Allison C. Mallory ^{1, 4}, Diana V. Dugas ^{2, 4}, David P. Bartel * ^{1, 3}, and Bonnie Bartel * ²

¹ Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142 USA
² Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77005 USA
³ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA
^* Correspondence:
Bonnie Bartel (713) 348-5602 (phone);  (713) 348-5154 (fax); bartel@rice.edu
and David P. Bartel (617) 258-5287 (phone);   (617) 258-6768 (fax);  dbartel@wi.mit.edu
⁴ These authors contributed equally to this work.

MicroRNAs (miRNAs) are 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2.

Results:

Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected.

Conclusions:

These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.

MicroRNAs are small regulatory RNAs (for review, see [1]) that were first discovered in animals [2–6] and more recently found in plants [7–9]. In animals, miRNAs are processed from imperfectly paired stem-loop precursor RNAs of approximately 70 nt, and some of the mature 21 nt miRNAs are reported to pair with target mRNAs to specify posttranscriptional repression of these targets [2, 3, 10–15].

In plants, miRNAs also derive from imperfectly paired stem-loops; however, these predicted precursors appear more variable in length than animal miRNA stem-loop precursors [7, 16]. Plant miRNA accumulation requires the nuclear activity of the RNaseIII DICER-LIKE 1 (DCL1) and to a lesser degree HEN1 and HYL1, nuclear-localized proteins with double-stranded RNA binding domains [7, 9, 17–20]. In addition, proper miRNA function requires AGO1 [21, 22], a protein with PAZ and PIWI domains that is conserved in most eukaryotes [23]. Like animal miRNAs, the accumulation of many plant miRNAs is not ubiquitous but, instead, varies in different organs and tissues and at different developmental stages, indicating that miRNA accumulation is spatially and temporally regulated [7, 8]. In addition, many of the miRNAs isolated from the dicot Arabidopsis are conserved in the monocot rice and other plant species, implying conserved evolutionary roles for plant
miRNAs [7, 16, 24].

Many regulatory targets of plant miRNAs have been confidently predicted based on their extensive, evolutionarily conserved complementarity to the miRNAs [25]. Of the 15 miRNA families identified thus far in plants, all have at least one predicted mRNA target, and most are predicted to target multiple mRNAs [7–9, 25–27]. Experimental data, often the detection of an mRNA fragment diagnostic of miRNA-directed cleavage, have confirmed the identities of targets for 11 miRNA families [19, 26–32]. These data also support the idea that most plant miRNAs act within an RNA-induced silencing complex (RISC) to direct cleavage of their target messages, although in one case diminution of target protein without a corresponding reduction in target mRNA indicates that plant miRNAs can also act to inhibit productive translation [31, 32].

Plant miRNAs have a striking propensity to target messages encoding transcription factors involved in cell fate determination [25]. Consistent with the prediction that miRNA pathways regulate plant development, plant mutants defective in miRNA accumulation, such as dcl1, hen1, hyl1, and ago1, exhibit pleiotropic developmental phenotypes including floral development defects and leaf morphology alterations [19–22, 33–35]. The importance of this regulation is underscored by the observation that dcl1 null mutations confer embryo lethality [36]. In addition, plants expressing a viral protein that alters miRNA accumulation and activity have developmental defects [29, 37]. Specialized developmental functions of specific plant miRNA families have recently been reported for the miR172, miR159/JAW, and miR165/166; these functions include roles in flowering time and floral organ identity [31, 32], rosette leaf curvature [26], and organ polarity [21, 38, 39], respectively. Two miRNAs, miR162 and miR168, are likely to influence development through feedback regulation of the miRNA pathway itself [22, 27].

miR164 is potentially transcribed from two loci, MIR164a and MIR164b [7], and is predicted to target a subset of genes in the NAC-domain transcription factor gene family [25]. This family, named after the founding members, petunia no apical meristem (NAM), Arabidopsis ATAF1 and ATAF2, and Arabidopsis CUP-SHAPED COTYLEDON2 (CUC2) [40, 41], is restricted to plants and has more than 100 members in Arabidopsis [42]. Five Arabidopsis NAC-domain messages have a miR164 complementary site with three or fewer mismatches to miR164 (Figure 1A) [25]. These include NAC1, CUC1, CUC2, At5g07680, and At5g61430. The miR164 complementary sites are located in the last exon of the mRNAs, downstream of the conserved NAC domain [25]. Of these predicted miR164 targets, CUC1 and CUC2 mRNAs have been shown to be cleaved within the miR164 complementary site [29].

                        Figure 1. miR164 Complementary Sites in Plant NAC-Domain Genes.

                        (A) Predicted pairing between miR164 and the miR164 complementary sites of Arabidopsis
                        mRNAs CUC1, CUC2, NAC1, At5g07680, and At5g61430 and of the petunia NAM
                        (GenBank accession X92205) and snapdragon CUC (GenBank accession AJ568269) mRNAs.
                        Watson-Crick base pairing between the mRNA and miR164 is indicated by black boxes,
                        whereas mismatches and G:U wobbles are unshaded. Mismatches and G:U wobbles with CUC1
                        are indicated by an X or black circles, respectively. The protein sequence encoded by the miR164
                        complementary site of each mRNA is listed. The open reading frames of each predicted
                        miR164-target are depicted (rectangle), showing the position of each conserved NAC domain
                        (gray box) and miR164 complementary site (asterisks).

                        (B) Illustration of the CUC1 genomic clone used to transform Arabidopsis. Locations of the
                        intergenic sequences (solid lines), intron sequences (dashed lines), 5' and 3' UTRs (black
                        boxes), NAC domain (gray boxes), remainder of the CUC1 open reading frame (open boxes),
                        start codon (ATG), stop codon (TGA), and miR164 complementary site (asterisk) are all
                        indicated. Positions of the BamH1 and EcoR1 sites used for cloning and the downstream gene
                       At3g15180 are also shown. Messenger RNA segments from CUC1 and 5mCUC1 are shown
                        paired with miR164, with the Nsp1 restriction site unique to 5mCUC1 indicated.

