miR156 and miR390 Regulate tasiRNA Accumulation andDevelopmental Timing in Physcomitrella patensC W OA

Sung Hyun Cho,a Ceyda Coruh,a,b and Michael J. Axtella,b,1

a Department of Biology, Penn State University, University Park, Pennsylvania 16802b Plant Biology PhD Program, Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania 16802

microRNA156 (miR156) affects developmental timing in flowering plants. miR156 and its target relationships with members ofthe SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family appear universally conserved in land plants, but thespecific functions of miR156 outside of flowering plants are unknown. We find that miR156 promotes a developmental changefrom young filamentous protonemata to leafy gametophores in the moss Physcomitrella patens, opposite to its role as aninhibitor of development in flowering plants. P. patens miR156 also influences accumulation of trans-acting small interferingRNAs (tasiRNAs) dependent upon a second ancient microRNA, miR390. Both miR156 and miR390 directly target a singlemajor tasiRNA primary transcript. Inhibition of miR156 function causes increased miR390-triggered tasiRNA accumulationand 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 regulatorynetwork controlled by miR156, miR390, and their targets controls developmental change in P. patens. The broad outlines andregulatory logic of this network are conserved in flowering plants, albeit with somemodifications. Partially conserved small RNAnetworks thus influence developmental timing in plants with radically different body plans.

INTRODUCTION

Plants produce both a multicellular haploid entity (the gameto-phyte) and a multicellular diploid entity (the sporophyte) duringtheir sexual life cycles. Mosses and other bryophytes havemorphologically complex, photosynthetic gametophytes withattached simple nonphotosynthetic sporophytes. By contrast,other lineages, including flowering plants, have simple, micro-scopic gametophytes enclosed by, and nutritionally dependentupon, a morphologically complex sporophyte. The extent towhich genes with ancestral roles in the development of complexgametophytes were recruited to control complex sporophyticdevelopment is unclear as both positive (Menand et al., 2007)and negative (Tanahashi et al., 2005) examples have been de-scribed. The question is particularly acute for microRNAs(miRNAs) because several miRNA families are conserved in awide range of land plants, including both sporophyte-dominatedand gametophyte-dominated species (Cuperus et al., 2011). Theregulatory targets of these conserved miRNA families are, asa general rule, also conserved, and the majority of the mostancient plant miRNA families regulate mRNAs encoding tran-scription factors (Axtell and Bowman, 2008). The functions ofmany of these miRNAs and their targets have been intensively

investigated in several flowering plants and frequently found toaffect various aspects of sporophytic development (Axtell andBowman, 2008). Global reduction of miRNA accumulation inDICER-LIKE 1A (DCL1a) mutants of the moss Physcomitrellapatens, whose life cycle is dominated by a morphologicallycomplex gametophyte, has pleiotropic effects on gametophyticdevelopment (Khraiwesh et al., 2010). However, the functionsof specific ancient plant miRNA families in P. patens or othergametophyte-dominated basal plant lineages remain unclear.miR156 is a master regulator of developmental transitions in

flowering plants. miR156 accumulation in flowering plants istemporally regulated, with high levels in early embryos, anda progressive reduction as the plant ages (Wu and Poethig,2006; Wang et al., 2009). Conversely, the accumulation of itsSQUAMOSA PROMOTER BINDING PROTEIN- LIKE (SPL) tar-gets generally increases with age in flowering plants (Wu andPoethig, 2006; Wang et al., 2009). SPL genes in flowering plantspromote adult leaf morphology and flowering (Poethig, 2009;Huijser and Schmid, 2011). miR172-mediated regulation ofAPETALA2 (AP2) family genes has a reciprocal relationship withmiR156/SPL: miR172 levels increase with plant age, whilemiR172-targeted AP2 family genes, several of which serve torepress adult traits, decrease (Huijser and Schmid, 2011). InArabidopsis thaliana, this coordination is achieved in part bydirect stimulation of miR172 transcription by miR156-targetedSPL9 and SPL10 (Wu et al., 2009). Genetic screens for Arabi-dopsis vegetative developmental mutants (Hunter et al., 2003,2006; Peragine et al., 2004; Fahlgren et al., 2006) have identifieda third small RNA pathway that influences vegetative devel-opmental transitions. miR390 stimulates production of trans-acting small interfering RNAs (tasiRNAs) that target AUXINRESPONSE FACTOR3 (ARF3)/ETTIN and ARF4 mRNAs. Lossof tasiRNA-mediated ARF3/4 regulation accelerates vegetative

1 Address correspondence to mja18@psu.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Michael J. Axtell (mja18@psu.edu).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.112.103176

The Plant Cell, Vol. 24: 4837–4849, December 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

developmental transitions, implying that ARF3/ETTIN and ARF4promote these changes. The miR390/tasiRNA/ARF regulatorysystem also influences organ polarity in flowering plants (Garciaet al., 2006; Nogueira et al., 2007). The miR156/SPL regulatoryinteraction appears universal among land plants (Arazi et al.,2005; Axtell et al., 2007), and the miR390/tasiRNA/ARF cascadeappears nearly universal (Axtell et al., 2006) save for an apparentsecondary loss in the lycophyte lineage (Banks et al., 2011),while the miR172/AP2 interaction appears to have emergedlater, after the divergence of the moss and lycophyte lineages(Axtell and Bartel, 2005).

