MpmiR319 promotes gemma/gemma cup formation in the liverwort Marchantia polymorpha

Abstract miRNAs were initially detected as ~20–22 nt sequences in plants. For decades miRNA-mediated regulation of target gene expression has been characterized in many cases. The sequences of miR159/319 family miRNAs are conserved among land plants. The roles of miR159/319 have been comprehensively characterized in development of dicot and monocot plants. However, their biological function in bryophytes remains enigmatic. A model bryophyte Marchantia polymorpha also encodes miR319 at two loci and expresses the miRNA. We used a CRISPR/Cas9 system to edit genome sequences at MpMIR319a and/or MpMIR319b loci. The mutant lines developed relatively few gemma cups and gemmae, suggesting that MpmiR319 targets MpRKD or MpR2R3-MYB21 transcripts and suppresses their expression. We constructed miR319-resistant MpRKD (mMpRKD) and MpR2R3-MYB21 (mMpR2R3-MYB21) by decreasing the complementarity to miR319. Introduction of mMpRKD resulted in gemma/gemma cup-less liverwort mutants, but mMpR2R3-MYB21 did not. Transcription fusion constructs between the MpRKD promoter and β-glucuronidase showed that the gene is expressed in the rim and bottom of gemma cups. We found that the mir319a/mir319b double mutant could form gemma cups but of different sizes in a unpredictable arrangement when planted on vermiculite. These results together suggest that miR319 guides the formation of gemma cups/gemmae in standard positions in collaboration with MpRKD.