Julian I. Schroeder - US grants

Research indicates that calcium ions and signal-modulated ion channels may play a central role in plant cellular signal transduction. Guard cells provide an ideal system for the study of plant signaling, as these cells respond dynamically to hormone and light stimuli in regulating gas exchange. Recent data suggest that calcium regulation of ion channels in the plasma membrane of guard clls provides a basis for physiologically- observed calcium dependent stomatal movements. Preliminary results show that abscisic acid induces increases in cytosolic calcium, which in turn may trigger stomatal closing, thus protecting plants from desiccation. The goals of the proposed research are to achieve an accurate understanding of the molecular mechanisms by which cytosolic calcium and other ions regulate inward rectifying potassium channels and by which mechanisms abscisic acid exerts control over these processes. Isolated guard cells will be studied by simultaneous patch clamp analysis and photometric measurements of cytosolic calcium, allowing the direct investigation of effects of cytosolic and extracellular modulators on signaling events. The knowledge gained from these studies will contribute substantially to our understanding of the molecular mechanisms controlling gas and water exchange in the leaves of plants and will provide a quantitative description of biophysical and biochemical regulation of ion channels in higher plant cells.

This is a Presidential Young Investigator Award to Dr. Julian Schroeder of the Biology Department at the University of California, San Diego. Dr. Schroeder's overall research goal is the elucidation of molecular mechanisms of ion transport and primary events in environmental and hormonal signal transduction in higher plant cells. His pioneering work in the application of techniques of electrophysiology to plant cell biology has had a major international impact. Dr. Schroeder also shows great promise as a teacher and mentor in training the next generation of scientists.

DESCRIPTION (provided by applicant): Abscisic acid (ABA) is a central stress hormone in Arabidopsis that mediates rapid cellular responses, down- regulates cell proliferation and causes cell cycle arrest. Early signal transduction networks that down-regulate cell proliferation are of key importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. The long-term goal of this research is to achieve a new and quantitative understanding of the network of events that mediate early abscisic acid signaling in the potent Arabidopsis guard cell system. We will characterize newly identified key cellular signaling mechanisms hypothesized to control the recently revealed early ABA receptor signaling core consisting of ABA receptors, PP2C protein phosphatases and SnRK2 & calcium (Ca2+)-dependent protein kinases, which mediate downstream ABA signaling. Specific Aims: I. Regulatory proteins of eukaryotic PP2Cs and ABA receptor interactors are not well-understood. We will characterize the functions of a newly identified proposed early receptor-PP2C control loop that can counteract monomeric ABA receptor-induced ligand-free leaky PP2C signaling. This includes the PP2C interacting and regulating ROP10 & 11, ABA receptor-interacting GDP/GTP exchange factor 1 and the OST1 protein kinase. II. How the universal second messenger Ca2+ mediates specific responses in eukaryotic cells is a key question in cell signaling research and disease. Our recent results point to a Ca2+ sensitivity priming mechanism, in which ABA primes Ca2+ sensors switching them from an inactivated Ca2+-insensitive state to a Ca2+-responsive primed state. Priming enables a specific Ca2+ response and this novel mechanism may be used by diverse eukaryotic Ca2+-signaling pathways. We will investigate the hypothesis that the PP2Cs directly inactivate the Ca2+-dependent kinase CPK6. In addition, cross-regulation of CPK6 and the OST1 protein kinase in ABA activation of SLAC1 anion channels will be investigated. We will identify the ABA-induced Ca2+ specificity signaling mechanisms using biochemical and dynamic in vivo cell signaling analyses and via parallel functional analyses of the reconstituted multi-component ABA signalosome in Xenopus oocytes. III. In a new chemical genetics screen of 9600 compounds, we have identified a small molecule DFPM that down-regulates ABA signaling. DFPM activates intracellular effector-triggered signaling and thereby rapidly (<3 min) deactivates ABA responses at the level of Ca2+ signaling in guard cells. New chemical genetic mutants showing strong insensitivity to DFPM-regulated ABA/Ca2+ signaling have been isolated. Selected mutants and the underlying mechanisms will be characterized to elucidate new elements and mechanisms mediating DFPM-induced rapid interference with ABA/Ca2+ signaling. This research will result in a new understanding of PP2C regulation and Ca2+ specificity signaling mechanisms, which are fundamental to numerous cell signaling processes and disease states, and will reveal novel mechanisms by which newly identified ABA receptors and regulation mechanisms control ABA signaling.

