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miR156 and miR390 Regulate tasiRNA Accumulation and
Developmental Timing in Physcomitrella patens
C W OA
Sung Hyun Cho,a Ceyda Coruh,a,b and Michael J. Axtella,b,1
a Department
b Plant
of Biology, Penn State University, University Park, Pennsylvania 16802
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 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.
INTRODUCTION
Plants produce both a multicellular haploid entity (the gametophyte) and a multicellular diploid entity (the sporophyte) during
their sexual life cycles. Mosses and other bryophytes have
morphologically complex, photosynthetic gametophytes with
attached simple nonphotosynthetic sporophytes. By contrast,
other lineages, including flowering plants, have simple, microscopic gametophytes enclosed by, and nutritionally dependent
upon, a morphologically complex sporophyte. The extent to
which genes with ancestral roles in the development of complex
gametophytes were recruited to control complex sporophytic
development is unclear as both positive (Menand et al., 2007)
and negative (Tanahashi et al., 2005) examples have been described. The question is particularly acute for microRNAs
(miRNAs) because several miRNA families are conserved in a
wide range of land plants, including both sporophyte-dominated
and gametophyte-dominated species (Cuperus et al., 2011). The
regulatory targets of these conserved miRNA families are, as
a general rule, also conserved, and the majority of the most
ancient plant miRNA families regulate mRNAs encoding transcription factors (Axtell and Bowman, 2008). The functions of
many of these miRNAs and their targets have been intensively
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Michael J. Axtell (mja18@
psu.edu).
C
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white in the print edition.
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www.plantcell.org/cgi/doi/10.1105/tpc.112.103176
investigated in several flowering plants and frequently found to
affect various aspects of sporophytic development (Axtell and
Bowman, 2008). Global reduction of miRNA accumulation in
DICER-LIKE 1A (DCL1a) mutants of the moss Physcomitrella
patens, whose life cycle is dominated by a morphologically
complex gametophyte, has pleiotropic effects on gametophytic
development (Khraiwesh et al., 2010). However, the functions
of specific ancient plant miRNA families in P. patens or other
gametophyte-dominated basal plant lineages remain unclear.
miR156 is a master regulator of developmental transitions in
flowering plants. miR156 accumulation in flowering plants is
temporally regulated, with high levels in early embryos, and
a progressive reduction as the plant ages (Wu and Poethig,
2006; Wang et al., 2009). Conversely, the accumulation of its
SQUAMOSA PROMOTER BINDING PROTEIN- LIKE (SPL) targets generally increases with age in flowering plants (Wu and
Poethig, 2006; Wang et al., 2009). SPL genes in flowering plants
promote adult leaf morphology and flowering (Poethig, 2009;
Huijser and Schmid, 2011). miR172-mediated regulation of
APETALA2 (AP2) family genes has a reciprocal relationship with
miR156/SPL: miR172 levels increase with plant age, while
miR172-targeted AP2 family genes, several of which serve to
repress adult traits, decrease (Huijser and Schmid, 2011). In
Arabidopsis thaliana, this coordination is achieved in part by
direct stimulation of miR172 transcription by miR156-targeted
SPL9 and SPL10 (Wu et al., 2009). Genetic screens for Arabidopsis vegetative developmental mutants (Hunter et al., 2003,
2006; Peragine et al., 2004; Fahlgren et al., 2006) have identified
a third small RNA pathway that influences vegetative developmental transitions. miR390 stimulates production of transacting small interfering RNAs (tasiRNAs) that target AUXIN
RESPONSE FACTOR3 (ARF3)/ETTIN and ARF4 mRNAs. Loss
of tasiRNA-mediated ARF3/4 regulation accelerates vegetative
The Plant Cell Preview, www.aspb.org ã 2012 American Society of Plant Biologists. All rights reserved.
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developmental transitions, implying that ARF3/ETTIN and ARF4
promote these changes. The miR390/tasiRNA/ARF regulatory
system also influences organ polarity in flowering plants (Garcia
et al., 2006; Nogueira et al., 2007). The miR156/SPL regulatory
interaction appears universal among land plants (Arazi et al.,
2005; Axtell et al., 2007), and the miR390/tasiRNA/ARF cascade
appears nearly universal (Axtell et al., 2006) save for an apparent
secondary loss in the lycophyte lineage (Banks et al., 2011),
while the miR172/AP2 interaction appears to have emerged
later, 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 that
miR156 promotes the transition from tip-growing protonemal
growth to production of leafy gametophores. Interestingly,
miR156-mediated promotion of this vegetative transition is
contrary to the inhibitory role of miR156 upon developmental
transitions in flowering plants. In addition, P. patens miR156 and
miR390 both directly target the same major tasiRNA primary
transcript, and perturbation of miR156 influences accumulation of tasiRNAs and their target mRNAs. Overexpression of
MIR390C resulted in slower gametophore production, accompanied by reduction of tasiRNA targets. Finally, we describe
a gene regulatory network in P. patens in which a set of miRNAs,
tasiRNAs, and three transcription factor families converge to
control developmental timing. This network is partially conserved in flowering plants, suggesting that the regulatory networks for developmental transition present in modern species
descended and diversified from those present in an ancient
ancestor.
