miR156 and miR390 regulate tasiRNA accumulation and developmental timing in Physcomitrella patens

Abstract

microRNA156 (miR156) affects developmental timing in flowering plants. miR156 and its target relationships with members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family appear universally conserved in land plants, but the specific functions of miR156 outside of flowering plants are unknown. We find that miR156 promotes a developmental change from young filamentous protonemata to leafy gametophores in the moss Physcomitrella patens, opposite to its role as an inhibitor of development in flowering plants. P. patens miR156 also influences accumulation of trans-acting small interfering RNAs (tasiRNAs) dependent upon a second ancient microRNA, miR390. Both miR156 and miR390 directly target a single major tasiRNA primary transcript. Inhibition of miR156 function causes increased miR390-triggered tasiRNA accumulation and decreased accumulation of tasiRNA targets. Overexpression of miR390 also caused a slower formation of gametophores, elevated miR390-triggered tasiRNA accumulation, and reduced level of tasiRNA targets. We conclude that a gene regulatory network controlled by miR156, miR390, and their targets controls developmental change in P. patens. The broad outlines and regulatory logic of this network are conserved in flowering plants, albeit with some modifications. Partially conserved small RNA networks thus influence developmental timing in plants with radically different body plans.

Figure 1.

Accumulation of miR156 and Its Targets during P. patens Gametophyte Development. Accumulation of P. patens miR156 and target SBP genes was assessed using qRT-PCR. Relative accumulation levels relative to the 1-week sample were plotted. Error bars indicate sd from three biological replicates. Analyses of significant differences are shown in Supplemental Figure 1 online. [See online article for color version of this figure.]

Figure 2.

miR156 Promotes Bud and Leafy Gametophore Formation. (A) Schematic of the MIM156 overexpression construct. pro, promoter; NosT and CaMVT, Nos and cauliflower mosaic virus terminator, respectively; At4, At4 gene from Arabidopsis; 108, 108 targeting locus from the P. patens genome (Schaefer and Zrÿd, 1997). (B) to (F) Delay of bud and gametophore production in MIM156 lines. Four-week-old plants (four independent MIM156 lines [B] to [E] and the wild type (WT; [F])] are shown. Bars = 3 mm. (G) to (H) Increased accumulation of miR156 targets in MIM156 plants at the time of maximal bud initiation. RNAs from spore-germinated 3-week-old tissues (G) and 5-week-old tissues (H) were analyzed by qRT-PCR as in Figure 1. Expression relative to the wild type was plotted. Error bars indicate sd from three biological replicates. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01).

Figure 3.

SBP3 Represses Bud and Leafy Gametophore Formation. (A) Accumulation of SBP transcripts during the P. patens life cycle. Expression levels relative to EF1α in a sample were plotted. Error bars indicate sd from three biological replicates. Analyses of significant differences are shown in Supplemental Figure 4 online. (B) to (D) Opposite effects of miR156 and SBP3 upon bud formation. Blended protonemal tissues of the wild type (WT) (B), MIM156-2 (C), and ΔSBP3 (D) were plated on cellophane overlaid media and incubated for 2 weeks. Arrowheads in (B) indicate buds. Bars = 1 mm. (E) Rates of bud and gametophore appearance. Seven-day-old protonemal tissues were inoculated on media. The numbers of buds and subsequent gametophores in four colonies were counted every 2 days. Error bars represent sd of four replicates. Significant differences from the wild type on each day were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01).

Figure 4.

miR156 Influences P. patens tasiRNA Accumulation and Function. (A) The annotated TAS6a/TAS3a primary transcript including miRNA target sites, locations of functional tasiRNAs, exons (rectangles), and central intron. (B) Small RNA gel blot analysis from 3-week-old ΔSBP3, MIM156-2, and MIM156-3 plants. The numbers indicate the intensities relative to U6 signals. WT, the wild type. (C) Stem loop qRT-PCR of tasiARF and tasiAP2 siRNAs from 3-week-old plants. Error bars indicate sd from three biological replicates. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01). (D) qRT-PCR of tasiARF and tasiAP2 target mRNAs in 3-week-old plants. Error bars indicate sd from three biological replicates. a, 1s14_392V6.1; b, 1s280_72V6.1; c, 1s6_75V6.1; d, 1s5_432V6.1; e, 1s74_86V6.1. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01). (E) Stem loop qRT-PCR of miR156 and miR390 from 3-week-old plants. Error bars indicate sd from three biological replicates. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01).

