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.
