RESEARCH 2002
RESEARCH 2001
RESEARCH 2000

E. Philip Lehman
Georgetown University
Washington, D.C.
Dr. Michael Schläppi, Mentor

Within the past five years, researchers identified a gene in the mustard plant Arabidopsis thaliana that is believed to increase a plant’s ability to withstand temperatures below the freezing point. Named EARLI1, the gene stably responds to vernalization and long-day photoperiods, and transiently to cold shock. Using transgenic A. thaliana plants infected by Agrobacteria and selected on kanamycin, it is possible to study the function and regulation of EARLI1.

It is hypothesized that the expression of the EARLI1 gene would alter the amount of damage sustained by a plant placed in sub-zero temperatures. This hypothesis can be tested via an ionic leakage assay, which calculates the total amount of a plant’s ions released to its surroundings upon being placed in a freezing environment. The overexpression construct contains a gene that confers resistance to the antibiotic kanamycin, and only seedlings that have been successfully genetically transplanted may grow on kanamycin-containing agar plates. Furthermore, another gene construct can be made to include the production of a protein, luciferase, whose presence can be confirmed via luminescence. Luciferase is produced under the control of the EARLI1 promoter and only appears when the gene for EARLI1 is transcribed. In a similar fashion, a construct with luciferase can be made for a gene, named SOC1, which encodes for a transcription factor. SOC1, like EARLI1, also responds to temperature and light.

When the ionic leakage assay was applied to wild type Columbia 0 plants, it was shown that decreased temperatures evoke larger degrees of ionic leakage. Plants acclimated in the cold (4?C) for at least one week exhibit significantly decreased amounts of leakage, suggesting the presence of cold naturally provides protection against freezing for a wild type plant. Thus, it was suggested that plants that contained overexpression of the EARLI1 gene might show less damage than their non-acclimated wild type plants at any given temperature. However, preliminary data from plants overexpressing EARLI1 displayed an increased leakage relative to wild type plants. 

The luciferase assay clearly demonstrated that the levels of EARLI1 are directly correlated with the presence of cold, and that varying levels of light also induce varying production of EARLI1 protein. In one experiment, plants placed in a 4?C refrigerator showed increasing levels of EARLI1 production over time, only to have EARLI1 levels decrease upon removing the plant from the cold. In a second experiment, long day (16h) SOC1 and EARLI1 lants resulted in target protein levels up to three times greater than their short day (8h) counterparts over a fourteen-day period. This provides evidence that the presence of cold and long day photoperiods are integral environmental factors responsible for the activation of the EARLI1 and SOC1 genes.