                        (C) miR164 cleavage sites in At5g61430, At5g07680, and NAC1 mRNAs determined by RNA
                        ligase mediated 5' RACE. At the left is the agarose gel showing the nested PCR products that
                        were cloned and sequenced for each gene. At the right, the frequency of 5' RACE clones
                        corresponding to each cleavage site (arrows) is shown as a fraction, with the number of clones
                        matching the target message in the denominator.

Here, we report biological roles for miR164-directed regulation of target NAC-domain transcription factor mRNAs. Arabidopsis plants transformed with a miR164-resistant version of the CUC1 target display embryonic, vegetative, and flower organ defects, including cotyledon orientation defects, rosette leaf abnormalities, and sepal and petal number and positioning defects. Furthermore, plants ectopically expressing miR164 exhibit cotyledon and floral organ fusion phenotypes overlapping with cuc1 cuc2 double mutant defects [40]. These plants exhibit additional organ fusion phenotypes that likely result from the misregulation of other miR164 targets. Indeed, we detect mRNA fragments diagnostic of miR164-directed cleavage of NAC1, At5g07680, and At5g61430 in wild-type plants. Together, these results identify a role for miR164-directed regulation and show that proper miR164-directed regulation of NAC-domain genes is essential for normal plant morphogenesis.

Results and Discussion:

 A miR164-Resistant Version of CUC1 Alters Embryonic, Vegetative, and Floral Development

CUC1 mRNA is found in most tissues assayed [43], whereas CUC2 mRNA is most abundant in flowers and seedlings [43], and NAC1 mRNA accumulates to the highest level in roots [45]. We used quantitative RT-PCR to compare mRNA levels of these three miR164 targets to each other and to additional predicted miR164 targets encoded by At5g07680 and At5g61430. All five genes were highly expressed in stems and buds but differentially expressed in other organs and seedlings (Figure 2A) . NAC1 was highly expressed in roots and seedlings, and At5g61430 expression also was high in roots. RNA blot analysis indicated that miR164 accumulates in all organs and developmental stages analyzed (Figure 2B), raising the prospect that it could be modulating the expression of these messages in many tissues of the plant.

                 Figure 2. Accumulation of miR164 and miR164-Targeted NAC-Domain mRNA in Arabidopsis Tissues

                 (A) miR164 target mRNAs accumulate differentially in Arabidopsis tissues. The indicated
                 NAC-domain mRNAs were quantified in total RNA prepared from root, rosette leaf, stem, cauline
                 leaf, bud (includes a mix of buds and inflorescence meristems), flower, silique (includes embryonic
                 tissues), and 12-day-old seedling tissues by using quantitative real-time RT-PCR with primers and
                 probes designed around the miRNA complementarity sites and normalized to the level of 18S rRNA
                 in the sample. TUB4 was used as a nontarget control. mRNA levels are displayed in arbitrary units,
                 and error bars represent the standard deviations of three PCR replicates of a single reverse
                 transcription reaction.

                 (B) miR164 accumulates differentially in Arabidopsis (Col-0) tissues. RNA gel-blot analysis of 30 mg
                 RNA from (A) with a DNA probe complementary to miR164. The positions of ³²P-labeled RNA
                 oligonucleotides are noted on the left. The blot was stripped and reprobed with an oligonucleotide
                 complementary to U6 as a loading control.

We used an in vitro assay that relies on miRNA-programmed RISC endogenously present in wheat-germ extract [30] to determine whether miR164-directed cleavage of 5mCUC1 RNA is reduced. Specific cleavage was detected for the wild-type (wt) CUC1 RNA, but not for the 5mCUC1 RNA (data not shown), although in both cases most of the RNA remained uncleaved after a 60 min incubation, suggesting that these extracts have only low levels of miR164-programmed RISC or perhaps that the sequence of this miRNA has diverged in wheat.

To assess the in vivo consequences of disrupting miR164 regulation of CUC1, we transformed wt Arabidopsis plants with 5mCUC1 and control CUC1 genomic constructs under the control of the native CUC1 5' and 3' regulatory sequences (Figure 1B) and analyzed the resultant transformants for developmental defects. None of the 62 control CUC1 primary transformants displayed any morphological abnormalities. In contrast, 34 of 74 5mCUC1 transformants displayed striking floral organ phenotypes that included one to four extra petals and one or two missing sepals (Figures 3D–3F) ; these phenotypes were observed in 5mCUC1 progeny as well (Figures 3G–3N). In wt plants, petals and reproductive structures are encased within two pairs of sepals as buds develop (Figures 3G, 3I, and 3K). In 5mCUC1 plants, the altered sepal number and position, delayed sepal formation or maturation, and the proliferation of petal organs caused immature floral buds to expose petals and reproductive structures throughout development (Figures 3H, 3J, and 3L–3N). Moreover, mature flowers of 5mCUC1 plants appeared larger than wt because of their open, claw-like shape (Figures 3E and F). Although floral organ development was altered in the 5mCUC1 plants, flower phyllotaxy on the primary stem and branches appeared normal. 5mCUC1 plants had reduced fertility compared to the control CUC1 plants; however, all but the most severely affected plants produced viable seed, indicating that both male and female reproductive structures were at least partially functional.

                 Figure 3. Developmental Abnormalities in 5mCUC1 Plants

                 (A–C) Vegetative development of 5mCUC1 plants and control CUC1 plants. (A) 10-day-old T2 control
                CUC1 (left) seedling with wild-type phenotype and 5mCUC1 (right) seedling with upright cotyledon
                 petioles. (B) 23-day-old T2 control CUC1 (left) and 5mCUC1 (right) plants. (C) Magnified view of a
                 23-day-old T2 5mCUC1 plant.

