Sarah M. Assmann - US grants

9316319 Assmann Application of Ca++ to plants prevents stomatal opening. One effect of Ca++ is to inactivate the inward K+ channels that mediate K+ uptake. Evidence has recently been obtained that this activation results from Ca++ activation of a phosphatase analogous to animal protein phosphatase 2B. The proposed whole-cell patch clamp experiments will test whether the protein phosphatase 2B acts " downstream" of abscisic acid and/or activated G-protein and/or inositoltriphosphate in a signal transduction pathway. Parallel biochemical studies utilizing guard cell protoplasts and guard cell protein extracts will determine whether abscisic acid, Ca++ and protein phosphatase 2B cause dephosphorylation of the same protein, as would be predicted if protein phosphatase 2B is a second mesenger for the abscisic acid response. Microinjection experiments on intact guard cells will address the possibility that protein phosphatase 2B is, alternatively or in addition, a second messenger for other environmental signals that prevent stomatal opening. %%% Stomatal guard cells are vital control points in the regulation of photosynthetic CO2 uptake and transpirational water loss. Stomatal appertures are regulated by osmotic swelling and shrinking of guard cells, driven by ion fluxes across the guard-cell plasma membrane. Several reports implicate Ca++ as one of the second messengers for abscisic acid, a plant hormone that prevents stomatal opening and also reduces inward K+ current. In this proposal complementary electrophysiological, cell biological and biochemical experiments are outlined to test the hypothesis that environmental signals which regulate stomatal opening act on inward K+ channels via a phosphatase 2B homologue. Results from these studies will provide insight into the second messenger systems utilized by plants to translate environmental signals into alterations of ion channel activity. Results will also be of interest to environmental physiologis ts who would like to understand how the external and internal environments affects stomatal apertures and thereby impacts plant productivity. ***

9416039 Assmann In the stomatal complex of maize (Zea mays), light stimulates uptake of K+ and Cl- into guard cells but simultaneously triggers loss of K+ and Cl- from subsidiary cells. How this exquisite level of cellular differentiation is achieved, such that the same physical stimulus from the environment elicits completely opposite ion transport processes in the two cell types, is the focus of this proposal. Experiments will utilize refinement of microscale (single cell) techniques of laser-assisted electrophysiology and molecular biology to analyze cellular function and gene expression. In guard cells, light activates a H+ ATPase which extrudes H+, thereby creating an electrical gradient for passive uptake of K+ through K+ selective ion channels. Guard cell and subsidiary cell protoplasts of maize will be patch-clamped, and their K+ and H+ ATPase currents will be analyzed and tested for light regulation. Techniques of differential display, and of cDNA synthesis within an individual living cell by reagents introduced via the patch- clamp pipette in whole cell configuration will be used to produce probes for genes that are differentially expressed in the two cell types. Probes will be used to screen libraries to obtain full length sequences. For all experiments guard cell and subsidiary cell protoplasts will be released using a recently developed microsurgery technique, in which a laser is used to burn a hole in the cell wall, and the protoplast is ten released through the hole. The laser microsurgery method obviates the use of cell-wall digesting enzymes to release protoplasts, which may induce gene expression and alter cell physiology and viability. Methods developed in this research should allow the patch-clamp and molecular study of single cells that have been inaccessible by previous approaches. %%% Plants take up CO2 from the atmosphere and use the sun's energy to fix CO2 into carbohydrates. CO2 enters plant leaves through tiny pores in the leaf surface called stomata. Water also exits the leaf through stomata. It is therefore important that stomatal apertures are tightly controlled, so that plants neither starve because of inadequate CO2 uptake, nor wilt because of excessive water loss. Stomatal apertures are regulated by pairs of guard cells which define and border each stomatal pore. Guard cell regulation of pore aperture requires uptake and loss of K+ from the guard cells. In corn, when K+ is not needed by the guard cells, it is stored in neighboring cells called subsidiary cells. The same environmental signals that cause guard cells to take up K+ trigger subsidiary cells to lose this ion, which the guard cells then have access to. How this exquisite level of cellular specialization is achieved is the purpose of t his study. An electrophysiological technique called patch-clamping will be used to study how light regulates the fluxes of K+ and other ions across the cell membranes of guard cells and subsidiary cells. Techniques of molecular biology will be applied o study how these two cell types differ in which genes they express. Understanding the mechanism of stomatal control is important, in order to learn which responses to target , in breeding or molecular genetic manipulations, to increase plant productivity or decrease plant sensitivity to draught. Such knowledge may also be important in deciding what the best conditions are for growing crop plants in space to support a manned space station. ***

Signal transduction is emerging as an important principle underlying much of plant development and adaptation to the environment. Signaling often involves multiple levels of response, from organ, tissue and cell interactions to events at the level of membranes, cytoplasm and gene expression. In our view, the most informative analyses of signal transduction involve multidisciplinary approaches where the signal is characterized through its multiple levels and forms of expression (genetic, biochemical, physical, physiological, etc.) We propose to establish a graduate training program with innovations in our instructional approach and research guidance with faculty from six (6) departments to pursue collaborative research across multiple levels of signal transduction. Toward this goal, we will develop a problem-based learning course to explore multiple facets of plant signal transduction. Graduate students will engage in multidisciplinary research by attacking research problems at the interface of two (or more) research efforts. An annual Distinguished Investigator seminar/workshop series and concluding international symposium will provide additional opportunity for student development. Our program will enrich the background and "tool-kit" available to the next generation, as well as the current generation, of researchers to investigate the mechanisms by which plants develop and react to their environment.

