II. Program Tasks — Ground-based Research Element: NSCORT

NSCORT. Calcium, Signaling and Gravity: An Integrated Molecular; Cellular and Physiological Approach to Plant Gravitational Biology

Administrator: Eric Davies, Ph.D. Department of Botany, Box 7612 College of Agriculture and Life Sciences North Carolina State University Raleigh, NC 27695-7612

Principal Investigators: Nina S. Allen; North Carolina State University Wendy F. Boss; North Carolina State University Christopher S. Brown; Dynamac Corporation Joan L. Huber; North Carolina State University Steven C. Huber; North Carolina State University Gloria K. Muday; Wake Forest University Dominique Robertson; North Carolina State University Ronald R. Sederoff; North Carolina State University William F. Thompson; North Carolina State University Edward B. Tucker; Baruch College, City University of New York Ross W. Whetten; North Carolina State University Eric Davies; North Carolina State University

NOTE: This NSCORT represents 12 individual tasks with 12 principal investigators.

Funding: UPN/Project Identification: 199-93-17-14 Solicitation: 94-OLMSA-04

Initial Funding Date: 1996 Expiration: 2000

Students Funded Under Research: 15 Post-Doctoral Associates: 7

FY 1997 Funding: $999,789

Phone: (919)515-2727 Fax: (919)515-3436 E-mail: eric_davies@ncsu.edu Congressional District: NC - 4

Task Description: This program in gravitational biology involves 9 faculty members from North Carolina State University, Wake Forest University, Baruch College, and the Dynamac Corporation through the Kennedy Space Center. The overall goal is to study calcium as a central focal point in the gravity response. The group uses an integrated molecular, cellular, and physiological approach to plant gravitational biology.

The precise modulation of calcium homeostasis will be acheived using transgenic technologies and monitored using sophisticated imaging techniques to verify the specificity and extent of transgenic expression. These efforts, in combination with our expertise in local and long-distance signalling, will make a major contribution to understanding the fundamental role of calcium in orchestrating the transduction of the gravity stimulus into an autopoietc (self-regulated) response.

The project brings together experts in a range of specially-selected fields to address a single major research problem, i.e., the fundamental role of calcium in regulating gravity-stimulated signal transduction in plants. The expertise to be called on includes molecular biologists to produce transgenic plants with altered calcium homeostasis (Thompson, Robertson, Sederoff); cell biologists to image calcium and other components of the

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signal transduction pathway (Allen, Tucker); physiologists to study signal transduction (Boss, Davies, Muday); and biochemists (Brown, Huber, Huber) to study calcium-modulated carbon/nitrogen metabolism. By fostering interdisciplinary collaborations among these diverse laboratories, the proposed program will create a multi-faceted approach to the study of plant gravitational biology.

The NSCORT is a consortium of institutions including North Carolina State University (College of Agriculture and Life Sciences and School of Forestry), Wake Forest University, Baruch College (City University of New York), NASA's Kennedy Space Center and Dynamac Corporation (which runs the Life Science Support contract at KSC). Faculty, staff and/or students from all of the institutions participate in various aspects of the program. It consists of three major components: Education, Outreach, and Research. Considerable progress has been made in all three areas.

Education: The NSCORT program offered the graduate-level course "Gravitational and Space Biology" to 15 graduate and advanced undergraduate students (Fall 1996). This course originated from the NC State University campus and was available on a real-time basis at 5 other campus locations throughout the state via the NC Regional Electronics Network microwave system. A new undergraduate level course "Space Biology" was initiated and taught to 16 students (Fall 1997) at the NC State University campus. A portion of this course was taught remotely from the Kennedy Space Center using real time audio and video link-ups. Experts in space biology (including Astronaut/Physician Dr. Chuck Brady) were brought in to expose students to current activities and opportunities in the field.

Outreach: A summer workshop for high school teachers entitled "Plants and Gravity" was offered. Ten teachers from five states spent time at the NC State University campus interacting with project leaders and research associates in the program. Some of the teachers have utilized the experience to develop new curricula at their schools. Additionally, a two day workshop was sponsored and held at the NC State University campus to train teachers for the educational opportunities associated with the Collaborative Ukrainian Experiment. As a group, the NSCORT participated in two separate science and technology symposia that offered middle school students, particularly girls, an opportunity to conduct hands-on experiments in a wide range of scientific areas. The NSCORT offered activities and tours at the North Carolina Science Olympiad and the National Science Olympiad. NSCORT scientists presented demonstrations and activities during science fairs at two local elementary schools and hosted a workshop on plant gravitational and space biology for middle and high school students attending the Imagination Station Summer Science Camp.

