Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species.

Abstract

Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.

Full text

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Endogenous and Synthetic MicroRNAs Stimulate
Simultaneous, Efficient, and Localized Regulation
of Multiple Targets in Diverse Species
W
John Paul Alvarez,a,bIrena Pekker,aAlexander Goldshmidt,aEyal Blum,aZiva Amsellem,aand Yuval Esheda,1
aDepartment of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
bSchool of Biological Sciences, Monash University, Victoria 3800, Australia
Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by
microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a
systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant
combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-
miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of
their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in
developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones
also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-
based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and
tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic trans-
formations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-
specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial
transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for reg-
ulated expression of multiple related genes in diverse species.
INTRODUCTION
A major component of pattern formation in plants involves
complex interplay between transcription factors (TFs) expressed
in precisetemporal and spatialdomains andmodifiers thatactto
maintain and refine their expression boundaries. TFs are usually
expressed at low levels and guide the activity of many down-
stream effectors. Evolutionary expansion of TF families in plants
(Riechmann et al.,2000) hasmeant thatfunctional redundancyis
a common theme in plant genomes. The majority of recently
identified plant microRNAs (miRNAs) impose sequence-based
simultaneous downregulation of developmentally important TFs
(Llave et al., 2002a; Rhoades et al., 2002). Indirect evidence
suggests that plant miRNAs have the potential to act efficiently
to eliminate or clear cells of their target gene activities (Bartel,
2004). For instance, a large reduction in the activity of miRNA-
regulated targets, either at the RNA or protein level, is evident
upon ectopic miRNA expression (Aukerman and Sakai, 2003;
Palatnik et al., 2003; Achard et al., 2004; Chen, 2004; Li et al.,
2005; Schwab et al., 2005). Complementing these observations,
strong dominant phenotypes are induced by target genes upon
release of miRNA-guided regulation by mutations in their miRNA
bindingsites,andmisexpressionofmiRNA-resistanttargetswith
mutations at the miRNA binding site results in much stronger
phenotypes than those obtained with native transcripts (re-
viewed in Chen, 2005). However, there is also evidence for
quantitative action of plant miRNAs in quenching, as opposed
to clearing, of the target gene activity. Thus, miRNA-resistant
mutants are inherited in an incompletely dominant manner and
are still subject to some miRNA-directed cleavage (Tang et al.,
2003; Mallory et al., 2004a), and mutations in miRNA genes can
result in increased levels of target gene expression (Baker et al.,
2005).
Despite molecular indications that miRNAs can be very effi-
cient, direct phenotypic evidence derived from comparing mul-
tiple mutant combinations with the effects of ectopic expression
of the corresponding miRNA is limited. Unlike animal miRNAs,
which simultaneously negatively regulate dozens of targets (Lim
et al., 2005), plant miRNAs appear to have more limited target
sets(Schwabetal.,2005),whichareusuallymultiplemembersof
the same gene family or even members of a single monophyletic
clade of a larger family (Bartel and Bartel, 2003). These qualities
can be used to facilitate the comparison of multiple mutant
combinations with the effects of ectopic expression of the
corresponding miRNA. In the absence of such a comparison,
the prevailing model for high specificity and potency of plants
miRNAs has yet to be verified. For example, in one case, ectopic
1To whom correspondence should be addressed. E-mail yuval.eshed@
weizmann.ac.il; fax 972-8934-4181.
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: Yuval Eshed
(yuval.eshed@weizmann.ac.il).
WOnline version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.105.040725.
The Plant Cell, Vol. 18, 1134–1151, May 2006, www.plantcell.org ª 2006 American Society of Plant Biologists

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miR164 induced cotyledon fusion and meristem arrest, mimick-
ing the double mutant phenotype of two out of the five NAC
domain genes targeted by this miRNA (Aida et al., 1997; Jones-
Rhoades and Bartel, 2004; Laufs et al., 2004; Mallory et al.,
2004b; Baker et al., 2005). However, overexpression of the
miR165/6 failed to mimic the seedling arrest and production of a
cylindrical monocot-like radial shoot observed in multiple mu-
tants of the five miR165/6 PHABULOSA (PHB)-like targets
(Emery et al., 2003; Li et al., 2005; Prigge et al., 2005; Williams
et al., 2005a). The failure to recapitulate the phenotype in this
latter case may reflect the inability of strong 35S:miR165/6
embryos to survive after transformation. Such a proposal could
be confirmed by more precise viability-independent expression
of the miR165/6.