                 (D–N) Comparison of floral organs of representative control CUC1 plants and 5mCUC1 plants.
                 Vertical views of a T1 control CUC1 (left) and 5mCUC1 (right) floral apex (D). Vertical views of
                  a control CUC1 T1 flower with four petals and four sepals (E) and a 5mCUC1 T1 flower with six
                  petals and two sepals

                 (F). Scanning electron micrographs (SEM) of a T2 control CUC1 (G) and a 5mCUC1 (H)
                 inflorescence. Note the altered number and position of developing sepals in 5mCUC1. Horizontal view
                 SEM of T2 control CUC1 (I) and 5mCUC1 (J) floral buds and vertical view SEM of T2 control CUC1
                 (K) and 5mCUC1 (L) stage-12 flowers. Note the absence of a sepal pair in the 5mCUC1 buds causes
                 buds to open. Vertical view SEM of a young T2 5mCUC1 flower (M) and horizontal view SEM of a
                 more mature 5mCUC1 flower (N). Petals (P) and sepals (Se) are indicated. Scale bars = 100 mm.

                        Figure 4. CUC1 mRNA in 5mCUC1 Flowers

                        (A) Phenotypic 5mCUC1 plants accumulate increased steady-state levels of CUC1 mRNA. The
                        indicated mRNAs were analyzed by using real-time quantitative RT-PCR with primers and
                        probes designed around the miRNA complementarity site of CUC1 and normalized to the level of
                        18S rRNA in the sample. Total RNA was prepared from a mix of buds and inflorescence
                        meristems of wt Col-0 plants, control CUC1 1-18 and 6-20, and 5mCUC1 M-20 and A-20
                        transformants lacking phenotypes, 5mCUC1 M-18 and B-19 transformants displaying flower
                        phenotypes, and 5mCUC1 A-32 and P-18 transformants displaying both rosette leaf and flower
                        phenotypes. TUB4 is included as a nontarget control. mRNA levels are displayed in arbitrary
                        units, and error bars represent the standard deviations of three PCR replicates.

                        (B) Relative levels of CUC1 and 5mCUC1 mRNAs in transgenic plants. The same RNA used in
                        (A) was reverse transcribed, and the resulting cDNA was PCR amplified to completion, then
                        digested with Nsp1, which cuts the 5mCUC1 amplicon, but not the CUC1 amplicon (Figure 1C)
                        or heteroduplex molecules that result from annealing of CUC1 and 5mCUC1 strands.
                        Agarose-gel separation and ethidium-bromide staining revealed the full-length PCR product (330
                        bp) and the Nsp1 digestion fragments (220 bp and 110 bp). DNA gel-blot analysis (not shown)
                        was used to quantitate the 330 bp and 220 bp DNAs, and the amount of 5mCUC1 relative to
                        CUC1 (correcting for heteroduplex) is shown below each lane.

mir164b Mutant Plants Have Reduced miR164 Accumulation but Retain Sufficient miR164 to Prevent
Developmental Abnormalities

Mutant plants that do not express miR164 would be expected to display phenotypes that overlap with 5mCUC1 phenotypes. Therefore, we searched the Salk Institute Genomic Analysis Laboratory collection [46] for plants with disruptions in MIR164a and MIR164b, two genomic loci with the potential to encode miR164 [7]. Although no mutants were found with insertions near MIR164a, a mutant (designated mir164b-1) was found with a T-DNA insertion within the MIR164b foldback. Sequencing the left border junction revealed that the T-DNA was inserted in the loop of the predicted hairpin precursor. The reduced miR164 accumulation in this line (Figure 5A) indicated that it may be a miR164b null allele. Although the level of miR164 accumulation was reduced approximately 15-fold in mir164b mutant seedlings (Figure 5A), we did not observe any developmental abnormalities in the mutant plants, indicating that the reduction in miR164 level at this early developmental stage is not sufficient to impact development. In accordance with this result, quantitative RT-PCR analysis of miR164 target mRNA levels in mir164b mutant seedlings did not reveal consistent changes in mRNA steady-state levels of the five predicted miR164 targets (data not shown). The T-DNA insertion in miR164b-1 is located within the loop of the miR164b precursor; thus, it is likely that this line is a null allele and it supports the hypothesis that MIR164a is at least partially redundant with MIR164b. Interestingly, a miR164-like sequence (miR164c) was cloned from Arabidopsis that differs from the sequence of miR164a and miR164b by a single adenine-to-guanine substitution at the 3' end (M. Jones-Rhoades, B. Reinhart, B.B., and D.P.B., unpublished data); therefore, miR164c may also contribute to miR164 target gene regulation.

Figure 5. Reduced miR164 Accumulation miR164b Mutant Plants and Increased miR164 Levels in 35S:MIR164b Plants.

                 (A) RNA gel-blot analysis of 30 mg total RNA prepared from 7-day-old wt Col-0 and miR164b-1
                 (SALK_136105) seedlings.

                 (B) Progeny of 35S:MIR164b plants that display organ fusion phenotypes have increased seedling
                 miR164 accumulation. RNA gel-blot analysis of 30 mg total RNA prepared from 7-day-old wt Col-0
                 seedlings and 7-day-old seedling progeny of 35S:MIR164b plants. Line 7 displayed no fusion
                 phenotypes; line 2 displayed stem-pedicle and weak sepal fusions; line 27 displayed stem-pedicle,
                 stem-leaf, and sepal fusions; and line 8 displayed stem-pedicle, stem-leaf, sepal, stamen, and rosette
                 leaf fusions.

                 (C) 35S:MIR164b plants that display organ fusion phenotypes have increased miR164 accumulation in
                 flowers. RNA gel-blot analysis of 9 mg total RNA prepared from a mix of buds and inflorescence
                 meristems of wt Col-0 and 35S:MIR164b plants. Line 81 displayed no fusion phenotypes; line 61
                 displayed stem-pedicle, stem-leaf, and sepal fusions; and line 8D displayed stem-pedicle, stem-leaf,
                 sepal, stamen, and rosette leaf fusions. The positions of ³²P-labeled RNA oligonucleotides are noted.
                 Blots were stripped and reprobed with oligonucleotides complementary to miR160 and U6 as loading
                 controls.

