Functional Analysis of 26-Hydroxybrassinolide, the Theoretical Product of BAS1/CYP72B1

Brassinosteroids are plant steroid hormones that play important roles in many developmental processes, including promotion of cell elongation, leaf and chloroplast senescence and floral induction. One brassinosteroid mutant, bas 1-D, overexpresses the BAS 1 gene, with a resulting phenotype that is a dark green, slow-growing dwarf with epinastic leaves, short stems and petioles, and delayed senescence.

The BAS 1 gene product is a cytochrome P450, CYP72B 1. P450s are hydroxylases involved in many different biochemical processes including steroid biosynthesis and catabolism. CYP72B 1 is thought to hydroxylate brassinolide, the most active brassinosteroid. In brassinolide feeding experiments, it has been observed that the bas 1-D mutant, which overexpresses CYP72B 1, has no detectable brassinolide and accumulates 26-hydroxybrassinolide.

The current hypothesis is that 26-hydroxybrassinolide is a less active form of brassinolide. The purpose of this research is to determine the activity of 26-hydroxybrassinolide. In doing so, a better understanding of the biochemical aspects of the action of BAS 1 can be gained.

Seeds of Col-0 (wildtype), bas 1-D, and BAS1antisense were grown in phytagar with 0, 0.1, 1.0, 10, 100, or 1000 nM of either brassinolide or 26-hydroxybrassinolide. After six days, the hypocotyls were measured to determine the relative effects of the brassinosteroids on hypocotyl elongation.

In all cases, hypocotyl length of brassinolide-grown seedlings greatly increased at the 10, 100, and 1000 nM levels, whereas 26-hydroxybrassinolide-grown seedlings showed only a slight increase in hypocotyl length at the 1000 nM level.

The data supports the hypothesis that 26-hydroxybrassinolide is a less active form of brassinolide. It can therefore be suggested that the BAS 1 gene product hydroxylates brassinolide to hydroxybrassinolide in order to regulate hypocotyl growth.