John Tallman - US grants

This application focuses upon the purification and characterization of the complex of proteins involved in the psychopharmacological actions of the benzodiazepines. Using a benzodiazepine (3-aminoclonazepam), which can be coupled to a solid matrix, a method for the purification of the benzodiazepine receptor protein is being developed. Along with the benzodiazepine receptor, it is possible to copurify both high and intermediate affinity binding sites for Gamma-aminobutyric acid. The kinetics, stoichiometry and molecular properties of these sites are investigated. Monoclonal antibodies against the purified complex are being prepared and several methods for detecting different properties of these antibodies are being developed. The physical locus of action of these antibodies will be compared to their pharmacological activities with the goal of defining the proximity of the sites of action of various drugs. The purified proteins will be reconstituted into a model system to directly study their action. It is expected that by a careful examination of the biochemical parameters of receptor function, greater reliability in predicting the actions of drugs which act at these sites can be made at a preclinical level. Changes (desensitization) in the complex due to chronic benzodiazepine administration or overactivity of GABA will also be studied in the model membrane system. By studying the molecular structure of the GABA-benzodiazepine receptor complex, we expect to gain greater insight into the pharmacology of psychoactive compounds such as diazepam, chlodiazepoxide and flurazepam. These compounds are among the most widely prescribed drugs in the world for the treatment of generalized anxiety and sleep disorders. Our studies will provide insights into the etiology of these conditions and the development of more effective therapy.

The most obvious signs of plant senescence are the withering and eventual death of flowers, fruits, leaves and roots. Senescence is regulated by complex processes involving environmental, physiological and genetic factors whose mechanisms are incompletely known. In this project, a unique model will be developed for the study of plant senescence, with the eventual goal of elucidating the molecular mechanisms. Plant senescence is important to the overall economy of the plant, fruit and grain ripening, crop production, and post-harvest storage of crops. Chloroplasts in mesophyll cells of Nicotiana glauca lose functionality during leaf senescence, but those in stomatal guard cells do not. In this project, mesophyll and guard cells will be cultured in ways that preserve the unique properties of the differentiated cell types. To optimize cell survival and preservation of physiological properties unique to each cell type, a variety of nutrient, osmotic, pH, light, and cell density conditions will be tested. Once cultures are established, conditions will be identified for fusing protoplasts from the two cell types and for culturing fusion hybrids. The functionality of guard cell chloroplasts in fusion hybrids will be monitored by microfluorospectrophotometry to determine whether senescing mesophyll cells contain factors capable of initiating guard cell chloroplast senescence.***//

The long-term objectives of this research are to understand the molecular basis of the actions of benzodiazepines and related compounds which act at the GABA/benzodiazepine receptor complex. These drugs are among the most widely prescribed compounds for the treatment of anxiety and sleep disturbances. They are also used as antiepileptic agents. The approach used in this proposal has been to isolate the proteins which are part of the GABA- benzodiazepine receptor complex, prepare antibodies to these proteins and identify the proteins biochemically and immunocytochemically. In addition, molecular probes to the and delta beta subunits of GABA/benzodiazepine receptor and other receptor molecules are being prepared to identify mRNA in cultured cells and from tissue for these receptors. The match between synthesis of mRNA and receptor number under conditions where receptor synthesis is induced and during development is to be studied. The effect of chronic administration of anxiolytics and antidepressants on receptor dynamics will be studied by comparing mRNA and receptor synthesis. The techniques to be used for mRNA determination are northern blotting and the levels of mRNA are quantitated with radiolabeled probes. ln this way, a clear relationship between receptor turnover and drug action will be established. The correlation of these changes with the therapeutic action of these drugs and their use in anxiety disorders and depression will be examined.

This award provides funds to the Natural Science Division at Pepperdine to continue a successful REU Site. The funds will enhance an already active program of undergraduate research in biology. Students will be taught the common elements of the research process and increase their appreciation for the diversity of biological subdisciplines. In addition, the program provides mechanisms for recruiting students from traditionally underrepresented groups in science and from schools where students may not have opportunities to experience biological research. Each summer the program will begin with a six-day research orientation workshop at the Murchison Science Center during which students will be introduced to the uses and limitations of specific research tools and techniques and immersed in the research process (literature review, hypothesis formulation, and hypothesis testing). After the orientation workshop, students will pursue individual research projects under the direction of faculty in either cellular biochemistry, behavioral ecology, plant physiological ecology or ecophysiology of marine intertidal animals. All research projects will be designed to provide students with insight into how scientists formulate research questions, design experiments, collect and analyze data, and communicate their results to the scientific community. Over the course of the summer, visiting scientists will hold special research seminars in each research area, giving students an opportunity to discuss their own data with other scientists. Each research group will hold weekly literature seminars to discuss current journal articles pertaining to their research interests. The program will conclude each summer with a student research symposium in early August. Follow-through will include student participation at local and regional undergraduate research conferences. In cases of special merit, students will be encouraged to present their results at national meetings and to prepare their results for publication in a scientific journal.