RESULTS
Temporal Regulation of miR156 and Its Targets in P. patens
miR156 accumulation in flowering plant sporophytes is temporally regulated, with high levels in early embryos and a progressive reduction as the plant ages (Wu and Poethig, 2006;
Wang et al., 2009). Conversely, the accumulation of its SPL
targets generally increases with age in flowering plants (Wu and
Poethig, 2006; Wang et al., 2009). Stem loop–mediated quantitative RT-PCR (qRT-PCR) (Varkonyi-Gasic et al., 2007) indicated that miR156 accumulation is also temporally regulated
in P. patens gametophytes (Figure 1; see Supplemental Figure 1
online). Young plants comprised solely of tip-growing, filamentous protonemata tissues (chloronema and caulonema; 1 to 2
weeks after germination from spores; see Supplemental Figure 2
online) have relatively little miR156 accumulation. Young buds
and early gametophores with one or two phylloids are most
enriched in 3-week old plants (see Supplemental Figure 2 online), with patches of buds on top of the lawn of filamentous
protonemata (Huijser and Schmid, 2011). Buds are the intermediate tissues between protonemata and gametophores.
Thus, we define these plants as undergoing the developmental
transition from young protonemata to adult leafy gametophores.
In these plants, the accumulation of miR156 reaches its maximum level (Figure 1; see Supplemental Figure 1 online). miR156
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.]
levels then gradually decline from their 3-week maximum as the
leafy gametophores mature (Figure 1; see Supplemental Figure
1 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. patens
miR156-targeted SBP genes are in the same gene family as the
miR156-targeted SPL genes in Arabidopsis and other flowering
plants; to be consistent with the prior literature [Riese et al.,
2007], we use the SBP acronym when referring to the P. patens
members of this gene family.) Like miR156, the SBP targets
were minimally expressed in 1- to 2-week-old plants. Increased
accumulation of SBP targets became apparent in older plants at
time points when miR156 accumulation levels were declining
from their maximum values (Figure 1; see Supplemental Figure 1
online).
miR156 Promotes Gametophore Production in P. patens
via SBP3
A miRNA target mimic (Franco-Zorrilla et al., 2007; Todesco
et al., 2010) designed to reduce miR156 function was overexpressed in wild-type P. patens (Figure 2A). Four independent
MIM156 transgenic lines had delayed bud initiation and reduced
numbers of leafy buds and subsequent gametophores (Figures
2B to 2F). In general, phenotypic severity was positively correlated 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). Mature
miR156 accumulation was also inhibited in MIM156 plants to
varying degrees (see Supplemental Figure 3B online). We noted
that the magnitude of reduction of mature miR156 accumulation
was not perfectly correlated with MIM156 transcript levels nor
with phenotypic severity. The MIM method is based on the
The Moss miR156-miR390-tasiRNA Network
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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).
sequestration of miRNA by overexpressed decoy targets, and
MIM-induced reductions in mature miRNA levels have been
inconsistently observed (Franco-Zorrilla et al., 2007; Todesco
et al., 2010). Therefore, the amount of functional inhibition of
miR156 in individual MIM156 lines should not be directly inferred
from accumulation of mature miR156 alone. In 3-week-old
MIM156 plants, accumulation of SBP3, SBP6, and SBP13 was
elevated approximately two-fold (Figure 2G). By 5 weeks, accumulation of these three miR156 target mRNAs in MIM156
plants was indistinguishable from the wild type (Figure 2H). We
noted that the magnitude of target upregulation was similar in the
weak MIM156-1 line and the strong MIM156-2 and MIM156-3
lines (Figure 2G). This suggests that there are other factors besides miR156 that act as a buffer for regulating the accumulation
of SBP mRNAs in P. patens. The timing of target upregulation in
the MIM156 plants coincided with both the maximal accumulation of miR156 and with the maximal initiation of leafy buds in the
wild type. Collectively, these data indicate that miR156 functions
to promote the vegetative developmental transition from protonemata 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; see
Supplemental Figure 4 online). We therefore hypothesized that
misregulation of SBP3 could be a major component of the
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MIM156-associated phenotypes. Deletion of the SBP3 locus was
achieved using a gene replacement strategy (see Supplemental
Figure 5 online). The resulting DSBP3 null mutant had a phenotype opposite to that seen in MIM156 plants: increased initiation
and numbers of leafy buds relative to the wild type (Figures 3B to
3E). SBP3 is therefore a negative regulator of bud and leafy gametophore formation in the P. patens gametophyte. We conclude
that elimination of single miR156 target, SBP3, is sufficient to
produce a phenotype opposite to that caused by reduction of
miR156 activity, implying that SBP3 is a miR156 target of major
importance.
miR156 Suppresses Accumulation and Function of
miR390-Dependent tasiRNAs
Several distinct small RNA pathways influence the rate and
timing of leafy gametophore formation in P. patens. Loss of the
Figure 3. SBP3 Represses Bud and Leafy Gametophore Formation.