Soils and waters with high levels of toxic metals such as cadmium (Cd), arsenic (As), lead (Pb) and mercury (Hg) are detrimental to human and environmental health. These four metals are among the Superfund;'s top five priority hazardous substances. Studies suggest that uptake of heavy metals into plant via the root system could provide a potent and cost effective approach for toxic metal removal and remediation of soils and waters. In plants and fungi, phytochelatins are major heavy metal chelating and detoxifying thiolate peptides, that form complexes with and detoxify heavy metals, including Cd, Zn, Pb, Hg and based on recent research also As. The enzyme phytochelatin synthase (PCS) produces phytochelatin, thus functioning as a major catalytic metal detoxification mechanisms in plants. However genes encoding phytochelatin synthases, had not yet been identified. We have recently cloned a new gene family (PCS) encoding phytochelatin synthases in plants and fungi. Expression of PCS cDNAs in S. cerevisiae dramatically enhance resistance to cadmium. Disruption of the PCS genes in S. pombe and Arabidopsis thaliana produces increased heavy metal sensitivity. Recombinant PCS proteins synthesize phytochelatins in vitro. We will test the hypotheses that stress-signaling pathways contribute to PCS induction and detoxification and that transgenic expression of PCS genes can, together with other metal-interacting mechanisms, enhance heavy metal hyper-accumulation and removal by plants. To test these hypotheses we will: (I) Characterize signaling mechanisms that induce PCS expression. (II) Characterize PCS expression and localization in Brassica juncea, which is one of the major plant species being studied for heavy metal biomediation. (III) Pursue transgenic over-expression in plants of PCS together with associated metal detoxification mechanisms to test for enhanced heavy metal tolerance and accumulation and (IV) provide selected transgenic lines to Phytotech Inc to include in field trials on super fund sites. (V) Pursue novel genetic activation-tagging screens in Arabidopsis and Cd-induced microarray analyses to identify new genes and pathways involved in heavy metal accumulation in plants. Results from these studies could play a central role in the development of future phytoremediation strategies for heavy metal uptake and biological removal of heavy metals form contaminated soils and waters.

Stomatal pores are formed by guard cell pairs in the epidermis of leaves. Stomata regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control transpirational water loss of plants. A network of signal transduction mechanisms in guard cells sense and transduce CO2, water status, light and other environmental stimuli to regulate stomatal apertures for optimization of CO2 influx, water loss and plant growth under diverse conditions. Elevated CO2 concentrations in leaves cause stomatal closure, whereas reduced CO2 concentrations result in stomatal opening. Photosynthesis and respiration cause CO2 concentration changes in leaves. Moreover, atmospheric [CO2] is predicted to double within the present century and these ambient CO2 increases reduce stomatal apertures of different plant species by up to 40 %. However, relatively little is known about the molecular signal transduction mechanisms in guard cells that mediate CO2-induced stomatal movements. A study suggests that cytosolic calcium ([Ca2+]cyt) elevations contribute to CO2-induced stomatal movements. New findings in the P.I.'s laboratory show that changes in [CO2] modulate cytosolic [Ca2+]cyt elevations in Arabidopsis guard cells. The guard cell CO2 response provides an opportune system to analyze hypotheses that cytosolic Ca2+ patterns contribute to signal transduction in plants. The long term goal of this research is to achieve an understanding of the molecular mechanisms that mediate CO2 signal transduction in guard cells. The research will investigate the hypothesis that CO2 modulation of the [Ca2+]cyt elevation pattern in guard cells contributes to CO2-induced signal transduction during stomatal closing and/or opening. To test this hypothesis, studies with the following specific aims will be pursued: Characterize CO2 responses of wildtype guard cells. Examine the effects on CO2 signal transduction of known mutants that affect Ca2+ and/or abscisic acid signaling at distinct points within the guard cell signaling network, to characterize genetic mechanisms that affect CO2 signaling and to determine which components are specific to defined signaling pathways. Gain insight into how [Ca2+]cyt is transduced and specifically define the roles of selected guard cell-expressed CDPKs in stomatal responses. Characterize new CO2 signaling mutants. The P.I. will further pursue outreach efforts through public forums and through research and career training and preparation of undergraduate and high school students. Understanding the molecular mechanisms by which CO2 modulates stomatal apertures is fundamental to understanding the regulation of gas exchange between plants and the atmosphere, will help to predict effects of atmospheric CO2 elevation on stomata and may also contribute to future engineering of crop plants and plant carbon sinks in the face of changing environmental conditions.

