Brassinosteroid-regulated gene expression

Abstract

Major brassinosteroid (BR) effects such as BR-induced growth are mediated through genomic pathways because RNA synthesis inhibitors and protein synthesis inhibitors interfere with these processes. A limited number of BR-regulated genes have been identified hitherto. The majority of genes (such as BRU1, CycD3, Lin6, OPR3, and TRIP-1) were identified by comparisons of BR-treated versus control-treated plants. However, altered transcript levels after BR application may not reflect normal physiological events. A complementary approach is the comparison of BR-deficient plants versus wild-type plants. No artificial treatments interfere with endogenous signaling pathways, but a subset of phenotypic alterations of phytohormone-deficient plants most probably is secondary. To identify genes that are subject to direct BR regulation, we analyzed CPD antisense and dwf1-6 (cbb1) mutant plants. Both show a mild phenotype in comparison with BR-deficient mutants such as cpd/cbb3, det2, and dwf4. Plants were grown under two different environments to filter out BR deficiency effects that occur only at certain environmental conditions. Finally, we established expression patterns after BR treatment of wild-type and dwf1-6 (cbb1) plants. Ideally, a BR-regulated gene displays a dose-response relationship in such a way that a gene with decreased transcript levels in BR-deficient plants is BR inducible and vice versa. Expression profile analysis of above ground part of plants was performed by means of Affymetrix Arabidopsis Genome Arrays.

Figure 1

Technical variability of gene expression analysis using Affymetrix Arabidopsis Genome Arrays. One single wild-type RNA isolation was used for the synthesis of four independently labeled samples that were hybridized to four Arabidopsis Genome Arrays. A, Number of genes called present in each of the four hybridizations according to the absolute call algorithm. B, Number of genes consistently called present in different hybridizations. C, Variation coefficients of expression values of the 4,616 genes termed present in all four hybridizations. Basis of the calculations are average difference values that serve as a relative indicator of the level of expression of a transcript. Variation coefficients (in %) were calculated by determining the ratios of the sds to the means for the 4,616 genes, multiplied by 100.