Constitutive Overexpression of MIR164b Recapitulates cuc1 cuc2 Double Mutant Phenotypes

Because plants expressing a miR164-resistant CUC1 mRNA have altered development, we expected that proper miR164 expression would also be required for normal development. To investigate this hypothesis, the stem loop of the MIR164b gene was cloned behind the CaMV 35S promoter and introduced into wt Arabidopsis plants. Misexpressed miR164 was expected to increase target mRNA cleavage, especially in cells that normally do not express the miRNA, and thereby phenocopy the organ separation defects seen in the cuc1 cuc2 double mutants, including fused cotyledons, sepals, and stamens [40, 43]. Indeed, although most 35S:MIR164b primary transformant seedlings had a wild-type appearance (Figures 6A and 6D) , several displayed partially fused cotyledon petioles (Figures 6B and 6C), similar to the cuc1 cuc2 mutants [40]. Mild cotyledon petiole fusion was correlated with the first true leaves appearing asymmetrically from the shoot apex (Figures 6E and 6F). More dramatic and frequent fusion phenotypes were observed in adult plants. 58 of 85 35S:MIR164b primary
transformants displayed partial or complete sepal fusion (Figures 6Q and 6R; compare to Figures 6L and 6M), and 16 of 85 displayed some degree of stamen fusion (Figure 6S; compare to Figure 6N). These results demonstrate that misexpression of MIR164b can recapitulate certain cuc1 cuc2 double mutant phenotypes, indicating that control of miR164 dosage and localization is necessary for proper CUC activity.

                          Figure 6. Fusion Phenotypes of 35S:MIR164b Plants

                          (A–C) Ten-day-old 35S:MIR164b seedlings displaying no fusion (A) or partial cotyledon
                          petiole fusion (B and C).

                          (D–F) Same seedlings as in (A–C) at 13 days old, displaying true leaf formation to one side of
                          the partially fused cotyledon petioles (E and F).

                          (G) 13-day-old 35S:MIR164b seedling displaying fused cotyledon petioles and no SAM.

                          (H) 29-day-old wt Col-0 plant.

                          (I–K) 35S:MIR164b plants (34 days old) displaying rosette leaf fusion (arrow in [J]) or
                          leaf-stem fusions (arrows in [I] and [K]).

                          (L–P) Wild-type (Col-0) plants. Buds (L) and flowers (M) display separated sepals and six
                          distinct stamen (N) imaged by SEM (arrows denote the points of separation). Pedicles emerge
                          from the inflorescence stems (O) and separated flower parts abscise from the fertilized siliques
                          (P).

                          (Q–U) 35S:MIR164b T1 plants display floral organ fusion defects. Buds (Q) and flowers (R)
                          have various extents of sepal fusion, and removal of sepals and petals exposes fused stamen
                          ([S]; arrows denote points of separation). 35S:MIR164b plants display varying degrees of
                          pedicles fused to stems ([T]; arrow highlights the altered angles between pedicle and stem;
                          compare to [O]). Fused sepals abscise late from 35S:MIR164b siliques ([U]; arrow indicates
                          dried sepals firmly attached to the silique). Occasional stamen-carpel fusions are observed
                          (arrowhead in [U]). The scale bar in (A) represents 1 mm and applies to panels (A)–(C). The
                          scale bar in (D) represents 1 mm and applies to panels (D)–(G). The scale bars in (I) and (J)
                          represent 1 cm, the scale bars in (L)–(N) and (Q)–(S) represent 500 mm, and the scale bars in
                          (O), (P), (T), and (U) represent 1 mm.

miR164-Directed Cleavage of NAC1, At5g07680, and At5g61430 Could Explain Additional Organ-Separation
Defects

Interestingly, 35S:MIR164b plants display organ fusion phenotypes not reported in cuc1 cuc2 mutants [40, 43, 44, 48]. These 35S:MIR164b phenotypes include fused rosette leaves (13/85 T1 plants; Figure 6J), leaf-stem fusions (28/85 T1 plants; Figures 6I and 6K; compare to Figure 6H), stem-pedicle fusions (25/85 T1 plants; Figure 6T; compare to Figure 6O), and occasional stamen-silique fusions (Figure 6U). Floral organs of affected plants did not abscise normally after fertilization but, rather, remained on the parent plant (52/85 T1 plants; Figure 6U; compare to Figure 6P), perhaps due to sepal separation defects (Figures 6Q and 6R).

The organ separation defects in the 35S:MIR164b plants that are not observed in cuc1 cuc2 could be explained by miR164-directed cleavage of additional targets. In addition to CUC1 and CUC2, at least three Arabidopsis NAC-domain genes, NAC1, At5g07680, and At5g61430, have extensive complementarity to miR164 ([25]; Figure 1A). To determine if these mRNAs are also targeted for cleavage in Arabidopsis, we amplified cleavage products from these mRNAs from wt Arabidopsis Col-0 RNA by using the RNA ligase-mediated 5'-RACE protocol that was used to demonstrate miR164-directed cleavage of CUC1 and CUC2 messages [28, 29]. RNA sequences with 5' termini corresponding to the center of the miR164 complementary site were consistently detected for all three genes (Figure 1C), indicating that NAC1, At5g07680, and At5g61430 are in vivo miR164 cleavage targets.

To assess whether the fusion phenotypes that we observed correlated with the level of miR164 overexpression, we examined miR164 levels in progeny seedlings of 35S:MIR164b transformants with strong and weak fusion phenotypes. We found that progeny of plants with a variety of fusion defects accumulate miR164 to a level ~9-fold higher than wt, whereas progeny of 35S:MIR164b plants without fusion phenotypes accumulated miR164 to a level similar to wt plants (Figure 5B). This correlation between miR164 levels and phenotypic severity was observed in RNA prepared from both seedlings (Figure 5B) and floral buds and meristems (Figure 5C), indicating that the miR164 overexpression persisted through development.

