Wendy Boss

Funding is requested under the intergovernmental personnel act for salary plus 28% fringe less 15% cost share for Dr. Wendy F. Boss, who will serve as a program director in the networks and regulation cluster of the molecular and cellular biology division at the National Science Foundation.

Overview: North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art, high resolution confocal laser-scanning microscope (CLSM) for the simultaneous imaging of multiple gene products and their distribution in both fixed and live tissues. The only high resolution confocal on campus is a Leica SP1 Laser Scanning Microscope which was purchased in 1996. The Leica SP1 CLSM has been well maintained and while it has served the needs of faculty and students for many years, it employs outdated technology and does not meet our present and growing needs. None of the CLSMs on the NCSU campus are capable of the high resolution, high sensitivity, dynamic and fixed-specimen imaging required to monitor low levels of multiple fluorophores or to analyze their distribution within samples. This means faculty must drive to the University of North Carolina, Duke or to the Research Triangle Institute to gain access to a state-of-the-art CLSM to conduct their research. Our faculty are actively training graduate and undergraduate students and participate in high school and middle school outreach programs; however, on-campus training and outreach are constrained by the lack of a state-of-the-art CLSM. Furthermore, because of the lack of infrastructure, we are no longer competitive in hiring new faculty. A number of strong job candidates have specifically noted the lack of suitable imaging facilities as a major determinant in their taking jobs at other universities and turning down offers by NCSU. Instrumentation: A Leica SP5 CLSM. The Leica SP5 CLSM can monitor dynamic changes in the spectral properties of specimens as well as the subcellular distribution of low levels of multiple fluorophores not detectable with the Leica SP1 CLSM. Furthermore, a Leica SP5 will enable us to monitor protein-protein interactions using Fluorescence Energy Transfer (FRET) and dynamic changes in protein redistribution in response to stimuli using Fluorescence Recovery After Photobleaching (FRAP). Neither FRET nor FRAP are possible with the Leica SP1. Broader Impacts: A new CLSM is essential to reveal the biological relevance of ongoing cellular, biochemical and genetic studies at NCSU. Impacted projects include but are not limited to research focused on: genes that regulate cellular development, particularly stem cells, in both animals and plants; new molecules involved in neuron-glial interactions; mechanisms regulating protein distribution and signaling in plants in response to abiotic and biotic stresses, and mechanisms regulating wound repair in animal cells. The new CLSM will be housed in the Cellular Molecular Imaging Facility (CMIF), a University-wide facility that serves as a focal point for imaging training and research. The CO-PIs will be the co-directors of the facility. Dr. Eva Johannes, the Assistant Director of the CMIF, will maintain and manage the equipment and supervise and train new users as well as teach the course on imaging. The CLSM will have immediate and broad impacts on the training of undergraduates, graduates and post graduates at NCSU as well as undergraduates at Meredith College (near-by woman?s college) who take the imaging course. Furthermore, the new CLSM will be used by a diverse group of PIs who provide role models as they mentor undergraduates in independent research and participate in summer REU programs involving students from non-research I and minority universities. The new CLSM will also be highlighted during the tours and hands on activities for prospective students and local school children conducted by the CMIF. For example, CMIF is a focal point of the ?Expanding your Horizon? program aimed at attracting 7th grade girls from around the State of North Carolina to a career in science, mathematics and engineering.

Dole Fresh Flowers, headquartered in Miami, Florida, is a leader in the cut flower industry. One of the advantages of a vertically integrated company is the ability to reduce costs while improving cut flower quality. However, cut flower vase life remains a primary concern. The overall goal of this project is to increase postharvest life of cut flowers, especially roses, through alteration of the production, harvest, processing and shipping processes and through genetic manipulation. This project will increase consumer confidence in cut flowers and allow suppliers and retailers to offer a guaranteed minimum vase life. The project will 1) Conduct a step-by-step evaluation of Dole Fresh Flowers? current rose production, harvest, processing and shipping processes with the intent to maximize postharvest life. 2) Maximize the vase life of currently-available rose cultivars through modified atmosphere storage, improved floral preservative performance, use of temperature-time indicators, development of retailer handling protocols, and third-party verification of vase-life guarantees. 3) Develop a robust set of tools and protocols for the genetic engineering of cultivated rose with the long-term objective of developing a distinctive rose with enhanced postharvest life, aroma and disease resistance, while still retaining high productivity, cut stem characteristics and flower color.

Providing life support for human exploration is a major challenge as we venture to Mars and beyond. Our hypothesis is that we can revolutionize life forms by selectively expressing in plants extremophile genes that will collectively enable functional life in inhospitable environments. For our phase I proposal, we demonstrated for the first time that an archaeal gene, Pyrococcus furiosus superoxide reductase, could be translated in a model eukaryotic system to produce a functional enzyme which retained the properties of the original archaeal protein. In our phase II proposal, we will extend our studies to the whole plant and will include a suite of genes from the P. furiosus reactive oxygen species (ROS) detoxification pathway that together will confer resistance to oxidative stress. Furthermore, we will increase reduced glutathione by introducing the glutathione reductase (GR) gene from a psychrophilic extremophile Colwellia psychrerythraea in order to address a major problem facing earth-based organisms on Mars, stabilizing protein structure during the rapid changes in temperature that will be encountered even in enclosed green houses. We will cross plants producing the heat stable ROS and cold functional GR enzymes to generate a hybrid tolerant of extreme environments, including cold, drought, heat and radiation. After each transgenic plant has been characterized biochemically, the plants will be distributed to NASA-funded labs for more complete physiological testing in space environments. In addition, we will work with these investigators to introduce the genes into breeding programs to generate crop plants to be grown in space. Finally, our road map includes working with an honors undergraduate class to investigate the use of novel genetic approaches to generate plants resistant to radiation damage to DNA.

