The regulation of pH is a critical homeostatic function of plant cells. In addition to acting as the primary cationic species responsible for energizing the plasma membrane, protons likely act as an important regulator and messenger. Despite this importance, few studies have thoroughly described cytosolic pH patterns as the plant cell progresses through the cell cycle. To investigate pH in plant cells, I chose Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells as a model system. My research has two aims. First, I will measure and report the interphase cytosolic pH of BY-2 cells. Next, I will assay the cytosolic pH as BY-2 cells progress through mitosis and cytokinesis. I hypothesize that pH patterns are be temporally or spatially associated with structures such as the mitotic spindle or the phragmoplast. To investigate cytosolic pH in BY-2 cells, I will develop a cytosolic pH reporter based on a pH sensitive ratiometric fluorescent dye. This dye will be able to resolve both temporal and spatial changes in pH throughout the cytosol while imposing a minimal amount of stress on BY-2 cells. I found that pH-GFP, a variant of eGFP, had qualities of a robust pH reporter. To introduce the dye, explored biolistic bombardment, Agrobacterium mediated transient transformation, and polyethylene glycol mediated transformation as methods for introducing the pH-GFP gene into BY-2 cells. I observed very few transformation events using these methods and my observations did not support these approaches as suitable for introducing pH-GFP into BY-2 cells.Master of Science (M.S.