DESCRIPTION (provided by applicant): Soils and waters with high levels of toxic heavy metals such as cadmium, arsenic, lead and mercury are detrimental to human and environmental health. These 4 metal(loid)s are among the Superfund's top 7 priority hazardous substances. Recent research and applications indicate that uptake of heavy metals into plants via the root system and accumulation of heavy metals in plant shoots could provide a cost effective approach for toxic metal removal and remediation of heavy metal-laden soils and waters. However many genes, mechanisms and pathways that function in heavy metal over-accumulation in plants remain to be identified and characterized. Phytochelatins are major heavy metal and metalloid chelating and detoxifying thiolate peptides in plants. In recent research we have made advances at understanding mechanisms that contribute to heavy metal detoxification and transport in plants, including isolation of phytochelatin synthase genes, characterization of mechanisms for root to shoot transfer of cadmium, isolation of heavy metal accumulation Arabidopsis mutants, development of a novel microarray-based rapid mutant cloning approach and microarray-based identification of putative transporter genes that may contribute to heavy metal transport. The investigators will test the hypotheses that, phytochelatins affect long distance root to leaf vascular transport of toxic metals; characterization of new toxic metal accumulation mutants will lead to identification of rate-limiting steps that function in plant heavy metal accumulation; and heavy metal sensing and signal transduction mechanisms in plants are important for plant heavy metal resistance and accumulation. To test these hypotheses, the proposed project will, in Specific Aims 1 and 2, characterize novel physiological and molecular mechanisms of root to shoot transport of heavy metals and phytochelatins using physiological, genomic, biochemical and membrane transport analyses. By pursuing a new high-throughput screening approach in collaborative research, the investigators have identified Arabidopsis mutants that affect the accumulation of toxic metals in leaves. In Specific Aim 3, a newly developed genomic microarray-based rapid mutant mapping and cloning approach will be used to isolate selected heavy metal accumulation mutant genes and characterize the underlying mechanisms. Specific Aim 4 will be to characterize heavy metal biosensing and transduction mechanisms in plants using a luciferase reporter screen. Trials with contaminated soils from Superfund sites will be pursued in collaboration with Edenspace Corp (in Specific Aim 5), to assess the feasibility of monitoring bioavailable heavy metals and of hyperaccumulating heavy metals and metalloids into plant roots and shoots using transgenic and mutant plants generated in this research.