The plant epidermis contains microscopic pores called stomata through which gas exchange with the environment occurs. Through the stomata, carbon dioxide is taken up for photosynthesis and water vapor and oxygen are lost. Stomatal apertures are regulated by pairs of guard cells which border and define the stomatal pores. Guard cells regulate stomatal apertures by osmotic swelling and shrinking, driven by uptake of ions and production of organic solutes (stomatal opening) or loss of ions and catabolism of organic solutes (stomatal closure). The plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure when plants are droughted or otherwise stressed. A few years ago, the PI's laboratory used biochemical methods to identify in guard cells an ABA-activated, Ca2+-independent kinase (ABA-activated protein kinase; AAPK). This serine/threonine kinase is activated within one minute by physiological concentrations of ABA and is detected in guard cells but not in epidermal or mesophyll cells (Li and Assmann (1996) Plant Cell 8: 2359-2368). These characteristics suggested that AAPK could play an important role in triggering the rapid changes in guard cell solute content that drive stomatal closure upon ABA exposure.
This project represents a request for additional funding for research related to that of PI's current NSF grant MCB 98-74438, initiated in March of 1999. Under the first year of funding of MCB 98-74438, the PI's laboratory succeeded in cloning the cDNA encoding AAPK, starting from AAPK peptide sequence obtained by mass spectrometric analysis of the purified guard cell protein. The PI's group has shown that biolistic transformation of guard cells with a dominant negative version of AAPK ("AAPK(K43A)") blocks ABA-induced stomatal closure. The PI's laboratory also has shown that AAPK(K43A) inhibits ABA-activation of a class of guard cell anion channels through which anion loss normally occurs during ABA-induced stomatal closure. This research has been published (Li et al., (2000) Science 287: 300-303). The research for which funding is sought under this request is the identification of proteins that interact with AAPK. The following approaches are proposed and prioritized: interaction cloning with labeled AAPK; yeast two-hybrid analysis; immunoprecipitation; and use of mass spectrometric analysis to identify the covalent modification of AAPK that occurs when ABA activates the kinase. Elucidation of the AAPK signal transduction pathway by these methods will increase understanding of hormonally-activated cellular signaling in plants and may provide an entry-point for biotechnological manipulation of stomatal responses to enhance ABA-induced stomatal closure when water is limiting, or to reduce ABA-induced stomatal closure and thus stomatal limitation of photosynthesis when water is abundantly available (e.g. during irrigation).

This is a POWRE project to allow the PI to take a sabbatical leave to work in the
laboratory of Dr. Michael Sussman at the University of Wisconsin, Madison. This fits the criteria of a POWRE award since: 1) this research concerns an area of plant science completely different from the PIs research focus on stomatal guard cells; 2) this research will provide a critical exposure to the new fields of plant genomics and proteinomics, including "hands-on" experience with a number of techniques which will subsequently be applied to the PI's own research area; 3) also under the auspices of the POWRE award, the PI will work with Prof. Edgar Spalding (also at the University of Wisconsin, Madison) on the improvement of an undergraduate laboratory exercise in plant electrophysiology, with the goal of incorporating this laboratory into her own teaching.
Dr. Sussman's laboratory is in the process of identifying T-DNA null mutants in all of the 35 -40 genes in the Arabidopsis thaliana genome that encodes calmodulin-domain kinases (CDPKs). Preliminary data from Dr. Sussman's laboratory suggest that CDPKs 10 and 11 may be involved in plant responses to saline conditions, with cdpk10/11 mutant plants showing improved survival when plants are grown on elevated NaCl in the absence of K+. Salinity is an important agronomic problem, affecting >7% of arable land world-wide. The experiments will test two hypotheses regarding CDPKs 10 and 11 and plant response to salinity: Hypothesis 1 It is known that plant K+ deficiency is a major aspect of salt stress. It is therefore hypothesized that wild type CDPKs 10 and 11 (and/or other CDPKs) reduce salt tolerance by down-regulating a K+ uptake transporter; in the cdpk 10/11 mutants such down-regulation is missing, and so salt tolerance is improved. This hypothesis will be tested by: 1) measuring growth, K+ uptake, and Na+ uptake in wild type vs. cdpk 10/11 mutant plants grown under limiting vs. sufficient K+ conditions; 2) measuring the same factors in triple mutants that I will produce between cdpk 10/11 andthe K+ uptake channels akt1 and kat1; and 3) determining that complementation of the cdpk 10/11 mutant with the wild type CDPK 10/11 genes restores the wild type phenotype. Hypothesis 2 The presence of Ca2+ can reduce the deleterious effects of NaCl on plant growth and K+ status. It was recently shown that the SOS3 protein has homology to the regulatory subunit of the Ca2+-activated phosphatase, calcineurin, and is necessary for this Ca2+ effect (Liu and Zhu, 1998, Science 280: 1943). sos3 mutant plants are deficient in K+ uptake. It is therefore hypothesized that SOS3/calcineurin and CDPK 10/11 target the same K+ transporter (either directly or through a signaling cascade), with wild type SOS3/calcineurin upregulating that K+ transporter under saline conditions and wild type CDPK10/11 downregulating that K+ transporter. To test this hypothesis: 1) growth, K+ and Na+ uptake will be evaluated in cdpk 10/11 mutants vs wild type plants under saline conditions with different levels of Ca2+; 2) phosphorylation assays will be conducted to identify (as spots on 2-D gels) root proteins whose phosphorylation status is enhanced by wild type CDPKs 10/11 and reduced by wild type SOS3. Such proteins will be sequenced and identified using the technique of tandem mass spectrometry

