The AtHB1 Transcription Factor Controls the miR164-CUC2 Regulatory Node to Modulate Leaf Development.

ABSTRACT

The presence of small tooth-like indentations, or serrations, characterizes leaf margins of Arabidopsis thaliana plants. The NAC family member CUP-SHAPED COTYLEDON 2 (CUC2), which undergoes post-transcriptional gene silencing by three micro-RNA genes (MIR164A, B and C), controls the extension of leaf serration. Here, we analyzed the role of AtHB1, a transcription factor (TF) belonging to the homeodomain-leucine zipper subfamily I, in shaping leaf margins. Using mutants with an impaired silencing pathway as background, we obtained transgenic plants expressing AtHB1 over 100 times compared to controls. These plants presented an atypical developmental phenotype characterized by leaves with deep serration. Transcript measurements revealed that CUC2 expression was induced in plants overexpressing AtHB1 and repressed in athb1 mutants, indicating a positive regulation exerted by this TF. Moreover, molecular analyses of AtHB1 overexpressing and mutant plants revealed that AtHB1 represses MIR164 transcription. We found that overexpression of MIR164B was able to reverse the serration phenotype of plants overexpressing AtHB1. Finally, chromatin immunoprecipitation assays revealed that AtHB1 was able to bind in vivo the promoter regions of all three MIR164 encoding loci. Altogether, our results indicate that AtHB1 directly represses MIR164 expression to enhance leaf serration by increasing CUC2 levels.