| Description: | Analysis of brassinosteroid (BR) biosynthesis and signal transduction, has shown that BRs are essential for normal plant development. We identified a BR-insensitive mutant in Arabidopsis, named bri1, with severe pleiotropic effects on development including dwarfism, de-etiolation, male sterility and altered leaf morphology (Clouse et al., 1996. Plant Physiol. 111:671-678). The BRI1 gene was cloned by Li and Chory (1997, Cell 90:929-938) and shows strong sequence homology to leucine-rich receptor kinases which function at the cell surface to transduce extracellular signals. We propose to identify genes that involve the BRI1 signal transduction pathway using microarray technology. Genes that require BRI1 for expression will have reduced hybridization intensity in the mutant while genes normally repressed by an active BRI1, will show elevated hybridization signal in the mutant compared to wildtype. Some of these genes may be directly involved in the BR response, while others may be secondary genes. Because bri1 is male sterile, a segregating F2 population (Col x bri1) will be used. Seeds will be surface sterilized and vernalized for 48 hours at 4 C in the dark. Seeds from mutant and wildtype will be placed on top of agar plates with half-strength MS media plus sucrose. Plates will be placed vertically in the dark for 4 days (seedlings will grow on agar surface). Col-0 seedlings will then be scraped into liquid half-strength MS media with sucrose. Mutant seedlings (easily distinguished from wildtype at this stage by their short hypocotyl) will also be rapidly placed in shaking liquid media. Col-0 and bri1 seedlings will be shaken in the light for an additional 7 days, after which seedlings will be rapidly vacuum filtered to remove culture media and frozen in liquid nitrogen. Poly A+ RNA will be isolated from mutant and wild-type tissue using the protocol suggested on the AFGC website. RNA will be used for first-strand cDNA synthesis by AFGC with reverse transcription in the presence of Cy3 (wildtype) or Cy5 (mutant) fluorescent dyes. The entire experiment will be repeated for the second array, reversing the dyes. The power of this approach has been shown in studies of yeast sporulation transcription factor Ndt80. Comparison of a strain lacking Ndt80 vs. wildtype on microarrays identified about 150 genes that required activity of this transcription factor for their expression (Chu et al., 1998, Science 282:699-705). |
NASCArrays Information at The BAR
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