Abstract: Mark Guiltinan (#0215923)

A grant has been awarded to Pennsylvania State University under the direction of Dr. Mark Guiltinan to develop a Plant Growth Facility consisting of six growth chambers and a small greenhouse. The facility center will provide a physical home for the graduate programs, and an organizing role for the enhancement of Plant Sciences research, teaching and outreach programs.
The objectives of the center will be to: provide state-of-the-art research facilities for a core group of resident plant scientists, including current faculty and new hires; provide collaborative space in the new building to members of the Plant Science Center; develop and enhance collaborative interactions among plant scientists and with researchers
in other fields, at Penn State and elsewhere; improve the Plant Physiology and Ecological and Molecular Plant Physiology graduate education programs, and; develop outreach programs for the general public and for pre-college students.

This center will support research in plant sciences for diverse studies, but will be particularly important to the growing number of plant scientists using the model plant species, Arabidopsis thaliana. The completion of the Arabidopsis genome sequence, along with major new funding programs in plant genomics at the NSF and USDA,
have brought us to a new era in plant research, requiring high throughput facilities for functional analysis of genes.

Penn State has made a priority effort to enhance the life sciences; enhancement of the Plant Sciences Center will help us move forward on this path, by providing necessary facilities and maximizing laboratory space in the main building. This will in turn help to attract top faculty candidates who will be concerned about availability of such facilities. The facility will provide for the first time, an interdisciplinary, inter-college facility for high quality environmental growth of plants to support the many plant scientists at Penn State. It will also serve our teaching and outreach programs, providing a facility available to all for the growth of plants.

Heterotrimeric G proteins couple multiple and diverse signals (e.g. light, pathogens, hormones) recognized by receptors to downstream effectors in a cell-specific manner. In Arabidopsis, hypothesized G-protein involvement in multiple signal transduction and developmental processes can be assessed using the tools developed by this project. The gene set comprises 16 heptahelical membrane proteins (candidate G protein-coupled receptors), one canonical Ga subunit, one Gb, and two Gg subunits (of the heterotrimeric complex), and three 'extralarge' Ga-related proteins (XLGs). The resources/tools that will be developed are: 1) GUS constructs allowing localization of expression for the gene set and; 2) constructs allowing visualization of physical interactions between members of the gene set in vivo in real time and with spatial dimensions using fluorescence energy transfer (FRET) between cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged proteins. The GUS constructs will be available at the conclusion of the first year of funding, and the YFP and CFP translational fusion constructs will be available at the conclusion of the second year of funding.

Vector maps, images of expression levels and patterns, protocol updates, and instructions for obtaining material will be posted weekly at the following URL, which will be TAIR-linked: www.plantbiology.unc.edu/ Constructs will be available for order via the webpage. For each translational fusion, three stable transgenic lines will be deposited in the stock center following validation.

The significance of the proposed work in relation to the overall 2010 project objectives is twofold. Function of the members of the gene family that is the target of this project will be determined as follows. Expression patterns of the genes will be evaluated in plants. The role the gene projects have in root cell proliferation and guard cell signaling will be evaluated, and novel small molecules that modulate plant G protein signaling pathways, as assessed by the FRET 'readout', will be identified. Tools developed by the project will allow the community to perform direct in vivo tests of hypothesized G protein involvement in any signal transduction or developmental context. As insights are gained into use of plant cell in vivo FRET analysis for monitoring the interaction between G protein complexes and G protein-coupled receptors, this tool will be made available to other plant research groups for evaluation of in vivo protein:protein interactions in other systems. In addition, the YFP constructs and lines will be compatible with BRET analysis being developed by the von Arnheim/Johnson 2010 project (Univ. Tennessee).

The broader impact of the proposed research includes pre- and postdoctoral training by direct mentorship of the two Principal investigators, and a hands-on tutorial on FRET measurements during a G protein workshop organized by the PIs. The workshop will be advertised in particular at small liberal arts colleges and minority institutions and undergraduate students will be eligible to apply for travel grants.

