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Characterization of proteolytic events during senescence of vegetative and reproductive plant tissues

Paul Thompson Stephenson, University of Massachusetts Amherst


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.

Subject Area

Botany|Botany|Cellular biology

Recommended Citation

Stephenson, Paul Thompson, "Characterization of proteolytic events during senescence of vegetative and reproductive plant tissues" (1998). Doctoral Dissertations Available from Proquest. AAI9841925.