Deciphering the role of TFs and their miRNA regulators in
pattern formation can benefit greatly from spatial and temporal
manipulationoftheiractivities.Thisway,earlydeleteriouseffects
can be bypassed and the autonomy of specific miRNA-induced
perturbations examined. Such tissue-specific silencing using
RNA interference (RNAi) has been successfully applied in plants
(Watson etal., 2005),but RNAi has alsobeen observed to induce
systemic spread of gene silencing (reviewed in Voinnet, 2005).
Notably, the common complexes involved in miRNA and short
interfering RNA (siRNA) biogenesis and processing raises the
possibility of a systemic component to miRNA-mediated regu-
lation. In support of such a role, different miRNAs have been
found in conducting phloem sap, a possible conduit for their
systemic spread (Yoo et al., 2004). However, while unequivocal
proof for long distance translocation of silencing signals was
perceived through grafting experiments between silenced and
nonsilenced transgenic tobacco (Palauqui et al., 1997), no such
evidence has been provided for miRNA signals. Moreover, in
mature Arabidopsis thaliana tissues, there is a strict overlap
betweenpremiR171promoterandmiR171activitymonitoredby
a sensor construct, suggesting that this miRNA maintains strict
spatial autonomy (Parizotto et al., 2004).
Many plant miRNAs appear to have a long coevolutionary
history with their targets, extending back to moss and lycopods
(Floyd and Bowman, 2004; Axtell and Bartel, 2005). The pairing
structure of the miRNA and nearly perfect complementary
miRNA* can be highly conserved. Though sequence conserva-
tion also occurs outside the domain of the miRNA and its
complement, it is the general predicted structure of the pre-
miRNA foldback, rather than its sequence per se, that is con-
served between the distantly related Arabidopsis and rice (Oryza
sativa) (Reinhart et al., 2002). This raises the question whether
pre-miRNAs retain functional conservation between distant spe-
cies. Such conservation would involve efficient miRNA biogen-
esis, including correct spatial recognition and processing of
the miRNA/miRNA* from the pre-miRNA foldback structure
(reviewed in Chen, 2005). Likewise, the coevolutionary history
of miRNA and their targets also raises the question of whether
unique characteristics define particular mRNAs as targets.
Evidence from metazoans suggests that this is not the case, as
the miRNA sequence and its complement in the foldback struc-
turecanbesubstitutedandtargetnoveltranscripts(Dickinsetal.,
2005). Similarly, analysis in Arabidopsis demonstrated that the
miRNA and its complement miRNA* domains of the pre miR171
backbone could be substituted to produce a novel miRNA that
successfully targeted green fluorescent protein (GFP) (Parizotto
et al., 2004). However, the capability and efficacy of the manip-
ulated pre-miRNA in targeting multiple endogenous genes not
normally targeted by miRNA regulation has yet to be examined.
Inordertoexploretheextentandautonomyofdownregulation
that can be induced by miRNAs, we have chosen an experimen-
tal platform that uses sets of target genes for which complete
conventional mutants are also available. Furthermore, miRNA
ectopic expression has been brought under the control of in vivo
constitutive ortissue-specificectopic expression ofpre-miRNAs
guided by RNA Pol II promoters. This system enabled us to
demonstrate that miRNAs have the potential to impose full or
partial phenocopies of multiple mutants in several independent
assays. These observations enabled us to expand the range of
miRNA control by custom-designed pre-miRNAs that can stim-
ulate phenocopy of mutations in genes not naturally regulated
in this manner. Endogenous and synthetic Arabidopsis pre-
miRNAs were functionally conserved in tomato (Solanum lyco-
persicum) and tobacco (Nicotiana tabacum), and tissue-specific
miRNA misexpression produced phenotypes that are limited to
the area of expression. Hence, these data suggest that miRNA
activities are quantitative and, at least for distances greater than
few cells, do not act outside of their domain of expression.