Stomata are biological valves in the aerial organs of plants that regulate simultaneously photosynthesis and transpiration. The opening and closing of stomata are affected by the shrinking or swelling of "guard cells" which form their edges. The chloroplasts of the guard cells that surround the stomatal pore do not senesce (yellow) at the same time as those in mesophyll tissue of the same leaf. The long range goal of the project is to culture stomatal guard cells in ways that preserve their unique physiological properties, including the functionality of their chloroplasts. Such cultures could be used to study the induction and regulation of chloroplast senescence. This laboratory has established protoplasts of stomatal guard cells of Nicotiana glauca in culture. However, optimal conditions for cell survival have not yet been identified. A variety of culture conditions will be evaluated to identify those that are optimal for cell survival. To determine whether any of the unique properties of guard cells are maintained in culture, some of the physiological and biochemical properties of freshly isolated guard cell protoplasts will be compared to those of protoplasts of cultured cells. The same experiments will be performed with guard cell protoplasts from senescing (chlorotic) leaves: 1) to evaluate their viability relative to those of guard cells of non-senescing leaves, and 2) to determine whether there are any differences in the properties of guard cells of the two types of leaves that might explain why the stomata of senescing leaves fail to open in response to light. This research is directed at developing techniques for culturing stomatal guard cells from leaves under conditions that not only permit their survival but also maintain their differentiated properties. The availability of such cultured cells would greatly facilitate further research on the physiological function of these cells which regulate the passage of water and gases across the surfaces of leaves. Since the chlorophyll-containing organelles, chloroplasts, of guard cells behave differently from the chloroplasts of other cells under some physiological conditions, these cells would provide an excellent model system in which to study the regulation of the functional state of chloroplasts. Since this research will be conducted at an undergraduate institution it will also provide an excellent opportunity for undergraduate students to gain first hand experience in research in plant cell biology.

MCB-9417761 Tallman The long-term objective of the project is to elucidate the molecular mechanisms that regulate senscence of cells and chloroplasts of plant leaves. Guard cells which flank stomata fail to undergo a senescence (aging) process like that which takes place in cells of the underlying tissue (mesophyll). Thus a system to culture individual guard cells has been developed to study the induction and regulation of senescence. Guard cells protoplasts (GCP, guard cells isolated with cell walls removed) have been successfully cultured for a week at 36-40o C, but lower temperatures were not effective at maintaining the guard cells as guard cells, but allowed redifferentiation and callus formation. The following model is suggested to account for the temperature sensitivity: 1) At lower temperatures (24-34oC) the plant hormone auxin induces synthesis of ethylene. 2) Ethylene synthesis results in cellular senescence or a growth response that results in callus formation. 3) Ethylene synthesis is inhibited at higher temperatures. 4) At higher temperatures, abscisic acid (ABA) counteracts the auxin/cytokinin response, thereby preventing redifferentiation of GCP. To test this model the following experiments are done: 1) The GCP are cultured at the higher temperature in ABA to test if ABA itself could induce dedifferentiation. 2) Examine the levels of ethylene produced by GCP at lower temperatures. 3) Test whether other conditions which which suppress ethylene action also allow the GCP to maintain a non-senescent state. This is a project supported through the Research at Undergraduate Institutions (RUI) program. Over the prior award period of three years there were 26 undergraduates involved in research. A similar level of involvement is expected in this award. %%% The long-term objective the project is to elucidate the molecular mechanisms that regulate the decline during aging (senescence) of cells and chloroplasts of plant leaves. Guard cells wh ich flank stomata fail to undergo an aging process like that which takes place in cells of the underlying tissue (mesophyll). Thus a system to culture individual guard cells has been developed to study the induction and regulation of senescence. In this set-up, guard cells protoplasts (GCP, guard cells isolated with cell walls removed) are successfully cultured for fairly long periods (days) at elevated temperatures (36-40o C). Lower temperatures are not effective at maintaining the guard cells as guard cells, but allow redifferentiation and general cell growth or callus formation. The hypothesis is that the plant growth regulator, ethylene, causes the cells to senesce at lower temperatures, but is not produced by the guard cells at higher tempertures. Also at lower temperatures, other growth regulators like auxin and cytokinin are active, resulting in higher growth rates and cellular dedifferentiation. These hypotheses are tested by measuring the levels of ethylene produced by the cells at different temperatures and by examining if exogeneously added growth regulators such as abscisic acid interfere with the aging process. The study leads to a better understanding of why guard cells fail to senesce and the mechnisms that induce and regulate senescence in plant cells. ***

