| Multiple mechanisms coordinate responses to the plant hormones auxin and brassinosteroids; yet, many questions remain about how these signals are integrated at the transcriptional level. Here, we report multiple levels of transcriptional cross-regulation. First, there is direct interaction between components of the signaling pathways. ARF2, an auxin-regulated repressor, is likely controlled by BIN2, a brassinosteroid-regulated kinase. BIN2-phosphorylation of ARF2 negatively regulates DNA binding and repressor activity. In addition, arf2 mutants show a constitutive brassinosteroid response. This suggests BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs, leading to synergistic increases in transcription. Second, we have shown that auxin and brassinosteroid signaling pathways converge on the same cis-regulatory elements. A region containing these elements showed enhanced binding by brassinosteroid- and auxin-regulated activators BES1 and MP/ARF5 following treatment with auxin or brassinosteroids. This suggesting that cross-regulation is converging on transcriptional complexes. Finally, brassinosteroids appear to modify chromatin structure and transcriptional elongation at shared target genes. BES1 interacts with IWS1, involved with transcriptional elongation, and ELF6/REF6, proposed to promote open chromatin. To determine whether brassinosteroids directly mediate DNA accessibility, we are performing qPCR on chromatin mutants following hormone treatment and luciferase assays on the reporter lines crossed to chromatin mutants. ChIP assays on RNA Pol II following hormone treatment will be used to determine whether brassinosteroids promote RNA Pol II traveling. This work supports a model where brassinosteroids are priming auxin-induced gene expression: (1) by promoting binding of BES1 to open chromatin in auxin-responsive promoters (2) by inactivating repressor ARFs leading to increased binding by activator ARFs and (3) by promoting recruitment of transcriptional activator complex components, activating transcription. These results refine our understanding of the molecular mechanism for auxin-brassinosteroid synergism, as well as increase our understanding of how the cross-regulation of hormones modifies plant development. |