(A) Accumulation of SBP transcripts during the P. patens life cycle. Expression levels relative to EF1a 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
DSBP3 (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).
P. patens–specific miRNA miR534a leads to elevated accumulation of two Blade on Petiole (BOP) target mRNAs and accelerated production of leafy gametophores (Saleh et al., 2011).
Accumulation of the miR534a-targeted BOP mRNAs was not
significantly affected in 3-week-old MIM156 plants, suggesting
that the influences of miR156 and miR534a on bud formation are
separate (see Supplemental Figure 6A online). P. patens DDCL3
mutants, in which 22- to 24-nucleotide small interfering RNAs
(siRNAs) derived from intergenic/repetitive elements are lost,
also have an accelerated leafy gametophore formation phenotype (Cho et al., 2008). Accumulation of SBP3, SBP6, and SBP13
was not detectably altered in 3-week-old DDCL3 plants, suggesting that the influence of DCL3 upon P. patens leafy bud
formation 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 gametophores (Talmor-Neiman et al., 2006). RDR6 is required for the
accumulation of TRANS-ACTING SIRNA3 (TAS3) family tasiRNAs
derived from non-protein coding RNA precursors processed by
miR390-directed slicing (Axtell et al., 2006; Talmor-Neiman et al.,
2006). Out of the large population of P. patens TAS3-derived
tasiRNAs, two are known to direct slicing of target mRNAs in
trans: tasiAP2, which directs slicing of mRNAs encoding an ERFlike single N-terminal AP2 domain, and tasiARF, which directs
slicing of mRNAs encoding B3 and ARF domains (Talmor-Neiman
et al., 2006; Axtell et al., 2007). Although there are six P. patens
TAS3 loci (TAS3a-f), TAS3a contributes the majority of tasiAP2
and tasiARF production (see Supplemental Table 1 online) and
dominates the overall small RNA profile from this family (Arif et al.,
2012). We and our colleagues recently discovered another
family of P. patens TAS loci, TAS6, characterized by miR156- and
miR529-directed slicing and close genomic proximity to TAS3
loci (note that miR156 and miR529 are related in sequence) (Arif
et al., 2012). Degradome analysis provided evidence that both
miR156 sites are sliced, and phased siRNAs dependent upon
RDR6 and DCL4 arise from the region between the two miR156
sites (Arif et al., 2012). A small RNA gel blot demonstrated that
TAS6a small RNA accumulation was also dependent upon RDR6
and DCL4 (see Supplemental Figure 7 online). The opposite
phenotypes of DRDR6 and MIM156 plants, as well as the close
proximity of miR156-sliced TAS6 loci to miR390-sliced TAS3
loci, suggested that the functions of P. patens miR156 and
miR390 may be directly intertwined. In support of this hypothesis, cDNA cloning revealed that TAS6a and TAS3a share a
single primary transcript, with a central 256-nucleotide-long intron separating the two tasiRNA generating regions (Figure 4A).
TAS6b/TAS3f and TAS6c/TAS3d also occur in tandem arrangements that suggest origins from a common primary transcript (Arif
et al., 2012).
We hypothesized that miR156 affects tasiRNA accumulation
via interaction with the TAS6a/TAS3a primary transcript. To test
this hypothesis, we first examined accumulation of small RNAs
in two independent MIM156 plants with a high level of MIM156
accumulation. Accumulation of small RNAs from the TAS6a
region was reduced in MIM156 plants, while small RNAs from
the TAS3a region were increased in MIM156 plants (Figure 4B).
The specific tasiAP2 and tasiARF small RNAs responded similarly
The Moss miR156-miR390-tasiRNA Network
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 DSBP3, 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).
to the total TAS3a small RNA population, with an increased
accumulation in MIM156 plants (Figure 4C). Consistent with the
tasiRNA accumulation patterns in MIM156 plants, tasiAP2 and
tasiARF targets generally had reduced accumulation levels
in MIM156 plants (Figure 4D). These data are consistent with a
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hypothesis where miR156 influences the production of tasiARF
and tasiAP2 production through an interaction with TAS6a-TAS3a
precursor.
Surprisingly, we found that DSBP3 plants had an increased
accumulation of miR156 (Figure 4E). The basis for the increased
accumulation of miR156 in DSBP3 plants is not clear. One
hypothesis consistent with this observation is that the SBP3
transcription factor normally acts a negative regulator of MIR156
transcription. TAS3a small RNA accumulation in the DSBP3
plants was not changed (Figures 4B and 4C), but two of the
three tasiAP2 target mRNAs accumulated at lower levels relative
to the wild type (Figure 4D). Indeed, the reductions in some of
the tasiAP2 targets observed in DSBP3 plants were similar
to those observed in the MIM156 lines, despite the opposite
morphological phenotypes (Figure 3), opposite miR156 accumulation patterns (Figure 4E), and unrelated tasiRNA accumulation patterns (Figure 4C) observed in these three lines.