Together, our results strongly suggest that proper miR164-directed gene regulation of CUC1, CUC2, and at least one other NAC-domain gene is essential for the separation of adjacent embryonic, vegetative, and floral organs and implicate miR164 as a common regulatory factor in the mechanism(s) controlling organ separation. The cuc1 cuc2–like fusions in 35S:MIR164b plants (Figure 6), the increase in CUC1 mRNA steady-state levels in 5mCUC1 plants with aberrant phenotypes (Figure 4), the detection of miR164-directed mRNA cleavage products in wild-type Arabidopsis (Figure 1), and the increase in CUC2 mRNA accumulation in mutants defective in miRNA biogenesis and function [22], all suggest that miR164 is regulating its target genes, at least in part, by mediating mRNA cleavage. However, we cannot rule out the possibility that miR164, in addition to directing mRNA degradation, may also repress translation, as reported for miR172 [31, 32].

miRNA Regulation of Lateral Organ Boundaries in Plants

miR164 is conserved between Arabidopsis and rice [7], and miR164 targets are found in the NAC-domain family in other plants as well. For example, rice encodes a NAC-domain mRNA (OsNAC2) with a two-mismatch miR164 complementary site [25]. In addition, examination of the petunia NAM  [41] and snapdragon CUPULIFORMIS (CUP) mRNAs [49] reveals that each contain a miR164 complementarity site with three mismatches (Figure 1A). The petunia nam mutant displays cotyledon fusions and usually lacks a SAM [41]. nam shoots that develop display altered numbers of second and third whorl floral organ primordia [41]. The snapdragon cup mutant displays defects in SAM formation and dramatic cotyledon, leaf, inflorescence, and floral organ fusions [49] reminiscent of the diverse organ fusions observed in the 35S:MIR164b plants (Figure 6). It is likely that several genes similar to CUC1/2, NAM, and CUP regulate lateral organ boundaries and that the exact division of labor among the genes varies depending upon the extent and nature of gene duplication in various plant lineages. The occurrence of miR164 complementary sites in these NAC-domain genes implicated in lateral organ boundary formation suggests that the miR164-based regulation of the process uncovered here will be general to flowering plants.

    There are over 100 NAC-domain genes in Arabidopsis, but only some of these are known to be miR164 target genes. One miR164 target is NAC1, which is important in lateral root development; others are Arabidopsis CUC1, CUC2, At5g07680, and At5g61430 (Figure 1C) [29]. Another likely miR164 target, At5g39610, which is closely related to At5g07680 and At5g61430, has a miR164 complementarity site with only four mismatches (including one G:U wobble) and is among the miR164 targets predicted in a recent computational analysis (M. Jones-Rhoades and D.P.B., unpublished data). However, other closely related genes of unknown function (e.g., At3g29035) and CUC3, which has a function partially redundant with CUC1 and CUC2 [50], lack extensive complementarity to miR164. It appears that miR164 regulation of these genes has either been lost over the course of evolution or does not involve extensive complementarity between the miRNA and the messages.

In plants, organ primordia derive from stem cells in the meristematic regions. These pluripotent stem cells develop into determinate organs through a series of coordinated cell-cell signaling events [51]. During embryogenesis, CUC1 and CUC2 are expressed in a few apical cells as early as the globular stage, and as the embryo develops, expression spreads to numerous cells located between the two cotyledon primordia [40, 43, 48]. This CUC1 and CUC2 expression is thought to define boundaries between developing cotyledons, perhaps by restricting cell proliferation, and thereby helping to establish bilateral symmetry [40, 43, 48]. CUC1 and CUC2 also are required for adjacent floral organ separation, suggesting that an analogous boundary definition process occurs during floral development [40, 43, 48], and our data point to other NAC-domain genes mediating additional organ separation events. The identification of miR164 as an integral regulator of a subset of NAC-domain genes, including CUC1 and CUC2, adds a new layer of complexity to the gene regulatory mechanisms that control cell differentiation and organ development in plants.

Perturbing miR164-directed CUC1 or CUC2 regulation in the 5mCUC1 and 35S:MIR164b plants would likely misregulate genes that rely directly or indirectly on proper CUC1 or CUC2 expression. For example, CUC1 or CUC2 is required for expression of SHOOT MERISTEMLESS (STM) [44, 48], which encodes a class I knotted-like homeobox (KNOX) protein that functions in meristematic and interprimordial regions to maintain cells in an undifferentiated state and to prevent these cells from proliferating [52–54]. Loss of STM results in partial cotyledon and floral organ fusions and reduced or absent SAM formation, reminiscent of the defects in 35S:MIR164b plants (Figure 6) [54, 55]. Genetic interactions imply that STM maintains the SAM by inhibiting expression of ASYMMETRIC LEAVES1 (AS1), a MYB-domain transcription factor [56]. as1 mutants develop abnormal rosette leaves with outgrowths or lobes [56], a phenotype similar to that of 5mCUC1 plants
(Figures 3B and 3C). Thus, misexpression of genes known to be downstream of CUC1 or CUC2 is likely to contribute to the cotyledon, leaf, and floral organ defects that we observed (Figures 3 and 6).

Interestingly, the phenotypes observed in the 5mCUC1 plants did not overlap with phenotypes reported for 35S:CUC1 plants, which include lobed cotyledons and ectopic adaxial meristems [43, 44]. The CaMV 35S promoter likely directs higher and more widespread expression than the CUC1 promoter [57], and thus miR164 may not be at sufficient levels in all relevant tissues to counter the message expressed in 35S:CUC1 plants, even though miR164 is present in many organs (Figure 2B). Similarly, plants expressing 35S:NAC1 also have developmental abnormalities [45]. The fact that 35S promoter-driven expression of CUC1 and NAC1 confers developmental anomalies indicates that miRNA-mediated regulation and other forms of posttranscriptional control, such as ubiquitin-mediated proteolysis [58], may not be sufficient for proper development and suggests that transcriptional regulation of both genes is also necessary. Indeed, CUC1 transcriptional control was
demonstrated when in situ hybridization analysis of embryonic CUC1 mRNA accumulation [43] showed CUC1 expression closely resembling the embryonic pattern of CUC1 promoter-driven green fluorescent protein (GFP) accumulation [57], even though the GFP reporter does not contain a miR164 complementary site. Nonetheless, the cotyledon orientation defects observed in 5mCUC1 plants (Figure 3A) reveal a clear role for miR164 during embryonic development that may be too subtle to detect by comparing mRNA in situ analysis with that of GFP reporters, suggesting that miRNAs also may be playing roles in other cases where posttranscriptional regulation has not been suspected. The striking abnormalities observed during floral development of 5mCUC1 plants (Figure 3) also illustrate the importance of miRNA-mediated postembryonic regulation of CUC1. Notably, the dual control of CUC1 and NAC1 by both transcriptional and posttranscriptional mechanisms distinguishes these miR164 targets from several mRNAs targeted by the miR156/157, miR159/JAW, miR165/166, and miR172 families, which do not confer dramatic phenotypes when driven by the 35S promoter but, in the cases tested, do
confer dramatic phenotypes when miRNA-resistant versions are expressed [26, 32, 38, 59].

