Abstract
Diverse leaf forms can be seen in nature. In Medicago truncatula, PALM1 encoding a Cys(2)His(2) transcription factor is a key regulator of compound leaf patterning. PALM1 negatively regulates expression of SGL1, a key regulator of lateral leaflet initiation. However, how PALM1 itself is regulated is not yet known. To answer this question, we used promoter sequence analysis, yeast one-hybrid tests, quantitative transcription activity assays, ChIP-PCR analysis, and phenotypic analyses of overexpression lines and mutant plants. The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) functions as a direct transcriptional repressor of PALM1. MtARF3 physically binds to the PALM1 promoter sequence in yeast cells. MtARF3 selectively interacts with specific auxin response elements (AuxREs) in the PALM1 promoter to repress reporter gene expression in tobacco leaves and binds to specific sequences in the PALM1 promoter in vivo. Upregulation of MtARF3 or removal of both PHANTASTICA (PHAN) and ARGONAUTE7 (AGO7) pathways resulted in compound leaves with five narrow leaflets arranged in a palmate-like configuration. These results support that MtARF3, in addition as an adaxial-abaxial polarity regulator, functions to restrict spatiotemporal expression of PALM1, linking auxin signaling to compound leaf patterning in the legume plant M. truncatula.
Highlights
Leaves are initiated from the flanks of the shoot apical meristem (SAM)
We show that AUXIN RESPONSE FACTOR3 (ARF3) binds to putative auxin response elements (AuxREs) in the promoter region of PALM1 and represses expression of reporter gene activities in transcription activity assays in tobacco leaves
The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) interacted strongly with the PALM1 promoter sequence in yeast one-hybrid assays (Figure 1B)
Summary
Leaves are initiated from the flanks of the shoot apical meristem (SAM). The class I Knotted-like homeobox transcription factor genes (KNOXIs), which are required to promote and maintain the meristematic activity of SAM, are downregulated at the incipient sites of leaf primordia (Jackson et al, 1994; Lincoln et al, 1994; Long et al, 1996). Genetic studies show that impairment of the tasiR-ARF pathway enhances the defects of the asymmetric leaves (as1) mutant in leaf adaxialabaxial partitioning and margin development (Garcia et al, 2006; Xu et al, 2006). AS1 and the tasiR-ARF pathway negatively regulate ARF3 expression at the transcriptional and post-transcriptional level, respectively It is still not clear whether and how ARF3 regulates leaf patterning in M. truncatula that forms dissected leaves. We show that ARF3 binds to putative auxin response elements (AuxREs) in the promoter region of PALM1 and represses expression of reporter gene activities in transcription activity assays in tobacco leaves
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