INDETERMINATE DOMAIN PROTEIN binding sequences in the 5′-untranslated region and promoter of the SCARECROW gene play crucial and distinct roles in regulating SCARECROW expression in roots and leaves

Abstract

SCARECROW (SCR) and SHORT-ROOT (SHR), which belong to the GRAS transcription factor family, are key regulators of root and leaf growth and development. Despite the importance of SCR expression for proper plant development, the mechanism of SCR regulation has not been clarified. A previous study showed that an INDETERMINATE DOMAIN transcription factor, JACKDAW (JKD), is essential for the expression of SCR in combination with SCR and SHR. In this study, we characterized possible binding sequences of INDETERMINATE DOMAIN PROTEIN in the 1.5kb upstream region of SCR. Mutation in a binding sequence 340bp upstream of the ATG increased transcriptional activation by JKD in transient assays using Arabidopsis cultured cells. However, the activity was not enhanced by SCR/SHR. Histochemical analysis of promoter activity in transgenic Arabidopsis plants carrying a fusion of the promoter and the β-glucronidase reporter gene showed that mutation of the -340bp sequence eliminated most of the promoter activity, indicating that this sequence was indispensable for SCR expression. Promoter deletion of downstream sequences from -280bp lost the enhanced activity by SCR/SHR in transient assays and activity in root tips and the bundle sheath (BS) in plants. Conversely, mutation at -480bp did not significantly influence transcriptional activity in transient assays. However, most of SCR expression was lost except for the root tip in plants. The sequences around -1kb appeared to regulate SCR expression negatively in plants. Together, these INDETERMINATE DOMAIN PROTEIN binding sequences have crucial and distinct functions in regulating SCR expression.