These data demonstrate that tasiAP2 and tasiARF accumulation levels are not the sole contributors to the accumulation
levels of their respective target mRNAs. Additionally, accumulation levels of the AP2 and ARF target mRNAs cannot be the
sole determinant of bud initiation rate, as plants with opposite
phenotypes (MIM156 and DSBP3) have the similar magnitude of
down-regulation.
Because MIM156 plants are developmentally retarded compared with the wild type at 3 weeks of age, it was possible that
the observed differences in small RNA and target accumulation
were due to different tissue compositions, instead of direct
molecular effects. Arguing against this possibility, we observed
similar differences in TAS3a and TAS6a small RNA accumulation
and tasiAP2 and tasiARF target accumulation in 7-week-old
plants (see Supplemental Figure 8 online), a time point at which
all lines are more similar morphologically. TAS6a produces
at least one functional tasiRNA (tasiZNF), which targets a zinc
finger domain mRNA (Arif et al., 2012). The accumulation of the
tasiZNF target was not significantly affected in MIM156 plants
(see Supplemental Figure 9 online), though accumulation of
small RNAs from TAS6a was reduced (Figure 4B), which suggests that dysregulation of tasiZNF does not contribute to the
MIM156 phenotype. Curiously, the tasiZNF target was significantly upregulated in DSBP3 (see Supplemental Figure 9 online).
We suspect this is an indirect effect caused by the loss of the
SBP3 transcription factor.
miR390 Represses Formation of Buds and
Leafy Gametophores
We next examined the expression patterns of miR390, tasiARF,
and tasiAP2 during gametophyte development (Figure 5A; see
Supplemental Figure 10A online). After spore germination,
miR390 accumulation declined slightly, reaching a minimum at
the 3-week-old samples when buds and young gametophores
are enriched and then gradually increased again later in development. This pattern is the opposite to that of miR156 (Figure
1). tasiARF and tasiAP2 also exhibited temporal expression
patterns, although they were distinct from that of miR390 (Figure
5A; see Supplemental Figure 10A online). The targets of tasiARF
and tasiAP2 were also temporally regulated, with many of them
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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 qRTPCR. 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).
The Moss miR156-miR390-tasiRNA Network
reaching peak accumulation levels 2 or 3 weeks after spore
germination (Figure 5B; see Supplemental Figure 10B online).
The patterns of target accumulation were largely unrelated to
those of their corresponding tasiRNAs, especially at early developmental time points. Therefore, tasiAP2 and tasiARF are not
the only factors that regulate the overall accumulation levels of
their targets during development.
To test if miR390 directly affects the developmental transition, we generated plants overexpressing P. patens MIR390C
under the control of the Ubiquitin promoter. In the MIR390Coverexpressing plants (OE), miR390 accumulated ;64-fold
higher relative to the wild type, with no detectable effect upon
miR156 accumulation (Figure 5C). The MIR390C OE plants exhibited a slower gametophore transition, with a significant deviation from the wild type at the 16th day after inoculation (Figure
5D; see Supplemental Figure 11 and Supplemental Table 2 online).
Accumulation of tasiAP2 and tasiARF was elevated in MIR390C
OE plants (Figure 5E), probably due to the higher availability of
RDR6 and DCL4 substrates produced by miR390-guided TAS3a
cleavage. The higher accumulation of tasiRNAs was correlated
with reduction of transcript accumulation from four out of five
of the tasiRNA targets (Figure 5F). We hypothesize that the one
exception to this trend, the tasiAP2 target 1s74_86V6.1 (Figure
5F), is due to factors counteracting the effect of tasiAP2, consistent with the notion that tasiAP2 accumulation is not the sole
determinant of target accumulation levels. Interestingly, neither
tasiZNF accumulation nor the accumulation of its target mRNA
were significantly different in MIR390C OE plants (Figures 5E
and 5G). Thus, miR156 affects tasiRNA accumulation from both
TAS6a and TAS3a (Figure 4), but miR390 only affects TAS3a.
Overall, these data show that miR390 represses the bud formation in P. patens and affects accumulation of TAS3a-derived
tasiRNAs and their targets.