Here, we report a functional study of P. patens miR156,a miR156-targeted target SPL gene, and miR390. We find thatmiR156 promotes the transition from tip-growing protonemalgrowth to production of leafy gametophores. Interestingly,miR156-mediated promotion of this vegetative transition iscontrary to the inhibitory role of miR156 upon developmentaltransitions in flowering plants. In addition, P. patens miR156 andmiR390 both directly target the same major tasiRNA primarytranscript, and perturbation of miR156 influences accumula-tion of tasiRNAs and their target mRNAs. Overexpression ofMIR390C resulted in slower gametophore production, accom-panied by reduction of tasiRNA targets. Finally, we describea gene regulatory network in P. patens in which a set of miRNAs,tasiRNAs, and three transcription factor families converge tocontrol developmental timing. This network is partially con-served in flowering plants, suggesting that the regulatory net-works for developmental transition present in modern speciesdescended and diversified from those present in an ancientancestor.

RESULTS

Temporal Regulation of miR156 and Its Targets in P. patens

miR156 accumulation in flowering plant sporophytes is tempo-rally regulated, with high levels in early embryos and a pro-gressive reduction as the plant ages (Wu and Poethig, 2006;Wang et al., 2009). Conversely, the accumulation of its SPLtargets generally increases with age in flowering plants (Wu andPoethig, 2006; Wang et al., 2009). Stem loop–mediated quan-titative RT-PCR (qRT-PCR) (Varkonyi-Gasic et al., 2007) in-dicated that miR156 accumulation is also temporally regulatedin P. patens gametophytes (Figure 1; see Supplemental Figure 1online). Young plants comprised solely of tip-growing, filamen-tous protonemata tissues (chloronema and caulonema; 1 to 2weeks after germination from spores; see Supplemental Figure 2online) have relatively little miR156 accumulation. Young budsand early gametophores with one or two phylloids are mostenriched in 3-week old plants (see Supplemental Figure 2 on-line), with patches of buds on top of the lawn of filamentousprotonemata (Huijser and Schmid, 2011). Buds are the in-termediate tissues between protonemata and gametophores.Thus, we define these plants as undergoing the developmentaltransition from young protonemata to adult leafy gametophores.In these plants, the accumulation of miR156 reaches its maxi-mum level (Figure 1; see Supplemental Figure 1 online). miR156

levels then gradually decline from their 3-week maximum as theleafy gametophores mature (Figure 1; see Supplemental Figure1 online).The three P. patens SQUAMOSA PROMOTER BINDING

PROTEIN (SBP) targets of miR156 (SBP3, SBP6, and SBP13)(Arazi et al., 2005; Riese et al., 2007; Addo-Quaye et al., 2009)also accumulated in a temporal fashion. (Note that the P. patensmiR156-targeted SBP genes are in the same gene family as themiR156-targeted SPL genes in Arabidopsis and other floweringplants; to be consistent with the prior literature [Riese et al.,2007], we use the SBP acronym when referring to the P. patensmembers of this gene family.) Like miR156, the SBP targetswere minimally expressed in 1- to 2-week-old plants. Increasedaccumulation of SBP targets became apparent in older plants attime points when miR156 accumulation levels were decliningfrom their maximum values (Figure 1; see Supplemental Figure 1online).

miR156 Promotes Gametophore Production in P. patensvia SBP3

A miRNA target mimic (Franco-Zorrilla et al., 2007; Todescoet al., 2010) designed to reduce miR156 function was overex-pressed in wild-type P. patens (Figure 2A). Four independentMIM156 transgenic lines had delayed bud initiation and reducednumbers of leafy buds and subsequent gametophores (Figures2B to 2F). In general, phenotypic severity was positively corre-lated with the accumulation of the MIM156 target mimic RNA,with the two stronger MIM156 expressers (MIM156-2 and -3)more delayed and the two weaker expressers (MIM156-1 and -4)less delayed (see Supplemental Figure 3A online). MaturemiR156 accumulation was also inhibited in MIM156 plants tovarying degrees (see Supplemental Figure 3B online). We notedthat the magnitude of reduction of mature miR156 accumulationwas not perfectly correlated with MIM156 transcript levels norwith phenotypic severity. The MIM method is based on the

Figure 1. Accumulation of miR156 and Its Targets during P. patensGametophyte Development.

Accumulation of P. patens miR156 and target SBP genes was assessedusing qRT-PCR. Relative accumulation levels relative to the 1-week samplewere plotted. Error bars indicate SD from three biological replicates. Anal-yses of significant differences are shown in Supplemental Figure 1 online.[See online article for color version of this figure.]

4838 The Plant Cell

sequestration of miRNA by overexpressed decoy targets, andMIM-induced reductions in mature miRNA levels have beeninconsistently observed (Franco-Zorrilla et al., 2007; Todescoet al., 2010). Therefore, the amount of functional inhibition ofmiR156 in individual MIM156 lines should not be directly inferredfrom accumulation of mature miR156 alone. In 3-week-oldMIM156 plants, accumulation of SBP3, SBP6, and SBP13 waselevated approximately two-fold (Figure 2G). By 5 weeks, ac-cumulation of these three miR156 target mRNAs in MIM156plants was indistinguishable from the wild type (Figure 2H). Wenoted that the magnitude of target upregulation was similar in theweak MIM156-1 line and the strong MIM156-2 and MIM156-3

lines (Figure 2G). This suggests that there are other factors be-sides miR156 that act as a buffer for regulating the accumulationof SBP mRNAs in P. patens. The timing of target upregulation inthe MIM156 plants coincided with both the maximal accumula-tion of miR156 and with the maximal initiation of leafy buds in thewild type. Collectively, these data indicate that miR156 functionsto promote the vegetative developmental transition from proto-nemata to leafy gametophores in P. patens.Relative accumulation of SBP3 was much higher than that

of SBP6 and SBP13 in wild-type P. patens (Figure 3A; seeSupplemental Figure 4 online). We therefore hypothesized thatmisregulation of SBP3 could be a major component of the

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-oldtissues (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).