Figure 5.

Overexpression of MIR390C Represses Bud and Leafy Gametophore Formation. (A) Expression pattern of miR390, tasiARF, and tasiAP2 small RNAs in wild-type plants at the indicated ages, measured by stem loop qRT-PCR. The relative accumulation levels to the 1-week sample were plotted. Error bars indicate sd from three biological replicates. Analyses of significant differences are shown in Supplemental Figure 10A online. (B) Expression pattern of the target mRNAs of tasiARF and tasiAP2 in the samples as in (A), measured by qRT-PCR. The relative accumulation levels to the 1-week sample were plotted. Error bars indicate sd from three biological replicates. a, 1s14_392V6.1; b, 1s280_72V6.1; c, 1s6_75V6.1; d, 1s5_432V6.1; e, 1s74_86V6.1. Analyses of significant differences are shown in Supplemental Figure 10B online. (C) Overexpression of MIR390C. RNAs from protonemata tissues of the wild type (WT) and MIR390c OE were analyzed by stem loop–mediated qRT-PCR. Error bars indicate sd from three biological replicates. Significant differences from wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01). (D) Rates of bud and gametophore appearance. Seven-day-old protonemal tissues of the wild type and MIR390C OE were inoculated on media. The numbers of buds and subsequent gametophores in 16 colonies were counted every 2 d. Error bars represent the se values for those 16 colonies. Significant differences from wild type on each day were analyzed by t test (*P ≤ 0.05). The raw data for day 16 is in Supplemental Table 2 online. (E) Stem loop qRT-PCR of tasiARF, tasiAP2, and tasiZNF in 3-week-old plants. Error bars indicate sd from three biological replicates. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01). (F) Accumulation of tasiARF and tasiAP2 target mRNAs in 3-week-old plants measured by qRT-PCR. Error bars indicate sd from three biological replicates. a to e are as in (B). Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01). (G) Accumulation of the tasiZNF target mRNA 1s286_43V6.1, measured by qRT-PCR. Error bars indicate sd from three biological replicates. No significant differences relative to the wild type were found (t test, P > 0.05).

Figure 6.

Cytokinin Treatment Has Mostly Negligible Effects upon RNA Accumulation Levels of Members of the miR156-miR390-tasiRNA Pathway. (A) Stem loop qRT-PCR of the indicated mature miRNAs from wild-type P. patens treated with 5 μM BAP or with a mock control. Error bars represent sd from three biological replicates. No significant difference from the wild type was found (P > 0.05 by t test). (B) qRT-PCR of the indicated miR156-targeted SBP mRNAs from wild-type plants treated with 5 μM BAP or with a mock control. Error bars represent sd from three biological replicates. No significant difference from the wild type was found (P > 0.05 by t test). (C) Stem loop qRT-PCR of the indicated tasiARF and tasiAP2 siRNAs from the samples as in (A). Error bars represent sd from three biological replicates. No significant difference from the wild type was found (P > 0.05 by t test). (D) As in (B) for tasiARF and tasiAP2 target mRNAs. a, 1s14_392V6.1; b, 1s280_72V6.1; c, 1s6_75V6.1; d, 1s5_432V6.1; e, 1s74_86V6.1. Significant differences from the wild type were analyzed by t test (*P ≤ 0.05 and **P ≤ 0.01).

Figure 7.

Comparison of Small RNA/Target and Genetic Networks Controlling Developmental Transitions between P. patens and Arabidopsis. (A) Small RNA–target interactions in P. patens. Colored arrows indicate small RNA targeting, open rectangles indicate open reading frames, gray regions indicate protein domains, and filled rectangles indicate regions of phased siRNA production. Not to scale. (B) Genetic interactions controlling bud formation and leafy gametophore formation in P. patens. Interactions in red differ when compared with Arabidopsis; those in black are conserved. Dotted lines indicate hypothetical interactions. (C) to (D) As in (A) and (B), respectively, for Arabidopsis. Empty triangle in (C) reflects the nonslicing miR390 site found in Arabidopsis TAS3 loci.