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

After microRNAs (miRNAs) were initially detected as approximately 21-nt sequences in plants, miRNA-mediated regulation of target gene expression was characterized in many cases. The sequences of miR159/319 family miRNAs are highly conserved and their development-related roles have been comprehensively characterized in dicot and monocot plants. However, their effects in basal land plants remain relatively unknown. We used a CRISPR/Cas9 system to edit genome sequences at MpMIR319a and/or MpMIR319b loci. Mutant mir319b lines developed relatively few gemma cups, while mutant mir319a lines produced relatively few gemmae in each gemma cup. Earlier 5′-RACE or degradome analyses suggested that MpmiR319 targets MpRKD and MpR2R3-MYB21 transcripts and suppresses expression. To determine whether these candidate genes influence the mir319a/mir319b phenotype we constructed miR319-resistant MpRKD (mMpRKD) and MpR2R3-MYB21 (mMpR2R3-MYB21) by decreasing the complementarity to miR319. We also introduced the genes into wild-type liverwort and screened for phenotypic changes. We observed that mMpRKD resulted in gemma/gemma cup-less liverwort mutants, but mMpR2R3-MYB21 did not. No major adverse effects were detected during male and female reproductive development induced by far-red illumination. These results indicate that miR319 influences gemma formation mainly by repressing MpRKD expression rather than MpR2R3-MYB21 expression.