The Moss miR156-miR390-tasiRNA Network 4839

MIM156-associated phenotypes. Deletion of the SBP3 locus wasachieved using a gene replacement strategy (see SupplementalFigure 5 online). The resulting DSBP3 null mutant had a pheno-type opposite to that seen in MIM156 plants: increased initiationand numbers of leafy buds relative to the wild type (Figures 3B to3E). SBP3 is therefore a negative regulator of bud and leafy ga-metophore formation in the P. patens gametophyte. We concludethat elimination of single miR156 target, SBP3, is sufficient toproduce a phenotype opposite to that caused by reduction ofmiR156 activity, implying that SBP3 is a miR156 target of majorimportance.

miR156 Suppresses Accumulation and Function ofmiR390-Dependent tasiRNAs

Several distinct small RNA pathways influence the rate andtiming of leafy gametophore formation in P. patens. Loss of the

P. patens–specific miRNA miR534a leads to elevated accumu-lation of two Blade on Petiole (BOP) target mRNAs and accel-erated production of leafy gametophores (Saleh et al., 2011).Accumulation of the miR534a-targeted BOP mRNAs was notsignificantly affected in 3-week-old MIM156 plants, suggestingthat the influences of miR156 and miR534a on bud formation areseparate (see Supplemental Figure 6A online). P. patens DDCL3mutants, in which 22- to 24-nucleotide small interfering RNAs(siRNAs) derived from intergenic/repetitive elements are lost,also have an accelerated leafy gametophore formation pheno-type (Cho et al., 2008). Accumulation of SBP3, SBP6, and SBP13was not detectably altered in 3-week-old DDCL3 plants, sug-gesting that the influence of DCL3 upon P. patens leafy budformation is also separate from that conditioned by miR156(see Supplemental Figure 6B online).P. patens DRNA DEPENDENT RNA POLYMERASE (RDR6)

plants also have an accelerated production of leafy gameto-phores (Talmor-Neiman et al., 2006). RDR6 is required for theaccumulation of TRANS-ACTING SIRNA3 (TAS3) family tasiRNAsderived from non-protein coding RNA precursors processed bymiR390-directed slicing (Axtell et al., 2006; Talmor-Neiman et al.,2006). Out of the large population of P. patens TAS3-derivedtasiRNAs, two are known to direct slicing of target mRNAs intrans: tasiAP2, which directs slicing of mRNAs encoding an ERF-like single N-terminal AP2 domain, and tasiARF, which directsslicing of mRNAs encoding B3 and ARF domains (Talmor-Neimanet al., 2006; Axtell et al., 2007). Although there are six P. patensTAS3 loci (TAS3a-f), TAS3a contributes the majority of tasiAP2and tasiARF production (see Supplemental Table 1 online) anddominates the overall small RNA profile from this family (Arif et al.,2012). We and our colleagues recently discovered anotherfamily of P. patens TAS loci, TAS6, characterized by miR156- andmiR529-directed slicing and close genomic proximity to TAS3loci (note that miR156 and miR529 are related in sequence) (Arifet al., 2012). Degradome analysis provided evidence that bothmiR156 sites are sliced, and phased siRNAs dependent uponRDR6 and DCL4 arise from the region between the two miR156sites (Arif et al., 2012). A small RNA gel blot demonstrated thatTAS6a small RNA accumulation was also dependent upon RDR6and DCL4 (see Supplemental Figure 7 online). The oppositephenotypes of DRDR6 and MIM156 plants, as well as the closeproximity of miR156-sliced TAS6 loci to miR390-sliced TAS3loci, suggested that the functions of P. patens miR156 andmiR390 may be directly intertwined. In support of this hypoth-esis, cDNA cloning revealed that TAS6a and TAS3a share asingle primary transcript, with a central 256-nucleotide-long in-tron separating the two tasiRNA generating regions (Figure 4A).TAS6b/TAS3f and TAS6c/TAS3d also occur in tandem arrange-ments that suggest origins from a common primary transcript (Arifet al., 2012).We hypothesized that miR156 affects tasiRNA accumulation

via interaction with the TAS6a/TAS3a primary transcript. To testthis hypothesis, we first examined accumulation of small RNAsin two independent MIM156 plants with a high level of MIM156accumulation. Accumulation of small RNAs from the TAS6aregion was reduced in MIM156 plants, while small RNAs fromthe TAS3a region were increased in MIM156 plants (Figure 4B).The specific tasiAP2 and tasiARF small RNAs responded similarly

Figure 3. SBP3 Represses Bud and Leafy Gametophore Formation.

(A) Accumulation of SBP transcripts during the P. patens life cycle. Ex-pression levels relative to EF1a in a sample were plotted. Error barsindicate SD from three biological replicates. Analyses of significant dif-ferences 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), andDSBP3 (D) were plated on cellophane overlaid media and incubated for2 weeks. Arrowheads in (B) indicate buds. Bars = 1 mm.(E) Rates of bud and gametophore appearance. Seven-day-old pro-tonemal tissues were inoculated on media. The numbers of buds andsubsequent gametophores in four colonies were counted every 2 days.Error bars represent SD of four replicates. Significant differences from thewild type on each day were analyzed by t test (*P # 0.05 and **P # 0.01).

