The regulation of DWARF4 expression is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis

Abstract

Mutants that are defective in brassinosteroid ( BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis ( Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4: beta-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4: GUS expression was negatively regulated by BRs. DWF4: GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4: GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4: GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSI-NOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.