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.

Keywords: Marchantia polymorpha; Gemma; R2R3MYB transcription factor; RKD transcription factor; gemma cup; liverwort; miRNA; notch.

Fig. 1.

Loci of MIR319a and MIR319b and mutants obtained in this study. (A) Primary MIR319a or MIR319b RNAs are predicted to be transcribed from two annotated loci in the M. polymorpha genome. (B) The wild-type (WT) MIR319a locus sequence targeted by gRNA#1 is presented. Mutants #5 and #10 were analyzed further. (C) The WT MIR319b locus sequence targeted by gRNA#3 or #4 is presented. Mutants #11 and #15 from the gRNA#3-treated pool and #6 and #9 from the gRNA#4-treated pool were analyzed further. In the superscript ge-x, x indicates the gRNA species used to create the mutant. (D) Changes in the MIR319a and b genomic loci in the double knockout line miR319ab #100. The MIR319a locus had a 5 bp deletion and the MIR319b locus had a 1 bp insertion, as did mir319a^ge-1 #10 and mir319b^ge-3 #15. We named the line mir319a^ge-1b^ge-3#100, or mir319 ab#100, for brevity in this work.

Fig. 2.

Phenotypes of miR319 mutants. (A) Appearance of miR319 mutant thalli. Scale bar=1 cm. WT, Tak-1 wild-type plant; a #5, mir319a^ge-1 #5, a #10, mir319a^ge-1 #10; b #11, mir319b^ge-3 #11; b #15, mir319b^ge-3 #15; b #6, mir319b^ge-4 #6; b #9, mir319b^ge-4 #9; ab#100, miR319a^ge-1b^ge-3 #100. (B) Disruption of the MpMIR319a and/or b loci decreased the number of gemma cups. Five plants were analyzed and the mean number of gemma cups per thallus is presented. Tukey–Kramer’s honestly significant difference test was performed to analyze the variance. Statistically significant differences are indicated by different letters (a, b, c; P<0.05). (C) Number of gemmae formed in each gemma cup. *P<0.05 (Tukey method).

Fig. 3.

Exon–intron structures of MpRKD and MpR2R3-MYB21. The exon–intron structures of MpRKD and MpR2R3 genes in the wild-type form (or sensitive form, with prefix g) and miR319-resistant form (with prefix m) of (A) MpRKD and (B) mMpR2R3-MYB21 genes. Vertical bars indicate conventional base pairing, whereas double dots indicate G–U pairs. AUG and UGA are initiation and termination codons, respectively, of the ORF. 5′ UTR and 3′ UTR refer to the untranslated regions.

Fig. 4.

Effect of the miR319-resistant mMpRKD gene. Introduction of the miR319-resistant mMpRKD gene suppressed gemma cup formation in thalli, but the introduction of gMpRKD and mMpR2R3MYB21 genes did not. Scale bar=1 cm. ab #100, miR319 a^ge-1b^ge-3 #100; WT, Tak-1.

Fig. 5.

Effect of the wild-type RKD gene. Introduction of the wild-type RKD gene (gMpRKD) induced normal gametophyte development in Tak-1 (A, B) and Tak-2 (G, H). Introduction of the miR319-resistant mutant RKD gene (mMpRKD) into Tak-1 induced the production of antheridiophores with stalked receptables with irregular asymmetrical shapes (C, D) and antheridiophores with exposed antheridia (E, F). Introduction of mMpRKD into Tak-2 induced the production of archegoniophores with short digits (I, J) and archegonia (H, K). Scale bars: 5 mm (A, C), 1 mm (B, D–G, I, J), 0.1 mm (H, K).

Fig. 6.

Transcription fusion analysis of promoters in the Tak-1 background. The marker GUS gene was placed under the promoters of MIR319a (A–D), MIR319b (E–H), MpRKD (I–L), and MpR2R2-MYB21 (M–P), and introduced. On day 3 (A, E, I, M), day 6 (B, F, J, N), day 9 (C, G, K, O), and day 12 (D, H, L, P), the respective thalli were collected and GUS expression (indigo blue) was visualized. Photos with lowercase letters show vertical sections (dotted lines) of the corresponding uppercase plant specimen.

Fig. 7.

Growth of wild-type Tak-1 and mir319ab#100 plants. Appearance of gemma cups and gemmae formed on Tak-1 (w1, w2) and mir319ab#100 (ab #100-1, ab #100-2) plants. Appearance of thalli of Tak-1 (w3) and mir319ab#100 (ab #100-3) grown from gemma for 21 d on plates. Insets show 14-day-old thalli of the corresponding plantlet.