4840 The Plant Cell

to the total TAS3a small RNA population, with an increasedaccumulation in MIM156 plants (Figure 4C). Consistent with thetasiRNA accumulation patterns in MIM156 plants, tasiAP2 andtasiARF targets generally had reduced accumulation levelsin MIM156 plants (Figure 4D). These data are consistent with a

hypothesis where miR156 influences the production of tasiARFand tasiAP2 production through an interaction with TAS6a-TAS3aprecursor.Surprisingly, we found that DSBP3 plants had an increased

accumulation of miR156 (Figure 4E). The basis for the increasedaccumulation of miR156 in DSBP3 plants is not clear. Onehypothesis consistent with this observation is that the SBP3transcription factor normally acts a negative regulator ofMIR156transcription. TAS3a small RNA accumulation in the DSBP3plants was not changed (Figures 4B and 4C), but two of thethree tasiAP2 target mRNAs accumulated at lower levels relativeto the wild type (Figure 4D). Indeed, the reductions in some ofthe tasiAP2 targets observed in DSBP3 plants were similarto those observed in the MIM156 lines, despite the oppositemorphological phenotypes (Figure 3), opposite miR156 accu-mulation patterns (Figure 4E), and unrelated tasiRNA accu-mulation patterns (Figure 4C) observed in these three lines.These data demonstrate that tasiAP2 and tasiARF accumula-tion levels are not the sole contributors to the accumulationlevels of their respective target mRNAs. Additionally, accu-mulation levels of the AP2 and ARF target mRNAs cannot be thesole determinant of bud initiation rate, as plants with oppositephenotypes (MIM156 and DSBP3) have the similar magnitude ofdown-regulation.Because MIM156 plants are developmentally retarded com-

pared with the wild type at 3 weeks of age, it was possible thatthe observed differences in small RNA and target accumulationwere due to different tissue compositions, instead of directmolecular effects. Arguing against this possibility, we observedsimilar differences in TAS3a and TAS6a small RNA accumulationand tasiAP2 and tasiARF target accumulation in 7-week-oldplants (see Supplemental Figure 8 online), a time point at whichall lines are more similar morphologically. TAS6a producesat least one functional tasiRNA (tasiZNF), which targets a zincfinger domain mRNA (Arif et al., 2012). The accumulation of thetasiZNF target was not significantly affected in MIM156 plants(see Supplemental Figure 9 online), though accumulation ofsmall RNAs from TAS6a was reduced (Figure 4B), which sug-gests that dysregulation of tasiZNF does not contribute to theMIM156 phenotype. Curiously, the tasiZNF target was signifi-cantly upregulated in DSBP3 (see Supplemental Figure 9 online).We suspect this is an indirect effect caused by the loss of theSBP3 transcription factor.

miR390 Represses Formation of Buds andLeafy Gametophores

We next examined the expression patterns of miR390, tasiARF,and tasiAP2 during gametophyte development (Figure 5A; seeSupplemental Figure 10A online). After spore germination,miR390 accumulation declined slightly, reaching a minimum atthe 3-week-old samples when buds and young gametophoresare enriched and then gradually increased again later in de-velopment. This pattern is the opposite to that of miR156 (Figure1). tasiARF and tasiAP2 also exhibited temporal expressionpatterns, although they were distinct from that of miR390 (Figure5A; see Supplemental Figure 10A online). The targets of tasiARFand tasiAP2 were also temporally regulated, with many of them

Figure 4. miR156 Influences P. patens tasiRNA Accumulation andFunction.

(A) The annotated TAS6a/TAS3a primary transcript including miRNAtarget sites, locations of functional tasiRNAs, exons (rectangles), andcentral intron.(B) Small RNA gel blot analysis from 3-week-old DSBP3, MIM156-2, andMIM156-3 plants. The numbers indicate the intensities relative to U6signals. WT, the wild type.(C) Stem loop qRT-PCR of tasiARF and tasiAP2 siRNAs from 3-week-oldplants. Error bars indicate SD from three biological replicates. Significantdifferences 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-oldplants. 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 byt 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 differencesfrom the wild type were analyzed by t test (*P # 0.05 and **P # 0.01).

The Moss miR156-miR390-tasiRNA Network 4841

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. Therelative accumulation levels to the 1-week sample were plotted. Error bars indicate SD from three biological replicates. Analyses of significant differencesare 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 tothe 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. Thenumbers 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. Significantdifferences from the wild type were analyzed by t test (*P # 0.05 and **P # 0.01).

4842 The Plant Cell

reaching peak accumulation levels 2 or 3 weeks after sporegermination (Figure 5B; see Supplemental Figure 10B online).The patterns of target accumulation were largely unrelated tothose of their corresponding tasiRNAs, especially at early de-velopmental time points. Therefore, tasiAP2 and tasiARF are notthe only factors that regulate the overall accumulation levels oftheir targets during development.

To test if miR390 directly affects the developmental transi-tion, we generated plants overexpressing P. patens MIR390Cunder the control of the Ubiquitin promoter. In the MIR390C-overexpressing plants (OE), miR390 accumulated ;64-foldhigher relative to the wild type, with no detectable effect uponmiR156 accumulation (Figure 5C). The MIR390C OE plants ex-hibited a slower gametophore transition, with a significant de-viation from the wild type at the 16th day after inoculation (Figure5D; see Supplemental Figure 11 and Supplemental Table 2 online).Accumulation of tasiAP2 and tasiARF was elevated in MIR390COE plants (Figure 5E), probably due to the higher availability ofRDR6 and DCL4 substrates produced by miR390-guided TAS3acleavage. The higher accumulation of tasiRNAs was correlatedwith reduction of transcript accumulation from four out of fiveof the tasiRNA targets (Figure 5F). We hypothesize that the oneexception to this trend, the tasiAP2 target 1s74_86V6.1 (Figure5F), is due to factors counteracting the effect of tasiAP2, con-sistent with the notion that tasiAP2 accumulation is not the soledeterminant of target accumulation levels. Interestingly, neithertasiZNF accumulation nor the accumulation of its target mRNAwere significantly different in MIR390C OE plants (Figures 5Eand 5G). Thus, miR156 affects tasiRNA accumulation from bothTAS6a and TAS3a (Figure 4), but miR390 only affects TAS3a.Overall, these data show that miR390 represses the bud for-mation in P. patens and affects accumulation of TAS3a-derivedtasiRNAs and their targets.