Stomata are microscopic pores in the otherwise gas-impermeable surface of leaves. Each stomatal pore (stoma) is flanked by two opposing guard cells that change in shape in response to environmental signals (e.g. light quality and intensity, intercellular leaf carbon dioxide levels, and apoplastic concentrations of abscisic acid [ABA]). The shape changes of guard cells allow the dimensions of each stoma to be varied as an integrated response to prevailing environmental conditions, thus regulating the rate of gas exchange between leaves and the air, and, therefore, rates of transpiration and photosynthesis. A great deal is known about the guard cell signal transduction mechanisms that result in stomatal changes. Because guard cells are highly specialized to transduce such signals, historically they have been considered among the most highly differentiated plant cell types. Recent experiments have shown, however, that in response to hormonal signals, guard cell protoplasts (GCP) cultured at 32C can reenter the cell cycle, de-differentiate, and divide to produce callus from which plants can be regenerated; thus guard cells are totipotent. These data suggest that guard cells are not as far from a meristematic state as was once thought, and that like other plant cells, they must be actively maintained in their differentiated state. Little is known about the signal transduction pathways or genes that might be involved in maintaining guard cells in their differentiated state in situ. Elevated temperature and ABA are the only signals required for maintenance of cultured GCP in their differentiated state in vitro. Several lines of evidence suggest a model where heat shock proteins (HSP) might be involved in maintaining the differentiated state. The working hypothesis is that HSP inhibit hormone-dependent re-entry of cultured GCP into the cell cycle by limiting translation of the transcripts induced by hormones. More specifically, it is hypothesized that at 38C activation of the heat shock transcription factor HSF results in production of HSP that reduce production of cyclin-dependent kinases and cyclin D3. Because at 32C inhibitors of ethylene synthesis also prevent cultured GCP from completing the cell cycle, it is further hypothesized that at 32C the plant hormone auxin stimulates the production of aminocyclopropane-1-carboxylic acid (ACC) synthase and/or ACC oxidase, resulting in increased production of ethylene. It is envisaged that ethylene then acts through a two-component regulatory system to activate a MAPK pathway that is required for cells to re-enter and/or complete the cell cycle. Following this basic hypothesis, it would be expected that transcripts for ACC synthase and ACC oxidase are either not produced or not translated at 38C and that MAPK is, therefore, not activated at 38C. The initial experiments to be conducted are aimed at elucidating the primary characterisctics of the responses of guard cells to elevated temperature. Among the characteristics to be studied are: 1) the effects of the length of exposure to 32C and 38C on the capacity of GCP to re-enter the cell cycle; 2) an analysis of the reversibility/inter-convertiblility of cell fates; 3) whether other conditions such as nutrient starvation can mimic the effects of cuture at 38C; and 4) the point in the cell cycle to which GCP cultured at 38C are able to proceed. Further experiments aimed at exploring the plausibility of the model described will include investigation of: 1) whether genes for cyclin-dependent kinase homologs, cyclin D3, ACC synthase, ACC oxidase, and MAPK are transcribed at 32C, at 38C, or both; 2) whether known HSP are produced at 38C, but not at 32C; 3) whether kinetics of HSP production are correlated with kinetics of loss of responsiveness of cultured GCP to auxin and/or cytokinin; and 4) whether MAPK is activated by an ethylene-dependent mechanism. Investigation of the proteins required to maintain plant cells in a differentiated state could provide insight into the question of a universal mechanism involving a small number of expressed proteins that maintain differentiated states in multicellular organisms. Such studies could also tell us whether the expression of these proteins makes some plant cells recalcitrant to culture. If marker proteins for GCP maintained in the differentiated state can be identified, it should eventually make it possible to clone the genes for these proteins such that they can be expressed in plant tissues under the control of selected promoters for control of plant cell differentiation in response to various environmental conditions.

A computerized physiology laboratory is supporting investigative, inquiry-based learning in a required, sophomore-level introductory core biology course, Physiological Dynamics in Animals and Plants (PDAP). The course provides students with an understanding of how divergent animal and plant physiological systems with similar adaptive functions possess a unity of form and function at the cellular level. It is adapted from an existing lab course that was developed at Harvey Mudd College. Signal transduction is used as a paradigm to unify plant and animal physiology. The laboratory is the first in a three-year sequence of experiences to develop students' scientific process skills in preparation for a capstone senior research thesis and introduces students to important data acquisition and communication technologies. For the first six weeks, students employ Biopac and Qubit systems in a series of animal and plant laboratory exercises of increasing technical sophistication. In a weekly recitation session, students learn to use web-based course management software for peer review and develop proposals for a research project. Two-student teams then carry out experimental investigations of their own design, reporting weekly to professors and peer review teams through an "electronic lab notebook. " In the final two weeks students participate in a "mini-symposia." Expected student learning outcomes are being assessed longitudinally with the help of a professional consultant. Results and course materials are being disseminated through presentations at professional meetings, articles in pedagogical journals, and through the course web site. It is expected that students will acquire the ability to design and interpret physiological studies, that they will develop an appreciation for parallels between animal and plant systems, and that the program will increase student participation in physiology. Broader impacts: The grant is serving a diverse K-12 population and enhance teacher preparation by providing equipment to outreach programs that train teachers in technology or employ biology majors as mentors in K-12 programs.