With the completion of plant genome sequences and with numerous large data sets emerging, an essential need has arisen to train students in plant systems biology at the interface of computational genomics, systems modeling and plant sciences. This entirely new interdisciplinary program will provide a unique training environment for graduate students and will position them at the frontier of systems biology to address major challenges facing plant scientists and agricultural biotechnology. The program will include focused mentoring of each student in two labs by two advisors from distinct disciplines. An entirely new curriculum, made possible through new faculty recruitment, will train students in post genomic plant sciences, proteomics, systems biology and network modeling and will include rigorous professional career preparation. A dynamic outreach program will assist in the recruitment of underrepresented students. Industry internships will draw upon the local active biotechnology arena. There are several broader impacts of this project. Interdisciplinary training in plant systems biology will stimulate innovations in the nation's agricultural and biotechnology industries, address global needs to feed the growing population and contribute to reducing environmental impacts of agriculture. Fresh water and food shortages are predicted to grow substantially in the coming decades. Plant biotechnology and molecular breeding will provide powerful contributions toward solving these problems. This comprehensive educational program will train graduate students at the interface of systems modeling and plant sciences and will have far-reaching impacts by producing highly trained scientists who will emerge as leaders in fields arising from the revolution in genomics information. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): Soils and waters with high levels of toxic heavy metals such as cadmium, arsenic, lead and mercury are detrimental to human and environmental health. These 4 metal(loid)s are among the Superfund's top 7 priority hazardous substances. Recent research and applications indicate that uptake of heavy metals into plants via the root system and accumulation of heavy metals in plant shoots could provide a cost effective approach for toxic metal removal and remediation of heavy metal-laden soils and waters. However many genes, mechanisms and pathways that function in heavy metal over-accumulation in plants remain to be identified and characterized. Phytochelatins are major heavy metal and metalloid chelating and detoxifying thiolate peptides in plants. In recent research we have made advances at understanding mechanisms that contribute to heavy metal detoxification and transport in plants, including isolation of phytochelatin synthase genes, characterization of mechanisms for root to shoot transfer of cadmium, isolation of heavy metal accumulation Arabidopsis mutants, development of a novel microarray-based rapid mutant cloning approach and microarray-based identification of putative transporter genes that may contribute to heavy metal transport. The investigators will test the hypotheses that, phytochelatins affect long distance root to leaf vascular transport of toxic metals;characterization of new toxic metal accumulation mutants will lead to identification of rate-limiting steps that function in plant heavy metal accumulation;and heavy metal sensing and signal transduction mechanisms in plants are important for plant heavy metal resistance and accumulation. To test these hypotheses, the proposed project will, in Specific Aims 1 and 2, characterize novel physiological and molecular mechanisms of root to shoot transport of heavy metals and phytochelatins using physiological, genomic, biochemical and membrane transport analyses. By pursuing a new high-throughput screening approach in collaborative research, the investigators have identified Arabidopsis mutants that affect the accumulation of toxic metals in leaves. In Specific Aim 3, a newly developed genomic microarray-based rapid mutant mapping and cloning approach will be used to isolate selected heavy metal accumulation mutant genes and characterize the underlying mechanisms. Specific Aim 4 will be to characterize heavy metal biosensing and transduction mechanisms in plants using a luciferase reporter screen. Trials with contaminated soils from Superfund sites will be pursued in collaboration with Edenspace Corp (in Specific Aim 5), to assess the feasibility of monitoring bioavailable heavy metals and of hyperaccumulating heavy metals and metalloids into plant roots and shoots using transgenic and mutant plants generated in this research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our goal is the analysis of regulatory mechanisms underlying the activation of a kinase family found in Arabidopsis and some protists like the malaria causing parasite Plasmodium falcipare. For this cause we perform auto- and cis-phosphorylation analyses in response to various stimuli in planta.

Stomata are the pores on the surface of leaves that 1) regulate the diffusion of carbon dioxide from the atmosphere into leaves for photosynthetic carbon fixation and 2) control the transpirational water loss of plants. Guard cells sense carbon dioxide concentration, water status, light and other environmental stimuli and integrate these to regulate stomatal apertures for optimization of carbon dioxide influx into plants, water loss and plant growth under diverse conditions. For example, elevated carbon dioxide concentrations in leaves cause stomatal closure, whereas reduced carbon dioxide concentrations result in stomatal opening. The concentration of atmospheric carbon dioxide is predicted to double within the present century: carbon dioxide at these increased levels is known to reduce the stomatal apertures of various plant species by up to 40%. This will have profound effects on global gas exchange between plants and the atmosphere and the efficiency of plant water use. However, relatively little is known about the molecular signal transduction mechanisms that mediate carbon dioxide-induced stomatal movements. Using the model plant Arabidopsis, the PI has shown that knock-out mutants in genes encoding carbonic anhydrase show an impaired carbon dioxide-induced stomatal movement response. The hypothesis that these proteins function in early carbon dioxide control of gas exchange regulation will be investigated. In this project, the genetic, molecular, cellular and physiological mechanisms by which these proteins mediate the stomatal response to carbon dioxide concentrations will be characterized.

Broader Impacts: The P.I. will pursue outreach efforts through public forums and through research and career training and preparation of high school students and undergraduate students. Underrepresented minority students will be trained to pursue supervised independent research projects. In addition the P.I. is training and preparing post doctoral and graduate scientists for advanced independent careers in research, technology and science education. Understanding the molecular mechanisms by which carbon dioxide modulates stomatal conductance is fundamental to understanding the regulation of gas exchange between plants and the atmosphere, will help to predict effects of atmospheric carbon dioxide elevation on plants, and may also contribute to future engineering of water use efficiency or leaf heat stress avoidance in crop plants and plant carbon sinks in the face of the continuing atmospheric carbon dioxide rise and climate change.