Research: In order to take an integrated approach to studying graviperception and response, six postdoctoral fellows were hired. These individuals possess skills in molecular biology, electrophysiology, biochemistry, and cell biology. Four graduate students are currently in the program and one or two more will be recruited. The physical set-up of the laboratories is complete, including the renovation of the electrophysiology laboratory and the acqisition of a Leica DMRXA confocal microscope with a rotatable stage. This piece of equipment will allow us to examine the real time in vivo imaging response of graviresponding plant tissue.

Focus has been placed on the maize pulvinus and the pine seedling compression wood systems as models to dissect biochemical and morphological response to gravity. Initial characterization is complete and detailed studies are underway. To understand the molecular genetic component of the gravity response in plants, we have chosen three proteins (calreticulin, calmodulin, and phosphatidylinositol kinase) to clone. These clones will then be used to transform Arabidopsis plants for studies on the influence of altered calcium homeostasis in the gravity response.

This research will determine the mechanisms by which plants perceive and respond to several environmental stimuli, especially gravity. It will provide a fundamental understanding of basic plant processes, especially at the cellular, molecular, and developmental levels. A deeper understanding of how plants respond to gravity and other environmental conditions will improve our understanding of how they grow in various space conditions (Earth orbit, Mars) and how their growth can be modified to maximize yields on Earth. More applied work on specific plants should yield valuable by-products of enhanced paper quality (pine seedling system and its formation of compression wood) and yield of seed grains (reorientation of com plants blown over in strong winds).

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FY97 Publications, Presentations, a n d Other Accomplishments: Allen, N.S. "Confocal microscopy and GFP imaging." (lecture) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 - September 4, 1997).

Allen, N.S. "Darkfield and phase microscopy, the basics." (lecture) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 - September 4, 1997).

Allen, N.S. "Deconvolution, what is it and where should you use it." (lecture) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 - September 4, 1997).

Allen, N.S. "From Leeuwenhoek to electronic light microscopy." (keynote address) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 -September 4, 1997).

Allen, N.S. "Resolution. Abbe's diffraction experiments." (lecture) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 - September 4, 1997).

Allen, N.S. "Polarization and DIC microscopy, theory and practice." (lecture) Dept. of Medical Microbiology, Linkoping University, Linkoping, Sweden. Video Microscopy Short Course (August 25 - September 4, 1997).

Allen, N.S. "Tip growth, an overview." Cytonet Conference, Breckenridge, CO (1997).

Allen, N.S. and Bennett, M.N. "Electro-optical imaging of F-actin and endoplasmic reticulum in living and fixed plant cells" in "Science of Specimen Preparation for Microscopy and Microanalysis." Edited by: Malecki, M. and Roomans, G. SMI Press, AMF O'Hare, Chicago, IL (In Press).

Allona, I., Quinn, M., Sun, Y-H., Shoop, E., Swope, K., St. Syr, S., Retzel, E., and Whetter, R. "Analysis of gene expression during xylem differentiation in loblolly pine." (Abstract) Plant Physiol., 114(3), 11 (1997).

Boss, W.F., Perera, I.Y., Ransom, W.D., Stevenson, J.M., Brglez, I., Heilmann, I.H., and Gross, W. "Wrapping about the cytoskeleton." (Abstract) Plant Physiol., 114(3), 15 (1997).

Brady, S.R., Dixon, M.W., Cyr, R.J., Fisher, D.D., and Muday, G.K. "BY2 protoplasts as a model for NPA sensitive auxin transport and gravitropism." (Abstract) ASGSB Bull., 10(1), 78 (1996).

Brown, C.S. "Plant growth and physiology in space." Baruch College, Manhattan, NY (March 4, 1997).

Brown, C.S. "Plant metabolism in space." University of North Carolina-Pembroke, Pembroke, NC (March 13, 1997).

Brown, C.S. "The Collaborative Ukrainian Experiment: Scientific overview." Presented to the 12th Man In Space Symposium, Washington DC (June 13, 1997).

Brown, C.S. (co-authors - Sanwo, M.M., Stryjewski, E.C., Peterson, B.V., Johnson, C.F., Piastuch, W.C., Hilaire, E., and Guikema, J.A.) "The interaction of microgravity and ethylene on soybean growth and metabolism." Presented to the 12th Man In Space Symposium, Washington DC (June 11, 1997).

Brown, C.S., Sanwo, M.M., Hilaire, E., Guikema, J.A., Stryjewski, E.C., and Piastuch, W.C. "Starch metabolism and ethylene production in space-grown soybean seedlings." (Abstract) ASGSB Bull., 10, 34 (1996).

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Brown, C.S., Sanwo, M.M., Stryjewski, E.C., Peterson, B.V., Johnson, C.F., Piastuch, W.C., Hilaire, E., and Guikema, J.A. "Carbohydrate metabolism and growth in space-grown soybean." (Abstract) Plant Physiol., 114(3), 91 (1997).

