RUI: Vacuolar Transport of Allelochemical Conjugates During Defense Responses.

One of the main functions of the plant cell vacuole is to store allelochemicals. These allelochemicals act as a chemical defense mechanism that helps protect the cell from attacks by other organisms. Allelochemical production presents somewhat of a dilemma for the cell since in order to be effective the plant must produce these compounds in relatively high concentrations. However, since many of these allelochemicals are non-specific toxins, high concentrations in the cell may result in autotoxicity. The plant cells appear to have resolved this dilemma by first conjugating the allelochemical to a water-soluble molecule such as glutathione (GSH) or glucose and sequestering the conjugate in the vacuole. These conjugates may be recognized and transported into the vacuole by a subclass of ATP-binding cassette transporters known as multidrug resistance-associated proteins (MRPs). In order to provide more information regarding the vacuolar transport of allelochemicals, this project has three general objectives. Objective 1 is to determine if glucose or GSH conjugates of trans-cinnamic acid (CA), para-coumaric acid (PC) and salicylic acid (SA) are localized in the vacuole of Arabidopsis cells. This will be determined through in vivo labeling techniques and TLC/HPLC analysis of vacuolar contents. CA, PC and SA were chosen for this study because they are allelochemicals found in virtually all plants. Objective 2 is to develop a simple method for the synthesis of the glucose conjugates of SA, CA and PC. For these compounds, these are the most common conjugates found in plant cells and a facile, high-yield synthesis will provide useful substrates for detailed vacuolar transport studies. Objective 3 is to determine how the vacuolar transport of both glucose and GSH conjugates changes during a defense response. A defense response will be initiated in Arabidopsis cell suspension cultures by the addition of a fungal elicitor or known signal molecules of plant defense (e.g. SA, ethylene, jasmonate, superoxide, and H2O2). Arabidopsis cell suspension cultures will be used for this study because of the molecular tools available and the ease in which the treatments can be added. Changes in the transcription of Arabidopsis MRPs (AtMRPs) in response to the treatments will be followed through dot-blot analysis using the AtMRP expressed sequence tags as probes. Vacuolar transport activity using both glucose and GSH conjugates will be compared to the changes in AtMRP transcription and measured using isolated tonoplast vesicles from the treated cells. These changes in transport activity will also be compared to changes that occur in allelochemical production and conjugation during a defense response. If vacuolar sequestration of allelochemicals is an important aspect of a plant's defense response, then changes in allelochemical production and conjugation should be accompanied by changes in vacuolar transport. The results from these studies should provide considerable insight into the importance of vacuolar sequestration of allelochemicals during a plant cell's defense response. In the future, manipulation of vacuolar sequestration through biotechnology may be able to enhance the plants own natural defense mechanisms, which would have great agricultural significance with regard to pest control.