SUMMARY (See instructions): Soils and waters with high levels of toxic heavy metal(loid)s such as arsenic, cadmium and mercury are detrimental to human and environmental health. These three metal(loid)s are among the Superfund's top 7 priority hazardous substances. Research and applications indicate that uptake of heavy metals into plants via the root system and accumulation of heavy metals in plant shoots could provide a cost effective approach for toxic metal removal and remediation of heavy metal-laden soils and waters. However important genes and pathways that function in heavy metal over-accumulation in plants remain to be identified. In recent research we have made major advances at understanding key mechanisms that function in heavy metal detoxification, transport and accumulation in plants. We will combine powerful genomic, genetic, biochemical and physiological approaches to test new central hypotheses by pursuing the following Specific Aims: L Characterize newly identified vacuolar membrane transporters that function in uptake and accumulation of phytochelatin-heavy metal(loid) complexes into plant vacuoles and analyze their bioremediation potential. \ l Understanding the control of heavy metal accumulation and distribution in roots and shoots is critical for engineering of plants for bioremediation. Determine the mechanisms by which a new peptide transporter mutant, opt3, causes hyper-accumulation in roots and under-accumulation of cadmium in plant leaves. iii Our recent research has shown that heavy metal-chelating and detoxifying thiols undergo long distance transport in plants. However, the plasma membrane transporters for uptake of glutathione (GSH) and phytochelatins (PCs) remain unknown. Using a high-throughput screen we have now identified OPT4 as a putative GSH & PC-Cd transporter. We will characterize 0PT4 and additional transporters to determine their underlying GSH/PC-Cd/As transport mechanisms and their functions in heavy metal distribution in plants. IV. The mechanisms and transcription factors that mediate rapid heavy metal-induced gene expression in plants remain largely unknown, but are important for heavy metal resistance and accumulation. The genes of newly isolated mutants impaired in cadmium-induced gene expression will be identified and their functions characterized. Furthermore, a potent genome-wide approach has been developed and will be pursued to identify and characterize key transcription factors and repressors that control cadmium- and arsenic-induced gene expression. We will work closely with the RTC and CEC in sharing our advances and foremost in translational analyses of our findings for their potential in phytoremediafion of contaminated soils and waters.

Plant leaves have thousands of microscopic adjustable pores in their leaf surface, called stomata. These stomatal pores in the surface of leaves open and close to regulate the necessary uptake of carbon dioxide into plants from the air. However, these stomatal pores also are the main pathway by which plants lose water, by evaporation. A typical plant loses 200 to 500 water molecules through these stomatal pores for every carbon atom that is absorbed (assimilated) by the plant for growth. The opening and closing of stomata is regulated by signals that include the concentration of carbon dioxide (CO2) in the air. The concentration of CO2 in the air is now 50% higher and rising, compared to only 150 years ago, meaning that plants could theoretically more efficiently take up CO2 from the air, while losing less water. However, important mechanisms and genes that mediate this agronomically relevant CO2 response of stomatal pore aperture regulation remain unknown. This project will characterize newly found key genes and proteins and define cellular networks through which elevated carbon dioxide controls the closing of stomatal pores and how low CO2 controls the opening of stomatal pores. This research can develop the knowledge necessary for the breeding of plants with improved growth properties and enhanced water use efficiency. The ability to manipulate the response of stomatal pores to carbon dioxide is important for unfavorable weather conditions, agricultural ground water depletion and droughts that are becoming more frequent in several of the major agricultural regions in the US as well as globally. The scientists will pursue an outreach program with research internships, professional preparation and mentoring with the public Preuss School for disadvantaged high school students in San Diego County, as well as training and professional preparation of visiting underrepresented summer research interns with UC San Diego's ENLACE program and with Howard University. Project personnel will be active within community outreach work that brings science and innovation close to the public and the investigators will participate in a recently launched outreach program through presentations and discussions with underrepresented students at inner city high schools in San Diego.

