Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis

Abstract

Auxin regulates nearly all aspects of plant growth and development including cell division, cell elongation and cell differentiation, which are achieved largely by rapid regulation of auxin response genes. However, the functions of a large number of auxin response genes remain uncharacterized. Paclobutrazol Resistance (PRE) proteins are non-DNA binding basic helix-loop-helix transcription factors that have been shown to be involved in gibberellin and brassinosteroid signaling, and light responses in Arabidopsis. Here, we provide molecular and genetic evidence that <i>PRE6</i>, one of the six <i>PRE</i> genes in Arabidopsis, is an auxin response gene, and that PRE6 is involved in the regulation of auxin signaling. By using quantitative RT-PCR, we showed that the expression level of <i>PRE6</i> was increased in response to exogenously applied IAA. GUS staining results also showed that the expression of <i>GUS</i> reporter gene in the <i>PRE6p:GUS</i> transgenic seedlings was elevated in response to auxin. Phenotypic analysis showed that overexpression of <i>PRE6</i> in Arabidopsis resulted in auxin-related phenotypes including elongated hypocotyl and primary roots, and reduced number of lateral roots when compared with the Col wild type seedlings, whereas opposite phenotypes were observed in the <i>pre6</i> mutants. Further analysis showed that <i>PRE6</i> overexpression plants were hyposensitive, whereas <i>pre6</i> mutants were hypersensitive to auxin in root and hypocotyl elongation and lateral root formation assays. By using protoplasts transfection, we showed that PRE6 functions as a transcriptional repressor. Consistent with this, the expression of the auxin response reporter <i>DR5:GUS</i> was decreased in <i>PRE6</i> overexpression lines, but increased in <i>pre6</i> mutants. When co-transfected into protoplasts, ARF5 and ARF8 activated the expression of the <i>PRE6p:GUS</i> reporter. Chromatin immunoprecipitation assays showed that ARF5 and ARF8 can be recruited to the promoter regions of <i>PRE6</i>. Taken together, these results suggest that <i>PRE6</i> is an auxin response gene whose expression is directly regulated by ARF5 and ARF8, and that PRE6 is a transcriptional repressor that negatively regulates auxin responses in Arabidopsis.