In plants as well as in animals, the genetic code is contained within the organism's DNA. Every cell in a multicellular organism has exactly the same DNA, and it is the regulation of which genes within that DNA are used, or "expressed", that makes one cell different from another. This regulation occurs at many levels. DNA is transcribed (transcription) into another type of nucleic acid, RNA, which is further processed (cut, spliced, modified) before its translation into protein. Thus, regulation of gene expression can occur at the level of transcription, at the level of mRNA processing and stability, and at the level of translation. The plant hormone, abscisic acid (ABA), plays an integral role in the ability of plants to acclimate to environmental stresses such as drought, cold, and salinity, which seriously compromise crop productivity both in the U.S. and world-wide. It has been known for some time that ABA plays an important role in the regulation of gene transcription. Results from a few laboratories are now beginning to show that ABA also plays an important role in RNA metabolism. In the present research, the roles of 3 RNA binding proteins, "AtAKIPs" in the regulation of ABA sensitivity, RNA metabolism, and drought tolerance will be investigated. Experiments will be conducted in the model organism, Arabidopsis thaliana. ABA sensitivity, drought tolerance, and RNA stability will be assessed in Arabidopsis transgenic plants that have altered amounts of each of these 3 proteins. AtAKIP RNA targets also will be identified, and computational and experimental approaches will be applied to identify consensus motifs within these RNAs that are necessary and sufficient for the RNA to bind to the AtAKIP. Once such motifs are identified, their presence will be assessed on a genome-wide basis both in Arabidopsis and in other organisms, to further scientific insight into the mechanisms of hormonally-mediated effects on RNA metabolism. The investigators are actively involved in outreach education to elementary schools. Dr. Assmann has developed teaching modules to introduce plant biology to kindergartners and has developed a video about plant gravity sensing. Dr. Bevilacqua is involved in career-site activities for "take your child to work" day and has created chemistry demonstrations for local elementary schools. The investigators will use information obtained from the present research in the development of new laboratories for an honors undergraduate biology course, and to expand the biological aspects of the curriculum of a graduate Nucleic Acids physical chemistry class.

The 16th Penn State Symposium in Plant Physiology, entitled "RNA Biology: Novel Insights from Plant Systems," will be held on May 18th-20th, 2006, at Pennsylvania State University. The field of RNA biology has experienced enormous growth in recent years, and plant biologists have been at the forefront of many of the important discoveries in this field. The Symposium program has been designed to bring together plant biologists in the RNA field, key scientists who study RNA function in non-plant systems, and RNA chemists. The Symposium will thus facilitate interactions and foster collaborations between plant biologists and other RNA biologists and chemists. In addition, the Symposium will provide educational and training opportunities for students and young scientists. Through press releases prepared by the Penn State Eberly College of Science Office of Information, information from the symposium will be disseminated to the general public.

Intellectual Merit. The research team will use modern biological tools to understand signal transduction processes of an important plant hormone, abscisic acid (ABA), in guard cells. Guard cells are highly specialized plant epidermal cells that enclose tiny pores called stomata. Stomatal movements (enabled by turgor changes in guard cells) control both CO2 uptake for photosynthesis and transpirational water loss, and thus play important roles in plant growth and acclimation to environmental stresses. ABA is a key indicator of drought stress. It induces stomatal closure via an intricate intracellular signaling network comprised of proteins and metabolites, thereby promoting plant water conservation. This research will analyze the roles of many proteins, especially those subject to reduction and oxidation (redox) modifications, in guard cell ABA signal transduction. Dynamic changes in key intracellular metabolites in guard cells upon ABA treatment will also be characterized. The project is expected to identify novel redox-regulated proteins and metabolites and put them into functional context of ABA signal transduction. The resources from this project will be distributed via a publicly accessible web interface and FTP sites for maximum scientific impact. The project is expected to provide comprehensive knowledge of regulatory mechanisms underlying stomatal movements that will help to develop crops with enhanced drought tolerance and improved productivity.