Cytokinin Application Does Not Strongly Affect
Accumulation of miR156, miR390, tasiRNAs, or
Their Targets
Cytokinin induces caulonemal cells to differentiate into buds in
P. 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 of
bud and gametophore formation, we tested whether cytokinin
treatment affects accumulation of these small RNAs and/or their
targets. Application of benzylaminopurine (BAP; e.g., cytokinin
B) to protonemata for 24 h accelerated bud formation (see
Supplemental Figure 12 online), without causing significant
changes in miR156 or miR390 accumulation (Figure 6A). Cytokinin also had negligible effects upon accumulation of the
three SBP targets of miR156 (Figure 6B) and upon tasiARF
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and tasiAP2 accumulation (Figure 6C). Similarly, the accumulation of the two tasiARF target mRNAs was unaffected by BAP
treatment. By contrast, tasiAP2 targets responded with a two- to
fourfold reduction in mRNA accumulation after cytokinin treatment (Figure 6D). Overall, these data indicate that cytokinin
treatment does not substantially affect the miR156-miR390tasiRNA network, at least at the level of RNA accumulation. The
one exception is the tasiAP2 target mRNAs, which appear to
respond independent of any changes in tasiAP2 accumulation;
these genes may serve as an integration point where the cytokinin-induced and miR156/miR390 pathways intersect. We conclude that the cytokinin-induced bud formation pathway in
P. patens is largely independent of, or downstream from, the
miR156-miR390-tasiRNA network.
A miRNA and tasiRNA Regulatory Network Controlling
Developmental Timing
Review and synthesis of previously described miRNA and
tasiRNA targets (Arazi et al., 2005; Axtell et al., 2006, 2007;
Talmor-Neiman et al., 2006; Addo-Quaye et al., 2009) in light of
improved P. patens gene annotations revealed a densely connected regulatory network (Figure 7A; see Supplemental Figure
13, Supplemental Table 3, and Supplemental Data Set 1 online).
The conserved miRNAs miR156, miR390, and miR529 (closely
related in sequence to miR156), along with the P. patens–specific
miR1219 and two different TAS3-derived tasiRNAs, coordinately
regulate members of three distinct transcription factor families
(SBP, ERF-like AP2, and ARF). Each RNA target in this network
is regulated by two distinct small RNA families. Perturbation of
this network influences the gametophyte body plan by either
retarding (inhibition of miR156 function in MIM156 plants,
overexpression of miR390 in MIR390C OE plants) or accelerating (loss of a critical miR156 target in DSBP3 plants) the production of buds and leafy gametophores (Figure 7B). miR390
and miR1219 accumulation is unaffected in MIM156 and DSBP3
plants (Figure 4E; see Supplemental Figure 14A online), while
miR529 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 the
similarity between miR529 and miR156 is not extensive enough
to allow miR156 to also target the tasiAP2 target mRNAs: We
find no evidence for this interaction from degradome sequencing data [Addo-Quaye et al., 2009], and the alignment between
miR156 and the tasiAP2 target mRNAs has extensive mismatches, particularly in the critical 59 end of the alignment [see
Supplemental Figure 14C online].) Importantly, we don’t exclude
the possibility of the presence of other currently unknown
components in this network as well and in particular the possibility 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 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).
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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 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 mM 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).
factors and the small RNAs that target them. The molecular
phenotypes of the DSBP3 plants (increased miR156 accumulation and decreased tasiRNA target accumulation) hint
at the likelihood of yet to be discovered connections in this
network. In addition, it remains possible that TAS6a-derived
tasiRNAs might also affect developmental progression in
P. patens.
DISCUSSION
miR156 and miR390 Control tasiRNA Accumulation and
Function in P. patens
In this study, we functionally characterized P. patens miR156 and
miR390, two of the most ancient miRNAs in plants. MIM156
plants exhibited a delayed bud and gametophore formation
and a coincident induction of SBP targets. Deletion of a single
miR156 target, SBP3, resulted in a phenotype opposite to that
caused by inhibition of miR156, suggesting the SBP3 is a
miR156 target of major phenotypic importance. Overexpression
of MIR390C also impeded the developmental transition. Accordingly, the accumulation of tasiRNAs was elevated, and target
ARF and AP2 transcription factors were suppressed in MIR390C
OE plants. These data indicate that miR156 and miR390 oppositely regulate developmental timing in P. patens. Previously, involvement of two miRNAs in the same developmental process
was reported in Arabidopsis, in which the gradient of miR390 and
miR166 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 determinants at the adaxial region. Thus, miR166 and miR390 regulate
leaf asymmetry via reciprocal expression patterns. Functional
characterization of P. patens miR166 has not yet been reported. However, we have shown that P. patens miR390 and
miR156 converge upon the same pathway by regulating a single TAS precursor with opposite effects on the production of
key tasiRNAs and ultimately upon phenotype. We hypothesize
that this convergent regulation confers a fine tuning on levels of
tasiRNAs and their targets to ensure the optimal timing of the
process of bud formation.
The Moss miR156-miR390-tasiRNA Network
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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.