Cytokinin Application Does Not Strongly AffectAccumulation of miR156, miR390, tasiRNAs, orTheir Targets

Cytokinin induces caulonemal cells to differentiate into buds inP. patens (Cove and Knight, 1993; Schumaker and Dietrich,1997) and regulates the accumulation of miR534a (Saleh et al.,2011). Because miR156 and miR390 are involved in timing ofbud and gametophore formation, we tested whether cytokinintreatment affects accumulation of these small RNAs and/or theirtargets. Application of benzylaminopurine (BAP; e.g., cytokininB) to protonemata for 24 h accelerated bud formation (seeSupplemental Figure 12 online), without causing significantchanges in miR156 or miR390 accumulation (Figure 6A). Cyto-kinin also had negligible effects upon accumulation of thethree SBP targets of miR156 (Figure 6B) and upon tasiARF

and tasiAP2 accumulation (Figure 6C). Similarly, the accumu-lation of the two tasiARF target mRNAs was unaffected by BAPtreatment. By contrast, tasiAP2 targets responded with a two- tofourfold reduction in mRNA accumulation after cytokinin treat-ment (Figure 6D). Overall, these data indicate that cytokinintreatment does not substantially affect the miR156-miR390-tasiRNA network, at least at the level of RNA accumulation. Theone exception is the tasiAP2 target mRNAs, which appear torespond independent of any changes in tasiAP2 accumulation;these genes may serve as an integration point where the cyto-kinin-induced and miR156/miR390 pathways intersect. We con-clude that the cytokinin-induced bud formation pathway inP. patens is largely independent of, or downstream from, themiR156-miR390-tasiRNA network.

A miRNA and tasiRNA Regulatory Network ControllingDevelopmental Timing

Review and synthesis of previously described miRNA andtasiRNA targets (Arazi et al., 2005; Axtell et al., 2006, 2007;Talmor-Neiman et al., 2006; Addo-Quaye et al., 2009) in light ofimproved P. patens gene annotations revealed a densely con-nected regulatory network (Figure 7A; see Supplemental Figure13, Supplemental Table 3, and Supplemental Data Set 1 online).The conserved miRNAs miR156, miR390, and miR529 (closelyrelated in sequence to miR156), along with the P. patens–specificmiR1219 and two different TAS3-derived tasiRNAs, coordinatelyregulate members of three distinct transcription factor families(SBP, ERF-like AP2, and ARF). Each RNA target in this networkis regulated by two distinct small RNA families. Perturbation ofthis network influences the gametophyte body plan by eitherretarding (inhibition of miR156 function in MIM156 plants,overexpression of miR390 in MIR390C OE plants) or accelerat-ing (loss of a critical miR156 target in DSBP3 plants) the pro-duction of buds and leafy gametophores (Figure 7B). miR390and miR1219 accumulation is unaffected inMIM156 and DSBP3plants (Figure 4E; see Supplemental Figure 14A online), whilemiR529 accumulation is slightly reduced in MIM156 plants,possibly due to interaction with the miR156 target mimic RNA(see Supplemental Figure 14B online). (However, note that thesimilarity between miR529 and miR156 is not extensive enoughto allow miR156 to also target the tasiAP2 target mRNAs: Wefind no evidence for this interaction from degradome sequenc-ing data [Addo-Quaye et al., 2009], and the alignment betweenmiR156 and the tasiAP2 target mRNAs has extensive mis-matches, particularly in the critical 59 end of the alignment [seeSupplemental Figure 14C online].) Importantly, we don’t excludethe possibility of the presence of other currently unknowncomponents in this network as well and in particular the pos-sibility for significant feedback loops between transcription

Figure 5. (continued).

(F) Accumulation of tasiARF and tasiAP2 target mRNAs in 3-week-old plants measured by qRT-PCR. Error bars indicate SD from three biologicalreplicates. 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. Nosignificant differences relative to the wild type were found (t test, P > 0.05).

The Moss miR156-miR390-tasiRNA Network 4843

factors and the small RNAs that target them. The molecularphenotypes of the DSBP3 plants (increased miR156 accu-mulation and decreased tasiRNA target accumulation) hintat the likelihood of yet to be discovered connections in thisnetwork. In addition, it remains possible that TAS6a-derivedtasiRNAs might also affect developmental progression inP. patens.

DISCUSSION

miR156 and miR390 Control tasiRNA Accumulation andFunction in P. patens

In this study, we functionally characterized P. patensmiR156 andmiR390, two of the most ancient miRNAs in plants. MIM156plants exhibited a delayed bud and gametophore formationand a coincident induction of SBP targets. Deletion of a singlemiR156 target, SBP3, resulted in a phenotype opposite to thatcaused by inhibition of miR156, suggesting the SBP3 is amiR156 target of major phenotypic importance. Overexpression

of MIR390C also impeded the developmental transition. Ac-cordingly, the accumulation of tasiRNAs was elevated, and targetARF and AP2 transcription factors were suppressed in MIR390COE plants. These data indicate that miR156 and miR390 oppo-sitely regulate developmental timing in P. patens. Previously, in-volvement of two miRNAs in the same developmental processwas reported in Arabidopsis, in which the gradient of miR390 andmiR166 defines the leaf polar axis formation (Nogueira et al.,2007). While miR166 defines abaxial fates in leaf primordia,miR390 restricts the expression domain of abaxial determi-nants at the adaxial region. Thus, miR166 and miR390 regulateleaf asymmetry via reciprocal expression patterns. Functionalcharacterization of P. patens miR166 has not yet been re-ported. However, we have shown that P. patens miR390 andmiR156 converge upon the same pathway by regulating a sin-gle TAS precursor with opposite effects on the production ofkey tasiRNAs and ultimately upon phenotype. We hypothesizethat this convergent regulation confers a fine tuning on levels oftasiRNAs and their targets to ensure the optimal timing of theprocess of bud formation.