Petal and sepal whorls have different responses to the increased or expanded presence of the 5mCUC1 miR164-resistant mRNA, perhaps reflecting somewhat differing gene regulatory circuitry controlling the separation of developing petal and sepal primordia. We speculate that the increased number of petals and decreased number of sepals could both be the outcome of increased organ separation in these plants: in one case increased instances of separation and in the other, an increased degree of separation. In the petal whorl, loss of miR164-mediated CUC1 regulation may increase the instances of petal primordia separation, resulting in additional primordia, and ultimately additional petals. In the sepal whorl, loss of miR164-mediated CUC1 regulation may increase the degree of separation between sepal primordia, resulting in fewer sepal primordia. The discovery that miR164-directed mRNA cleavage is important in these processes of organ development and separation will facilitate their molecular characterization.

Conclusions:

Here, we demonstrate that miR164 regulates NAC-domain target genes in Arabidopsis. Perturbation of miR164-directed regulation causes development abnormalities in embryonic, vegetative, and floral organs, thus establishing miR164 as a key regulatory component essential for normal plant development and furthering the understanding of the biological roles of miRNA regulation in plants.

Experimental Procedures:

DNA Constructs and Transgenic Plants

CUC1 and 5mCUC1

The genomic sequence of CUC1 (At3g15170), including ~1.4 kilobases (kb) and ~0.7 kb of putative 5' and 3' regulatory sequences, respectively, was cloned as an ~3.7 kb BamH1-EcoR1 fragment into pBluescriptIISK+ (Stratagene) from the bacterial artificial chromosome F4B12. Site-directed mutagenesis was performed by using PfuUltra polymerase followed by Dpn1 digestion, as suggested by the manufacturer (Stratagene), to produce the 5mCUC1 sequence. The sequence of the primer pair used for CUC1 mutagenesis is 5mCUC1 forward
5'-CCGATCATCAATACCTTTGCGACGGAGCATGTTTCATGTTTTTCAAATAACTCTGCTGCTCATACCG-3'
and 5mCUC1 reverse
5'-CGGTATGAGCAGCAGAGTTATTTGAAAAACATGAAACATGCTCCGTCGCAAAGGTATTGATGATCGG-3'.

After mutagenesis, a 1.5 kb Pac1-Nco1 fragment spanning the mutagenized CUC1 miR164 complementary site was subcloned and used to replace the corresponding wild-type sequence of the original genomic CUC1 clone. This 1.5 kb fragment was sequenced to ensure that only the desired silent mutations were present. The control CUC1 and the 5mCUC1 ~3.7 kb BamH1-EcoR1 fragments were subcloned into the binary vector pGreenII0219 and then electroporated [60] into Agrobacterium tumefaciens strain GV3101::pMP90 [61]. Arabidopsis thaliana (Col-0 accession) was transformed by using the floral dip method [62]. The collected seeds were surface sterilized and plated on Bouturage No. 2 media (Duchefa Biochemie) containing 30 ?g/mL hygromycin for selection of transformants. Seedlings were grown under long-day conditions (16 hr light, 8 hr dark) at 20°C for about 14 days before transfer to Metromix 200 soil (Scotts), where they were grown at 20°C under long-day conditions.

35S:MIR164b

To make the 35S:MIR164b construct, genomic DNA from Col-0 plants was PCR amplified by using oligonucleotides MIR164b-C3  5'-GTCctcgagTCACGTTTTCAAATATCAAACCTAC-3' and MIR164b-C2
5'-TATgcggccgcTCTCCTGTCTAATACTCGCTAACC-3', which have flanking Xho1 or Not1 sites (lowercase). The resulting ~900 basepair (bp) product encoded the predicted miR164b hairpin precursor along with 385 bp of upstream and 355 bp of downstream sequence. The PCR amplification product was gel purified by using a Matrix Gel Extraction kit (Marligen Biosciences, Ijamsville, MD) and subcloned into the pCR4-TOPO vector (Invitrogen). The resultant clones were sequenced to identify PCR-derived errors, and a clone was selected with the expected sequence except for an A and T deleted 374 bp and 192 bp upstream of the foldback, respectively. The XhoI-NotI insert from this clone was ligated into Xho1-Not1-cut 35SpBARN [63] between the CaMV 35S promoter and the nos terminator. The resultant 35S:MIR164b plasmid was electroporated [60] into A. tumefaciens GV3101, which was used to transform Col-0 by using the floral dip method [62]. Transformants were identified on PN (plant nutrient medium) [64] plates containing 7.5 ?g/ml glufosinate ammonium (Crescent Chemical, Augsburg, Germany). Seedlings were grown under continuous light at 22°C for approximately 14 days
before transfer to Metromix 200 soil (Scotts), where they were grown in continuous light at 22°C. Transformation of each T1 plant was confirmed by PCR amplification of genomic DNA [65] with the primers 35S-F  5'-AAGGGATGACGCACAATCCCACTATCC-3' and MIR164b-C2, which yielded a 1 kb product from Col-0 (35S:MIR164b) plants.

mir164b

The mir164b mutant was identified from the SALK_136105 line generated by the SALK Institute Genomic Analysis Laboratory, La Jolla, CA [46]. The genotypes of plants segregating for this insertion obtained from the ABRC were determined by PCR-amplifying genomic DNA by using the primers MIR164b-1
5'-CAAAGAAATGTTGTTACCTGGTAATTAG-3' and a modified version of the LBb1 primer
5'-CAAACCAGCGTGGACCGCTTGCTGCAACTC-3'; http://signal.salk.edu ), which results in an ~700 bp product from the disrupted chromosome, or MIR164b-1 and MIR164b-4 5'-CCATAAAGCTTCAAAATTTACAGAGTTTCC-3',
which results in a 694 bp product from the wild-type chromosome. The former product was directly sequenced by using the LBb1 primer to determine the exact location of the T-DNA insert, which was found to be at position 84 within the 149 nt predicted foldback of MIR164b. Homozygous plants were compared to the wild-type Col-0 after growth on PN medium supplemented with 0.5% sucrose or in soil.