Brown, D. and Muday, G. "Testing the hypothesis that flavonoids rfegulate auxin transport in vivo." (Abstract) 8th International Conference on Arabidopsis Research. 4-8. (1997).

Brown, D.E., Reed, R.C., and Muday, G.K. "Polar auxin transport and gravitropism in Arabidopsis thaliana" (Abstract) ASGSB Bull., 10(1), 38 (1996).

Butler, J.A., Hu, S., Brady, S., Dixon, M.W., and Muday, G.K. "In vitro and in vivo evidence for actin association of the NPA binding protein." Plant J. (In Press).

Carter, C.N. and Allen, N.S. "Localization of putative L-type calcium channels in living higher plant cells using the fluorescent dye DM-BODIPY DHP." (Abstract) Plant Physiol., 114(3), 275 (1997).

Collings, D. (co-authors - Winter, H. and Allen, N.S.) "Cytoskeletal organization in gravistimulated maize stems." New England Society for Microscopy. Woods Hole, MA (May, 1997).

Collings, D., Winter, H., Huber, J.L., and Allen, N.S. "Structural analysis of gravistimulated maize stems." (Abstract) Plant Physiol., 114(3), 86 (1997).

Croxdale, J.G., Cook, M.E., Tibbitts, T.W., Brown, C.S., and Wheeler, R.M. "Structural aspects of potato tubers formed in space." (Abstract) ASGSB Bull., 10(1), 13 (1996).

Croxdale, J.G., Cook, M., Tibbitts, T.W., Brown, C.S., and Wheeler, R.M. "Structure of potato tubers formed in microgravity." J. Exp. Bot. (In Press).

Davies, E. (invited seminar) Department of Botany, University of Washington, Seattle (1997).

Davies, E. (invited speaker) University of California-Riverside Symposium on Post-trancriptional Regulation of Gene Expression (1997).

Davies, E. (plenary speaker) Molecular Biology of Plants under Environmental Stress, Poznan, Poland (1997).

Heilmann, I., Gross, W., and Boss, W.F. "Polyphosphoinositide metabolism in Galdieria sulphuraria." (Abstract) Plant Physiol., 114(3), 268 (1997).

Holzwarth, G., Webb, S.J., Kubinski, D.J., and Allen, N.S. "Improving DIC microscopy with polarization modulation." J. Microscopy (In Press).

Holzwarth, G., Webb, S.J., Kubinski, D.J., and Allen, N.S. "Improving DIC microscopy with polarization modulation." (Abstract) Mol. Biol. Cell, 7, 158A (1996).

Hu, S., Dixon, M.L., and Muday, G.K. "Development of a procedure to depolymerize the actin cytoskeleton for purification of associated proteins." (Abstract) ASGSB Bull., 10(1), 27 (1996).

Huber, J.L., Winter, H. and Huber, S.C. "Do Ca4* and protein phosphorylation play a role in the gravitropic response?" (Abstract) Plant Physiol., 114(3), 272 (1997).

Johnson, K.M., Brown, C.S., and Piastuch, W.C. "Examination of the expression patterns of genes involved in carbon metabolism in soybean seedlings grown in space." (Abstract) Plant Physiol., 114(3), 253 (1997).

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Luciano, R.L. and Muday, G.K. "Characterization of gravity dependent growth in Fucus zygotes." (Abstract) ASGSB Bull., 10(1), 26 (1996).

MacKay, J., O'Malley, D.M., Presnell, T., Booker, F.L., Campbell, M.M., Whetten, R.W., and Sederoff, R.R. "Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase." PNAS, 94, 8255-8260 (1997).

Patent Approved, U. S. Patent #: Undetermined. MacKay, O'Malley, Whetten, and Sederoff. "Method of altering lignin in trees."

Muday, G.K. "The role of auxin transport and the actin cytoskeleton in plant gravity response." Joint NSF/NASA NSCORT Meeting, Kennedy Space Center, FL (February 17, 1997).

Muday, G.K. and Reed, R.C. "Inhibition of auxin novement from the shoot into the root inhibits lateral root development in wild-type Arabidopsis thaliana and alf l- l ." (Abstract) 8th Intl. Conf. on Arabidopsis Res. 4:41 (1997).

Muday, G.K., Butler, J.H., Hu, S., Brady, S., and Dixon, M.W. "Actin association of the naphthylphthalamic acid binding protein from zucchini hypocotyls." (Abstract) ASGSB Bull., 10(1), 55 (1996).

O'Malley, D.M., MacKay, J.J., Whetten, R., Campbell, M., Liu, B-H., and Sederoff, R.R. "Genomic approaches to manipulating lignin composition and amount in wood." TAPPI Proceedings pp. 105-109 (1996).