A Partially Conserved Small RNA Network Regulates Bud
Formation in Mosses
The conserved miRNAs miR156, miR529, miR390, as well as the
nonconserved miRNA, miR1219, and TAS3-derived tasiRNAs
coordinate the regulation of three distinct transcription factor
families in P. patens. While this network is not precisely replicated in angiosperms, there are several intriguing parallels
(Figures 7C and 7D). The regulation of SBP domain mRNAs by
miR156 (Arazi et al., 2005; Schwab et al., 2005; Wu and Poethig,
2006; Wu et al., 2009) and the regulation of ARF mRNAs by
miR390-dependent, TAS3-derived tasiRNAs is shared by both
lineages (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), is
directly regulated by the products of miR156-targeted SBP
genes (Wu et al., 2009) and cooperates with miR156 to regulate
multiple facets of developmental timing in angiosperms (Huijser
and Schmid, 2011). In addition, miR156 appears to directly cooperate with miR172 to trigger-phased siRNA production from
a Medicago truncatula AP2 gene (Zhai et al., 2011). miR172 is
absent in P. patens; however, P. patens mRNAs containing an
ERF-like single AP2 domain are targeted by tasiRNAs, which are
themselves influenced by miR156. We did not find neighboring
miR156 and miR390 complementary regions (“neighboring” was
defined as occurring within 20 kb) in a survey of 19 seed plant
genomes (one lycophyte, four monocots, and 15 eudicots), suggesting that the TAS6/TAS3 arrangement may be unique to the
bryophyte lineage. Nonetheless, a potential connection between
miR156 and miR390 in Arabidopsis is suggested by the unexplained influence of the Arabidopsis miR390 tasiRNA pathway
upon the accumulation of the miR156 SBP domain target SPL3
(Peragine et al., 2004).
miR156, miR390, and the targets of miR156 influence vegetative developmental transitions in both mosses and flowering
plants. Interestingly, the directionality of miR156’s phenotypic
influence differs between flowering plants and mosses; it represses transitions in the former and promotes them in the latter. By contrast, miR390 represses developmental transitions
in both lineages. The tight regulatory network found in P. patens
converging upon interlocking small RNA regulation of SBP/SPL,
AP2, and ARF mRNAs appears to have been partially conserved
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during land plant diversification. Comparing the genes directly
regulated by SBP/SPL, AP2, and ARF transcription factors in
different plant lineages should be a productive avenue of future
research.
However, it is not yet clear if the tasiRNA-targeted ARF and
AP2 genes in P. patens directly regulate bud formation or,
alternatively, whether the developmental effects of MIR390
overexpression might instead be mediated by other unknown
components. We favor the hypothesis that the bud formation
defects seen upon overexpression of MIR390 are due to the
elevated levels of AP2 and/or ARF mRNAs that are apparent, but
we have not yet directly demonstrated this by analysis of the
relevant P. patens ARF and AP2 mutants. In addition, accumulation levels of the relevant tasiRNAs are not strongly correlated with those of their target mRNAs during gametophore
development (Figures 5A and 5B). One possible explanation for
this discordance is that the AP2 and ARF mRNA levels are
controlled by more factors other than just tasiRNAs. However,
we cannot exclude the possibility that other, currently unknown
genes, mediate the miR390-dependent phenotypes in P. patens.
affect the timing of bud formation in P. patens. Nonetheless, we
acknowledge that further investigation might yet uncover molecular links between these three pathways.
Why does P. patens devote so much regulatory capacity to
the control of bud development? The transition from filamentous, two-dimensional growth to the production of upright leafy
gametophores is a major developmental transition for mosses.
Bud formation and subsequent leafy gametophore growth will
only be successful if the protonematal network has become
extensive enough to anchor the growing plant and can provide
adequate nutrients, especially nitrogen, from the soil. Furthermore,
production of buds and leafy gametophores is a prerequisite for
the appearance of gametangia and subsequent attempts at
sexual reproduction. Perhaps the importance of the decision to
produce leafy buds and gametophores and the need to integrate
multiple endogenous and environmental stimuli has led to the
multiplicity of pathways that affect this trait.
Many Pathways Influence Bud Formation in P. patens
Oligonucleotides
Several previous studies reported the role of small RNAs or of
small RNA-related genes in the timing and rate of bud and leafy
gametophore development in P. patens, including miR534a
(Saleh et al., 2011), DCL3 (Cho et al., 2008), and RDR6 (TalmorNeiman et al., 2006; Axtell et al., 2007). In this study, we
demonstrated that a miR156- and miR390-regulated tasiRNA
network is also involved in this process. The requirement of
RDR6 for tasiRNA biogenesis integrates RDR6 into the miR156miR390-tasiRNA network; however, to the best of our current
knowledge, miR534a and DCL3 both act independently of the
miR156-miR390-tasiRNA pathway to regulate bud and leafy
gametophore production.
miR534a, a moss-specific miRNA targeting the BOP1 and
BOP2 transcription factor mRNAs, represses bud and leafy
gametophore formation (Saleh et al., 2011), which is opposite
to the role of miR156 and similar to the role of miR390. Cytokinin, which is critical for bud initiation in P. patens (Cove and
Knight, 1993; Schumaker and Dietrich, 1997), downregulates the
MIR534a promoter and causes a correlated increase in BOP1
and BOP2 accumulation (Saleh et al., 2011). Our data show that
reduction of miR156 function in MIM156 plants did not affect
accumulation levels of BOP1 or BOP2, and cytokinin treatment
had negligible effects on the accumulation of most of the RNAs
involved in the miR156-miR390-tasiRNA pathway. Thus, the
miR156-miR390-tasiRNA pathways and the cytokinin-miR534a
pathways appear to independently control bud and gametophore production.