Broader Impacts. This project will benefit society at large because a better understanding of ABA signal transduction will inform rational crop engineering for better agricultural yield and stress tolerance. Since protein redox regulation is a ubiquitous biological process, occurring in essentially every organism including plants, animals, and micro-organisms, the data, techniques and resources developed in the project will be of immediate value to a broad range of scientists and will be disseminated via a web interface and FTP sites, as well as in publications and at scientific conferences. The project will involve cross-disciplinary training of personnel, including high school students, undergraduate and graduate students, in the frontiers of modern biological sciences. Given that knowledge of large-scale and high-throughput protein analysis (proteomics) is still not widespread, a proteomics workshop will be offered to graduate students and post-doctorates nationwide. Graduate students and post-doctorates from the Chen and Assmann laboratories will participate in developing and running the workshop, thus gaining valuable experience in teaching outside the standard classroom setting. Students from under-represented groups will be recruited for the training opportunities. Overall, the training and outreach program is designed to help prepare the next generation of scientists for competitive careers in modern biology.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Much of biology is regulated by the specific and high-affinity binding of ligands to proteins and nucleic acids. In most cases, however, the interacting molecules are either unknown or poorly characterized. This project proposes to establish a calorimetry facility at the Pennsylvania State University that will have high-throughput and high-sensitivity isothermal titration (ITC) and differential scanning (DSC) calorimeters. This unique facility will enable detection of ligand and drug binding to proteins and RNA, which will lead to the discovery and characterization of a wide range of ligand-biopolymer interactions, as well as determination of effects of ligand and biopolymer modifications on RNA and protein stability. New breakthroughs are anticipated in the areas of plant biology, metabolism, and enzymology, among others. Training opportunities will be available to undergraduate, graduate, and postdoctoral personnel at the interface of biology and chemistry. Research and educational opportunities will also be provided to Penn State campuses outside the main University Park campus, including the Altoona and York campuses, which are primarily undergraduate institutions. Outreach will be provided to underrepresented students at the undergraduate and high school levels, including hands-on calorimetry experiments and tours of the facility. Lastly, a short course will be developed on biothermodynamics that will present theory and provide hands-on training in ITC and DSC to all users. Scientific discoveries, publications, and educational material will be disseminated to the public through a Penn State website.

1126373
Ferry

Mass spectrometry (MS) is one of the leading analytical tools exploited by the metabolomics community. Despite technological advances, it remains impossible for a single piece of MS instrumentation to provide all of the information sought in global analyses. As such, all of the centers of excellence in metabolomics utilize pipelines that exploit multiple analytical platforms (e.g., LC-MS, GCEI-MS, GC-CI-MS). The proposed AB Sciex TripleTOFTM 5600 is a high resolution, exact mass MS/MS platform that is specifically designed to provide the resolving power sought by the metabolomics community. This instrument provides a mass resolution of up to 40,000 full width at half height (FWHH) in both positive and negative ion modes, exact mass (1 ppm RMS) with a dynamic range of up to five orders of magnitude, a rapid acquisition rate (20 Hz) that fully exploits the resolving power of UPLC, and absolute sensitivity more usually associated with triple quadrupole instruments operating in Selected Reaction Monitoring (SRM) mode. In short, this instrument offers speed, accuracy and versatility to explore complex samples from a large user group with diverse needs.

G proteins, which are composed of three smaller proteins named Galpha, Gbeta, and Ggamma, are important and ubiquitous cellular proteins in animals, fungi, and plants. Signaling pathways that are coupled to G proteins allow these organisms to sense and respond appropriately to their environments. In plants, G proteins help plant cells to sense both pathogenic bacteria and water stress. One of the best understood cellular systems of G protein regulation in plants is the stomatal guard cell. Guard cells are highly specialized cells found in the outermost tissue layer (the epidermis) of the leaf. Pairs of guard cells regulate the opening and closing of microscopic pores called stomata, through which plants take up carbon dioxide from the air for photosynthesis but through which they also lose water vapor to the atmosphere, leading potentially to dehydration stress. The guard cells control the aperture of the stomata by changing their shape and volume, processes that are in turn controlled by the uptake of potassium and other solutes. Proteins (KAT1) in the guard cell membrane mediate the uptake of potassium which drives this stomatal opening. KAT1 protein activity is inhibited by the plant hormone abscisic acid (ABA) and by molecules produced by bacteria that cause plant disease. G proteins serve as "middle-men" that relay the signals from ABA and bacteria to the KAT1 proteins, but G proteins are only one component of the complex intracellular signaling networks underlying these responses. This project will elucidate in detail the networks of intracellular signaling proteins that relay signals (for example from ABA) to G proteins and on to KAT1. An integral component of this research is the development of new and broadly applicable computational tools for understanding and predicting cellular signaling networks.
BROADER IMPACTS: This research has significance for plant biology (and more broadly for society) because it will help develop crops with improved resistance to infection and to environmental stress. The computational approaches developed by this research will be broadly applicable to any cellular signaling network, regardless of organism. This project will provide interdisciplinary training to undergraduate and graduate students and post-doctoral fellows at the interface of cell and computational biology. Project personnel will work towards improving the representation of minorities in science by participating in outreach and mentoring activities at the annual SACNAS (Society for the Advancement of Chicano/a and Native American Scientists) conference.

This project is supported jointly by the Networks and Regulation and Mathematical Biology Programs.

Intellectual Merit. The research team plans to use modern biological tools to investigate the functions of metabolites in the processes of red light and carbon dioxide (CO2) regulated stomatal movement at the plant leaf surface. Stomata are microscopic pores enclosed by pairs of highly specialized epidermal cells called guard cells. Stomatal movements (enabled by volume changes in guard cells) control both CO2 uptake for photosynthesis and water loss from the plant to the atmosphere, and thus are closely related to plant growth, acclimation to environmental stresses such as drought, agricultural yield, bioenergy and human life. Red light and CO2 provide energy and the substrate for photosynthesis and biomass production. Red light and CO2 also regulate stomatal movement via intricate intracellular and extracellular signaling and metabolic networks. This research will elucidate and analyze these networks by studying the roles of metabolites synthesized by the guard cells or secreted by the major photosynthetic cells of the leaf in response to these signals. Dynamic changes of intracellular and extracellular metabolites upon red light and CO2 treatment will be characterized and correlated with the physiological output of stomatal movement. The project is expected to identify novel metabolites, place them in a functional context, and construct predictive molecular models of red light and CO2 signal transduction. The resources from this project will be distributed via a publicly accessible web interface for maximum scientific impact. The project is expected to contribute positively toward future biotechnology of enhanced plant yield and bioenergy.