RESULTS
Precise Ectopic Expression of Endogenous miRNA Can
Phenocopy Multiple Mutant Combinations
Plant miRNAs, such as miR164, have been ectopically ex-
pressed using the 35S promoter directly or through chemical
induction driving the stem foldback that constitutes the pre-
miRNA with various 59 and 39 endogenous additions (Figure 1A;
Guo et al., 2005). We wished to examine the ability of precise
spatialexpressionofmiRNAactivitytoeffectivelymonitormiRNA
potential. Transactivation of protein-encoding genes using the
LhG4-OP system has been effective in driving strong, specific
expression and obviating early deleterious effects of gene over-
expression (Moore et al., 1998). To assay the capacity of this
system to express pre-miRNAs, genomic fragments containing
the pre-miRNA foldback flanked by short 59 and 39 pre-miRNA
sequences were cloned behind an OP array followed by a TATA
box (see Supplemental Table 1 online). In the case of OP:
miR164b, the resulting T2 lines (see Methods for selection and
scoring of transgenic lines; summarized in Table 1) were trans-
activated using the PHB:LhG4 promoter, which is expressed in
the developing cotyledon primordia and throughout the apical
meristem (Figure 1C) to provide PHB?miR164b F1s (Figure
1E; ? denotes transactivation). Expression of miR164 under this
promoter mimicked the fused cotyledon and meristem arrest
phenotype of the cup-shaped cotyledon1 (cuc1) and cuc2 dou-
ble mutant, two of miR164’s six targets (Figure 1D). These ob-
servations indicate that expression of pre miR164b using the
transactivation systemcanefficientlydeplete cellsoftarget gene
activities.
To extend these observations to other Arabidopsis miRNAs,
we expressed OP:miR165b using the promoter of one of its
Mutant Phenocopy with MicroRNA 1135

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Figure 1. miRNAs Can Quantitatively Regulate Multiple Transcripts Simultaneously and Phenocopy Their Combined Loss of Function.
(A) A scheme of an endogenous pre-miRNA. The red and blue fragments will be cleaved by DICER-LIKE1 (DCL1) to generate the miRNA and miRNA*,
respectively.
(B) A 10-d-old wild-type seedling.
(C) The promoter of PHB drives GFP expression throughout the shoot apex in wild-type heart-stage embryos.
1136The Plant Cell

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target genes, the PHB:LhG4 driver. miR165/6 target the five
PHB-like genes that redundantly promote meristem establish-
ment and maintenance as well as differentiation of lateral organs
and the vasculature. phb phavoluta (phv) revoluta (rev) triple
mutants or loss of all of the five PHB-like genes result in seedling
arrest after the production of a cylindrical monocot-like radial
shoot in which the apical meristem activity is abolished (Figure
1F; Emery et al., 2003; Prigge et al., 2005). However, previous
overexpression studies with 35S:miR165 resulted in variable
seedling phenotypes in which the most extreme plants had small
leaves with some polarity defects (Li et al., 2005). By contrast,
PHB?miR165b plants gave rise to radial seedlings, phenocopy-
ing the multiple mutant combination (Figure 1G). These results
illustrate that specific miRNA expression can abolish target gene
activity and demonstrate the potential potency of precise pre-
miRNA misexpression as a vehicle for simultaneous downregu-
lation of multiple members of the same gene family.
35S-Driven miRNAs Can Faithfully Mimic
Loss-of-Function Phenotypes
It is likely that the inefficiency of 35S:miR165a in producing a
phenocopyof thePHB-likegenesreductionisduetotheirspatial
expression in early stage embryos and their importance in
embryogenesis. Auxin Response Factor6 (ARF6) and ARF8 are
the only predicted targets of miR167, and arf6 arf8 double
mutants are viable, late flowering, have dark green leaves, and
exhibit unexpanded 2nd and 3rd whorl floral organs (Nagpal
et al., 2005). To assay whether the 35S promoter can effectively
drive a miRNA-mediated reduction in these genes, we assayed
35S:pre miR167a transformants. Out of 20 independent T1
plants, eight had a similar phenotype to that reported for arf6
arf8 double mutants both vegetatively and in flowers, while the
remainder had a range of weaker phenotypes (Figure 1H, Table
1). No additional features than described for the double mutants
were noticed. Thus, depending on the miRNA and its targets,
elevated ectopic miRNA expression by the constitutive 35S
promoter can specifically reduce multiple target gene activities
to levels that parallel that of multiple loss-of-function mutants.
