Area(s) of Expertise
Research in my lab in collaboration with Dr. Imara Perera focused on the role of inositol phospholipids in regulating plant sensing and response to environmental stimuli including osmotic stress and gravity. We took a combinatorial approach that includes biochemistry, cell biology and molecular biology to characterize the enzymes involved in inositol lipid biosynthesis, to study their subcellular distribution and to genetically alter the flux through the PI signaling pathway. In addition, in collaboration with Dr. Amy Grunden, we took a molecular genetics approach to reduce the presence or reactive oxygen species in plants by expressing genes from Pyrococcus furiosus.
- Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway? , Planta (2015)
- Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis , Plant Cell (2013)
- Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects plant nuclear lipids and nuclear functions , Plant Physiology and Biochemistry (2012)
- Phosphoinositide signaling , Annual review of plant biology, vol 63 (2012)
- Basal signaling regulates plant growth and development , Plant Physiology (2010)
- Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato , Plant Biotechnology Journal (2010)
- Blue light induced changes in inositol 1,4,5-trisphosphate in Cuscuta campestris seedlings , Weed Research (Oxford) (2009)
- Expression of Pyrococcus furiosus superoxide reductase in arabidopsis enhances heat tolerance , Plant Physiology (2009)
- Phosphatidylinositol (4,5)Bisphosphate inhibits K+-Efflux channel activity in NT1 tobacco cultured cells , Plant Physiology (2009)
- Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains , Biochemical Journal (London, England : 1984) (2008)
Plants are continually challenged by environmental stresses that increase the production of reactive oxygen species (ROS, e.g., superoxide and hydrogen peroxide). ROS can induce a switch from primary to secondary metabolism and will ultimately lead to cell death. We have shown that expression of superoxide reductase (SOR) from the archaeal hyperthermophile Pyrococcus furiosus can dampen cytosolic ROS signals and foster robust plant growth under inhospitable conditions such as heat and drought. SOR is more efficient in removing ROS than the endogenous plant enzyme, superoxide dismutase. SOR is a reductase and results in a net loss of O2 making it an extremely effetive ROS scavenger. P. furiosus SOR is also functional from 4-100oC. Our hypothesis is that crop plants producing SOR pathway enzymes will have increased yield at high temperatures. ROS are difficult to study in vivo and the downstream effectors are not well characterized. We propose to use this novel synthetic system to dissect the mechanisms of ROS sensing and response and to increase biomass production in crop plants. Our aims are: 1) To transform tomatoes (cv. Micro-Tom) with both P. furiosus SOR and rubrerythrin reductase (Rr), the next enzyme in the SOR pathway. Rr reduces peroxide to water without producing oxygen and should be even more effective in scavenging ROS; 2) To characterize the effects of SOR and SOR/Rr expression on reproductive growth (fruitset) and fruit quality which are temperature sensitive in tomato. 3) To characterize downstream effectors of ROS-sensing using SOR and SOR/Rr-transgenic Arabidopsis plants.
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.
Phosphatidylinositol phosphate 5-kinases (PIP5Ks) are enzymes that synthesizes phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2 by phosphorylating PtdIns4P. The largest subfamily of plant PIP5Ks have an N-terminal extension not found in PIP5Ks from any other organism studied thus far. This unique N-terminal extension contains regions similar to the membrane occupation repeat nexus (MORN) motifs of animal junctophilins, proteins essential for selective coupling of ER rynodyne sensitive channels to calcium influx channels in the plasma membrane and of ARC3, a protein essential for chloroplast fission. We have shown that Arabidopsis PIP5Ks are the flux limiting step in the plant phosphoinositide (PI) pathway and that N-terminal MORN domain of the AtPIPK1 regulates enzyme activity as well as the subcellular distribution and can be phosphorylated by a calcium-independent protein kinase. Furthermore, PIP5K1 co-purifies with actin cytoskeleton and recruits phosphatidylinositol 4-kinase beta (PI4K?Ò1) to an F-actin fraction. The hypothesis to be tested in this work is that the plant PIP5K1 associated with actin filaments recruits and targets vesicles to selective intracellular membranes (e.g. the plasma membrane) through the PIP5K1 N-terminal MORN domain. Our specific aims are: 1) To characterize lipid binding and optimize binding conditions for the N-terminal MORN domain and full length AtPIP5K1 and AtPIP5K9 in the presence and absence of F-actin and PI4K?Ò1. 2) To characterize the effects of protein phosphorylation on vesicle binding and enzyme activity. This will include identifying phosphorylation sites that affect both vesicle binding and enzyme activity. 3) To identify target vesicles through in vitro vesicle-capture experiments using C-terminal fused AtPIP5K1 and AtPIP5K9 and by transient transformation using green fluorescent protein fusion proteins. 4) To complete the characterization of the type II putative PI4Ks The overall goal of this research is to characterize a plant-specific family of enzymes which combine the fundamental functions of a PI pathway enzyme (PtdInsP 5-kinase) with a membrane adhesion (MORN) domain. Because no other organism (including yeast, C. elegans and Drosophila) contains PIP5Ks that have N-terminal MORN motifs, the dual function of this plant specific protein will reveal new insights into vesicle targeting. This is high risk research that will impact our understanding of the fundamental differences in plant and animal trafficking and PI metabolism. The research will impact the careers of 3 graduate students, two of whom will graduate within the first year and a half of the proposal and at least 3 undergraduates a year who work with the group and who present their research at the undergraduate research symposium usually winning campus-wide awards. In addition, the transgenic cells and plants produced in this work are used in the undergraduate physiology lab to study the impact of altering the PI pathway on fundamental metabolism (respiration) and gravitropism. The undergraduate physiology course has launched several young scientists into careers in plant biology in the past few years.