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 mM BAP or with a mock control. Error bars represent SDfrom 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 mMBAP or with a mock control. Error bars represent SDfrom 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. Significantdifferences from the wild type were analyzed by t test (*P # 0.05 and **P # 0.01).

4844 The Plant Cell

A Partially Conserved Small RNA Network Regulates BudFormation in Mosses

The conserved miRNAs miR156, miR529, miR390, as well as thenonconserved miRNA, miR1219, and TAS3-derived tasiRNAscoordinate the regulation of three distinct transcription factorfamilies in P. patens. While this network is not precisely repli-cated in angiosperms, there are several intriguing parallels(Figures 7C and 7D). The regulation of SBP domain mRNAs bymiR156 (Arazi et al., 2005; Schwab et al., 2005; Wu and Poethig,2006; Wu et al., 2009) and the regulation of ARF mRNAs bymiR390-dependent, TAS3-derived tasiRNAs is shared by bothlineages (Adenot et al., 2006; Fahlgren et al., 2006; Garcia et al.,2006; Hunter et al., 2006; Axtell et al., 2007). In angiosperms,miR172 targets several mRNAs containing dual AP2 domains,including AP2 itself (Aukerman and Sakai, 2003; Chen, 2004), isdirectly regulated by the products of miR156-targeted SBPgenes (Wu et al., 2009) and cooperates with miR156 to regulatemultiple facets of developmental timing in angiosperms (Huijserand Schmid, 2011). In addition, miR156 appears to directly co-operate with miR172 to trigger-phased siRNA production froma Medicago truncatula AP2 gene (Zhai et al., 2011). miR172 is

absent in P. patens; however, P. patens mRNAs containing anERF-like single AP2 domain are targeted by tasiRNAs, which arethemselves influenced by miR156. We did not find neighboringmiR156 and miR390 complementary regions (“neighboring” wasdefined as occurring within 20 kb) in a survey of 19 seed plantgenomes (one lycophyte, four monocots, and 15 eudicots), sug-gesting that the TAS6/TAS3 arrangement may be unique to thebryophyte lineage. Nonetheless, a potential connection betweenmiR156 and miR390 in Arabidopsis is suggested by the un-explained influence of the ArabidopsismiR390 tasiRNA pathwayupon the accumulation of the miR156 SBP domain target SPL3(Peragine et al., 2004).miR156, miR390, and the targets of miR156 influence vege-

tative developmental transitions in both mosses and floweringplants. Interestingly, the directionality of miR156’s phenotypicinfluence differs between flowering plants and mosses; it re-presses transitions in the former and promotes them in the lat-ter. By contrast, miR390 represses developmental transitionsin both lineages. The tight regulatory network found in P. patensconverging upon interlocking small RNA regulation of SBP/SPL,AP2, and ARF mRNAs appears to have been partially conserved

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, grayregions 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 withArabidopsis; 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.

The Moss miR156-miR390-tasiRNA Network 4845

during land plant diversification. Comparing the genes directlyregulated by SBP/SPL, AP2, and ARF transcription factors indifferent plant lineages should be a productive avenue of futureresearch.

However, it is not yet clear if the tasiRNA-targeted ARF andAP2 genes in P. patens directly regulate bud formation or,alternatively, whether the developmental effects of MIR390overexpression might instead be mediated by other unknowncomponents. We favor the hypothesis that the bud formationdefects seen upon overexpression of MIR390 are due to theelevated levels of AP2 and/or ARFmRNAs that are apparent, butwe have not yet directly demonstrated this by analysis of therelevant P. patens ARF and AP2 mutants. In addition, accu-mulation levels of the relevant tasiRNAs are not strongly corre-lated with those of their target mRNAs during gametophoredevelopment (Figures 5A and 5B). One possible explanation forthis discordance is that the AP2 and ARF mRNA levels arecontrolled by more factors other than just tasiRNAs. However,we cannot exclude the possibility that other, currently unknowngenes, mediate the miR390-dependent phenotypes in P. patens.

Many Pathways Influence Bud Formation in P. patens

Several previous studies reported the role of small RNAs or ofsmall RNA-related genes in the timing and rate of bud and leafygametophore development in P. patens, including miR534a(Saleh et al., 2011), DCL3 (Cho et al., 2008), and RDR6 (Talmor-Neiman et al., 2006; Axtell et al., 2007). In this study, wedemonstrated that a miR156- and miR390-regulated tasiRNAnetwork is also involved in this process. The requirement ofRDR6 for tasiRNA biogenesis integrates RDR6 into the miR156-miR390-tasiRNA network; however, to the best of our currentknowledge, miR534a and DCL3 both act independently of themiR156-miR390-tasiRNA pathway to regulate bud and leafygametophore production.

miR534a, a moss-specific miRNA targeting the BOP1 andBOP2 transcription factor mRNAs, represses bud and leafygametophore formation (Saleh et al., 2011), which is oppositeto the role of miR156 and similar to the role of miR390. Cyto-kinin, which is critical for bud initiation in P. patens (Cove andKnight, 1993; Schumaker and Dietrich, 1997), downregulates theMIR534a promoter and causes a correlated increase in BOP1and BOP2 accumulation (Saleh et al., 2011). Our data show thatreduction of miR156 function in MIM156 plants did not affectaccumulation levels of BOP1 or BOP2, and cytokinin treatmenthad negligible effects on the accumulation of most of the RNAsinvolved in the miR156-miR390-tasiRNA pathway. Thus, themiR156-miR390-tasiRNA pathways and the cytokinin-miR534apathways appear to independently control bud and gameto-phore production.