DCL3, responsible for 22- to 24-nucleotide-long siRNA production from repetitive genomic regions, negatively affects
developmental timing (Cho et al., 2008; Arif et al., 2012). The
accumulation of the miR156-targeted SBP mRNAs was unchanged in DDCL3 plants, and the accumulation of miRNAs is
not affected in DDCL3 mutants (Cho et al., 2008). Thus, the
miR156-miR390-tasiRNA pathway, the cytokinin-miR534a-BOP
pathway, and the DCL3 pathway all seem to independently
METHODS
All oligonucleotide sequences are listed in Supplemental Table 4 online.
Construction of Vectors
For construction of the MIM156 vector, the At4 gene was amplified from
Arabidopsis thaliana and cloned into the pUJ3 vector. The pUJ3 vector
contained the Gateway cassette under the control of the maize (Zea mays)
Ubiquitin promoter and a hygromycin selection cassette, in which two
DNA fragments from the 108 locus are inserted at the flanking regions of
the expression and the selection cassette for targeting into the 108 locus
into the moss genome (Schaefer and Zrÿd, 1997). To create a miR156
target mimic, oligos were hybridized and ligated with the HindIII digested
pUJ3-At4 (Figure 2A). For the SBP3 knock out construct, two ;1-kb
regions 59 and 39 from the open reading frame of SBP3 were amplified and
inserted into the pUQ vector (Cho et al., 2008). The vector for MIR390c
overexpression was obtained by the insertion of pri-MIR390c region into
the BsrGI site in the pUJ3 vector.
Transformation and DNA Gel Blot Analysis
Physcomitrella patens transformation was performed as described (Cho
et al., 2008). Genomic DNAs were extracted using a Phytopure DNA
extraction kit (GE Healthcare) and used for genotyping via PCR. For DNA
gel blot analysis of DSBP3, the SphI-digested genomic DNAs were
blotted onto a Hybond NX nylon membrane (GE Healthcare) and hybridized following a standard protocol (Sambrook and Russell, 2001). As
a 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 the
GeneRacer kit (Invitrogen) following the manufacturer’s protocol.
Quantitative Real-Time RT-PCR
Total RNA for miRNA analyses were isolated using Tri-Reagent (SigmaAldrich) following the manufacturer’s instructions and transcribed with a
Superscript III reverse transcriptase (Invitrogen) essentially as previously
The Moss miR156-miR390-tasiRNA Network
described (Varkonyi-Gasic et al., 2007). DNase I (Fermentas)–treated total
RNAs (100 ng) were converted to cDNAs with Superscript III reverse
transcriptase, primed by miRNA-specific stem loop primers using a
pulsed RT reaction as described (Varkonyi-Gasic et al., 2007). As a reference, the primer for U6 was also included in the same reaction. A serial
dilution was included in the same reaction plate of real-time PCR to
calculate the PCR efficiency.
Total RNAs for mRNA analyses were isolated using an RNeasy plant
mini kit (Qiagen) and reverse transcribed with a QuantiTect reverse
transcription kit (Qiagen). EF1a was used as a reference. Real-time PCRs
were performed using a QuantiTect SYBR Green PCR kit (Qiagen) with an
ABI7300 teal-time PCR system (Applied Biosystems). PCR efficiencies
were calculated based on serial dilution and used to determine relative
accumulation levels by the using the efficiency corrected DDCt (cycle
threshold) method. Relative expression was normalized to the per-gene
wild-type values, except Figures 1, 5A, and 5B, which were normalized to
the 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 to
10 online). As a statistical analysis, Student’s t tests were performed with
two-tailed distribution.
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Measurement of Growth Rate and BAP Treatment
For measurement of growth rate, one to two filaments of 1-week-old
protonemal tissues were inoculated onto BCD media with (Figures 3E and
5D) or without (see Supplemental Figure 11 online) ammonium supplementation under standard growth conditions (25°C, 16 h day/8 h night)
(Ashton and Cove, 1977). Numbers of buds and gametophores were
counted every 2 d.
For BAP treatment, blended protonemata were grown on cellophane
overlaid BCD media with ammonium. BAP (5 mM; Sigma-Aldrich) dissolved in 40 mM NaOH was added on top of the protonemata and
incubated for 24 h. As a mock control, 40 mM NaOH treatment was
performed simultaneously.
Accession Number
Sequence data for the TAS6a/TAS3a primary transcript from this article
can be found in the GenBank/EMBL data libraries under accession number
JN674513.