Broader Impacts. This project is expected to generate results with broad societal impact, e.g., leading to rational plant metabolic engineering for enhanced stress tolerance, more food and bioenergy. The results will improve understanding of plant metabolism and signaling in guard cells and photosynthetic mesophyll cells, unravel the metabolic networks regulating stomatal function, and advance basic biological science. The techniques, data and resources developed during the project will be of immediate value to studies of other plant cells, pathways and species and will be disseminated via a web interface as well as in publications and at scientific conferences. The project will provide cross-disciplinary training of personnel, including high school students, undergraduate students, graduate students, and postdoctoral scientists, in the frontiers of modern biological sciences. Because large-scale and high-throughput metabolite analysis (metabolomics) is still in its infancy, a hands-on plant metabolomics workshop/symposium will be offered to graduate students and postdoctorates nationwide. Graduate students and postdoctoral scientists from the Chen and Assmann laboratories will be involved in developing and running the workshop, thus gaining valuable experience in teaching outside the standard classroom setting. Women and under-represented students will be recruited for the training opportunities. Overall, the training and outreach program is designed to integrate research and education to help prepare the next generation of plant scientists in the frontier areas of metabolomics, computational biology and the emerging disciplines of systems biology.

Stomata are pores on the leaf surface through which plants release oxygen and water vapor and take up carbon dioxide from the atmosphere. The size of the stomatal aperture (and hence the exchange of carbon dioxide and water vapor with the atmosphere) is controlled by specialized cells. These cells (called guard cells) change their shape in response to environmental conditions and are responsive to a range of signaling systems within the plant. The goal of this research is to understand how reactive oxygen species and changes in the reducing and oxidizing biochemistry in guard cells regulate an enzyme system (protein kinases) that controls stomatal movements in response to environmental cues. The function of stomata impacts the response of plants to environmental stress (including temperature and drought) and this research should lead to the rational breeding of improved (stress resistant) crop plants. The project will enable cross-disciplinary training of college and high school students (including women and members of underrepresented groups), in emerging fields of high-throughput science at the interface of biology and chemistry.

The project will use multiple approaches including molecular biology, biochemistry, genetics and analytical chemistry to tackle a critical problem in plant biology, i.e., the functions of reactive oxygen species and redox changes in regulating kinase activities and stomatal movements. The central hypothesis is that redox-dependent modification of cysteine residues in key protein kinases provides a versatile means of regulating kinase function in stomatal signaling. This hypothesis will be tested by pursuing two specific objectives: 1) To determine the cysteine modifications of Brassica napus sucrose non-fermenting related kinases. The experiments will enable comprehensive analysis of cysteine modifications in response to abscisic acid (a drought stress hormone) and a bacterial flagellin peptide that is involved in stomatal entrance of plant pathogens. 2) To determine cysteine modifications functional in stomatal movement and how they affect kinase phosphorylation. The experiments will identify the cysteine modifications essential for stomatal movement and analyze cysteine redox changes that regulate kinase activity. This project will reveal novel regulatory mechanisms underlying stomatal movements and will contribute to the emerging concept of redox modulation as a versatile mechanism by which cells regulate important signaling components and processes.

PI: Philip C. Bevilacqua (Penn State University)

CoPIs: David H. Mathews (University of Rochester) and Sarah M. Assmann (Penn State University)

Rice is a staple food for more than 1/3 of the world's population and the second most important crop in terms of global production. Given global population growth, limited resources of fresh water, and global warming, it is very important to develop stress-tolerant varieties of rice. This research has the potential to advance both basic and applied knowledge regarding how RNA structure regulates expression of diverse genes in response to abiotic stresses, information that may useful for crop improvement. In addition to the training of students and postdoctoral associates, this project will provide summer training for underrepresented high school students in math and sciences in collaboration with the SEECoS program (http://science.psu.edu/outreach/special-programs/seecos-summer-experience-in-the-eberly-college-of-science) at Penn State. This project will also contribute to a new training program for ethics at Penn State and to an ethics module on Genetic Manipulation. All project outcomes will be made available to the public. Sequence data will be available at http://rna.urmc.rochester.edu/ and through long term repositories such as the NCBI-SRA, Galaxy, and the Single Nucleotide Resolution Nucleic Acid Structure Mapping (SNRNASM; http://snrnasm.bio.unc.edu/)website. Biological materials generated in this project will be available on request or through the Dale Bumpers National Rice Resource Center.