Perera, I.Y., Stevenson, J.M., and Boss, W.F. "Investigation of phosphoinositide metabolism in gravistimulated maize pulvini." (Abstract) Plant Physiol., 114(3), 284 (1997).

Ralph, J., MacKay, J., Hatfield, R., Whetten, R.W., O'Malley, D.M., and Sederoff, R.R. "Abnormal lignin in a loblolly pine mutant." Science, 277, 235-239 (1997).

Ruegger, M., Dewey, E., Hobbie, L., Brown, D., Bernasconi, P., Turner, J., Muday, G., and Estelle, M. "Reduced NPA-binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects." Plant Cell, 9, 745-757 (1997).

Scott, A.C. and Allen, N.S. "The effect of carbonylcyanide m-chlorophenylhydrazone (CCCP) application on the gravitropic response in roots and stamen hairs." (Abstract) ASGSB Bull., 10(1), 17 (1997).

Sederoff, R.R. Sigma Xi invited lecture, BASF, Research Triangle Park, NC (1997).

Sederoff, R.R. Workshop presentation, Agricultural Biotechnology, Uppsala, Sweden (1997).

Sederoff, R.R. (invited seminars) Horticultural Research, Auckland, New Zealand (1996); Forestry Research Institute, Rotorua, New Zealand (1996); Department of Chemistry, University of Ohio (1997); ForBio Research Ltd. Brisbane (1997); University of Arizona, Tucson (1997); University of Stellenbosch, South Africa (1997).

Sederoff, R.R. (invited speaker) Forest Tree Workshop, Plant & Animal Genome 5 (1996); University of Chicago, Genetics Minisymposium (1997); Institute of Paper Science and Technology, Atlanta, GA (1997); Chinese Academy of Forestry, Beijing, China (1997); Nanjing Forestry Universty, Nanjing, China (1997); Presidents Circle, National Academy, Woods Hole, MA (1997); Molecular Genetics of Forest Trees, IUFRO, Quebec (1997); Internat. Soc. Plant Molecular Biology, Congress in Singapore (1997).

Sederoff, R.R. (symposium speaker) Swedish Plant Physiology Society, Uppsala (1997); Amer. Assoc. Plant Physiology, Vancouver (1997); Eucalypt: IUFRO meeting, Salvador, Brazil (1997); Forest Biotechnology Conf., Blomfontein, South Africa (1997).

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Tibbitts, T.W., Croxdale, J.G., Brown, C.S., and Wheeler, R.M. "Potato tuber growth and starch accumulation in space." (Abstract) ASGSB Bull., 10(1), 28 (1996).

Vian, A., Henry-Vian, C„ and Davies, E. "Molecular analysis in tomato of stree related genes in relation to electrical signal." (Abstract) Plant Physiol., 114(3), 278 (1997).

Winter, H., Huber, J.L., and Huber, S.C. "Sucrose metabolism during gravity induced cell elongation." (Abstract) Plant Physiol., 114(3), 143 (1997).

Winter, H., Huber, J.L., Collings, D., Allen, N.S., and Huber, S.C. "Carbohydrate metabolism in graviresponding maize pulvini." (Abstract) ASGSB Bull., 10(l):extra (1996).

Wyatt, S.E. "Gravity, calcium, signalling & Arabidopsis." Plant Physiology Seminar, Dept. of Botany, Duke University, Durham, NC (March, 1997).

Wyatt, S.E., Brown, D., Robertson, D., and Muday, G. "The gravitropic response of the inflorescence stems of Arabidopsis(Abstract) Plant Physiol., 114(3), 133 (1997).

Wyatt, S.E., Tsou, P-L., and Robertson, D. "Manipulation of calcium homeostasis with calreticulin." (Abstract) Plant Physiol., 114(3), 274 (1997).

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The NCSU-NSCORT is a t e a m of sc ien t i s t s f r o m N.C. S t a t e Universi ty and Wake Forest Universi ty .

M e m b e r s of the NCSU-NSCORT a r e listed below. Click on the i r n a m e for m o r e in format ion .

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JOAN L. HUBER 1996-1998

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GRADUATE STUDENTS

SANDRA ANTE, MS 2000

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Julie Bates Chad B r e n n e m a n La Shun Carroll Crystal Car t e r Jenny Chang Linda Cowan Tracey Crans ton Johna than Earp Julie Eledah Reathal Geary S t ephan ie Gupton Hea the r Hinkle Emily Holton Shiquan Hu J a m i e Huang R'amon Jacobs Melissa Keller Cass ie Larrieux Tad Leeds Marcus Lewis Randy Luciano

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