Supplemental Data
Small RNA Gel Blots
Total RNAs, including small RNAs, were isolated using Tri reagent (SigmaAldrich) following the manufacturer’s instructions. Small RNAs were
enriched as described (Blevins, 2010). Eight micrograms of small RNAs
were resolved in 15% Criterion TBE-Urea gel and transferred to the
Hybond-NX nylon membrane (GE Healthcare), followed by cross-linking
using a 1-ethyl-3-(3-dimethylamonipropyl) carbodiimide–mediated chemical cross-linking method (Pall and Hamilton, 2008). The probes for
TAS3a, TAS6a, and U6 were amplified from cDNA by PCR and radiolabeled with [a-32P]dCTP using an NEblot Kit (New England Biolabs) and
hybridized to the membrane at 37°C within PerfectHyb solution (SigmaAldrich). Membranes were washed twice with nonstringent washing buffer
(33 SSC, 5% SDS, and 25 mM NaH2PO4, pH 7.5) for 10 min and twice for
30 min at 42°C. The final wash was performed with the stringent wash
buffer (13 SSC and 1% SDS) for 5 min at 42°C. The oligo probes for
tasiAP2, tasiARF, miR529, miR1219, and U6 were radiolabeled with
[g-32P]dATP using T4 polynucleotide kinase (New England Biolabs) following the manufacturer’s protocol, hybridized to the nylon membrane at
42°C, and washed as in the double-stranded DNA probes at 50°C. The
radioactive signals were recorded onto phosphor screens (GE Healthcare)
and scanned using a STORM860 phosphor imager (Amersham Biosciences). Membranes were deprobed by washing in 1% SDS at 80°C for
30 min and hybridized with another probe. Image quantification was
performed with the ImageJ program (NIH).
Small RNA Target Analyses
P. patens degradome data (National Center for Biotechnology Information 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 the
genomic loci encompassing known TAS3 and TAS6 loci or, in the case of
TAS6a/TAS3a, the full-length primary transcript. Strong evidence of
slicing (thick lines in Supplemental Figure 13 online) reflects either prior
evidence from the literature in the form of gene-specific RNA ligase–
mediated 59 rapid amplification of cDNA ends, degradome-based evidence with a P value # 0.05, or both. Weak evidence for slicing (solid thin
lines in Supplemental Figure 13 online) reflects degradome data with a P
value > 0.05. Details are in Supplemental Table 3 and Supplemental Data
Set 1 online.
The following materials are available in the online version of this article.
Supplemental Figure 1. Accumulation of miR156 and Its Targets
during P. patens Gametophyte Development.
Supplemental Figure 2. The Life Cycle of Physcomitrella patens
Supplemental Figure 3. Functional Knockdown of miR156 by Target
Mimicry.
Supplemental Figure 4. Accumulation of SBP Transcripts during the
P. patens Gametophyte Development.
Supplemental Figure 5. Targeted Knockout of SBP3.
Supplemental Figure 6. Transcript Levels of Genes Related to the
Gametophore Transition in P. patens.
Supplemental Figure 7. Small RNA Gel Blots of TAS6a and TAS3a in
DDCL4 and DRDR6.
Supplemental Figure 8. miR156 Influences P. patens tasiRNA
Accumulation and Their Targets in Older Tissues.
Supplemental Figure 9. Transcript Level of the Target of tasiZNF in
DSBP3 and in MIM156s in 3-Week-Old Samples.
Supplemental Figure 10. Accumulation of miR390-Regulated
tasiRNAs and Their Targets
Supplemental Figure 11. Comparison of Bud and Leafy Gametophore Formation in MIM390C OE, DSBP3, and DRDR6-19 Plants.
Supplemental Figure 12. Cytokinin Treatment Accelerates Bud
Formation.
Supplemental Figure 13. Detailed Small RNA–Target Interactions in
P. patens.
Supplemental Figure 14. Accumulation of miR529 and miR1219 in
MIM156-2 and DSBP3 Plants.
Supplemental Table 1. Origins of P. patens tasiAP2 and tasiARF.
Supplemental Table 2. Bud Formation in MIR390C-Overexpressing
P. patens.
Supplemental Table 3. Summary of small RNA/Target Data.
Supplemental Table 4. Oligonucleotide Sequences Used in This
Study.
Supplemental Data Set 1. Small RNA Target Alignments and
Degradome Data.
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ACKNOWLEDGMENTS
This work was funded by a grant from the U.S. National Institutes of
Health-National Institute of General Medical Sciences (R01 GM84051) to
M.J.A. We thank Jo Ann Snyder for technical support, Zhaorong Ma and
Wolfgang Frank for insights on the TAS6 family, Rajendran Rajeswaran
for discussions and pilot experiments, and all members of the Axtell Lab
for many constructive discussions during this study.
AUTHOR CONTRIBUTIONS
M.J.A. and S.H.C. designed the research. S.H.C. and C.C. performed
research. 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 December
10, 2012; published December 21, 2012.
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Sung Hyun Cho, Ceyda Coruh and Michael J. Axtell
Plant Cell; originally published online December 21, 2012;
DOI 10.1105/tpc.112.103176
This information is current as of June 18, 2017
Supplemental Data
/content/suppl/2012/12/20/tpc.112.103176.DC1.html
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