CUC1 Transcript Preparation and In Vitro Cleavage Assay

Arabidopsis CUC1 transcripts containing the miR164 complementary site were generated by PCR amplification of CUC1 or 5mCUC1 genomic clones followed by in vitro transcription by using T7 RNA polymerase. The primer sequences used to generate CUC1 and 5mCUC1 template were
5'-GGCCGTAATACGACTCACTATAGGGGAGAAGGAGTGGTACTTCTTC-3' and
5'-CATTGCAAATGACGGAGGAGG-3'. Wheat germ lysate preparation, cap labeling, and in vitro cleavage assays were performed as described [30].

5' RACE

poly(A)⁺ RNA isolation, cDNA synthesis, non-gene-specific 5'-RACE amplifications, and gene-specific 5'-RACE
amplifications were performed as described [28, 29] by using the GeneRacer Kit (Invitrogen) with the exception that an additional nested gene-specific 5'-RACE amplification was performed. The following primers were used with the GeneRacer 5' Nested Primer:
NAC1 external 5'-CAGTGCTTGGAATACCGATGTCGGTCAG-3',
NAC1 internal 5'-GCTGACTGAGTAGAGCTCTGAGAACCATC-3';
At5g61430 external 5'-GATCAACCGGTCCAGTAGAGGAAGACGG-3',
At5g61430 internal 5'-CAGTGTTCATGTCAGTTGAAACTCCGG-3';
At5g07680 external 5'-GGTTCAAGATCAACCGGACCGGAAGAG-3',
At5g07680 internal 5'-CCCGGTTTCTTGCGAGATGCTCAATG-3'.

RNA Isolation and miRNA Gel-Blot Analysis

Total RNA was isolated as described [66] or by using RNeasy Plant Mini Kits (Qiagen, Valencia, CA). For RNA gel-blot analysis, total RNA was separated on a 15% denaturing polyacrylamide gel, electroblotted to nylon membrane, and hybridized with an end-labeled miR164 DNA probe, and then stripped and reprobed with miR160 and/or U6 DNA probes [7]. Hybridization signals were quantified with a Fuji phosphorimager.

Reverse Transcription and Quantitative Real-Time PCR

For each sample, 0.6 mg total RNA was treated with DNaseI (Amplification Grade, Roche Applied Science, Indianapolis, IN) and reverse transcribed in a 40 ml volume by using 400 units SuperScript III (Invitrogen, Carlsbad, CA) primed with a mixture of reverse primers (QRTR primers listed below; 2 mM each) for the NAC-domain genes, TUB4, and 18S rRNA. The resulting cDNA was diluted to 200 ml with water. Gene-specific primers and TaqMan probes were designed to span the miRNA-complementary site of each mRNA by using Primer Express software (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was carried out in triplicate 25 ml reactions by using the ABI Prism 7000 Sequence Detection System and TaqMan Universal PCR Master Mix (ABI) with primers at a final concentration of 0.5 mM each and using 10 ml diluted cDNA for the mRNAs or 0.001 ml for the 18S reaction. Primer pairs used were

CUC1-QRTF  5'-TCTGCCGGTTCTGCAATTG-3' and
CUC1-QRTR   5'-CATCGGTATGAGCAGCAGAGTT-3',

CUC2-QRTF  5'-CAGCCGTAGCACCAACACAA-3' and
CUC2-QRTR 5'-GTCTAAGCCCAAGGCCCCGTAGTA-3',

NAC1-QRTF   5'-AACCCTCTTCTTATCTCAGTGATGATC-3' and
NAC1-QRTR  5'-AGGTTCGAGTTTAAGGTTTGGTTCT-3',

At5g07680-QRTF   5'-CCACCTTTAACTGATTCTTCACCATA-3', and
At5g07680-QRTR   5'-AGCAATTGAGTATTGTTCCTCTAGTTTCA-3',

At5g61430-QRTF  5'-CCAAAACAGAACCGGTCTACGT-3' and
At5g61430-QRTR   5'-GAAGCAATTGAGTGTGGTTCCTT-3',

TUB4-QRTF   5'-CTGTTTCCGTACCCTCAAGC-3', and
TUB4-QRTR   5'-AGGGAAACGAAGACAGCAAG-3'.

The following probes were paired with the respective primer pairs at a final concentration of 0.2 mM: CUC1-probe  5'-TCCGATCATCAATACCT-3',
CUC2-probe 5'-AGCGCAATAACCGAGC-3',
NAC1-probe 5'-CTACATCATCAATGAGCA-3',
At5g-probe 5'-CTGCTTCTCCAACCAA-3',
and TUB4-probe 5'-CGCTAATCCTACCTTTGGTGATCTTAACCAT-3'.

The NAC-domain gene specific probes were 5' labeled with 6-FAM and 3' labeled with MGBNFQ (minor groove binder/nonfluorescent quencher). The TUB4 probe was 5' labeled with HEX and 3' labeled with TAMRA. The 18S primers and probe were from ABI (TaqMan Ribosomal RNA Control Reagents). PCR conditions were 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. cDNA amplification was monitored in real time by using ABI Prism 7000 Sequence Detection System software. Amplification of 18S rRNA was monitored as an endogenous control that was used to normalize template amounts using the
comparative C_T method (ABI Prism 7700 Sequence Detection System User Bulletin #2, http://www.appliedbiosystems.com).

