Functional Analysis of the BAS1 Gene and Its Product: CYP 72B1

Washington University, Saint Louis MO

Investigators

Abstract

Brassinosteroids are growth-promoting hormones involved in modulating plant plasticity in response to changes in the environment. Previous studies of brassinosteroids have concentrated on either the metabolic or reception pathways for these hormones. A novel gene affecting brassinosteroid responses in plants, CYP72B1, has recently been identified. This gene appears to be involved in brassinosteroid inactivation rather than biosynthesis or perception acting as a brassinosteroid hydroxylase that catabolizes the most active form of the hormone, brassinolide, into an inactive form, 26-hydroxybrassinolide. By regulating the active levels of brassinolide in certain tissues or at certain times, CYP72B1 may be acting as a modulator of brassinosteroid responses during plant development. One hypothesis is that CYP72B1 uses brassinolide as an enzyme substrate generating the less-active brassinosteroid, 26-hydroxybrassinolide. Heterologous expression of CYP72B1 in yeast coupled with feeding experiments using radiolabeled brassinolide will test whether CYP72B1 uses brassinolide as an enzyme substrate. Physiological analysis will also be used to further characterize 26-hydroxybrassinolide activity. A second hypothesis is that CYP72B1 expression affects growth in Arabidopsis by regulating brassinolide levels. A T-DNA knockout mutation in the CYP72B1 gene will allow a detailed analysis of the role this gene plays in development. A third hypothesis is that CYP72B1 activity is regulated transcriptionally and post translationally at the tissue specific level. RT-PCR experiments show that CYP72B1 transcripts accumulate greater in the rosettes vsersus the hypocotyls of developing seedlings. CYP72B1 transcript accumulation measurements will be expanded to many different tissues of both juvenile and adult plants. CYP72B1 translational fusions with reporter genes will also be used to examine CYP72B1 protein levels in different tissues during plant development. Together, this research will increase the understanding of CYP72B1's role in modulating brassinosteroid levels and perception during plant development. In addition to the training of one graduate student and one postdoctoral fellow, several undergraduates are likely to participate in this project. Findings from this research will be incorporated, when appropriate, into an undergraduate laboratory course on DNA manipulation taught by the Principal Investigator, Professor Michael Neff.