Many perennial plants that live in hot climates have evolved to survive heat extremes. It is assumed that the seemingly modest temperature increases of 5-6ºC predicted to accompany the next century of global climate change will have little effect on these plants. However, research on the effects of such temperature increases on plant cells growing near their thermal survival limits suggests otherwise, and has led to this project. Plants grow by the division of existing cells, and then enlarging the smaller, newly-formed cells. It has been shown that 5-6ºC temperature increases block the action of a critical growth hormone, auxin, that at lower temperatures activates genes required for plant cells to divide and grow. In this project it will be determined whether 5-6ºC temperature increases block auxin's ability to activate genes that control cell division and growth in intact plants. It will also be examined whether temperature increases block auxin action by inhibiting one particular enzyme called a Rac GTPase, an important protein in the chain of events that leads to the auxin-activated responses.

Broader Impacts:
This research will be conducted in the biology department of a small undergraduate college in which faculty members and students participate together in research. This project will encourage undergraduates to pursue research careers by: (1) recruiting and training two students each year to conduct research; (2) developing collaborative research projects between students; (3) creating a student-led science reading group; and (4) arranging student-hosted visits by prominent scientists who will consult with students and faculty on research. A regional faculty workshop will be held on the topic of integrating research into undergraduate teaching. This study has implications for conservation management of native, heat-tolerant plants and the animals that may depend on them. It may also find application in the development of strategies to genetically engineer heat-tolerant perennial crops and ornamentals.

Six research-productive faculty members from the biology, chemistry, and physics departments at Willamette University will purchase, install, maintain, and manage a shared Zeiss LSM-710 confocal microscope to add essential functionality to Willamette?s Core Imaging Facility and advance multidisciplinary research in the cellular, molecular, and biophysical sciences. Confocal microscopy is the method of choice when sensitive, high resolution, three-dimensional, time-resolved imaging is required to unambiguously localize and monitor simultaneous dynamic changes in cellular molecules, structures, and processes. Research by the PI and co-PIs requires submicron axial resolution, elimination of background out-of-focus light, and 3-D reconstruction for precise localization and accurate quantification of sub-cellular molecules. Spectral un-mixing of the signals is required for several investigators who work with dyes that emit at wavelengths close to that of specimen background (PI Coddington and co-PIs Fisher, Tallman). Further, the work of co-PIs Altman, Duncan, and Tallman requires imaging of sub-cellular protein movements in live cells and/or tissues. This necessitates outfitting the LSCM system with Definite Focus, allowing imaging of single proteins over extended time-scales without stage drift in the z-axis. Drs. Tallman and Altman?s research further requires cells and tissues to be maintained at precise temperatures and pH, which necessitates the environmental module extra to the stage. Thus, the specific capabilities that are required are rapid speed of acquisition; live cell and tissue imaging through time series analysis; laser power for photo bleaching purposes; and spectral separation capabilities. In summary, the Zeiss LSM 710 mounted on an inverted Axio Observer Z1 microscope optimally meets our research and training needs.

The NSF award is used to purchase a confocal microscope for six faculty members in the Biology, Chemistry and Physics departments at Willamette University, a primarily undergraduate institution. Three of the six co-PI?s are women, and four are pre-tenure junior faculty; all of whom serve a strongly female undergraduate student body in biological and STEM fields. Acquisition of this equipment allows Willamette researchers to answer a variety of highly relevant research questions including: how stress hormones regulate animal behavior; how genes and proteins control nervous system development and function; how pesticides interfere with animal development; and how heat stress and hormones control plant growth and reproduction. The microscope accelerates our faculty research programs to the level of disciplinary recognition required to sustain and/or compete for other external research grants. We also realize broader gains as the microscope becomes part of a larger imaging facility available for research use by faculty members at other small colleges in the area and a fulcrum for interdisciplinary and collaborative research. This state-of-the-art instrument attracts highly motivated undergraduates to science careers and provides opportunities for high-impact research training of undergraduates bound for graduate school and for immediate employment in the technical workforce.