Senescence in plants is a genetically controlled developmental event that enables the plant to eliminate individual cells, tissues, organs and even the entire plant in order to adapt to change in environmental conditions, reproductive needs, or pathogenic attack. An integral part of the cell death occurring during senescence is the selective degradation of proteins. Proteins may be degraded via the activity of several classes of enzymes, characterized by the different amino acids present at their catalytic sites, known as proteinases. Alternatively, protein degradation in eukaryotic cells can be accomplished through the action of an ATP dependent pathway known as the ubiquitin system, which is capable of selecting specific proteins for degradation by creating ubiquitin-protein conjugates that are then hydrolyzed by the 26S proteasome, a multisubunit proteinase. As a first step in determining whether the ubiquitin pathway was involved in the death of cells during vascular tissue development, experiments were conducted to localize the presence of ubiquitin and ubiquitin-protein conjugates to differentiating vascular tissues. Immunolabeling on tissue prints from cross sections of Phaseolus vulgaris petioles, Gossypium hirsutum hypocotyls and Coleus x hybridus internodes showed accumulation of ubiquitin-protein conjugates in regions of vascular tissues. Immunohistochemical labeling confirmed the presence of elevated levels of ubiquitin-protein conjugates in differentiating and regenerating xylem tissues of Coleus. In addition, labeling of ubiquitin-protein conjugates was observed in parenchymatous tissues and elevated levels of ubiquitin-protein conjugates was also observed in vascular cambia. The senescence of daylily (Hemerocallis hybrid cv Stella d'Oro) petals is accompanied by a rapid decline in protein over a 24 h time period. To determine how this loss of protein is regulated we conducted experiments to characterize and measure the activity of several classes proteinases. Treatment with class specific inhibitors indicated the presence of serine, cysteine, and metalloproteinases. The activity of these proteinases increased after flower opening and this increase in activity could be eliminated by treatment with the protein synthesis inhibitor cycloheximide. Ion leakage was delayed by treatment with inhibitors of the 26S proteasome, suggesting a link between protein hydrolysis and membrane permeability. Immunoblots labeling ubiquitin-protein conjugates and ubiquitin system enzymes revealed that overall profiles of ubiquitinated proteins do not change during senescence but that several enzymes of the ubiquitin system decline after flower opening. Northern analysis showed slight accumulation polyubiquitin message during petal senescence. We propose that senescence in daylily petals is controlled in part by the appearance of at least three classes of proteinases which may work in concert with the ubiquitin system.

A pollen tube is the cell that navigates sperm cells through the style and delivers them to the ovum, in a rapid and highly-polarized fashion. Underlying the process of pollen tube growth are a dynamic actin cytoskeleton and oscillating gradients and fluxes of ions. The work presented in this dissertation focuses on visualzing the actin cytoskeleton and gauging calcium dynamics in growing pollen tubes. Three different GFP-labeled actin-binding domains are used to probe the actin cytoskeleton of growing Lilium and Nicotiana pollen tubes. Each marker highlights different aspects of the actin cytoskeleton, whereas no single marker reveals the entire spectrum of actin present. Thus, GFP-ADF and GFP-talin reveal elements of the cortical fringe of actin, whereas GFP-fimbrin generously labels actin filaments in the shank of the pollen tube. Because high levels of expression inhibit pollen tube growth and cause structural aberrations of the actin cytoskeleton, I urge caution in their use. A steep tip-focused gradient of calcium that oscillates with the same periodicity as growth rate, is a consistent feature of growing pollen tubes. Conventional methods for monitoring calcium in growing pollen tubes rely on microinjection of fluorescent dyes, a procedure that is both invasive and technically challenging in the small pollen tubes of the model species Nicotiana tabacum and Arabidopsis thalaiana . Here, two transformable calcium indicators, yellow cameleon YC2.1 and ratiometric pericam, are introduced as effective indicators of cytoplasmic calcium in growing pollen tubes. Finally, mag-fura 2 is employed to measure the calcium concentration in the lumen of the endoplasmic reticulum ([Ca 2+]L) of growing Lilium formosanum pollen tubes. I provide evidence that calcium ions are periodically withdrawn from the apex and funneled into the endoplasmic reticulum. I thus support the hypothesis that the endoplasmic reticulum sequesters calcium in the clear zone and thereby confines the tip-focused gradient to the apex. The endoplasmic reticulum therefore emerges as a potential key player in the process of pollen tube growth.

