Investigation of Brassinolide Hormone Contribution to Cold Tolerance in Arabidopsis thaliana
The circadian clock is a ~24-hour endogenous timekeeper conserved across kingdoms. The cyclic expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) is an important component of the Arabidopsis thaliana circadian clock. Although light signaling is the main stimulus which regulates CCA1 expression, other factors such as temperature and phytohormone exposure can change the expression pattern of this gene to alter clock parameters. Brassinosteroids are a class of phytohormones that play a crucial role in plant growth and development and have been shown to inhibit the expression of CCA1. Brassinosteroids have also been used to increase cold tolerance across various crop plants. Because there is an interest in improving the cold tolerance of crop plants through use of this phytohormone, our goal was to investigate how exogenous brassinosteroid application and low temperature affect the expression pattern of CCA1, and thus the endogenous clock. We visualized the expression patterns of the CCA1 promoter (CCA1pro) in response to brassinosteroid treatment and low temperature in A. thaliana seedlings using a luciferase reporter construct. These transgenic seedlings were treated with 0.1 µM or 100 µM of 24-epibrassinolide (EBR) or a control solution (water and low concentration ethanol). The expression of CCA1pro was analyzed under control or low temperature conditions (12/12 hr photocycle), in recovery after cold (12/12 hr photocycle), and in free-running constant light conditions after entrainment in either control or low temperature conditions. We determined that 0.1 µM EBR did not significantly change the expression patterns of CCA1pro, but low temperature did. Additionally, 100 µM EBR significantly dampened the amplitude of CCA1pro expression under constant light conditions. Overall, we demonstrated that EBR and temperature affect CCA1pro expression in A. thaliana. This result implies that changes in temperature and brassinosteroid exposure can both induce significant changes to the circadian clock of A. thaliana. This will be an important consideration as efforts to use brassinosteroids to ameliorate cold response in various crop types expand, as unintended consequences could arise from alteration of expression of the endogenous timekeeper.