Precise Expression of miRNAs Can Reveal Novel
Mutation Patterns
Embryonic expression of either miR164 or miR165 resulted
in a seedling phenocopy of multiple mutants in the correspond-
ing target genes. While cuc1 cuc2 seedlings can be rescued
using tissue culture, phb phv rev or plants mutant for all five
PHB-like mutant genes do not develop beyond the seedling
stage. Because the CUC-like and PHB-like genes are active
throughout plant development, the use of tissue-specific miRNA
expression could reveal functions of these genes later in plant
development. To examine the utility of this approach, trans-
activationofselectedstrongOP:miR164bandOP:miR165blines
was performed using the promoter of the flower meristem gene
APETALA1 (AP1).
As shown in Figures 1I and 1J, the expression mediated by
AP1 promoter initiates transcript accumulation throughout
young floral meristems. In AP1?miR164b plants, the sepals
are completely fused, petals are absent, and stamens exhibit
fusion to each other and the gynoecium (cf. Figures 1L and 1K).
This phenotype is similar, albeit more severe than that observed
incuc1cuc2flowersgeneratedthroughtissueculture(Aidaetal.,
1997). In AP1?miR165b plants, only radial filamentous struc-
tures were observed in the place of flowers, consistent with the
miR165 effectively eliminating flower meristem function (Figure
1M). These findings are consistent with the observed seedling
phenotypes, indicating that the PHB-like genes are essential for
embryo and flower meristem maintenance. These observations
demonstrate that the transactivation system can be effectively
usedinconjunction withmiRNA-mediated lossof geneactivity in
specific cells at any stage of the plant’s life cycle.
Low Levels of miRNA Expression or High Levels of
Inefficient miRNA Suggest That Plant miRNAs Can
Act in a Quantitative Fashion
Tissue-specific transactivation of pre-miRNAs identified lines
producing a loss-of-function phenocopy but also uncovered
OP:miRNA lines that induced mild phenotypes (Table 1). For
Figure 1. (continued).
(D) Arabidopsis cuc1 cuc2 double mutant seedlings.
(E) F1 seedlings of OP:miR164b transactivated by PHB:LhG4.
(F) A monocot-like phv phb rev triple mutant seedling.
(G) A monocot-like PHB?miR165b seedling of comparable age.
(H) Whole shoot and flower (inset) of 35S:miR167a plant next to same age wild type display identical alterations found in arf6 arf8 double mutants (cf.
with Nagpal et al., 2005).
(I) Scanning electron micrograph of wild-type flowering apex.
(J) A cross section through AP1?HP-GFP flowering apex with expression throughout emerging flower meristems.
(K) Wild-type flower.
(L) Fused sepals and absent petals in AP1?miR164b flower.
(M) Flowering apex and filamentous flowers (inset) of strong AP1?miR165b plant.
(N) Normal sepals, radial petals, distorted stamens, and multiple carpels in a weak AP1?miR165b flower.
(O) Sequence alignment of the wild type and phv-1d mutant with corresponding miR165b and miR165bm6.
(P) Seedling expressing ANT?miR165b#4 results in radialized cotyledons and aborted meristem.
(Q) and (R) Adaxial surface of wild-type (Q) and ANT?miR165bm6 (R) leaves. Note the adaxial outgrowths of the transgenic leaf (arrow).
(S) Normal sepals, distorted stamens, and multiple carpels in a strong ANT?miR165bm6 flower.
FM, flower meristem; IM, inflorescence meristem; P, petal. Bars ¼ 3 mm in (B), (F), and (G) and 20 mm in (C), (I), and (J).