DCL3, responsible for 22- to 24-nucleotide-long siRNA pro-duction from repetitive genomic regions, negatively affectsdevelopmental timing (Cho et al., 2008; Arif et al., 2012). Theaccumulation of the miR156-targeted SBP mRNAs was un-changed in DDCL3 plants, and the accumulation of miRNAs isnot affected in DDCL3 mutants (Cho et al., 2008). Thus, themiR156-miR390-tasiRNA pathway, the cytokinin-miR534a-BOPpathway, and the DCL3 pathway all seem to independently

affect the timing of bud formation in P. patens. Nonetheless, weacknowledge that further investigation might yet uncover mo-lecular links between these three pathways.Why does P. patens devote so much regulatory capacity to

the control of bud development? The transition from filamen-tous, two-dimensional growth to the production of upright leafygametophores is a major developmental transition for mosses.Bud formation and subsequent leafy gametophore growth willonly be successful if the protonematal network has becomeextensive enough to anchor the growing plant and can provideadequate nutrients, especially nitrogen, from the soil. Furthermore,production of buds and leafy gametophores is a prerequisite forthe appearance of gametangia and subsequent attempts atsexual reproduction. Perhaps the importance of the decision toproduce leafy buds and gametophores and the need to integratemultiple endogenous and environmental stimuli has led to themultiplicity of pathways that affect this trait.

METHODS

Oligonucleotides

All oligonucleotide sequences are listed in Supplemental Table 4 online.

Construction of Vectors

For construction of the MIM156 vector, the At4 gene was amplified fromArabidopsis thaliana and cloned into the pUJ3 vector. The pUJ3 vectorcontained the Gateway cassette under the control of themaize (Zeamays)Ubiquitin promoter and a hygromycin selection cassette, in which twoDNA fragments from the 108 locus are inserted at the flanking regions ofthe expression and the selection cassette for targeting into the 108 locusinto the moss genome (Schaefer and Zrÿd, 1997). To create a miR156target mimic, oligos were hybridized and ligated with the HindIII digestedpUJ3-At4 (Figure 2A). For the SBP3 knock out construct, two ;1-kbregions 59 and 39 from the open reading frame of SBP3were amplified andinserted into the pUQ vector (Cho et al., 2008). The vector for MIR390coverexpression was obtained by the insertion of pri-MIR390c region intothe BsrGI site in the pUJ3 vector.

Transformation and DNA Gel Blot Analysis

Physcomitrella patens transformation was performed as described (Choet al., 2008). Genomic DNAs were extracted using a Phytopure DNAextraction kit (GE Healthcare) and used for genotyping via PCR. For DNAgel blot analysis of DSBP3, the SphI-digested genomic DNAs wereblotted onto a Hybond NX nylon membrane (GE Healthcare) and hy-bridized following a standard protocol (Sambrook and Russell, 2001). Asa probe, hptII fragment was amplified by PCR and radiolabeled with[a-32P]dCTP using an NEblot kit (New England Biolabs).

Rapid Amplification of cDNA Ends–PCR

The full-length cDNA of TAS6a and TAS3a precursor was cloned using theGeneRacer kit (Invitrogen) following the manufacturer’s protocol.

Quantitative Real-Time RT-PCR

Total RNA for miRNA analyses were isolated using Tri-Reagent (Sigma-Aldrich) following the manufacturer’s instructions and transcribed with aSuperscript III reverse transcriptase (Invitrogen) essentially as previously

4846 The Plant Cell

described (Varkonyi-Gasic et al., 2007). DNase I (Fermentas)–treated totalRNAs (100 ng) were converted to cDNAs with Superscript III reversetranscriptase, primed by miRNA-specific stem loop primers using apulsed RT reaction as described (Varkonyi-Gasic et al., 2007). As a ref-erence, the primer for U6 was also included in the same reaction. A serialdilution was included in the same reaction plate of real-time PCR tocalculate the PCR efficiency.

Total RNAs for mRNA analyses were isolated using an RNeasy plantmini kit (Qiagen) and reverse transcribed with a QuantiTect reversetranscription kit (Qiagen). EF1a was used as a reference. Real-time PCRswere performed using a QuantiTect SYBR Green PCR kit (Qiagen) with anABI7300 teal-time PCR system (Applied Biosystems). PCR efficiencieswere calculated based on serial dilution and used to determine relativeaccumulation levels by the using the efficiency corrected DDCt (cyclethreshold) method. Relative expression was normalized to the per-genewild-type values, except Figures 1, 5A, and 5B, which were normalized tothe per-gene week one value, and Figure 3A to the per-EF1a values,respectively, or as described (see Supplemental Figures 1, 3, 4, 6, and 8 to10 online). As a statistical analysis, Student’s t tests were performed withtwo-tailed distribution.

Small RNA Gel Blots

Total RNAs, including small RNAs, were isolated using Tri reagent (Sigma-Aldrich) following the manufacturer’s instructions. Small RNAs wereenriched as described (Blevins, 2010). Eight micrograms of small RNAswere resolved in 15% Criterion TBE-Urea gel and transferred to theHybond-NX nylon membrane (GE Healthcare), followed by cross-linkingusing a 1-ethyl-3-(3-dimethylamonipropyl) carbodiimide–mediated chem-ical cross-linking method (Pall and Hamilton, 2008). The probes forTAS3a, TAS6a, and U6 were amplified from cDNA by PCR and radio-labeled with [a-32P]dCTP using an NEblot Kit (New England Biolabs) andhybridized to the membrane at 37°C within PerfectHyb solution (Sigma-Aldrich). Membranes were washed twice with nonstringent washing buffer(33 SSC, 5% SDS, and 25 mM NaH2PO4, pH 7.5) for 10 min and twice for30 min at 42°C. The final wash was performed with the stringent washbuffer (13 SSC and 1% SDS) for 5 min at 42°C. The oligo probes fortasiAP2, tasiARF, miR529, miR1219, and U6 were radiolabeled with[g-32P]dATP using T4 polynucleotide kinase (New England Biolabs) fol-lowing the manufacturer’s protocol, hybridized to the nylon membrane at42°C, and washed as in the double-stranded DNA probes at 50°C. Theradioactive signals were recorded onto phosphor screens (GEHealthcare)and scanned using a STORM860 phosphor imager (Amersham Bio-sciences). Membranes were deprobed by washing in 1% SDS at 80°C for30 min and hybridized with another probe. Image quantification wasperformed with the ImageJ program (NIH).