Control reactions in which reverse transcriptase was omitted did not give amplification signals above the threshold. Direct sequencing of the CUC1 and CUC2 amplification products revealed that the CUC1 and CUC2 primer pairs were gene specific.

RT-PCR, Nsp1 Digestion, and DNA Gel-Blot Analysis

Total RNA was prepared from a mix of buds and inflorescence meristems of 57-day-old T2 5mCUC1, control CUC1, and  wt Col-0 plants as described [66]. 5 mg of total RNA was used for oligo-(dT)20 primed first strand cDNA synthesis followed by RNase H digestion as recommended by the manufacturer (ThermoScript RT system, Invitrogen). PCR amplification with 50 ng of cDNA as a template was performed to completion by using the following CUC1 primer pair:
CUC1 forward   5'-GCGTAGTTAGTAGAGAGACG-3' and
CUC1 reverse   5'-GAGGCAGAGAAGGTAGATTCG-3'.

To equalize the possibility of heteroduplex formation in the 5mCUC1 samples, the final PCR products were denatured and renatured. Nsp1 digestion of the 330 bp 5mCUC1 PCR product yielded ~110 bp and ~220 bp fragments. The wt CUC1 PCR product did not contain an Nsp1 restriction site and thus was not cleaved after
Nsp1 digestion. To monitor Nsp1 digestion efficiency, parallel reactions were conducted in which each reaction was spiked with a 1.2 kb DNA fragment containing an Nsp1 restriction site, which produced ~560 bp and ~600 bp fragments after digestion. This control DNA was cleaved to completion, indicating that the undigested fragments in the 5mCUC1 RT-PCR did not contain the Nsp1 site and derived from the endogenous CUC1 gene. DNA gel-blot analysis was performed as described [66]. Undigested and Nsp1-digested PCR amplified products were separated on a 2% agarose gel, blotted to nylon membrane, and hybridized with 32P end-labeled CUC1 reverse primer (above), which detects both the undigested CUC1 and 5mCUC1 330 bp PCR products and the 220 bp fragment generated by Nsp1 digestion of the 5mCUC1 PCR product. Hybridization signals were quantified with a Fuji phosphorimager. Heteroduplex DNA involving one strand of CUC1 hybridized to one strand of 5mCUC1 was assumed to be refractory to Nsp1 cleavage. The square root of the fraction cut by Nsp1 indicated the total fraction of 5mCUC1 present as either a homoduplex or one strand of a heteroduplex and was used to calculate the reported mRNA ratios.

Scanning Electron Microscopy

Plant tissues were fixed in 3% gluturaldehyde, 25 mM NaPO₄ (pH 6.8) overnight at 4°C with constant shaking, rinsed five times with 50 mM sodium cacodylate buffer (pH 7.0), and treated with 1% OsO₄, 50 mM sodium cacodylate (pH 7.0) for at least 6 hours at 25°C. Tissues were dehydrated through a graded ethanol series (25%, 50%, 75%, 90%, 95%, and 100% ethanol) and then critical point dried by using CO₂. Tissues were mounted on stubs and shadowed with gold and pallidium (4:1) before viewing through a Jeol 5600LV scanning electron microscope.

Acknowledgments:

We thank M. Jones-Rhoades, G. Tang, and P. Zamore for wheat germ extracts; M. Axtell for cDNA used in the RNA ligase-mediated 5' RACE reactions; P. Mullineaux and R. Hellens (John Innes Centre and The Biotechnology and Biological Sciences Research Council) for the pGreenII0129 binary vector; M.E. Lane for microscope use; and N. Watson of the W.M. Keck Foundation Biological Imaging Facility at the Whitehead Institute for scanning electron microscopy technical advice. The Salk Institute Genomic Analysis Laboratory generated the sequence-indexed T-DNA insertion mutant (mir164b), and the Arabidopsis Biological Resource Center at Ohio State University supplied mir164b seeds and the F4B12 clone. We thank H. Vaucheret and members of each Bartel laboratory for helpful advice and critical comments on the manuscript. This research
was supported by the National Institutes of Health (F32-GM071200, A.C.M.; T32-GM08362, D.V.D.; R24-GM069512, B.B. and D.P.B.), the G. Harold and Leila Y. Mathers Charitable Foundation (B.B.), the Robert A. Welch Foundation (C-1309, B.B.), and the Alexander and Margaret Stewart Trust (D.P.B.).

Note Added in Proof:

The data referred to as “M. Jones-Rhoades and D.P.B., unpublished data” are now in press:
Jones-Rhoades, M.W. and Bartel, D.P. (2004). "Computational identification of plant microRNAs and their targets, including a stress-induced miRNA." Molec. Cell, vol. 14, no. 6, pp. 787–799.
http://www.molecule.org/content/article/abstract?uid=PIIS1097276504003284

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NetworkEditor's Perspective: "MicroRNAs in a negative and positive sense":

In this new study by Allison Mallory, Diana Dugas, David Bartel, and Bonnie Bartel, we see both the positive and the negative effects of a microRNA species on the Arabidopsis embryo and its developed organs.
This same ambivalent response to microRNAs has been noted in the following 3 papers by Kuwabara, et al, by Persengiev, et al, and by Geiss, et al. Reference 4 by Czihak reminds us that RNA species have a similar inductive effect in early sea urchin embryos.

Additional References:

1. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells". Cell,  vol. 116, no. 6, pp.779-793  (19 March 2004).

2. Persengiev SP, Zhu X and Green MR, "Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)",  RNA, vol. 10, no. 1, pp. 12-18 (January, 2004).

3. Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, and Sen GC, "A Comprehensive View of Regulation of Gene Expression by Double-Stranded RNA-Mediated Cell Signaling", J. Biol. Chem. vol. 276, pp. 30178-30182 (2001).

4. Czihak G, "Evidence for Inductive Properties of the Micromere-RNA in Sea-urchin Embryos", Naturwissenschaften, vol. 52, no. 6, pp. 141-142 (1965).

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