be increased variability in the amounts and distribution of precipitation. This will result in profound impacts on global fresh water resources, over 65% of which are used for agriculture. There will be increased competition for water from municipal, industrial, and agricultural users.

Studies in Arabidopsis have shown that the control of stomatal aperture can be manipulated by modifying guard cell signal transduction components to improve drought tolerance and more sustainable water use of plants. However, ubiquitous expression of such genes may also confer undesired traits to the plants in addition to the desired trait of drought tolerance, because this manipulation would affect the whole plant. Cell-type specific genetic manipulation of such genes would be more effective.

The long-term goal of this research is to provide detailed functional knowledge of mechanisms regulating drought responses and water use efficiency, by using a systems approach to investigate cellular dynamic changes in response to abiotic stress signals. This knowledge will be used to manipulate guard cells towards developing practical strategies for improving water stress tolerance of B. napus and other crop plants.

To accomplish the goal, our lab, in collaboration with Sarah M. Assmann (Penn State University), Joel S. Bader (Johns Hopkins University), John K. McKay (Colorado State University), Scott C. Peck (University of Missouri, Columbia), and Julian Schroeder (University of California, San Diego), will analyze guard cell activities in response to drought, including dynamic changes in RNA molecules, proteins, and metabolites in the canola plant and develop a genome scale view to understand how cellular networks and hormones regulate the plant’s reaction under low water conditions. These data sets will be used, together with advanced genome sequencing approaches, to map genetic lines in Brassica napus and to identify natural variation in sensitivity to drought and in the speed at which water evaporates from stomates. Models generated from integrating this genomic, bioinformatic, and proteomic information will provide important information and a blueprint for improving water use efficiency and resistance to drought in crops.

These research activities will generate a new “systems biology” view of a single plant cell type that can be used to manipulate guard cells and to develop practical universal strategies for improving water stress tolerance in a variety of crop species. Each site will broaden the impact of this research by conducting active outreach activities, such as engaging high school students and undergraduates from under-represented groups in the project. All data sets, protocols, and biological resources will be released to the public through a project website (to be determined) and through the relevant long-term data repositories that include the Arabidopsis Biological Resource Center, the Multinational Brassica Genome Project, Gene Expression Omnibus, IntAct, and BioGRID.

Arabidopsis 2010 (Associomics): Towards a Comprehensive Arabidopsis Protein Interactome Map: Systems Biology of the Membrane Proteins and Signalosomes
Membrane proteins are essential for biological processes and involved in a variety of cellular functions. For example, cells utilize a network of signal transduction events that elicits cellular responses when an extracellular signal is received by membrane proteins at the cell surface. Despite recent progress and effort in proteomics, the interactions of membrane proteins have yet to be identified at the systems level. The goal of this Arabidopsis 2010 project is to radically expand our knowledge of the Arabidopsis plasma membrane protein interactome and its interface with key signaling proteins. In collaboration with Drs. Wolf Frommer (Carnegie Institution of Washington), Reka Albert (Penn State University), Sally Assmann (Penn State University), Seung Y. Rhee (Carnegie Institution of Washington), and Julian Schroeder (UC, San Diego), we pursue the analysis of membrane proteins and soluble signaling proteins on a genome-wide basis by adapting a high-throughput, quantitative, and cost-effective approach in parallel with bioinformatics methods to integrate complex protein interactions and diverse information. Go to http://www.associomics.org/ for more information.

Study of ABA Signaling and its Application to Develop Drought-Tolerant Crops
Water stress is one of the devastating environmental problems causing severe loss in agriculture. Federal Emergency Management Agency estimates that drought damage costs the US $6 to 8 billion annually. Plants are irrigated to avoid water stress during drought. In fact, 65% of global fresh water is used for plants, and fresh water is a scarce resource for agriculture. The use of drought-tolerant crops would lead to a reduction in crop losses and freshwater consumption. Abscisic acid (ABA) plays a central role in the protection of plants from various environmental stresses. Cellular ABA levels are precisely controlled to elicit adaptive responses in reaction to a changing environment. More effective genetic manipulation of drought hardiness can be achieved by providing plants with a mechanism by which the plants obtain rapid and transient increases of ABA in response to drought. Molecular manipulation to increase cellular ABA levels rapidly and transiently in response to drought can lead to the development of engineered crop plants with enhanced water stress tolerance and more sustainable water use. The goal of this research is to provide insights into ABA-regulated cellular processes by identifying novel molecular components in ABA homeostasis and signaling, which can lead to development of crops with improved water stress tolerance.

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