Fluorescent ratiometric imaging and spectrofluorometric analysis was used to study two signal transduction mechanisms in the stamen hair cells of Tradescantia virginiana. The first study determined the metabolic pathway necessary for the inactivation of Inositol 1,4,5-trisphosphate mediated calcium release from intracellular stores in the living plant cell. Tradescantia stamen hair cells, preloaded with the calcium sensitive ratiometric dye fura-2-dextran, were injected with analogs of Inositol 1,4,5-trisphosphate and cytosolic calcium levels monitored by ratiometric imaging. The injected analogs were selected due to their insensitivity to various kinases and phosphatases for which Inositol 1,4,5-trisphosphate is a substrate. We determined that the 5-phosphatase is the preferred pathway for inactivation of Inositol 1,4,5-trisphosphate in the living plant cell. The second study investigated cyclic ADP-ribose mediated calcium release in the intact plant cell and determined the presence of the metabolic machinery necessary to synthesize cyclic ADP-ribose from its precursor NAD+. Cyclic ADP-ribose was observed to cause calcium release in the stamen hair cells of Tradescantia that were preloaded with the calcium sensitive dye fura-2-dextran. Evidence of cyclic ADP-ribose synthesis was determined using two experimental techniques. Homogenates of the sea urchin Lytechnicus piclus were used as bioassays to detect cyclic ADP-ribose in extracts of Tradescantia stamen hair cells that were incubated with [special characters omitted]NAD+. Cyclic ADP-ribose synthesis was detected from fluorimetric analysis of the homogenate as the calcium sensitive dye fluo-3 was present in the homogenate to detect cyclic ADP-ribose mediated calcium release from sea urchin egg microsomes. We also determined cyclic ADP-ribose synthesis by injection of fura-2-dextran loaded stamen hair cells with [special characters omitted]NAD+ and observing a delayed calcium increase in the cytosol. These results establish the metabolic fate of inositol 1,4,5-trisphosphate in plant cells and demonstrate the biochemical capability for plant cells to synthesize cyclic ADP-ribose to mediate calcium release in plants.

The roles of the calcium binding messenger protein calmodulin and the kinesin-like calmodulin binding protein during cell division in plants were investigated employing microinjection of various probes in Tradescantia virginiana stamen hair cells. Fluorescently labeled calmodulin was found to distribute uniformly throughout the cytoplasm, and was not specifically colocalized with the microtubules of the dividing cell as has been reported earlier. Injected calmodulin redistributed during fixation as if for immunolocalization, suggesting that earlier reports were based on artifactual localizations, and consequently that the role of calmodulin in cell division should be reevaluated. Inhibition of calmodulin using a synthetic calmodulin binding peptide, prevented the actomyosin based cytoplasmic streaming and the late lateral expansion phase of cytokinesis, but did not affect the architecture of cytoplasmic strands or anaphase chromosome motion. Thus, although calmodulin is not localized specifically to the phragmoplast, it does play a regulatory role during cytokinesis. Microinjection of antibodies to the kinesin-like calmodulin binding protein (KCBP) that constitutively activate this microtubule motor protein, caused precocious nuclear envelope breakdown, metaphase arrest, and delays in cytokinesis. However, the antibodies did not inhibit chromosome motion. These results, in combination with earlier characterization of this kinesin, lead to the conclusion that KCBP is differentially involved in forming a converging bipolar spindle. Interfering with its regulation affects the dynamic transformations of the microtubule cytoskeleton between these phases. The results establish a regulatory role for calmodulin in cell division and address two possible downstream signaling pathways: inhibition of myosins involved in the lateral expansion of the phragmoplast, and inhibition of the spindle forming kinesin-like calmodulin binding protein.

The submersed aquatic plants Eurasian watermilfoil (Myriophyllum spicatum) and Hydrilla (Hydrilla verticillata) are a nuisance in waterways of the U.S. Biological control with plant pathogens is a proven method for the management of aquatic plants. An attempt was made to identify lectins of Eurasian watermilfoil and Hydrilla tissues in hopes of characterizing a means of specific attachment to fungal pathogens on the target plants. Four fungal isolates (Fusarium roseum, Macrophomena phaseolina, Colletotrichum gloeosporioides and Mycoleptodiscus terrestris) were evaluated for the ability to attach and infect Eurasian watermilfoil and Hydrilla. Lectins were isolated from total protein by affinity chromatography. Lectin activity was evaluated for agglutination of the human ABO blood groups and fungal mycelium. Eurasian watermilfoil lectin was evaluated for its ability to inhibit fungal growth. No specific attachment was detected on Hydrilla tissues. F. roseum attached specifically to Eurasian watermilfoil tissues. All four fungi were pathogenic on Hydrilla tissues after seven days in test tube bioassays. All but F. roseum were pathogenic on Eurasian watermilfoil. Lectins specific for $\alpha$-L-fucose were isolated from both Eurasian watermilfoil and Hydrilla. The Eurasian lectin has a molecular weight of 48 Kd. The Hydrilla lectin is a complex of two proteins with molecular weights of 67 Kd and 50 Kd. Both Eurasian and Hydrilla lectins agglutinated type O red blood cells. Hydrilla lectin has no affect on mycelial suspensions of the four fungi, while Eurasian lectin agglutinated all fungi except M. phaseolina. The existence of lectins in the aquatic plants Hydrilla verticillata and Myriophyllum spicatum has been demonstrated. Plant lectins play a role in plant-pathogen association through attachment and/or recognition. Where attachment is specific and the lectin agglutinates the fungus, disease resistance occurs. This evidence supports the theory that recognition through plant lectins is a host defense mechanism.