Small RNA Target Analyses

P. patens degradome data (National Center for Biotechnology Informa-tion Gene Expression Omnibus GSM410805; Addo-Quaye et al., 2009)were analyzed using CleaveLand 3.0.1 (http://axtell-lab-psu.weebly.com/cleaveland.html). Transcript models were the version 6.1 models,downloaded from Phytozome 7.0 in August, 2011, augmented with thegenomic loci encompassing known TAS3 and TAS6 loci or, in the case ofTAS6a/TAS3a, the full-length primary transcript. Strong evidence ofslicing (thick lines in Supplemental Figure 13 online) reflects either priorevidence from the literature in the form of gene-specific RNA ligase–mediated 59 rapid amplification of cDNA ends, degradome-based evi-dence with a P value# 0.05, or both. Weak evidence for slicing (solid thinlines in Supplemental Figure 13 online) reflects degradome data with a Pvalue > 0.05. Details are in Supplemental Table 3 and Supplemental DataSet 1 online.

Measurement of Growth Rate and BAP Treatment

For measurement of growth rate, one to two filaments of 1-week-oldprotonemal tissues were inoculated onto BCDmedia with (Figures 3E and5D) or without (see Supplemental Figure 11 online) ammonium supple-mentation under standard growth conditions (25°C, 16 h day/8 h night)(Ashton and Cove, 1977). Numbers of buds and gametophores werecounted every 2 d.

For BAP treatment, blended protonemata were grown on cellophaneoverlaid BCD media with ammonium. BAP (5 mM; Sigma-Aldrich) dis-solved in 40 mM NaOH was added on top of the protonemata andincubated for 24 h. As a mock control, 40 mM NaOH treatment wasperformed simultaneously.

Accession Number

Sequence data for the TAS6a/TAS3a primary transcript from this articlecan be found in the GenBank/EMBL data libraries under accession numberJN674513.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Accumulation of miR156 and Its Targetsduring P. patens Gametophyte Development.

Supplemental Figure 2. The Life Cycle of Physcomitrella patens

Supplemental Figure 3. Functional Knockdown of miR156 by TargetMimicry.

Supplemental Figure 4. Accumulation of SBP Transcripts during theP. patens Gametophyte Development.

Supplemental Figure 5. Targeted Knockout of SBP3.

Supplemental Figure 6. Transcript Levels of Genes Related to theGametophore Transition in P. patens.

Supplemental Figure 7. Small RNA Gel Blots of TAS6a and TAS3a inDDCL4 and DRDR6.

Supplemental Figure 8. miR156 Influences P. patens tasiRNAAccumulation and Their Targets in Older Tissues.

Supplemental Figure 9. Transcript Level of the Target of tasiZNF inDSBP3 and in MIM156s in 3-Week-Old Samples.

Supplemental Figure 10. Accumulation of miR390-RegulatedtasiRNAs and Their Targets

Supplemental Figure 11. Comparison of Bud and Leafy Gameto-phore Formation in MIM390C OE, DSBP3, and DRDR6-19 Plants.

Supplemental Figure 12. Cytokinin Treatment Accelerates BudFormation.

Supplemental Figure 13. Detailed Small RNA–Target Interactions inP. patens.

Supplemental Figure 14. Accumulation of miR529 and miR1219 inMIM156-2 and DSBP3 Plants.

Supplemental Table 1. Origins of P. patens tasiAP2 and tasiARF.

Supplemental Table 2. Bud Formation in MIR390C-OverexpressingP. patens.

Supplemental Table 3. Summary of small RNA/Target Data.

Supplemental Table 4. Oligonucleotide Sequences Used in ThisStudy.

Supplemental Data Set 1. Small RNA Target Alignments andDegradome Data.

The Moss miR156-miR390-tasiRNA Network 4847

ACKNOWLEDGMENTS

This work was funded by a grant from the U.S. National Institutes ofHealth-National Institute of General Medical Sciences (R01 GM84051) toM.J.A. We thank Jo Ann Snyder for technical support, Zhaorong Ma andWolfgang Frank for insights on the TAS6 family, Rajendran Rajeswaranfor discussions and pilot experiments, and all members of the Axtell Labfor many constructive discussions during this study.

AUTHOR CONTRIBUTIONS

M.J.A. and S.H.C. designed the research. S.H.C. and C.C. performedresearch. M.J.A. analyzed degradome and target prediction data. M.J.A.and S.H.C. wrote and edited the article with input from C.C.

Received July 23, 2012; revised December 7, 2012; accepted December10, 2012; published December 21, 2012.

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The Moss miR156-miR390-tasiRNA Network 4849

DOI 10.1105/tpc.112.103176; originally published online December 21, 2012; 2012;24;4837-4849Plant Cell

Sung Hyun Cho, Ceyda Coruh and Michael J. AxtellPhyscomitrella patens

miR156 and miR390 Regulate tasiRNA Accumulation and Developmental Timing in

 This information is current as of June 1, 2018

 

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