When plants are subjected to drought, molecular crowding within cells is enhanced and osmolyte and K+ concentrations increase. Likewise, when plants are stressed from temperature, RNAs in cells are thermodynamically affected. Taken together, these observations suggest that drought and temperature regulate RNA folding, and that RNA structure may play a central role in regulating gene expression and stress tolerance in rice. To evaluate the contribution of RNA structural changes to the regulation of gene expression in rice, this project will leverage, advance and apply recently developed in vivo and computational tools to reveal, on a genome-wide basis, how RNA folds in vivo in rice during drought and temperature stress.

The 20th Penn State plant Biology Symposium will focus on the important topic of "Plant Stress-Omics in a Changing Climate". Plenary sessions are focused on water and salinity, atmosphere (temperature, CO2, ozone), biotic/abiotic interactions and the use of novel tools. The conference is held at Penn State University, University Park takes place May 13-16, 2015. There will be two formal poster and two short talk sessions to feature research from talented junior scientists. The conference includes an impressive cadre of US and international speakers. The organizing committee and speaker list seems balanced in terms of broadening participation.

The workshop addresses an important topic in Plant Biology, namely how plants deal with increasing environmental stresses. Particularly noteworthy is a planned workshop preceding the meeting to provide training in novel mass spectrometric methods. These methods are necessary to determine metabolomics changes in response to various environmental stresses. The meeting is an ideal forum for students and postdocs to identify future mentors, and for established scientists to forge meaningful collaborations.

One of the major goals of plant biology is to find out how plant growth can be optimized to maximize crop production. The goal of this project is to develop a mechanistic understanding of how plants balance growth with water loss. The experiments described in this proposal will provide new insights into how crop plant production could be optimized under various growing conditions. The project relies on application to plant cells of techniques used in neuroscience in order determine how these cells respond to stimuli on the molecular level. Results of experiments performed on plants during the course of this project may in turn provide valuable information for future studies on how similar signaling pathways impact nervous system function in many different species of animals. The impact of these plant cellular responses on whole plant growth and water conservation will also be assessed. This innovative approach provides an unusual opportunity for students to gain cross-disciplinary training in both plant biology and neuroscience. The project will serve as a training ground for both graduate and undergraduate researchers, in some cases providing the latter with their very first research experience. Furthermore, it will support educational outreach efforts by the investigators targeted towards introducing science careers to underrepresented middle school and high school students.

The experiments outlined in this proposal are specifically targeted at defining the molecular mechanism(s) through which cellular oxidation state and gaseous messengers regulate plant guard cell K+ channels and stomatal apertures. The opening and closing of stomata by guard cells is mediated in large part by these channels and is sensitive to oxidation state and gases. However, the molecular pathways that connect these cellular signals to changes in K+ channel activity are not yet known. In this project, the investigators will test a novel hypothesis that oxidation state and gaseous messengers regulate K+ channel activity directly through a prosthetic group integral to the channels themselves. The hypothesis is based on the finding that gating of many CNBD family cation channels, including these plant K+ channels and several important classes of animal CNBD family channels, is regulated by similar prosthetic groups.

This research will study a specialized plant cell type's response to adverse environmental conditions to provide new insights into how signaling components within living cells interact with each other to affect cell function. This specialized cell type, the guard cell, occurs in the epidermis, where pairs of guard cells enclose microscopic pores called stomata, through which plants take up CO2 (the substrate for photosynthesis) and, inevitably, lose water. Resultant improved understanding of the regulatory mechanisms by which guard cells respond to drought and atmospheric carbon dioxide levels can assist in development of crop varieties with greater yield under a range of growing conditions. General insights into biological networks gained in this project will improve understanding of dysregulated cell signaling, with broad implications for physiology, agriculture, and biotechnology. Personnel, including undergraduate researchers, will be cross-trained in emerging approaches in both experimental and computational biology. The principal investigators will develop a new course to introduce first year undergraduates to biological networks and other mathematical biology concepts and encourage STEM participation.

Heterotrimeric G-proteins, composed of G-alpha subunits and G-beta-gamma dimers, comprise a ubiquitous signaling mechanism found in organisms as diverse as fungi, animals, and plants. This research will study G protein involvement in guard cell drought and CO2 signaling networks to address the fundamental question of whether and how G-alpha subunits compete for or partition their interaction with specific G-beta-gamma dimers. In plants, guard cells are the best understood single cell system of G protein regulation. In response to the hormone abscisic acid (ABA; an indicator of drought and other stresses), and to elevated concentrations of CO2, complex signaling networks are activated in guard cells that drive stomatal closure. This project will use genetic analyses to determine competition vs. partitioning of the four G-alpa subunits for the three Arabidopsis G-beta-gamma dimers during ABA and CO2 responses. The research will apply tests of protein-protein interaction to assess new candidate G protein interactors and to determine how G protein signaling interconnects with other known ABA and CO2 signaling components of guard cells. The project will develop new general reachability analysis and combinatorial logic methods to describe convergent or overlapping networks, and these methods will then be applied to the new experimental datasets obtained to create a new network model of the core components of G protein, ABA, and CO2 signaling. The new combinatorial logic methods for network construction developed will advance fundamental knowledge in systems biology: they will be applicable to directional signaling and genetic networks of any organism, and will enable a paradigm shift from assuming isolated linear pathways (e.g. as in classic epistasis analysis) to considering all network architectures consistent with a set of observations. Thus, the results from this research will have general implications for the common but complex phenomenon of cross-talk in biological systems.

