Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.
Extraction and purification of BRs from soybean seeds with different methods were investigated as described in . From Table 2 , the C18 cleanup did not appear to contribute a great deal in terms of sample purity and retention. Furthermore, such an SPE procedure is very time-consuming and labor intensive. The solvent partitioning with DCM is more efficient than solvent partitioning with ethyl acetate. The wash step with hexane to remove fat/lipids is not efficient for the recovery of BRs which may have been caused by the BRs transfer to hexane rather than remaining in the aqueous methanol solution. The recoveries of brassinolides are better than those of castasterones. The partition with DCM is the best cleanup method for all the BRs studied. If labeled isotopic isomers are applied to the method, better recovery efficiency should be achieved. Furthermore, this study mainly focused on LC-MS/MS instrumentation. Therefore, the cleanup procedures presented were designed to be simple and fast while taking advantage of the high sensitivity and selectivity of LC-MS/MS. As a result of this simple approach, it was determined that BRs solubilized in organic solvents without labour-intensive sample cleanup could still be identified by LC-MS/MS. Future studies could explore other methods to increase recovery and reduce the complexity of the sample matrix.