Project elements: ? This is a new REU site. ? Project title: REU Site: Synthetic Biology Undergraduate Research Experience ? Principal Investigator: Susan Carson ? Submitting Organization: North Carolina State University; Other Organizations: none ? Location of undergraduate research: North Carolina State University ? Main fields and sub-fields of research: Synthetic biology, biotechnology, molecular biology, plant biology, chemical and biomolecular engineering, biochemistry, genomics ? Number of undergraduate participants per year: 8 NSF-funded, 3 NCSU-funded ? This is a summer REU site. ? This is a 10 week program. ? The project includes a research ethics workshop. ? Student Applicant Point-of-Contact: Sue Carson 919-513-0330 firstname.lastname@example.org ? Web address will link from http://www.ncsu.edu/biotechnology. Project summary: Synthetic biology is defined as ?the design and construction of new biological parts, devices and systems, or the re-design of existing, natural biological systems for useful purposes? (http://syntheticbiology.org). It is a cutting-edge technology that will attract students from a wide variety of interests and disciplines to the field of plant biology. North Carolina State University (NCSU) has a strong core of plant biologists who are working with two aspects of synthetic plant biology: using recombinant plants as ?factories? to generate useful products, and developing model systems for studying fundamental biological mechanisms. The Biotechnology (BIT) Program and the Department of Plant Biology at North Carolina State University propose to establish an REU site to provide undergraduate students with a summer research experience in this area of synthetic biology, complemented by training in core laboratory skills in molecular biotechnology. Targeted student participants will include rising sophomores, juniors and seniors who have demonstrated interest in molecular biotechnology as it applies to synthetic biology. Our goals are to inspire and train students to pursue research to answer fundamental questions and for practical applications. The intellectual merit of the proposed program stems from the following: 1. The Department of Plant Biology at NCSU has a strong core of faculty engaged in cutting-edge synthetic biology research using plant hosts. The majority of mentors are from this core faculty, with important faculty collaborations from other departments including microbiology, chemical and biomedical engineering, biochemistry, and plant pathology. 2. In addition to the research component, the program will include a three-day laboratory-intensive workshop to familiarize students with critical techniques in the molecular biology lab in the first week of the program. This hands-on workshop will allow all students to master the most critical laboratory skills before beginning in their research labs. 3. All students will receive training in essential bioinformatics skills, including navigating GenBank, performing BLAST searches, and designing PCR primers. 4. Students will have multiple public-speaking opportunities to present their research in various formats, including oral presentations in group meetings, and a poster presentation at the campus-wide undergraduate research symposium at the end of the program. The broader impacts of the program will be to attract undergraduates (with special emphasis on underrepresented groups) to pursue graduate work and ultimately a career in an area of molecular biology, especially plant molecular biology, a discipline often overlooked by minority students. By complementing a traditional summer research experience with introductory laboratory skills training and coordinated advising, we hope to develop a new paradigm for successful undergraduate research programs.
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: · Elementary education through the web-based teaching tool ?Adventures of the Agronauts?; · College course distribution through the on-line course ?Space Biology? and the distribution of the CD set of the same name; · 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.