This project will use a combination of cell biological, biochemical, molecular genetic, mathematical modeling, genomic and systems biological approaches to identify new critical molecular components of the CO2 signaling network and characterize how this network operates to regulate stomatal pore apertures. The focus of this project is to identify how the CO2 stimulus is transmitted into the stomatal movement network, with these goals: (1) Biochemical mechanisms and network principles will be determined by which newly identified genes and the encoded proteins mediate early CO2 sensing and signal transduction. (2) New hypotheses will be investigated on how cell-to-cell signaling in leaves affects CO2 control of stomatal movements by combined computational modeling, genetics, metabolomics and molecular cell biology. (3) Newly isolated "chill" mutants that have cooler leaf temperatures and are defective in the dynamic CO2 response of grass stomata will be mapped and the underlying gene and protein of at least one rate-limiting gene will be isolated and its functions in stomatal movements of the specialized dumbbell-shaped guard cells of grasses will be determined.

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.

PI: Geoffrey Chang (University of California-San Diego)

Co-PIs: Erin Connolly (University of South Carolina), Leon Kochian and Miguel Piñeros (USDA-ARS/Cornell University), Michael Stowell (University of Colorado-Boulder), Julian Schroeder and Milton Saier (University of California, San Diego)

Plants need to take up nutrients from the soil and transport these nutrients to specialized cells throughout the whole plant. These nutrients are transported across plant cell membranes and organelle membranes inside of each plant cell. Transporters are proteins that mediate the passage of virtually every molecule and ion across plant cell membranes. Many natural variants of transport proteins have been shown to be important for key agronomic traits associated with plant growth and yield, and tolerance to abiotic and biotic stresses. However, the fundamental biochemical, functional, and structural characteristics of these proteins are largely unknown. To address these questions and the glaring need for new tools in this research area, we are establishing a new center called the Center for Research on Plant TransporterS (CROPS), devoted to the functional and structural characterization of plant transporters prioritized by their agronomic importance. This center will leverage an existing advanced research infrastructure to produce purified plant transporters on a large-scale and rapidly discover/generate novel single-domain antibodies that can be used as valuable tools and potentially transformative and enabling resources for the greater plant science community. In addition, CROPS will determine the molecular structures of key plant membrane transporters and their natural variants, providing a new framework for understanding their functions in the context of their in planta roles, underpinning agriculturally important traits in diverse crop species.

Our understanding of plant transporters has increased dramatically over the past quarter century. This has led to a growing awareness that plant transporters play important roles in many agronomic traits, such as efficient acquisition/use of water and nutrients and crop tolerance to adverse soil environments arising from salinity, acidity, and the presence of heavy metals. Natural genetic variation is the foundation for breeding a wide variety of agriculturally relevant traits. Understanding how genetic variation translates into transporter structural and functional changes is critical to provide for a more efficient exploitation of this variation in crop improvement. CROPS will focus on key questions regarding agronomic traits that relate to food security and sustainability under marginal conditions and human health/nutrition and food safety. Specifically, through external collaborations and internal expertise, CROPS will target key transporters including those involved in micronutrient acquisition, aluminum tolerance, phosphorous acquisition efficiency, nitrogen acquisition efficiency, salt tolerance and pathogen and insect resistance. As a first step, CROPS will establish an efficient pipeline based on proven technologies for the production of plant membrane transport resources for the plant scientific community. These resources include large quantities of purified plant transporter proteins, a powerful in vitro production platform for nanobodies, novel synthetic single-domain antibodies, and state-of-the-art structural determination techniques. CROPS has a strong commitment to providing access to all data and biological resources to the broader plant membrane transport community. Access to proteins, antibody reagents, and information about protein structure will be provided through a project website/database and by dissemination through long-term repositories. Specifically, all structural x-ray and EM structural data (GC and MS) will be deposited in the Protein Data Bank (http://www.pdb.org) and the EMDataBank (http://www.emdatabank.org). All plant and synthetic nanobody DNA sequences will be annotated and deposited in GenBank and Gramene. Plasmid constructs will be deposited in Addgene (https://www.addgene.org), a not-for-profit plasmid repository. With regard to outreach, CROPS will provide mechanisms for inviting the wider community to tap into its pipeline as a basis for their own research. Researchers will be invited to submit nominations for specific plant transporters of interest. Graduate students and postdocs will receive training and access to some of the most state-of-the-art techniques in rapid antibody (nanobody) evolution, membrane protein structural/functional biology, and molecular-based trait analysis for advancing independent careers. CROPS will have a strong emphasis on providing research training and laboratory experiences to high school and undergraduate students from underrepresented groups, with a focus on development of writing and presentation skills as well as mentoring students to take personal responsibility and gain scientific educational and project management skills.

