Abstract
MicroRNAs (miRNAs) play important roles in regulating flowering and reproduction of angiosperms. Mature miRNAs are encoded by multiple MIRNA genes that can differ in their spatiotemporal activities and their contributions to gene regulatory networks, but the functions of individual MIRNA genes are poorly defined. We functionally analyzed the activity of all 5 Arabidopsis thaliana MIR172 genes, which encode miR172 and promote the floral transition by inhibiting the accumulation of APETALA2 (AP2) and APETALA2-LIKE (AP2-LIKE) transcription factors (TFs). Through genome editing and detailed confocal microscopy, we show that the activity of miR172 at the shoot apex is encoded by 3 MIR172 genes, is critical for floral transition of the shoot meristem under noninductive photoperiods, and reduces accumulation of AP2 and TARGET OF EAT2 (TOE2), an AP2-LIKE TF, at the shoot meristem. Utilizing the genetic resources generated here, we show that the promotion of flowering by miR172 is enhanced by the MADS-domain TF FRUITFULL, which may facilitate long-term silencing of AP2-LIKE transcription, and that their activities are partially coordinated by the TF SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 15. Thus, we present a genetic framework for the depletion of AP2 and AP2-LIKE TFs at the shoot apex during floral transition and demonstrate that this plays a central role in floral induction.
Highlights
Transcription factors (TFs) and microRNAs are well-studied regulators of development in animals and plants
MIRs can be inactivated by different types of mutation that could be induced by clustered regularly interspaced short palindromic repeats (CRISPR), such as deletion of the coding sequences of the miRNA/miRNA duplex or mutagenesis of the sites required for DICER-LIKE1 processing of the pri-miRNA [15,16]
The expression patterns of the MIR172 genes were previously assessed by semiquantitative reverse transcription PCR (RT-PCR) in whole seedlings [22]; the expression of these genes in the shoot apical meristem (SAM) was not determined
Summary
Transcription factors (TFs) and microRNAs (miRNAs) are well-studied regulators of development in animals and plants. To understand the interactions between miRNAs and their targets, most research has been limited to the use of miRNA mimicry strategies or the expression of miRNA-resistant versions of the target genes [5,6,7,8,9,10] Both of these approaches have been useful but are subject to artefacts caused by off-target effects or ectopic expression, and they do not offer insights into the contribution of individual MIR family members to developmental regulatory networks. MIRs can be inactivated by different types of mutation that could be induced by CRISPR, such as deletion of the coding sequences of the miRNA/miRNA duplex or mutagenesis of the sites required for DICER-LIKE1 processing of the pri-miRNA [15,16] We follow both strategies to inactivate all 5 members of the MIR172 gene family in Arabidopsis thaliana
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