Mutant Phenocopy with MicroRNA 1137

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example, whena weak OP:miR165b line was transactivated with
the AP1:LhG4 promoter, the growth of the petals and stamens
was markedly affected and additional carpels were formed
(Figure 1N). Associations between phenotype strength and tran-
scription levels have been shown previously for ectopic miR164
and miR166g expression (Laufs et al., 2004; Williams et al.,
2005a). Likewise, assaying both the weak and strong OP:
miR165b lines with the same promoters revealed consistent
phenotype strengths restricted to the promoter’s expression
domain. These observations imply that when expressed in a
discrete group of cells, miR165 can act in a quantitative fashion.
We hypothesized that quantitative action of miRNA may
also be achieved by the generation of miRNA with lower homol-
ogy. This was based on the observation that mRNA of the
dominant phv-1d miRNA-resistant mutation was still cleaved
(our unpublished data; Tang et al., 2003). The phv-1d molecular
lesion is a G-to-A transition in the miR165/6 target region
opposite position 6 from the 59 end of the miR165/6 (Figure
Table 1. Endogenous and Synthetic miRNAs Examined in This Study
miRNA and
Its Pre-miRNA
Backbone
Transgenes and
Recipient Speciesa
Target Genes
Target Gene Function and
Loss-of-Function Mutant
Description
Frequency and Range of
Phenotypic Responses in
Independent Lines
StrongIntermediate Weak
miR164/pre
miR164b
Arabidopsis
OP:miR164bb
NAC domain TFs
CUC1, CUC2, NAC1,
At5g07680, At5g61430,
At5g39610
Embryonic meristem establishment,
organ separation, and lateral root
outgrowth (Figure 1D)
1000
tomato
OP:miR164bc
Arabidopsis
OP:miR165bb*
Tomato NACs
PHB-like class III
HD-ZIP TFs
PHV, CNA, REV,
ATHB8, PHB
281
miR165/pre
miR165b
Meristem establishment and
maintenance, adaxial differentiation
of lateral organs, and vasculature
patterning (Figure 1F)
2382
tomato
OP:miR165bc
Arabidopsis
OP:miR165ab*
Arabidopsis
OP:miR166gb*
Arabidopsis
OP:miR165m6c
Arabidopsis
35S:miR167ab
Tomato PHB-like
As for miR165b
130
miR165/pre
miR165a
miR166/pre
miR166g
miR165m6/pre
miR165b
miR167/pre
miR167a
As for miR165b
1722
As for miR165b As for miR165b
1633
As for miR165b but with reduced
homology toits targets
ARF TFs
ARF6, ARF8
As for miR165b
110
Promotion of flowering and
JA-mediated
flower organ maturation
(Nagpal et al., 2005)
Abaxial differentiation of lateral organs,
vascular development, flowering,
and cell growth (Figure 2D)
8 102
miR-ARF/pre
miR164b
Arabidopsis
35S:miR-ARFb
ARF TFs
ARF2, ARF3/ETT,
ARF4
983
tomato
35S:miR-ARFc
tobacco
35S:miR-ARFc
Arabidopsis
35S:miR-NGAab
OP:miR-NGAab
tomato
35S:miR-NGAc
340
260
miR-NGA/pre
miR164a
B3 domain TFs
NGA1, NGA2, NGA3, NGA4
Lateral organ growth
(Figures 3E and 3H)5
2
9
6
6
2
Weak homology with
NGA-like/BI934304
and BI934637
As for miR-NGAa
054
miR-NGA/pre
miR164b
Arabidopsis
35S:miR-NGAbb
As for miR-NGAa
794
Frequent silencing was observed in subsequent generations for the lines marked with asterisks.
aThe 10OP:miRNA lines were examined upon transactivation with a promoter LhG4 driver. Combinations with specific promoters are described in the
text. The 35S:miRNA lines were scored directly as T1s and showed consistent phenotypes upon cross with the wild type.
bPhenotype relative to loss-of-function phenocopy (close resemblance is strong).
cPhenotype scoring is relative to other transgenic lines carrying the same construct because corresponding mutants are not available.
1138 The Plant Cell