Summary Heterotrimeric G protein signaling pathways are of tremendous importance to human health. Mutation of G protein subunits causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. Indeed, G protein pathways are the targets of approximately a third of all drugs under clinical use. The Assmann laboratory has furthered fundamental understanding of heterotrimeric G protein signaling through elucidation of G protein-mediated signaling cascades and phenotypes. The emphasis of the parent award is on mechanisms of phospho-regulation of G protein signaling that are evolutionarily conserved but have been oft overlooked in mammalian systems. In this research, the model plant Arabidopsis is used as a facile system to investigate kinase-mediated phosphorylation of the canonical G protein ? (G?) subunit, GPA1, and to explicate the downstream signaling impacts of this phosphorylation; in particular, how phosphorylation biases interactions with downstream effector proteins. In parallel, relevance to human health is demonstrated through assessment of the impacts of analogous phosphorylation events on human G? subunits in vitro, using BODIPY-GTP binding and hydrolysis assays on the corresponding human phosphomimic mutants. This supplement requests funding for the purchase of a BioTek Synergy Neo2 plate reader to increase the throughput and reliability of the BODIPY- GTP activity assays and to allow the implementation of orthogonal methods, particularly transcreener assays, that will allow independent validation of these biochemical data. The dual monochromators of the Synergy Neo2 plate reader will allow for assay versatility with a multitude of fluorophores. The speed and sensitivity of the Synergy Neo2 far outpaces that of the extant obsolete Flx800 plate reader, allowing for finer timescale measurements of a greater number of G? variants. In addition, the capability of the Synergy Neo2 to assay luciferase activity will facilitate assessment of biased signaling arising from phosphorylation-dependent protein- protein interactions, as the Synergy Neo2 has the capability for high-throughput measurements of protein-protein interaction using the split-luciferase method. In summary, the proposed state-of-the-art plate reader will provide reliable, sensitive, and rapid data acquisition as well as entirely new capabilities to probe the molecular effects of G protein phosphorylation. Finally, the instrumentation will be available to other NIH-funded researchers at Penn State in the Biology and Chemistry Departments.

Ribonucleic acids (RNA) are essential molecules in living organisms, including in plants. RNA can serve roles as both an informational molecule (genetic code) and a functional molecule (perform and regulate chemical reactions). RNA can fold into complex shapes that can control whether it stays intact or is degraded. This in turn can control how a plant responds to environmental stresses it faces such as heat and cold. The research involves the development of new experimental technologies to investigate RNA structures one molecule at a time and new computational technologies of artificial intelligence wherein a computer learns patterns that can predict RNA structure and its variation. Rice is an important world-wide crop, and the research applies these technologies to rice varieties that are grown in different parts of the world. There are thousands of different varieties of rice adapted to local environments and their RNAs often differ from each other by relatively few changes. Some of these changes will alter the shape of the RNA and thus the response of that rice variety to stress. A major goal of these study is to identify those changes that alter RNA shape and thereby affect temperature tolerance. Once identified, these shape-shifters could be engineered into specific rice varieties to breed crops more resistant to stress. Aspects of the research will involve high school students and their teachers, and research results and methods will be disseminated in public outreach activities.

RNA structure is a primary determinant of gene expression. Individual copies of the same transcript can take on different structures as influenced by their microenvironment, but methods have been lacking to categorize this diversity. Single nucleotide polymorphisms (SNPs) also can affect RNA structure as “riboSNitches”; however, riboSNitches have not been studied in plants, and their conditionality on environmental conditions has not been assessed. Using rice (Oryza sativa) as the primary model system, the proposed research will develop new wet bench and computational approaches that will allow categorization of the mRNA “pan-structurome,” its consequent impacts on gene expression, and its functional association with respect to local climate conditions in rice landraces. Training will be provided to postdoctoral fellows, graduate students, undergraduates, and high school students and teachers. Broader Impacts will include development of the Oryza CLIMtools webtool to relate rice genotypes with climate variables and to identify beneficial structural haplotypes for use in development of elite rice cultivars. Impact will be broadened through technology including enhanced browser-based RNA structure-reactivity visualization and publicly available instructional screencasts. Collaborations with PUI Swarthmore College will engage undergraduate researchers in computational aspects of the project. Local high school students will perform whole plant physiological experiments, engaging a future generation of biologists and chemists. Finally, the 23rd Penn State Plant Biology Symposium, on RNA biology, will be organized, which will promote the global field of post-transcriptional gene regulation.

This award was co-funded by the Plant Genome Research Program in the Division of Integrative Organismal Systems and the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.