The response of plants to gravity involves a sensing mechanism, initiating a signal transduction pathway, which results in changes in gene expression. The only link between gravity response and transcriptional changes known so far is mediated by auxin. Using whole genome microarray technology, we have identified genes that respond exclusively to gravity stimulation but not to mechanical stimulation in the Arabidopsis thaliana root apex. The fastest transcriptional gravity-specific responses occur within less than 1 minute after gravity stimulation. The majority of those gravity-specific genes were not induced after gravity stimulation of transgenic Arabidopsis plants with reduced the levels of inositol-1,4,5-trisphosphate (IP3), a very early component of gravitropic signal transduction. These transgenic plants have a delayed and slow response to gravity. Most auxin-regulated genes did not show specificity to gravity stimulation and responded in the same pattern in wildtype and transgenic plants. Our hypothesis is that gravitropic regulation of transcription is mediated by two separate and independent branches of the signal transduction pathway: an IP3-mediated gravity-specific mechanism and an IP3-independent hormone-regulated pathway. We propose to use a cluster of gravity-specific co-regulated, fast and transiently expressed genes to further investigate the mechanism by which IP3- mediates gravity specific transcriptional changes. The molecular and physiological functions of these gene products are unknown. Identification of the transcriptional activators and repressors, and the functions of the proteins of the fast and transiently expressed gravity-specific genes will allow us to link signal transduction events to differential cell elongation and to determine key transcriptional regulators of the gravitropic response. Transcriptional activators that function within the short time we have described must be constitutive components of very early gravitropic signal transduction. Our first research objective is to investigate the functions of those genes that are specifically expressed within the first 2 min after gravity stimulation. We will use a reverse genetics approach (RNAi) and determine the location of transcripts and proteins on a cellular and subcellular level. Our second research objective is to identify the mechanisms by which IP3 regulates gravity specific changes in transcript abundance of genes. We will generate promoter-reporter gene constructs to monitor their responses in the whole plant and to different abiotic stimuli. In addition we will use genetic and biochemical approaches to identify the proteins that are binding to these promoters. Additionally, we remain committed to a comprehensive approach to the enhancement of science education at all levels and the public appreciation of science. We intend to build on our established strengths in this area. These include: ??A? Elementary education through the web-based teaching tool ?Adventures of the Agronauts?; ??A? College course distribution through the on-line course ?Space Biology? and the distribution of the CD set of the same name; ??A? General public interaction via lectures, workshops and activities conducted by scientists from our research laboratories. Through these activities we will actively promote the education and public outreach goals of NASA.

The proposed conference will focus on the mechanisms by which plants generate and use lipid-mediated signals to alter growth and development. A major goal of this conference is to bring together a diverse group of international plant biologists to advance our understanding of the plant lipid-mediated signaling interactome (protein network). The conference will consist of a combination of plenary and shorter lectures, poster sessions and two workshops. One workshop will be on lipidomics and nanotechnology applications for single cell analysis and the other will be on in vivo analysis of lipid signaling using fluorescent peptides and fluorescent lipids. There will be 30 invited speakers and we estimate that over 100 participants will attend. The speakers will be scientists working in the field of lipid signaling and signal transduction.

The proposed conference will focus on mechanisms by which plants generate and use lipid-mediated signals. Major goals of this conference are to advance our understanding of the plant lipid-mediated signaling networks and to integrate them into the nexus of plant signaling, to discuss and assess current and future methods for studying lipid signaling and to provide training and networking opportunities for young investigators, students and postdocs. We will be ?building connections? between signaling pathways, upstream and downstream signaling events and researchers. To this end, this proposal is a request for support for 14 student/postdoc travel awards. There has not been a conference on plant lipid-mediated signaling since 1989. We think this conference is timely and will have a positive and lasting impact on the field. There has been a lot of interest from the community. A copy of the conference flyer listing some of the speakers who will be attending is attached as a supplementary document. We are working to gather a consortium of support for the conference and hope that the USDA will provide the corner stone by funding travel for students and postdoc participants.

Genetic, biochemical and physiological data indicate that lipid signaling impacts plant responses to abiotic and biotic stress. The proposed conference will focus on the mechanisms by which plants generate and use lipid-mediated signals to alter growth and development. A major goal of this conference is to bring together a diverse group of international plant biologists to advance our understanding of the plant lipid-mediated signaling interactome (protein network). The conference will consist of a combination of plenary and shorter lectures, poster sessions and two workshops. One workshop will be on lipidomics and nanotechnology applications for single cell analysis and the other will be on in vivo analysis of lipid signaling using fluorescent peptides and fluorescent lipids. There will be 30 invited speakers and we estimate that over 100 participants will attend. The speakers will be scientists working in the field of lipid signaling and signal transduction. It is anticipated that students and postdocs will give poster presentations or talks.

This is a request for funds for travel to attend the Annual Meeting of The American Society of Plant Biologist (ASPB) to present the recent results of the thesis research project of Rafaelo Galvao. The ASPB meeting provides a unique opportunity for him to gain insights into his research from highly qualified investigators from many different parts of the country. In addition a meeting of such magnitude is the ideal occasion for establishing contacts and interactions and, more importantly, learning the cutting age research that is on going in the top plant biology research labs around the world. The title of the research that will be presented is: Characterization of the protein kinase activity of a putative Arabidopsis thaliana lipid kinase