Specialized pores (called stomata) in the leaves of plants open and close to regulate the uptake into plants of carbon dioxide from the air, and the loss of water (as vapor) from plant tissues out into the air. The opening and closing of stomata is regulated by signals that include the concentration of carbon dioxide in the air, but knowledge of how this is achieved is incomplete. This project will define the genes, proteins and networks of control involved in the regulation of stomata by carbon dioxide, and develop the knowledge necessary for the breeding of plants with improved growth properties and water use efficiency. The ability to manipulate these properties of plants is important for unfavorable weather conditions, climate changes, droughts becoming more frequent in some locations, and as carbon dioxide levels in the air increase. The investigators will pursue an outreach program involving research internships, professional preparation and mentoring with the public Preuss School for disadvantaged high school students in San Diego County as well as engaging with summer research interns from Howard University. Project personnel will be active within the San Diego Science Festival, a large annual event that brings science and innovation close to the public.

Some components (including carbonic anhydrase, anion channels and protein kinases) of the carbon dioxide signaling network that regulates stomatal aperture are known. However, the manner in which diverse signals, genes and proteins (a number of which are still to be uncovered) are integrated by guard cells into the network that controls stomatal aperture is not known. This project will use a combination of systems biology, biochemical, genetic and mathematical modeling approaches to identify additional critical molecular components of the signaling network and gain insight into the manner in which this network operates in guard cells to regulate stomatal aperture and how plants can improve their water use efficiency and drought resilience.

This award is supported jointly by the Cellular Dynamics and Function program in the Division of Molecular and Cellular Biosciences and by the Physics of Living Systems program in the Division of Physics.

Project Summary/Abstract Soils and waters with high levels of toxic metal(loid)s such as cadmium, lead, arsenic and mercury are detrimental to human and environmental health. These 4 heavy metal(loid)s are among the Superfund's top 7 priority hazardous substances. Many human diseases have been attributed to environmental contamination by heavy metals, including cancers and neurological disorders. Research and applications indicate that uptake of heavy metals into plant roots and accumulation of heavy metals could provide a cost effective approach for toxic metal removal and bioremediation of heavy metal-laden soils and waters. In recent research we have made major advances at understanding key mechanisms that function in heavy metal detoxification, transport and accumulation in plants. However important genes and pathways that function in heavy metal over- accumulation in plants remain to be identified. We will combine powerful genomic, genetic, biochemical and engineering approaches to test new central hypotheses by pursuing the following Specific Aims: Aim I. The regulatory mechanisms, transcription factors (TFs), and transcriptional network that mediate rapid heavy metal(loid)-induced transcriptional responses in plants remain largely unknown. Using a luciferase- based cadmium- and arsenic-induced reporter mutant screening approach we have isolated mutants in rapid Cd- and As-induced gene expression. New mutants in major Cd-/As-dependent repression and induction loci will be characterized and the underlying genes isolated and their functions determined. Collaborative research with Geoffrey Chang (Project 5) will pursue development of cost-effective innovative heavy metal toxicant nano-reporters in plants. Aim II. The many genetic redundancies in plant genomes cause major limitations in heavy metal response gene discovery. To address redundant gene function on a systems biology scale we have designed a genomic scale artificial microRNA (amiRNA) library for genome-wide knockdown of homologous gene family members which is leading to discovery of new genes and will be used to characterize key plant genes and network mechanisms that function in heavy metal accumulation, resistance and remediation. Aim III: Using genes identified in Specific Aims I and II and previous research, gene-stacking will be used to generate plants and investigate their enhanced heavy metal accumulation and root sequestration (phytostabilization) potential. Furthermore, by genomic investigation of plants that are being used for phytostabilization at semi-arid Superfund sites, the above advances will be used in collaboration with the University of Arizona Superfund Research Center to uncover mechanisms that render plants suitable for phytostabilization of toxic metal(loid)s. The proposed research will be leveraged to develop technologies for avoiding the growing problem of accumulation of heavy metals and arsenic in edible plant tissues.